16.1 Hemostasis: regulation and deregulation

Gert Müller-Berghaus, Lothar Thomas

16.1.1 Introduction and overview

The hemostasis system impedes the loss of blood and disturbance of blood flow through a balanced interaction of blood cells, blood vessels, plasma proteins and low molecular weight substances /1/.

Primary hemostasis: Following a vascular injury, the hemostasis is rapidly activated to initiate the formation of a platelet plug followed by a stable fibrin clot. During primary hemostasis platelets adhere to the extracellular matrix components at the side of injury, and to one onather, plugging vascular defects and often stopping bleeding.

Secondary hemostasis: The secondary hemostasis involves a series of enzyme-mediated activation of clotting factors occurring in distinct but overlapping steps of initiation, amplification, and propagation These stages of homeostasis take place on two main types of cells, tissue factor bearing cells and platelets, both of which control the coagulation process and regulate the regulation amount, and timing of thrombin generation. The ultimate goal is production of sufficient thrombin to convert fibrinogen to fibrin, and emesh the initial platelet plug in a stable fibrin meshwork.

The hemostasis system involves /2/:

• The system of blood vessels including the endothelial cells and subendothelial matrix of the vessel wall, vasoconstriction and the resulting reduction of blood flow
• Blood cells, especially thrombocytes (platelets)
• The plasma coagulation system with interacting platelets, coagulation factors and coagulation inhibitors
• The fibrinolytic system with its activators and inhibitors.

At rest the interaction of these components is a continuous perfectly organized series of events which includes a low level of continuing consumption and formation of all components. The interactions are activated by (e.g., vascular injury) in different ways:

• Normal regulation of hemostasis: activated platelets and plasma coagulation factors, come in contact with the subendothelial matrix causing platelet adhesion to the vessel wall. Platelet activation, the initial step of arrest of bleeding, is mediated by the von Willebrand factor. This leads to the formation of platelet aggregates, also referred to as platelet clot which seal the vascular leak (primary hemostasis). At the same time, the activation of the plasma coagulation system by factor VIIa takes place in combination with the tissue factor (TF) released from TF-containing cells as the cofactor.
• Deregulation of hemostasis: for example, in pathological conditions greatly activated hemostasis leads to the formation of cell aggregates and fibrin-rich clots in the blood vessels causing a reduction in blood flow and resulting in thrombosis and ischemia of tissues.

Hemostasis is subject to regulation similar to many other functional systems within the organism. Humoral and cellular systems, activators and inhibitors as well as positive and negative feedback mechanisms participate in this regulatory process. Defects in participating reactants or deregulation by activation or inhibition of the regulatory systems may result in hemostatic disequilibrium, thus followed by either hemorrhage (bleeding) or thrombosis (blood clot formation).

Hemostatic regulation takes place on the surfaces of TF-containing cells (e.g., on platelets or endothelial cells) or in injuries subendothelially, but not within the fluid phase. Physiologically, hemostatic reactants are activated to a small extent and thrombin is generated causing the conversion of minimal quantities of fibrinogen into fibrin. Fibrin, in turn, is subject to degradation by fibrinolysis. Fibrin synthesis is so minimal that under physiological conditions no blood clot formation is detectable by screening assays.

Interaction of the mechanisms

The vascular endothelium, platelets, plasma coagulation system and fibrinolytic system are not to be viewed as separate systems. The complexity of the reactions can be best exemplified by the hemostatic processes following vascular injury /2/:

• If a blood vessel is injured to such an extent that subendothelial structures are exposed, vasoconstriction will take place initially. Parallel to this, platelet adhesion to the subendothelial structures will occur.
• Platelet activation leads to the perpetuation of the reaction and platelet aggregates form
• Subendothelial structures also initiate the activation of the plasma coagulation system. Contact factors as well as the tissue factor, which is a component of the subendothelial extracellular matrix, are part of this activation process.
• Platelets, activated by subendothelial collagen, express membrane glycoproteins (GPs) such as the GP Ib/IX/V receptor that reacts with the von Willebrand factor (VWF), thus binding and activating the platelets.
• The expression of specific platelet membrane phospholipids represents a surface for complex formation between the reactants. These complexes trigger the initial step of coagulation factor activation.
• During the initiation phase of coagulation, small amounts of thrombin are produced on tissue TF-bearing cells.
• This initial thrombin generation is critical for fully activating adjacent platelets into a highly procoagulant state.
• During the amplification phase, thrombin participates in positive feedback loops and activates FXI, FV, and FVIII. These events induce a marked generation of thrombin and acceleration of the plasma coagulation system.
• If a platelet fibrin clot develops at an intact vessel wall site, it will soon be lysed since endothelial cells release tissue plasminogen activator at an increased rate, thus inducing fibrinolysis activation.
• The platelet fibrin clot formed initially remains intact at the site of the vessel wall injury since the damaged endothelial cells are unable of producing or releasing fibrinolysis activators in adequate quantities. The inevitably associated lack in local fibrinolytic activity around the site of a vascular injury causes the platelet fibrin clot to stay in place.

Refer to Fig. 16.1-1 – Interaction of platelets, plasmatic coagulation system and fibrinolytic system in vessel wall injury.

16.1.2 Vessel wall and endothelial cell

Endothelial cells produce substances with pro aggregatory and anti aggregatory effects, adhesive proteins, and substances with impact on the vascular tone.

Modulation of the vascular tension

Since the muscular layer of arteries and arterioles is thicker than that of veins and venules, the vasomotor reaction of vasoactive substances is greater on the arterial side than on the venous side. Endothelial cells synthesize, among others /2, 3/:

• Nitric oxide (NO), also referred to as endothelium-derived relaxing factor (EDRF). Besides its inhibitory effect on platelet aggregation, EDRF is a potent vasodilator. The release of EDRF from the endothelial cells is stimulated by bradykinin, histamine, acetylcholine, thrombin, vasopressin and ADP.
• Prostacyclin (PGI₂), likewise inhibits platelet aggregation and causes vasodilation
• High-molecular-weight kininogen from which bradykinin with its vasodilatory effects is produced
• Endothelin-1, thromboxane A₂ and angiotensin converting enzyme (ACE). The first two have a direct vasoconstrictor effect. ACE has an indirect vasoconstrictor effect since it transforms angiotensin I into the vasoconstrictor angiotensin II.

Refer to Fig. 16.1-2 – Modulation of vascular tone by vascular endothelium.

16.1.2.1 Interactions between the endothelium and platelets

Adhesive proteins are not only responsible for the adhesion of endothelial cells to the substratum but also contribute to the attachment of platelets to subendothelial structures following a vascular wall injury. Endothelial cells influence the functions of platelets by synthesizing the following activators and inhibitors of platelet aggregation /5/:

• Platelet-activating factor (PAF) released from endothelial cells following their stimulation. PAF also induces the adhesion and aggregation of platelets and stimulates the secretion of EDRF.
• Von Willebrand factor (VWF) which is mostly synthesized by endothelial cells and megakaryocytes. It mediates the adhesion of platelets to subendothelial connective tissue.
• Prostacyclin and EDRF. Both substances are the most potent inhibitors of platelet aggregation and have a vasodilatory effect.

16.1.2.2 Antiaggregatory effect of the endothelium

Endothelial cells exert an antiaggregatory effect by releasing enzymes such as, for example, nucleotidases. ATP originating from platelets or endothelial cells is catabolized into ADP by these nucleotidases with subsequent further breakdown into AMP and adenosine. Adenosine thus being formed can be taken up and eliminated by endothelial cells. ADP is a potent substance triggering platelet aggregation whereas adenosine is an inhibitor of platelet activation by raising the concentration of cAMP. In addition, adenosine also causes vasodilation. Through these processes, endothelial cells contribute to the local regulation of platelet aggregation and platelet disaggregation /6/.

Not only do endothelial cells act on platelets but, in reverse, platelets also influence the function of endothelial cells in many ways. Accordingly, serotonin and noradrenaline, after their release from stimulated platelets, may cause an isometric contraction of endothelial cells. Platelets also secrete substances such as vasopressin, ATP/ADP, PAF, and serotonin, which influence the metabolic capacity of endothelial cells, (e.g., the production of prostacyclin and EDRF) (Fig. 16.1-2 – Modulation of vascular tone by vascular endothelium).

16.1.2.3 Interactions between the endothelium and the coagulation system

Endothelial cells synthesize not only substances with a pro coagulant effect but also those with an anticoagulant effect. The release of these substances is regulated via a negative feedback mechanism.

Pro coagulant activity

Endothelial cells may exert their pro coagulant activities in different ways:

• The endothelial cells, similar to platelets, represent a surface to which coagulation factors bind, where enzyme-substrate complexes are formed and thus where complex formation of reaction partners occurs. Accordingly, prothrombinase, which is composed of F Xa, F Va and Ca²⁺, combines with prothrombin on the surface of endothelial cells /7/.
• A specific receptor of the endothelial cell membrane for F IXa. The receptor-F IXa complex activates F X in the presence of F VIII /8/.
• The F XII activator formed by the endothelial cell. It converts F XII into F XIIa which in turn may activate pre kallikrein and F XI, thus initiating the activation of the intrinsic pathway of the plasma coagulation system /9/.
• The formation of tissue factor (TF) and its expression on the subendothelial extracellular matrix, thus enhancing the plasmatic coagulation system /10/.
• The formation of vitronectin, an adhesive protein neutralizing heparin and heparin-like substances, thus reducing the inhibition of both F Xa and thrombin by antithrombin /11/.

Anticoagulant activity

Via feedback mechanisms, endothelial cells are involved in the modulation of anticoagulant activities. The endothelial cells produce thrombomodulin, heparan sulfate, tissue factor pathway inhibitor and dermatan sulfate which carry different activities.

The membrane receptor thrombomodulin is the most important inhibitor of the coagulation system, binds thrombin with high affinity and exerts the following effects:

• Thrombin is disabled from cleaving fibrinogen and also from activating F V, F VIII and F XIII
• Thrombin-mediated aggregation of platelets will not occur if thrombin is bound to thrombomodulin
• The binding of thrombin to thrombomodulin initiates a negative feedback mechanism which leads to the inhibition of F Va and F VIIIa. After binding to thrombomodulin, thrombin changes its specificity and is capable of activating protein C which together with protein S proteolytically breaks down F Va and F VIIIa. This negative feedback mechanism is an effective regulatory system suited to locally modulate the plasmatic coagulation.
• Thrombin bound to thrombomodulin can be inhibited by antithrombin (AT).

Refer to Fig. 16.1-3 – Anticoagulation activities of the endothelial cell.

Heparan sulfate on the surface of endothelial cells binds thrombin as well as antithrombin and, in a similar manner to heparin, accelerates the inactivation of thrombin. The binding of antithrombin to the endothelial cell membrane, as facilitated by heparan sulfate, may be inhibited by platelet factor 4 (PF4) which is released by platelets, because PF4 competes with heparan sulfate for binding to antithrombin.

The tissue factor pathway inhibitor (TFPI) is synthesized by endothelial cells and continuously released into the plasma. Together with F Xa and F VIIa/TF, it forms a quaternary complex and inhibits the extrinsic pathway of activation. Heparin injections lead to significant rises in plasma TFPI /12/.

Dermatan sulfate expressed on the surface of endothelial cells accelerates thrombin inhibition by heparin cofactor II.

Tissue factor

The TF, a transmembrane cell receptor with a MW of 47 kDa, is the activator of the extrinsic coagulation pathway. TF is activated by phosphatidylserine exposed in vascular injury and binds the activated F VII (F VIIa). The TF/FVIIa complex activates coagulation by proteolysis of F X and F IX. Relevant amounts of TF are only expressed by activated (and not by non-activated) platelets, endothelial cells and monocytes/macrophages.

16.1.3 Platelets

Platelets are very important for intact hemostasis since without them a defect in the vessel wall cannot be repaired. Activated platelets have the following functions /13/:

• In hemostasis the platelets adhere to the injured blood vessel wall, form platelet aggregates and thus stop the bleeding
• In pro coagulation the platelets participate in the formation of a platelet/fibrin clot to seal the damaged vessel wall
• In the inhibition of fibrinolysis. Activated platelets release the plasminogen activator inhibitor type 1 and thus participate in the inhibition of fibrinolysis around the area of the fibrin clot.

16.1.3.1 Platelet receptors

GP Ib/IX/V is the receptor for the VWF. A reduced amount of functional receptors (e.g., in uremia) leads to reduced adhesion of platelets to the sub endothelium.

GP IIb/IIIa (α_IIbβ₃-integrin), is the receptor for fibrinogen. A modified conformation of the receptor (e.g., in uremia) results in failure of the platelets to aggregate.

GP VI and α₂β₁-integrin, are the receptors for collagen.

16.1.3.2 Hemostatic function of platelets

Platelets play an important role in hemostasis and are the first to respond to vessel injury. Activation of platelets results in multiple subpopulations:

• Platelets with expression of the active α_IIbβ₃-integrin and a lack of phosphatidylserine (PS) exposure. These aggregating, or spread, platelets mediate clot retraction.
• PS exposing platelets with characteristic baloon shape.They contain increased cytosolic Ca²⁺ and enhanced ability to bind coagulation factors and promote thrombin generation. A subset of PS exposing platelets are coated with several procoagulant α-granule proteins on their surface, such as fibrinogen, FV and von Willebrand factor.

The susceptibility of a thrombus to fibrinolysis is influenced by the platelet count and fibrin structure. Platelets anchor to fibrinogen via the α_IIbβ₃-integrin; this binding interaction destabilizes the forming thrombus and initiates the process of clot retraction.

The hemostatic function of the platelets is to stop bleeding following tissue injury and terminate exposure of the sub endothelium. The line between these physiological functions and abnormal thrombosis is narrow. Thus, platelets contribute to the formation of atherothrombosis, a leading cause of mortality.

The degree to which the platelets contribute to fatal events by clot formation depends on the location of the clots in the circulation:

• Venous thrombosis is mostly based on the activation of plasma components inducing a pro coagulant status
• In arterial thrombosis, platelets play an important part in the vascular occlusion process in the presence of atheromatous plaques.

The continuum of platelet functions in hemostasis is differentiated into the initiation, extension and consolidation phases.

16.1.3.3 Platelet initiation phase

Following vascular injury, circulating platelets and F VIII bound to von Willebrand factor (VWF) multimers are marginated. The initial contact of the platelets and their binding to the sub endothelium of the blood vessel are mediated by collagen fibrils which bind and activate VWF molecules. Interaction between the platelet receptor GP Ib/IX/V and the VWF bound to subendothelial collagen fibrils leads to the adhesion and activation of the platelets. In the presence of high local VWF concentration, the VWF also binds to the platelet receptor α_IIbβ₃-integrin, which is the physiological binding site for fibrinogen. The platelets aggregate to form a clot at the site of injury. Supported by vasoconstriction, the clot formation impedes blood loss, a procedure also referred to as primary hemostasis.

The GP Ib/IX/V complex, the complex α_IIbβ₃-integrin and the TF are important high-affinity platelet receptors that initiate and extend plasmatic coagulation. GP VI is a specific low-affinity receptor that has potent signalling capacity.

The central event in the platelet aggregation is the binding of fibrinogen to GP IIb/IIIa. This represents the mechanism by which platelet-to-platelet interaction is mediated.

16.1.3.4 Platelet extension phase (propagation phase)

In contrast to platelet adhesion in the initiation phase, platelet aggregation only takes place if prior platelet activation and cell membrane alteration have occurred, (e.g., organization of the complex α_IIbβ₃-integrin has taken place). Because of its special molecular structure as a double molecule, fibrinogen is predestined for exerting a bridging effect between complexes α_IIbβ₃-integrin of two platelets.

The formation of a platelet aggregate at the site of vascular injury mediated by collagen and the exposed VWF is followed by clot formation with further platelets recruited from the bloodstream. The synthesis of small amounts of thrombin at the site of injury initiates further platelet activation and triggers a highly pro coagulatory state of the platelets. The highly active platelets, also referred to as coat-plts (collagen and thrombin activated platelets), are loaded with coagulation factors and have a high granule content. They are capable of attaching to each other, a process referred to as platelet aggregation. This process is activated by the coat-plts releasing soluble agonists such as ADP, thromboxane A₂ ,and adrenalin or binding agonists such as thrombin. Thus, platelet adhesion is enhanced.

16.1.3.5 Platelet consolidation phase

The clot consolidation phase is the last phase in the coagulation cascade. In this phase, signals are generated by integrins after binding fibrinogen. These signals trigger events promoting the growth and consolidation of the clot, such as the reorganization of the cytoskeleton, formation and stabilization of large platelet aggregates, development of a pro coagulatory surface and clot retraction. Thus, the inter platelet space is consolidated and the local concentration of platelet activators is increased.

16.1.3.6 Platelet-based formation of thrombin

The pro coagulatory function of the platelets is the ability to provide the primary surface for thrombin formation /13/. This allows the generation of fibrin and enables effective hemostasis. Thrombin also activates the platelets.

The platelet-based thrombin formation and coagulation phases are shown in Fig. 16.1-4 – Phases of platelet-induced coagulation.

Platelet aggregation is shown in Fig. 16.1-5 – Platelet adhesion and platelet aggregation at the site of vascular wall injury.

Different types of platelet storage granules are involved in the coagulatory function of platelets (Fig. 16.1-6 – Three types of platelet storage granules).

16.1.3.7 Platelet enhanced thrombin formation

Small amounts of thrombin are immediately produced at sites of vascular injury with exposed subendothelial structures (Fig. 16.1-4). During the coagulation initiation phase, F VII binds to platelet TF and is rapidly activated to F VIIa. The F VIIa/TF complex catalyzes the formation of F IIa, the activation of F X to F Xa and of F IX to F IXa. The activity of F Xa is restricted to TF containing cells because once dissociated from the platelet surface fluid-phase F Xa is immediately inhibited by the TF pathway inhibitor and by antithrombin. This does not apply to F IXa.

The two factors have different functions:

• F Xa remains bound to platelet TF and interacts with F Va forming the pro thrombokinase complex (F Xa/F Va) that generates small amounts of prothrombin on platelets
• F IXa does not remain bound to TF, but passes on to the surface of other activated platelets, where it binds to a specific platelet receptor and interacts with F VIIIa forming the tenase complex (F IXa/F VIIIa) that activates F X on the platelet surface.   

Small amounts of thrombin also induce the activation of platelet-bound F XI to F XIa and enhance thrombin formation by activation of F IX to F IXa. Factor IXa passes on to other platelets and contributes to the formation of tenase complexes on the platelet surface.

Large amounts of thrombin required for effective hemostasis are generated by association of F Xa, which is produced by platelet-bound tenase complex, (F VIIa, F IXa, Ca²⁺, phospholipids) and F Va to form the pro thrombokinase complex (F IIA, F VIIa; F IXa, F Xa).

Thrombin formation is not limited to platelets but also occurs on other cells, for example on activated cells of the vessel wall.

16.1.3.8 Platelet activation by thrombin

Thrombin is a potent platelet activator capable of inducing a whole range of platelet functions such as modified structure, TxA₂ synthesis, Ca²⁺ mobilization, protein phosphorylation and platelet aggregation.

The conversion of activated platelets into a pro coagulatory state is associated with specific biochemical and morphological changes. These changes are comparable with those of apoptotic cells and comprise the activation of caspase, proteolysis of the cytoskeleton, exposure of phosphatidylserine (PS) on the membrane surface, contraction of the plasma membrane, membrane blebbing and micro vesiculation. It is assumed that exposure of PS that is trans located from the platelets onto the cell membrane leads to functional conversion to a pro coagulatory surface.

This process is regulated by two different pathways /14/:

• Ca²⁺-dependent, caspase-independent pathways induced by agonists of the platelet function
• Bak/Bax-caspase-mediated pathways independent of platelet activation. Bak and Bax are essential mediators of the intrinsic pathway of cellular apoptosis. Caspases are cysteinyl aspartate specific proteases and important apoptotic enzymes. Caspases are subdivided into initiator caspases (caspase 8, 9) that contribute to triggering apoptosis and effector caspases (caspase 3, 7, 6) that cleave cellular proteins.

16.1.3.9 Negative regulation of platelet activation

A thrombus builds up gradually and can at any time stop growing and become consolidated. EDRF and prostacyclin play an important role in the negative regulation of the platelets. Platelet activation can also be inhibited by the adhesion molecule PECAM-1 (CD31), a potent inhibitor of thrombus formation.

PECAM inhibits the effect of:

• GP VI and GP Ib/IX/V complex, proteins involved in thrombin-mediated platelet activation
• Integrin α_IIbβ₃ (GP IIb/IIIa complex) that mediates platelet inside-out signalling.

16.1.3.10 Secretory function of platelets

Following platelet activation, the contents of the storage granules are secreted into the environment within 10–120 sec. Platelets contain the following morphologically different storage granules:

• ∂-granules secreting ADP, ATP and serotonin
• α-granules secreting VWF, thromboglobulin, platelet factor 4, fibrinogen, high-molecular-weight kininogen, F V, thrombospondin and cellular growth factors such as PDGF, EGF, TGF-β
• Lysosomes excreting acidic hydrolases.

Refer to Fig. 16.1-6 – Three types of platelet storage granules.

16.1.3.11 Aspirin effects on platelets

Platelets and endothelial cells synthesize eicosanoids in response to various stimuli. Stimuli for eicosanoid synthesis are the adhesion of platelets to collagen and the effect of agonists such as thrombin bound platelets and/or endothelial cells. Eicosanoids are oxygenated derivatives of arachidonic acid which contain 20 C-atoms. Eicosanoid synthesis is initiated by the release of arachidonic acid. Under the influence of cyclooxygenase, prostaglandin-endoperoxides are synthesized from arachidonic acid. These prostaglandin- endoperoxides are in subsequent metabolic reactions transformed into prostaglandins, thromboxanes or leukotrienes. Thromboxane and prostacyclin play important counterpart roles in the interaction between endothelial cells and platelets. Attempts have been undertaken to pharmacologically benefit from the differences between these two arachidonic acid derivatives. Aspirin alkylates a reactive serine within the cyclooxygenase, thus irreversibly inactivating this enzyme. Since platelets do not have a nucleus, they are unable to synthesize new cyclooxygenase; therefore, following treatment with aspirin, there is no synthesis of thromboxane and PGI₂. Endothelial cells, however, which preferably synthesize prostacyclin, are capable of producing cyclooxygenase again within a few hours after the intake of aspirin. Low-dose aspirin administration, therefore, makes sense because it blocks the synthesis of thromboxane within the platelets and at the same time allows the biosynthesis of prostacyclin within the endothelial cells to restart relatively fast.

Refer to Fig. 19.1-6 – Cytoplasmic membrane with receptors, G-proteins (α, β, γ) and bound phospholipase A2.

16.1.4 Plasma coagulation system

Two models of the coagulation system are described /1, 15/:

• The cascade model that contains two pathways (extrinsic and intrinsic pathway) acting independently of each other, involving a series of proteolytic reactions that converge to a common point of thrombin generation and fibrin deposition that result from FXa activation of prothrombin (Tab. 16.1-1 – Factors of the coagulation system).
• The alternative or cell based model. The model is based on the non-redundant function of thrombin generated on the surface of TF bearing cells and on platelets. For the formation of fibrin clots, it is essential that these thrombin generating reactions are localized to cells at the site of injury which is not accounted for in the cascade model or factor based assays. The cell based model is physiologically relevant as it recognizes the cells are pivotal to regulating the temporal and spatial activity of the coagulation proteins. This model highlights that assessment of thrombin generation rather than individual clotting factors, would provide a more complete picture of the blood clotting process in vivo.

16.1.4.1 Coagulation factors

Plasma coagulation factors are glycoproteins characterized by different levels and half-lives:

• The activated forms of factors II (prothrombin), VII, IX, X, XI and XII are serine proteases. They are present in blood as proenzymes and are transformed into their active form during the sequence of interactions in the formation of fibrin
• The activated factors V and VIII are not enzymes, they do, however, play an important role in the activation of the blood coagulation cascade
• The factors of the prothrombin complex (II, VII, IX and X) are synthesized by hepatocytes dependent on vitamin K. In the presence of vitamin K, postribosomal carboxylation of glutamic acid (glu) to γ-carboxyglutamic acid residues (gla) takes place. In the absence of vitamin K or in the presence of coumarin derivatives, the transformation into carboxylated proteins does not occur /18/.
• The von Willebrand factor (VWF), also a plasma factor with a pro coagulant effect, plays a critical role in platelet adhesion; in the circulating blood, it is present as a complex with F VIII. The VWF is synthesized by endothelial cells and megakaryocytes while F VIII is produced by the sinusoidal cells of the liver /19/.
• F XIII is considered to be one of the pro coagulant plasma factors. It is a transglutaminase which is primarily synthesized in the liver. It is responsible for cross linking of polymerized fibrin due to the development of covalent binding. Approximately 50% of the entire F XIII level in the blood is present within the cytosol of platelets /20/.

16.1.4.2 Activation of the plasma coagulation system

Blood coagulation is continually ongoing on a very low level /16/. Major activation take place when there is an injury or another stimulus which influences hemostasis. In this case the coagulation factors are measurably activated in the form of a cascade one after the other until the soluble plasma protein fibrinogen is transformed into a visible fibrinous clot. The plasma coagulation system is activated by the extrinsic pathway. The intrinsic pathway enhances the activated coagulation. The subdivision of the coagulation cascade into an intrinsic and an extrinsic activation system does not exist in vivo.

Refer to Fig. 16.1-7 – Cascade of plasma coagulation activation and associated positive and negative feedback mechanisms.

16.1.4.3 Extrinsic pathway

As a rule, the pro coagulatory activity of the tissue factor (TF) on the cell membrane is idle. It is activated by phospholipids in the event of vascular injury or cellular apoptosis. Phosphatidylserine of the inner cell membrane is externalized and exposed to enhance binding of TF and F VIIa as an initial coagulation complex triggering the coagulation cascade /17/. This complex activates F X to F Xa together with F IX by proteolysis. During this process, small amounts of prothrombin are converted into thrombin. Together with F Va, F VIIIa and F IXa, an enhancing loop is formed causing significant amplification of thrombin formation (Fig. 16.1-7 – Cascade of plasma coagulation activation and associated positive and negative feedback mechanisms).

The F Xa together with F Va represents prothrombinase, an enzyme-cofactor complex which converts prothrombin into thrombin /18/.

Significant potentiation of the initial stimulus which occurs in the event of a vessel wall injury or in case of the activation of cellular systems is caused by:

• The sequential activation of pro enzymes (inactive clotting factors) and involvement of enhancing loops
• The formation of coagulation factor complexes on endothelial cell and platelet surfaces, thus resulting in local concentrations of the individual factors which greatly exceed those observed in the plasma. The effect of F Xa is potentiated 300,000-fold by complex formation with prothrombin, F Va, phospholipids, and Ca²⁺.

16.1.4.4 Intrinsic pathway

In the event of vascular injury, the surface-sensitive coagulation factors F XII and F XI are activated by subendothelial structures, a process also referred to as contact activation. Contact activation is considered to be an enhancing loop. However, it is important to keep in mind that, according to clinical observation, only F XI deficiency but not F XII deficiency or a deficiency of pre kallikrein or high-molecular-weight kininogen results in hemorrhagic diathesis. In the intrinsic pathway, factors XI, IX, and X are activated in sequence. F VIIIa together with phospholipids and Ca²⁺ accelerates the activation of F X to F Xa. During this process, F IXa is the enzyme and F VIIIa is the cofactor. Circulating F VIII is bound to von Willebrand factor (VWF), which stabilizes F VIII and binds to the subendothelial matrix to mediate platelet adhesion at sites of injury, hence localizing at these sites. Following activation by F Xa or F IIa-mediated proteolysis via amplification and feed back loops, activated F VIIIa (F VIIIa which becomes detached from VWF) forms the F Xase complex with F IXa on the surface of platelets to potentiate F Xa generation /19/.

16.1.4.5 Regulation of the plasma coagulation system

Plasma coagulation is regulated by the following factors and mechanisms:

• Plasma inhibitors: serpins circulating in the blood as well as other inhibitors are responsible for the inactivation of activated coagulation factors
• Negative feedback mechanisms: parallel to the synthesis of coagulation factors with a proteolytic effect during the activation of the coagulation cascade, proteases are activated which proteolytically cleave co factors, thus down regulating the activation of the coagulation system via a negative feedback mechanism. The mechanism involving protein C is an example of this process
• Localization of the activation of the plasma coagulation system on cell surfaces: the activation of the coagulation system preferentially takes place on the surface of platelets and endothelial cells, whereby the activation of the blood coagulation cascade remains a local event
• Inhibition by coagulation end products: the circulating degradation products of fibrin and fibrinogen inhibit the polymerization of newly formed fibrin as well as the aggregation of platelets and lead to the increased release of plasminogen activators from the vascular wall
• Removal by the reticuloendothelial system: activated coagulation factors locally present in elevated concentrations are "washed out” by the blood stream and are subject to rapid removal from the circulation through the mononuclear phagocytic system in the liver and spleen.

16.1.4.6 Plasma coagulation inhibitors

Various inhibitors of the plasma coagulation system circulate in the plasma or are present within the platelets (Tab. 16.1-2 – Inhibitors of the plasma coagulation). These proteinase inhibitors limit the activation of the blood coagulation system by inhibiting key factors of the enhancing loops by complex formation. They do not completely block the blood coagulation cascade. Instead, they limit the systemic activation to areas where blood coagulation i.e., bleeding cessation, is required. All inhibitors of the blood coagulation cascade are serine proteinase inhibitors (serpins).

Inhibitors include:

• Antithrombin (AT)is the most important inhibitor of the plasma coagulation system. AT inhibits relatively slowly serine proteases, especially, however, F IXa, F Xa as well as thrombin (Fig. 16.1-8 – Cascade of plasma coagulation activation and associated essential inhibitors). In the presence of heparin, the rate of inhibition is significantly increased. This is the reason why AT is also referred to as heparin cofactor I. When thrombin is bound to fibrin, it can be inhibited neither by AT nor by the AT-heparin complex /20/.
• Heparin cofactor II (HC-II). Heparin accelerates the inhibition of serine proteases by this inhibitor. In contrast to AT, HC-II does not inhibit F Xa. Dermatan sulfate is capable of increasing the inhibitory effect of HC-II but not that of AT.
• Tissue factor pathway inhibitor (TFPI), which down regulates the activation of the extrinsic pathway of the coagulation system (Fig. 16.1-8 – Cascade of plasma coagulation activation and associated essential inhibitors). TFPI is synthesized by endothelial cells and directly inhibits F Xa and forms a quarternary complex with F VIIa/TF. The inhibitory effect of TFPI can be increased 5-fold by an injection of heparin since heparin promotes the release of TFPI from the endothelial cells. Approximately 10% of the TFPI circulating in the blood is stored within the platelets and is released from them following stimulation with thrombin.
• Further inhibitors of the plasma coagulation system include α₁-proteinase inhibitor (α₁PI), C1-esterase inhibitor (C1-Inh) and α₂-macroglobulin (α₂M). α₁PI and C1-Inh are involved in the inhibition of the intrinsic activation of the plasma coagulation system. α₂M is considered to be a secondary inhibitor or a back-up inhibitor which is capable of inhibiting kallikrein, thrombin and plasmin.

16.1.4.7 Negative feedback mechanisms of the coagulation system

Two important negative feedback mechanisms of the plasma coagulation system are:

• The protein C system which includes protein C, protein S, and thrombomodulin (Fig. 16.1-9 – Protein C system defects). Protein C and protein S are synthesized in the liver dependent on vitamin K. Protein C, together with thrombin, binds to the endothelial cell membrane receptor thrombomodulin, thus becoming activated protein C (APC). APC is an enzyme which, in conjunction with protein S, cleaves F Va and F VIIIa, thereby again down regulating the “upregulated” activation of the plasma coagulation system, since protein C can only exert its activity after being activated exclusively by thrombin. The protein C system is the most important negative feedback mechanism of the plasma coagulation system.
• Thrombin, which has two different modes of action: as part of a positive feedback mechanism, it inactivates factors V, VIII, and XI; via the protein C mechanism, it inactivates F Va and F VIIIa.

16.1.4.8 Modulation of the activation of the blood coagulation system on cell surfaces

Both platelets and endothelial cells represent an ideal surface for the activation of the blood coagulation system for the following reasons:

• The cells feature special receptors for the coagulation factors and express anionic phospholipids which function as binding sites for F IXa and F Xa
• Activated platelets expose receptors for F Va and F VIIIa
• Activated platelets release platelet factor 4 which neutralizes heparin, thus accounting for this additional prothrombotic effect exerted by platelets
• Non-activated, endothelial cells tend to reveal anticoagulant properties rather than pro coagulant activity. Non-activated endothelial cells express the membrane receptor thrombomodulin which inhibits thrombin and simultaneously activates protein C. Furthermore, AT which has bound to the heparan sulfate of the endothelial cell membrane may inhibit thrombin. The balance between the components with pro- and anticoagulant effects shifts, however, whenever endothelial cell function is impaired by various agonists, thus reflecting activation of the endothelial cell. Tissue thromboplastin, the strongest activator of the plasma coagulation system, may be expressed on cell surfaces following activation of the cells. In addition, on its surface, the activated endothelial cell provides phospholipids and binding sites to F IXa and F Xa, thus allowing the formation of activated coagulation factor complexes similar to the process observed in platelets.

16.1.4.9 Deregulation of the coagulation system

Hemorrhagic diathesis and thrombophilia are the most significant forms of deregulation of the plasma coagulation system.

Hemorrhagic diathesis

Hemmorhagic disorders may result from a decrease in the activity of fibrinogen, prothrombin, factors VII, IX, X, XI and XIII as well as factors V and VIII. Deficiencies in F XII, prekallikrein, and high-molecular-weight kininogen, however, do not cause hemorrhagic diathesis.

Thrombophilia

A thrombotic risk is associated with both an increase in F VIIa activity and elevated concentrations of fibrinogen and F VIII. The expression of tissue factor in many disease states has been recognized as the cause of thrombophilia. The negative feedback mechanism via the protein C system is of utmost importance for the modulation of hemostasis (Fig. 16.1-9 – Protein C system defects). Patients with deficiencies of protein C, protein S or thrombomodulin are afflicted by thrombophilia. APC resistance refers to the inability of activated protein C (APC) to cleave its substrates F Va and F VIIIa. This is caused either by a mutation of the substrates /21/, or APC by circumstances under which APC cannot reach the substrate due to inhibition by antibodies (e.g., as seen in the presence of lupus anticoagulant) /22/.

16.1.5 Fibrin formation and cross linking

Fibrinogen is the plasma protein with the highest plasma concentration in the coagulation system and plays a key role in the hemostatic system. Thrombin-catalyzed cleavage of fibrinopeptides A and B converts fibrinogen into fibrin, which spontaneously polymerizes and forms double stranded protofibrils that assemble into branched fibrin fibers, forming the fibrin clot. Low fibrinogen concentrations are associated with bleeding and high ones are associated with thrombosis /22/. The conversion of fibrinogen to fibrin is shown in Fig. 16.1-10 – Stepwise process of fibrin polymerization and degradation.

Fibrinopeptide cleavage

In two reactions which take place parallel to each other, thrombin initially cleaves off fibrinopeptide A from the fibrinogen molecule. A fibrin monomer remains which, due to a change in configuration, is able to polymerize with other fibrin monomers and, in the same way, also with fibrinogen. The fibrinopeptides are only cleaved off if desAA-fibrin has polymerized. The cleavage of fibrinopeptide B is not essential for the development of a fibrin clot.

Fibrin polymerization

Several fibrin monomers form protofibrils by polymerization which attach to each other by lateral association.

Soluble fibrin

In the presence of high concentrations of fibrinogen or fibrin degradation products, the fibrin polymerization is delayed or even inhibited, thus accounting for the fact that fibrin does not polymerize into a fibrin clot. Fibrin which remains in solution within the plasma is referred to as soluble fibrin (Fig. 16.1-11 – Soluble fibrin). Soluble fibrin is composed of fibrin oligomers, which do not polymerize into a fibrin clot since fibrinogen and/or fibrin degradation products block the fibrin polymerization sites /23/. In the case of limited proteolysis, soluble fibrin in the plasma represents an intermediary product in the formation of a fibrin clot .

Fibrin cross linking

A fibrin clot but also soluble fibrin may be crosslinked due to the action of F XIIIa. The cross linking process represents a transpeptidation (i.e., a covalent binding, between two γ-chains or two α-chains of adjacent fibrinogen or fibrin molecules). The covalent binding is an ε-(γ-glutamyl)-lysyl binding.

Other functions of fibrinogen

Fibrinogen is not only a component of fibrin-rich clots but is also involved in various other biological reactions as an adhesive molecule. Fibrinogen interacts with platelets, endothelial cells, macrophages and fibroblasts. During the process of platelet aggregation, platelets are linked to each other primarily by fibrinogen which binds to the glycoproteins of any two adjacent platelets. Endothelial cells also have a fibrinogen receptor which binds these cells on their contra luminal side to the extracellular matrix. In addition, fibrinogen has an impact on the agglutination of red blood cells and is involved in the process whereby bacteria and malignant cells adhere to cell surfaces and to the extracellular matrix.

Refer to Fig. 16.1-5 – Platelet adhesion and platelet aggregation at the site of vascular wall injury.

16.1.5.1 Transformation of fibrin into fibrin degradation products

If fibrin is formed in the intravascular or extravascular space as part of bleeding arrest or as part of intravascular coagulation or inflammatory reaction, it is degraded by proteolysis after having fulfilled its physiological function. Under these physiological conditions, this is referred to as fibrinolysis whereas the dissolution of a thrombus under pathological conditions is called thrombolysis. Fibrin can also be eliminated by cellular degradation and phagocytosis.

Fibrin is split by plasmin into fibrin degradation products (FDP). If plasmin is present in high concentrations, it may also transform fibrinogen into FDP. Different FDPs are formed depending on whether plasmin splits fibrin crosslinked or not crosslinked by F XIIIa, fibrinogen or soluble fibrin (Fig. 16.1-12 – Non-crosslinked soluble fibrin or fibrin polymer is converted by plasmin to non-crosslinked fibrinolytic degradation products).

FDP have anticoagulant properties due to the fact that they inhibit:

• Polymerization of fibrin by blocking the polymerization sites on the fibrin molecule, thus acting as a solvent mediator for soluble fibrin
• Platelet aggregation since they interfere with the binding of fibrinogen to platelets.

Fibrinolysis leads to the breakdown of:

• Fibrinogen into two D-fragments each plus one E-fragment since the symmetrical fibrinogen molecule is composed of two terminal D-domains and one central E-domain. These fragments are also referred to as D-dimers.
• Crosslinked fibrin into fragments of varying composition by plasmin. In the circulation only FDPs but no D-dimers are detectable; these degradation products are composed of crosslinked Y-fragments and D-domains, thus making Y-D the smallest crosslinked unit detectable in the plasma (Fig. 16.1-10 – Stepwise process of fibrin polymerization and degradation).

16.1.6 Fibrinolysis

Fibrinolysis is an essential physiological mechanism. The fibrinolytic system shares similarities with the coagulation system and like in coagulation requires activation steps for pro enzymes and cofactor functions. The central enzyme is plasminogen, a precursor of the serine protease plasmin.

There are two major pathways which trigger the activation of fibrinolysis:

• The clot formation, a fibrin dependent step
• The release of plasminogen activator inhibitor 1 (PAI-1) from activated platelets.

Fibrinogen has two important functions in hemostasis:

• It degrades fibrin-rich clots after they have fulfilled their physiological function
• It limits clot formation.

Consequently, this implies that fibrinolysis is involved in the wound healing process and recanalization of blood vessels obstructed by thrombus.

In the activation of fibrinolysis, a distinction is made between an intrinsic pathway via contact activation and an extrinsic one via plasminogen activators (Fig. 16.1-13 – Cascade of fibrinolysis activation).

16.1.6.1 Components of the fibrinolytic system

The intrinsic activation pathway of fibrinolysis involves the factors of the so-called contact activation, F XIIa, pre kallikrein and high-molecular-weight kininogen (HK). The components of the fibrinolytic system are listed in Tab. 16.1-3 – Components of the fibrinolytic system.

16.1.6.1.1 Plasminogen

Plasminogen is synthesized by hepatocytes and in its native form has glutamic acid at its N-terminal end, thus also being referred to as Glu-plasminogen. After the activation of Glu-plasminogen by plasmin, Lys-plasminogen with an N-terminal lysine is produced initially /23/. The cleavage of Glu-plasminogen into Lys-plasminogen results in a change in configuration, rendering Lys-plasminogen more susceptible to activation by plasminogen activators. The degradation of fibrin by plasmin enhances the binding of Glu-plasminogen to fibrin, thereby accelerating the thrombolytic process (positive feedback mechanism). By this process, thrombolysis becomes localized to the site of fibrin synthesis. Plasminogen binds to fibrin, α₂-antiplasmin, histidine-rich glycoprotein, thrombospondin, tetranectin, and the extracellular matrix via a region referred to as cringle structure.

16.1.6.1.2 Tissue-type plasminogen activator (t-PA)

Endothelial cells are the primary site of synthesis for t-PA. In addition, t-PA is synthesized by mesothelial cells, megakaryocytes and monocytes. In plasma, t-PA is present as a complex with plasminogen activator inhibitor type 1 (PAI-1); therefore, the concentration of free t-PA in plasma is only 20%. The half-life of t-PA is 4 min., which is similar to that of PAI-1.

Fibrin binds t-PA with high affinity. If fibrin is crosslinked by F XIIIa, the ability of t-PA to bind to fibrin is impaired. The explanation for this is that the binding site for t-PA on fibrin is not accessible under these circumstances or the binding site for t-PA is sterically blocked by the cross linking of α₂-antiplasmin to fibrin.

16.1.6.1.3 Urokinase

Urokinase cleaves plasminogen at the same site as t-PA. Urokinase, also referred to as urinary-type plasminogen activator (u-PA), is synthesized in the epithelial cells of the renal tubules and eliminated by urinary excretion. Urokinase is also synthesized by monocytes. Endothelial cells are capable of producing the precursor of urokinase known as pro-urokinase. If endothelial cells are stimulated, u-PA is secreted preferentially on the contra luminal side while t-PA secretion occurs on the luminal side.

Urokinase is produced from pro-urokinase which is also referred to as single-chain u-PA (scu-PA). Minimal concentrations of plasmin are sufficient to transform the single-chain molecule into a two-chain molecule or so-called two-chain u-PA (tcu-PA), of which only the latter is in fact urokinase. The half-life of u-PA is 5–10 min.

16.1.6.2 Activation of fibrinolysis

Under physiological conditions, continuous activation of fibrinolysis takes place since fibrinolytic degradation products are detectable also under resting conditions. An intrinsic and an extrinsic activation pathway are differentiated (Fig. 16.1-13 – Cascade of fibrinolysis activation).

Intrinsic activation pathway

If fibrin is deposited on endothelial cells, they move away from each other, thus allowing the exposed subendothelial connective tissue to initiate the process of contact activation /25/.

Extrinsic activation pathway

t-PA is continuously secreted at a basal rate in the extrinsic activation pathway. In the event of hypoxia or following neurohumoral stimulation, t-PA is released at an increased rate. Even strenuous physical exercise or venous compression will lead to the release of t-PA from endothelial cells. Glu-plasminogen, the native plasminogen, is transformed auto catalytically or by trace amounts of plasmin into Lys-plasminogen which has an increased affinity for fibrin and which is rapidly transformed further by t-PA in the presence of fibrin.

Urokinase is a second, important activator of the extrinsic fibrinolytic system. Because of the fact that only scu-PA is detectable in the plasma, it is assumed that t-PA and pro-urokinase complement each other in the activation of fibrinolysis. Although urokinase does not bind to fibrin, the activation of pro-urokinase to urokinase occurs much faster in the presence of fibrin. During basal fibrinolysis, minimal amounts of plasmin are possibly produced via t-PA; plasmin transforms pro-urokinase into urokinase. The activation of the contact system leads to the biosynthesis of kallikrein which is a very potent activator of pro-urokinase. The catalytic activity of urokinase is significantly increased when urokinase binds to the u-PA receptor which is detectable on many cells including, for example, endothelial cells and monocytes. Urokinase is involved in the fibrinolysis of fibrin clots since monocytes penetrate fibrin-rich clots and contribute to thrombolysis.

16.1.6.3 Regulation of fibrinolysis

The regulation of fibrinolysis reveals three characteristic features:

• There is continuous activation of the fibrinolytic system, thus resulting in basal fibrinolysis
• Beyond basal fibrinolysis, the fibrinolytic system can only be slowly activated
• If the inhibitors of fibrinolysis are neutralized, activation of the fibrinolytic pathway begins very rapidly and strongly.

Similarly to other biological systems, the fibrinolytic system is subject to regulation and under physiological conditions is in a dynamic steady state. The continuous activation of fibrinolysis is associated with the formation of plasmin.

Fibrinolysis regulation is also suggested by the observations that:

• Patients with congenital α₂-antiplasmin deficiency clinically present with a predisposition to hemorrhagic diathesis
• A disorder of fibrinolysis such as seen, for example, in dysfibrinogenemia-type thrombophilia results in elevated rates of thrombotic episodes.

Systematic examinations allow the conclusion that plasma coagulation and fibrinolysis do not take place systemically and that both are activated locally.

Several mechanisms and systems are involved in the regulation of fibrinolysis:

• The biosynthesis of t-PA is regulated
• Inhibitors circulating in the plasma induce an inhibition of fibrinolysis, thus contributing to its regulation
• Systemic activation is prevented by the localization of fibrinolysis
• Fibrinolysis is modulated by links existing between the fibrinolytic system and activation of the plasma coagulation system as well as the aggregation of platelets.

Fibrinolytic activators locally present in elevated concentrations are little effective because they are “washed out” by the blood stream and are subsequently removed from the circulation by the mononuclear phagocytic system in the liver and spleen.

Regulation of t-PA synthesis

The biosynthesis of t-PA is subject to a circadian rhythm and steroid hormones are involved in the regulation of t-PA synthesis. Stimuli and substances influencing the synthesis and activity of t-PA are:

• Intravenously administered acetylcholine which leads to a rise in fibrinolytic activity
• Epinephrine which releases t-PA from endothelial cells
• Bradykinin, whose production results from the cleavage of high-molecular-weight kininogen during the activation of the contact phase and which represents a potent vasodilator, also releases t-PA from endothelial cells.

In contrast to these stimulants, cAMP causes down regulation of the t-PA synthesis within the endothelial cells.

Modulation of the activation of fibrinolysis

Under physiological conditions, t-PA is a weak activator of plasminogen, as long as both components are not bound to fibrin. Even a 20–100-fold increase in the concentration of t-PA (e.g., following strenuous physical activity) does not lead to a systemic activation of fibrinolysis. The formation of free plasmin with subsequent fibrinolysis will not occur unless there is an excessive, 1,000–5,000-fold rise in the t-PA concentration, as is seen, for example, under therapeutic conditions. Since the body aims to prevent intravascular fibrin formation, t-PA and plasminogen are quickly bound in the event of fibrin production in order to generate plasmin for the degradation of fibrin. The resulting fibrin degradation products limit the further production of fibrin by inhibiting the polymerization of fibrin as well as the aggregation of platelets (Fig. 16.1-14 – Inhibition of fibrin polymerization and platelet aggregation by FDP).

PAI-1 causes additional modulation of fibrinolysis activation. PAI-1 prevents the activation of plasminogen in the circulation due to its molecular excess in contrast to the activators of plasminogen. The concentration of PAI-1 and thus the inhibition of local fibrinolysis in close proximity to a platelet fibrin clot can be increased since approximately 80% of the PAI-1 contained in the blood is stored within platelets which release it when they are activated. Thus, the degradation of the blood clot which is necessary for the wound closure can be prevented.

16.1.6.4 Inhibition of fibrinolysis

Several inhibitors are involved in the inhibition of the fibrinolytic system (Tab. 16.1-4 – Inhibitors of the fibrinolytic system). Most of these inhibitors belong to the family of serpins.

Regulation of fibrinolysis by inhibitors

The regulation of fibrinolysis by inhibitors takes place at several levels. Only about 50% of the plasminogen circulating in the blood are available for transformation into plasmin; the remaining portion is bound to histidine-rich glycoprotein and can only be released slowly for activation.

If activation of fibrinolysis occurs within the fluid phase, plasmin is quickly inhibited by α₂-antiplasmin (α₂-AP) since the binding of plasmin and t-PA to fibrinogen is minimal. If, however, fibrin is present, the affinities of the components of the fibrinolytic system to each other change. Plasminogen and t-PA bind to fibrin, allowing plasmin to be produced in minimal quantities. During the next step, plasmin activates pro-urokinase to urokinase and initiates further activation of fibrinolysis.

α₂-AP may be bound to fibrin by F XIIIa thus allowing it to be concentrated. α₂-AP bound to fibrin is an effective inhibitor of plasmin and contributes to the maintenance of the fibrin clot.

Localization of fibrinolysis

Plasmin which is bound to fibrin can only be partially inhibited by α₂-AP thus maintaining some fibrinolytic activity at the site of the fibrin clot. On the other hand, free plasmin can still be inhibited if α₂-AP is bound covalently to fibrin by F XIIIa.

Free plasmin released during the degradation of the fibrin-rich clot can effectively be inhibited in the circulation by α₂-AP, thus allowing fibrinolysis to remain focussed on the local area of the fibrin clot. Further localization of fibrinolysis is mediated by the platelets which release PAI-1 during the coagulation process.

The different regulation systems guarantee the localization of the fibrinolytic process to the fibrin clot and stabilization of the clot by inhibiting fibrinolysis. Due to this mechanism, fibrinolysis is initiated locally and only with a delay, allowing on the one hand fibrin to fulfill its function as a vascular occluding agent and as a component in inflammatory reactions and on the other hand to be slowly broken down again.

Release of t-PA and activation of the blood coagulation cascade

Thrombin increases the release of t-PA from endothelial cells. The concentration of t-PA in the circulation can be raised even more significantly by an injection of F Xa. The potentiation of plasmin formation after the binding of t-PA to fibrin leads to plasmin being formed at the site of its substrate (i.e., fibrinolysis is achieved very specifically and locally). Plasmin breaks down fibrin into fibrin degradation products (FDPs). If plasmin is present in a high concentration, it can also convert fibrinogen into FDP.

In classic thrombolytic therapy with urokinase or streptokinase, FDPs are preferentially produced.

Clearance by the reticulo-endothelial system

Activated components of the fibrinolytic system as well as enzyme-inhibitor complexes are removed from the circulation by the reticuloendothelial system with various half-lives. If the circulation or the hepatosplenic function are impaired, fibrinolytic activity remains elevated, as is typically observed in severe liver disease.

16.1.6.5 Deregulation of the fibrinolytic system

If the balance between activators and inhibitors is disturbed within the fibrinolytic system, either thrombophilia or a predisposition to bleeding may ensue.

Hemorrhagic diathesis

In severe liver disease, the inhibitors α₂AP and histidine-rich glycoprotein are decreased. The inhibitor PAI-1 evidently is not sufficient to adequately compensate for the pro fibrinolytic effect of t-PA. In orthotopic liver transplantation, massive hyper fibrinolysis occurs during the anhepatic phase. The lack of fibrinolysis inhibitors after removal of the liver is most likely the explanation for these findings. Systemic activation of fibrinolysis is also observed in conjunction with surgery involving organs rich in pro fibrinolytic components, such as the lung and the liver.

Thrombophilia

A decrease in contact activation of the fibrinolytic system causes an increased risk of thrombosis. Accordingly, homozygous F XII deficiency is associated with the risk of thrombophilia /29/.

In the absence of fibrin, t-PA is capable of only slowly activating plasminogen. In the presence of fibrin, the catalytic activity increases about 1,000-fold, involving the formation of a ternary complex between t-PA, plasminogen and fibrin. If the binding sites for plasminogen or t-PA are absent or defective due to a mutation (e.g., as in dysfibrinogenemia-type thrombophilia) the fibrinolytic system cannot effectively be activated. These patients have an increased risk of thrombosis.

The posttraumatic or postoperative reduction in the fibrinolytic activity is probably mediated by the increased synthesis of PAI-1 which occurs as a result of increased cytokine release following tissue injury. The reduction of fibrinolytic activity may possibly represent a protection against tissue dissolution of a fresh hemostatic vascular occlusion. At the same time, it also explains, however, the increased occurrence of postoperative venous thrombosis.

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16.2 Hemorrhagic diathesis overview

Hans D. Bruhn, Lothar Thomas

Disorders of hemostasis result in an abnormal bleeding tendency. They may affect /1/:

• The blood-clotting system leading to coagulation disorders
• The fibrinolytic pathway causing hyper fibrinolysis-induced hemorrhagic disorders
• The platelet count (thrombocytopenia) and the function of platelets (thrombocytopathy): in both cases, the hemostatic system may be dysfunctional causing bleeding predisposition
• The blood vessel wall causing vascular disorders and bleeding predisposition.

Diagnostic investigations of hemorrhagic disorders must detect the various possible causes of a bleeding predisposition. From a differential diagnostic point of view, the following clinical observations must be emphasized:

• Local or widespread hemorrhages (hematomas, suffusions) occur in conjunction with coagulation disorders
• Mucocutaneous petechial hemorrhages (purpura) are associated with thrombocytopenia and thrombocytopathy
• Hemarthroses occur mostly in conjunction with the two coagulation disorders hemophilia A and hemophilia B and are rather rare in thrombocytopenia.

In many cases, the clinical impression provides important information regarding the cause of a bleeding tendency and diagnostic investigations allow differential diagnostic conclusions and final diagnosis.

Further differential diagnostic details are presented in Tab. 16.2-1 – Differential diagnosis of hemorrhagic diathesis based on the clinical picture.

It is important in connection with a clinically manifested predisposition to bleeding to selectively take the patient’s history. Detailed questions in the anamnesis of bleeding should provide information as to whether predisposition to bleeding is familial, as in most hemophilias, or whether it is only the patient who is afflicted. It is also important to know whether the patient recently developed a bleeding tendency, for example under the influence of drugs, and had not presented with any such symptoms in previous years /2/.

16.2.1 Disorders of the plasma coagulation system

Disorders in the synthesis of blood coagulation factors are based on congenital or acquired defects which cause hematomas, suffusions, hemarthroses, deep hematomas, hematuria and bleeding into body cavities.

16.2.1.1 Congenital coagulation disorders

Coagulation disorders are subdivided into:

• Type I: a coagulation factor is not synthesized or synthesized at a reduced rate because of complete or almost complete deletion of the gene. Functional blood coagulation tests and immunochemical assays indicate the same degree of reduction.
• Type II: a variant of the affected coagulation factor is produced with an absent or altered function caused by a minor alteration in the gene. Immunochemical assays indicate low level and possibly even normal level results in comparison to functional tests.

Type I and type II are different in the inheritance patterns but not in their clinical symptomatology (Tab. 16.2-2 – Inheritance patterns of hemorrhagic disorders).

Prevalence: 1 in 8,000 individuals; hemophilia A, hemophilia B and the von Willebrand disease make up 94% of these cases.

16.2.1.1.1 Hemophilia A and B

A distinction must be made between patients with /3/:

• Absent or decreased F VIII protein
• Qualitative disorders where F VIII protein concentration is relative high in relation to the clotting activity.

Hemophilia A and B are inherited in an X-linked recessive pattern or occur spontaneously. The absence or a lack of activity of F VIII:C (hemophilia A) or F IX (hemophilia B) result in hemorrhagic disorders which are characterized by a prolongation of the activated partial thromboplastin time (aPTT) in conjunction with a normal prothrombin time (PT) and a normal bleeding time. The half-life time in plasma for F VIII is 10–12 hours and for F IX 16–18 hours /4/.

The nomenclature and functions of F VIII are listed in Tab. 16.2-3 – Nomenclature and functions of F VIII.

The incidence of X-linked hemophilia A is 1 in 5000 males. The incidence of hemophilia B is 5-fold lower.

Hemophilia A

Hemophilia A can be caused by a multitude of genetic defects. Defects in the gene F8C have been identified. The most frequent mutations in F8C are intron 22 and intron 1 inversions, which occur in 50% and 30% of patients, respectively /5/. The X-chromosomal mode of inheritance in hemophilia A is the reason why only men are clinically affected by the disease whereas women, as carriers, transmit the gene to their offspring. In the differential diagnosis, distinction must be made between hemophilia A and von Willebrand syndrome which in addition leads to a disorder in platelet adhesion and aggregation.

The analysis of individual factors (using plasma deficient in F VIII) allows the determination of the coagulation activity of F VIII (F VIII:C).

Hemophilia A can be subdivided into different degrees of severity depending on the residual activity of F VIII (Tab. 16.2-4 – Classification of hemophilia A and B by severity).

An overview of the desirable F VIII:C levels in the event of various types of bleeding is presented in Tab. 16.2-5 – Therapeutic desirable minimal values in hemophilia A and B depending on the type of bleeding. Specific treatment consists of substituting the missing coagulation factor. The F VIII target concentration and the duration of therapy must be adapted to the given situation. As a rule of thumb for dose calculation, 1 unit of F VIII concentrate/kg body weight increases the F VIII plasma level by 2%. The maximum F VIII concentration is reached immediately after injection.

Detection of female gene carriers: Daughters of hemophiliacs (hemophilia A) and mothers of more than one hemophilic son are obligatory hemophilia gene carriers. In order to detect these carriers, the ratio between functional and immunochemical factor determination is used. The ratio of F VIII:C/F VIII R:Ag may decrease to 0.5 in female gene carriers. The diagnosis can also be verified by molecular biological tests.

Substitution of F VIII

The decrease in F VIII activity is bi-exponential with a short initial half-life of 4–6 h and a second phase with a half-life of 14 h. Mean half-life is 12 h. The F VIII therapy dose should be administered at intervals of 8–12 h. In their consensus recommendations, the German Hemophilia Society recommends a mean initial dose of 20–40 units/kg body weight in hemarthrosis and muscular hemorrhage in patients with severe hemophilia A and 50–70 units/kg body weight in severe to life-threatening hemorrhage. For maintenance therapy, half of the initial dose is administered every 8–12 h until cessation of bleeding, depending on the type of bleeding disorder and the response to therapy.

According to some authors, the initial dose requirement is calculated based on the formula (Dose requirement = A × G × F), with A indicating the desired increase in units, G indicating the body weight in kilograms and F representing a factor of 0.6 in hemophilia A and 1.0 in hemophilia B. Monitoring of the plasma levels of F VIII or F IX or the aPTT are necessary during therapy.

Gene therapy

Gene therapy is a suitable treatment of hemophilia because a minimal expression of F VIII already leads to major improvement of the bleeding and gene expression can be evaluated easily by measuring F VIII concentrations in plasma. According to studies in comparison to previous substitution therapy the number of bleedings reduced to 53–96% in gene therapy. Permanent concentration of F VIII are reported for 6 years /20/.

Hemophilia B

Hemophilia B is a disease with an X-chromosomal recessive mode of inheritance. The diagnosis is based on a prolonged aPTT and the subsequent individual factor analysis. In this test, plasma deficient in F IX is supplemented by patient plasma and the aPTT measured.

Substitution of F IX

In the event of bleeding, F IX substitution is performed employing the same dose as in hemophilia A although the different half-lives result in dissimilar dose intervals. Prophylactic F IX treatment in hemophilia B consists of administering F IX at a dose of 18 units/kg body weight once a week. As a rule of thumb for dose calculation in hemophilia B, 1 unit of F IX concentrate/kg body weight increases the F IX concentration by 1%. The maximum F IX concentration is reached immediately after injection. Contrary to F VIII, F IX has a longer half-life (17 h); therefore, dose intervals are 12–24 h.

Gene therapy

Gene therapy is a suitable treatment of hemophilia because a minimal expression of F IX already leads to major improvement of the bleeding and gene expression can be evaluated easily by measuring F IX concentrations in plasma. According to studies in comparison to previous subsitution therapy the number of bleedings reduced to 53–96% in gene therapy. Permanent concentration of F IX are reported for 8 years /20/.

16.2.1.1.2 Inhibitors to F VIII and F IX

Specific inhibitors of coagulation factors are a rare phenomenon associated with a high risk of bleeding.

F VIII antibodies

F VIII antibodies occur as allo- and autoantibodies. In patients with severe hemophilia, F VIII alloantibodies lead to complete inhibition of the F VIII activity; there is a linear correlation between antibody concentration and the logarithm of residual F VIII activity. Even in the presence of high levels, autoantibodies do not completely block the F VIII activity.

In patients with hemophilia A, F VIII is inhibited by alloantibodies and very rarely by autoantibodies. The risk of inhibitor formation is 20–30%; this type of complication can already occur after an average of 10–15 days of therapy /6/. In rare cases, inhibitors may develop in patients after long years of therapy. The development of an inhibitor compromises the ability to effectively manage hemorrhage because the plasma F VIII concentration does not increase following the infusion of F VIII concentrate.

The incidence of clinically relevant F VIII autoantibodies is 1 to 1 million. The risk of bleeding in affected patients is higher than 80%. F VIII autoantibodies are detected at an increased rate during pregnancy, in rheumatoid arthritis, malignancies, drug-induced, in systemic lupus erythematosus and other autoimmune diseases.

The treatment of patients with bi-specific antibody emicizumab results in reduction of the rate in bleedings in patients with F VIII hemophilia. The antibody binds with one tethering arm to the ligand F IXa and with the other to the ligand F Xa. During this step the F IXa mediated activation of F X and formation of the tenase complex (F VIIIa, F IXa, phospholipids and Ca²⁺) is initiated. In this way the blood clotting cascade works without F VIII /5/.

F IX antibodies

F IX antibodies occur as allo- and autoantibodies. F IX- alloantibodies are detected in hemophilia B patients at an incidence of 1–4%. The inhibitors are oligo- or polyclonal IgG antibodies. Autoantibodies to F IX are rarer than autoantibodies to F VIII. Their occurrence is associated with pregnancy, surgery and autoimmune disease.

Laboratory findings in hemophilia

Isolated prolongation of the aPTT is the main finding. Differentiation from genuine F VIII or F IX deficiency is achieved mixing patient plasma with normal plasma. In order to determine the presence of F VIII and/or F IX inhibitors, the diluted and undiluted patient sample is mixed with normal plasma and then analyzed to find out whether F VIII and/or F IX are inactivated by the patient plasma. A patient sample producing a residual F VIII activity of 50% of the normal value is considered to contain 1 Bethesda unit of F VIII inhibitor/mL by definition. See Section 16.15 – Analysis of individual coagulation factors).

16.2.1.1.3 von Willebrand syndrome (VWS)

The VWS is caused by genetic alterations in the gene VWF that lead to a decrease or qualitative defect of the VWF protein. Predisposition to bleeding results from the loss of two key functions of the VWF protein:

• Binding and consolidation of F VIII
• Binding of platelets to the subendothelial matrix.

The differentiation between the VWF and hemophilia is shown in Tab. 16.2-6 – Differentiation between hemophilia A and von Willebrand syndrome. For further information, see Section 16.18 – von Willebrand factor (VWF).

16.2.1.1.4 Rare hereditary coagulation factor defects

In general, all coagulation factors of the intrinsic and extrinsic pathways may be congenitally decreased, thus resulting in a predisposition to bleeding /7/. These deficiencies include the following: F XI, pre kallikrein (Fletcher factor), high-molecular-weight kininogen (Fitzgerald trait), F VII, F II, F X, F V, fibrinogen, fibrinogen variants (dysfibrinogenamia), F XIII, α₂-antiplasmin.

Hereditary F XI deficiency

This rare disease the prevalence is 1 : 1,000,000. F XI deficiency is autosomal recessively inherited. The bleeding tendency occurs under posttraumatic or postoperative conditions. The half-life of F XI of 60–80 h must be taken into account when administering fresh frozen plasma.

Congenital afibrinogenemia

The prevalence of this rare disorder is 1–2 per 1,000,000. Carriers may have umbilical cord hemorrhage in the first days of life or secondary hemorrhage following venipuncture. Fibrinogen concentrates can be used for therapy to raise fibrinogen concentration to 500–1,000 mg/L.

Hereditary F XIII deficiency

This disorder has a prevalence of 1 : 5,000,000. Major clinical symptoms include wound healing deficits and cerebral hemorrhage. F XIII concentrates are used for therapy.

16.2.1.1.5 Hemophilia-like disorders

Homozygous deficiency states of F II, V, VII and X may cause hemorrhagic diathesis as in hemophilia. Prothrombin concentrates are used for therapy in such cases, and fresh frozen plasma is used in F V deficiency.

16.2.1.1.6 Factor XII deficiency

In many cases, significantly prolonged aPTT points to the correct diagnosis. Subsequent individual factor analysis then reveals F XII deficiency. Acquired F XII deficiency is often based on hepatic damage, including drug-induced damage.

Contrary to other factor deficiencies, congenital F XII deficiency does not imply predisposition to bleeding but rather to thrombosis. F XII deficiency was for the first time described in the patient Hageman. It induced pulmonary embolism in the patient’s older age. Increased activity of the fibrinolytic pathway has been discussed as the underlying cause of thrombophilic tendency in patients with F XII deficiency.

16.2.1.1.7 Pre kallikrein and HMWK deficiency

Clinically, there is no bleeding tendency despite significant changes in aPTT.

Plasma pre kallikrein

Plasma deficient in pre kallikrein is suited for analysis. An amidolytic assay is also available where a pre kallikrein activator converts pre kallikrein to kallikrein. Kallikrein cleaves chomogenic tripeptide substrate releasing p-nitroaniline which is measured at 405 nm /8/.

High molecular weight kininogen (HMWK)

Plasma deficient in HMWK is difficult to prepare. A new easier and reproducible HMWK assay was developed using a chromogenic substrate which is incubated with an excess of F XI and F XIIa in the presence of kaolin in order to form activated F XIa. The formed F XIa is measured under the selected assay prerequisites using the chromogenic substrate glu-Pro-Arg-p-nitroanilide /9/.

16.2.1.1.8 Inhibitor deficiency

α₂-antiplasmin deficiency is an autosomal recessive hereditary disease of the hemophilia type that may cause severe hemorrhagic diathesis.

16.2.1.1.9 Other inhibitor deficiency conditions

Autosomal dominant hereditary defects of antithrombin, heparin cofactor II, protein C and protein S cause a predisposition to the development of thromboses due to a inhibitor deficit resulting in inadequate neutralization of coagulation activation. Homozygous protein C deficiency becomes manifest immediately after the delivery of an affected newborn in the form of purpura fulminans. Factor concentrates and fresh frozen plasma are employed for the treatment. Refer to:

• Tab. 16.1-2 – Inhibitors of the plasma coagulation pathway
• Fig. 16.1-8 – Cascade of plasma coagulation activation and associated essential inhibitors.

16.2.1.2 Acquired disorders of the coagulation system

For disorders of hemostasis following organic diseases, refer to:

• Section 16.5 – Hemostasis in trauma, inflammation and peri operative setting
• Section 16.6 – Hemostasis in liver disease
• Section 16.7 – Hemostasis in renal disease
• Section 16.8 – Hemostasis in tumor patients.

16.2.1.2.1 Vitamin K deficiency and hemostasis

If vitamin K is deficient (e.g., in maldigestion and malabsorption) or if vitamin K antagonists (coumarins) are present, the synthesis of the factors II, VII, IX, X, protein C and protein S is incomplete. As a result of this, no carboxyl group is incorporated in the glutamic acids at the N-terminal end of the factor peptide chains. The coagulation state in vitamin K deficiency is characterized by a decrease in factors II, VII, IX and X in conjunction with normal F V activity. The activities of protein C and protein S are also reduced.

Vitamin K deficiency is observed in conjunction with total parenteral nutrition, impaired absorption, for example, malabsorption, biliary atresia, biliary fistulas and antibiotics-induced changes in the intestinal flora. For treatment, vitamin K is perorally administered in a dose of 10–20 mg; in the case of absorption problem, it is administered parenterally. The intravenous administration of vitamin K may in rare cases cause severe allergic reactions. Therefore, an accurate medical history should be obtained concerning the possibility of such an allergy.

The onset of the effect of perorally or parenterally administered vitamin K takes 36 h or longer and is measurable as clinically relevant improved hemostasis. Hence, parenteral administration of a factor concentrate is the preferred approach in acute hemorrhagic events involving prolonged, vitamin K deficiency induced prothrombin time.

16.2.1.2.2 Coumarins and hemostasis

Coumarins block the vitamin K dependent carboxylation of the coagulation factors II, VII, IX and X as well as protein C and protein S. Depending on their half-life, F VII and protein C decline at first, followed by a decrease in factors X, IX and II. F V remains normal.

Antibiotics can have a similar effect as coumarins by destroying the vitamin K forming intestinal flora, for example β-lactam antibiotics and/or destroying vitamin K epoxide hydrolase (cephalosporins), thus interfering with the synthesis of vitamin K dependent coagulation factors. Inhibition of the hepatic vitamin K epoxide reductase leads to a coumarin-like inhibition of the synthesis of vitamin K dependent coagulation factors.

Coumarin therapy monitoring

Therapy is usually monitored by determining the prothrombin time (PT) and calculating the INR value. The therapeutic range of the PT is defined as 15–30% of normal. A range of 35–45% is recommended in specific indications (low-dose oral anticoagulation) in non-rheumatic atrial fibrillation and long-term venous thromboprophylaxis.

INR value: for monitoring of coumarin therapy using the INR value, see Section 16.10 – Prothrombin time (PT) and Section 16.28 – Monitoring of anti thrombotic therapy.

Risk of bleeding under coumarin therapy

The total risk is 2–3% per year and the risk of cerebral hemorrhage is 0.2–0.4%. In hemorrhagic diathesis induced by coumarins, orally administered vitamin K only leads to slow normalization of the coagulation value. Therefore, administration of fresh frozen plasma together with a prothrombin complex concentrate is recommended.

16.2.1.2.3 Heparin and hemostasis

Heparin therapy is monitored by aPTT. In the treatment of thromboses, a 1.5–2.5-fold prolongation of the aPTT is considered to be the therapeutic range. Heparin can be neutralized by protamine chloride.

Excessive heparin doses resulting in a predisposition to bleeding are possible (e.g., in conjunction with the treatment of thromboses, extra corporeal circulation, procedures requiring cardiopulmonary bypass and hemodialysis). Elevated heparin concentrations prolonging the aPTT are also measured in urticaria pigmentosa. Predisposition to bleeding and thrombosis in heparin-induced thrombocytopenia. See Section 17.5 – Heparin-induced thrombocytopenia).

16.2.1.2.4 Immunologically mediated coagulation disorders

Immunologically mediated coagulation disorders are caused by antibodies (IgG or IgM) which display two different modes of action. They either inactivate a coagulation factor or a receptor at the platelet membrane as part of a time-dependent reaction (neutralizing inhibitors) or they interfere with one of the phases of coagulation (interfering inhibitors), thus causing the clinical picture of severe hemophilia (see Section 16.15 – Analysis of individual coagulation factors).

Neutralizing inhibitors

They are found in hemophilia A, rheumatoid arthritis, lupus erythematosus, ulcerative colitis, polymyositis, bronchial asthma, monoclonal gammopathies, mycosis fungoides, pemphigus, bullous dermatitis, penicillin-related hypersensitivity angiitis and lymphomas.

Interfering inhibitors

Lupus anticoagulants: they are immunoglobulins directed against membrane phospholipids (e.g. found after viral or drug-induced tissue damage). These interfering inhibitors do not cause bleeding but they commonly induce arterial or venous thromboses and in women may lead to spontaneous abortion.

Lupus anticoagulants are found in autoimmune diseases such as systemic lupus erythematosus, lymphoproliferative diseases and infections (see Section 16.22 – Antiphospholipid syndrome.

16.2.1.2.5 Disseminated intravascular coagulation

Disseminated intravascular coagulation (DIC) is an acquired coagulation disorder due to the systemic intravascular activation of the coagulation system along with the formation of thrombi in the microcirculation and secondary hyper fibrinolysis. This processes causes the consumption of coagulation factors and platelets and, with hyper fibrinolysis, the onset of hemorrhagic diathesis. The formation of micro thrombi within life-sustaining organs and hemorrhagic diathesis lead to organ damage and impairment of vital functions due, for example, to respiratory insufficiency (acute respiratory distress syndrome/shock lung) and renal insufficiency.

Characteristic changes in hemostasis parameters are depicted in Fig. 16.2-1 – Characteristic changes in hemostasis parameters in a severe consumption of clotting factors.

For further information, see Section 16.5.3 – Diagnostics in trauma and peri operative setting.

16.2.1.2.6 Kasabach-Merritt syndrome

It is a form of DIC with a chronic course presenting in small infants with the triad of giant hematoma, thrombocytopenic purpura and afibrinogenemia, as a result of the stasis.

16.2.1.2.7 Waterhouse-Friderichsen syndrome

It is characterized by an extremely dramatic course and in 90% of the cases can be traced back to the presence of meningococcal sepsis. Untreated, it results in death within a few hours. Therapeutically, heparin, thrombolytics or antibiotics and, in the case of extremely pronounced adrenal insufficiency, even corticosteroids have all been employed.

16.2.1.2.8 Fulminant purpura

This disorder is a form of DIC with symmetrical necroses involving the extremities and the trunk; it occurs following infectious diseases and is characterized by the simultaneous presence of high fever, leukocytosis, circulatory decompensation and shock.

Hereditary thrombotic thrombocytopenic purpura is a rare autosomal recessive disorder caused by ADAMTS13 mutations that result in the absence or severe deficiency of the plasma metalloprotease ADAMTS13. The protein is required for cleavage of newly synthesized von Willebrand factor multimers. The patient’s age may help to distinguish between hereditary TTP and acquired TTP. Acquired TTP is much less common in young children than in adults. The presence of a functional ADAMTS13 or an increased anti-ADAMTS13 IgG antibody titer argues against the diagnosis of of hereditary TTP /18/.

16.2.2 Thrombocytopathy associated disorders

Thrombocytopathies are disorders of platelet function that may be associated with mild hemorrhagic diathesis. However, hemostasis can be severely impaired depending on the type of platelet dysfunction and the additive effect of other, concurrently present hemostatic disorders. Congenital and acquired thrombocytopathies are distinguished.

Congenital thrombocytopathies are rare, but are often associated with a strong predisposition to bleeding (Tab. 16.2-7 – Congenital thrombocytopathies). Refer also to Section 17.7 – Inherited platelet-based bleeding disorders).

Acquired thrombocytopathies are common and usually characterized by mild predisposition to bleeding (Tab. 16.2-8 – Acquired thrombocytopathies).

16.2.3 Vascular hemorrhagic diathesis

Vascular disorders triggering hemorrhagic diathesis must be differentiated into congenital and acquired forms. A predisposition to bleeding in vascular disorders can derive from circumscribed morphological alterations involving the blood vessel walls, on changes in vascular permeability or on changes in vessel fragility.

Diagnostically, vascular hemorrhagic disorder may be present in cases with:

• Prolonged bleeding time in conjunction with a normal coagulation profile (PT, aPTT)
• Normal platelet count and function
• Normal von Willebrand factor.

The following tests can be used for differentiating vascular hemorrhagic diathesis from platelet-induced hemorrhagic diathesis.

Bleeding time

The subaqueous bleeding time according to Marx is a screening test. In this test, the fingertip is pricked with a sterile vaccination lancet and then immersed into a cup with sterilized water (37 °C), with the blood flowing into the surrounding water like a thread. The sudden end of the thread represents the end of the bleeding time.

Upper reference interval: up to 4 min.

Clinical significance: the bleeding time comprises the reaction of the vessel wall, the number and function of the platelets and the presence of the von Willebrand factor.

Rumpel-Leede test

To perform the test, a blood pressure cuff is applied around the patient’s upper arm and inflated to 10 mm Hg above the patient’s diastolic blood pressure for 5 min. The test is positive if clearly visible petechia are detected in the cubital fossa after removing the cuff /10/.

Clinical significance: vascular disorder can be discussed if the number and function of the platelets are normal and analyses do not point to von Willebrand syndrome.

16.2.4 Diagnostic tests for disorders of hemostasis

If a predisposition to bleeding is suspected, the following diagnostic tests must be performed depending on the patient’s bleeding history /11/:

• Tests for primary hemostasis, comprising activation, adhesion and aggregation of platelets
• Tests for the coagulation system, and importantly familial history (pointing to congenital disorders)
• Drug history because acquired tendency to bleeding is usually induced by drugs.

The first step is to perform basic diagnostic screening assays if there is no positive bleeding history. This applies, for example to preoperative preparation: if the bleeding history is positive, further diagnostic testing is required. This also applies to all occasions when the basic test results are within the reference interval.

Important basic and extended diagnostic tests are listed in Tab. 16.2-9 – Diagnostic tests for hemorrhagic diathesis.

16.2.5 Findings in disorders of the plasma coagulation system

Findings in disorders of plasma coagulation are listed in Tab. 16.2-10 – Tests and findings in suspected coagulopathy /15/.

16.2.6 Fibrinolysis therapy monitoring

The proenzyme plasminogen in human blood is activated to plasmin by therapeutically administered activators such as urokinase, streptokinase or tissue-type plasminogen activator for thrombolytic therapy of arterial or venous vascular occlusions /16/.

The characteristic features of thrombolytic agents are presented in Tab. 16.2-11 – Characteristics of thrombolytic agents.

The indications for thrombolytic therapy are shown in Tab. 16.2-12 – Indication for fibrinolysis therapy.

The contra indications are listed in Tab. 16.2-13 – Contra-indications of thrombolytic therapy.

Tissue plasminogen activator (t-PA)

t-PA preferentially activates plasminogen which is bound to fibrin, thus exerting mostly localized fibrinolytic activity at appropriate dose for therapeutic solubilization of a clot. At higher doses, t-PA also induces systemic fibrinolytic activity.

Urokinase and streptokinase

As therapeutic fibrinolysis activators, they cause a systemic activation of plasminogen which subsequently exerts its effect on the coagulation factors. Fibrinogen, F V and F VIII and to a lesser degree factors II, VII, IX, X and kininogen are cleaved.

Systemic administration of urokinase causes the conversion of plasminogen to plasmin. The administration of streptokinase causes the formation of streptokinase-plasminogen complexes. Plasmin is formed by modifying the conformation of plasminogen due to autocatalytic proteolytic activation. The resulting plasmin effect leads to a decrease in fibrinogen with a simultaneous increase in fibrinogen degradation products. Fibrinogen degradation products have an inhibitory effect on coagulation since they can interfere with the reaction between thrombin and fibrinogen. This effect of the fibrinogen degradation products leads to a prolongation of the thrombin time (TT). The aPTT may be prolonged in a similar way; while in general this also applies to the PT, but the prolongation is less pronounced /17/.

The TT has the highest sensitivity regarding therapeutically induced fibrinolytic and/or thrombolytic activity. Hence, the TT determination should have priority in fibrinolytic/thrombolytic therapy monitoring over all other tests. However, the aPTT and PT also provide important information on the therapy-related changes in the hemostasis system.

Tests for monitoring fibrinolytic therapy

The following tests should be performed before and during thrombolytic therapy:

• Complete blood count including the platelet count, PT, aPTT, fibrinogen, blood group determination and urinalysis
• 4 h after the start of therapy and then, afterwards, twice each day, monitoring should be performed by the following assays: TT, aPTT, possibly fibrinogen and urinalysis
• Often, a heparin infusion is applied simultaneously with thrombolytic therapy, therefore possibly making it necessary for therapeutic reasons to assess the thrombolytic activity separately from the heparin effect. In order to do so, the reptilase (batroxobin) time can be determined. Both enzymes have a thrombin-like effect, however, they are not affected by heparin. For this reason, they are suited to assist in the differentiation between fibrin degradation products which have accumulated at a higher rate due to thrombolysis and the effect of thrombin inhibitors such as heparin and hirudin which do not prolong the batroxobin time. Besides thrombolytic therapy, low fibrinogen concentrations and dysfibrinogenemias may prolong the batroxobin time.

None of the available laboratory tests (i.e., neither the TT nor the aPTT) can predict the success or failure of thrombolytic therapy or indicate impending bleeding. However, a lack of TT prolongation suggests inadequate activation of the fibrinolytic system during thrombolytic therapy, while significant prolongation of the aPTT to more than 2 min. or of the PT to more than 60 sec indicates at least a predisposition of the hemostatic system to possible bleeding.

Under such circumstances, further tests should be performed, for example:

• Plasminogen concentration analysis; congenital or acquired plasminogen deficiency, possibly dysplasminogenemia
• Determination of the anti-streptokinase titer in the case of low-dose streptokinase therapy; during high-dose streptokinase therapy, the anti-streptokinase titer is usually easily masked.

Local causes for bleeding

Bleeding (e.g., resulting from atherosclerotic changes in cerebral blood vessels) can, due to the nature, not be detected by any laboratory test but may lead to the feared complication of cerebral hemorrhage in patients undergoing thrombolytic therapy, especially those > 60 years of age. Cerebral hemorrhage is primarily feared to occur within the scope of acute lysis of a recent myocardial infarction, especially in concurrent administration of a thrombolytic agent and an anticoagulant such as heparin.

In general, local causes for bleeding which may complicate thrombolytic therapy (e.g., gastric ulcer, renal stones, malignant tumor, diabetic retinopathy) are not detected by any laboratory test. In this regard, the information obtained during the medical history and clinical analysis prior to start of therapy is often more important than any of the laboratory tests.

Prior to the start of thrombolytic therapy, the following should be taken into account:

• Existing therapy with inhibitors of platelet function (e.g., aspirin, clopidogrel) since these medications may significantly increase the risk of bleeding during thrombolytic therapy
• Predisposition to bleeding known to the patient.

A possible bleeding tendency during thrombolytic therapy is indicated by:

• Markedly decreased fibrinogen levels (≤ 0.5 g/L)
• Marked prolongation of the PT
• Marked prolongation of the aPTT and TT.

16.2.7 Coumarin therapy monitoring

Coumarin derivatives, as vitamin K antagonists, block the γ-carboxylation of the coagulation factors II, VII, IX and X during their synthesis in the hepatocytes. This results in the loss of the Ca²⁺-binding ability of these factors, thus causing the formation of functionally inactive factors (PIVKA; protein-induced by vitamin K absence).

Depending on the half-life of the vitamin K dependent coagulation factors, it ranges from 6 h in F VII to 70 h in F II, the coagulability of plasma decreases due to this anticoagulant effect. Adequate and stable anticoagulation is usually not reached until 3–4 days after initiation of therapy.

The coagulation inhibitor protein C, also a vitamin K dependent protein, decreases rapidly, like F VII, because of its short half-life (7 h) whereas prothrombin (70 h) and F X (50 h) decline more slowly. Accordingly, during the first few days of oral anticoagulant therapy, a state of blood hyper coagulability may develop, especially in the case of preexisting protein C deficiency, leading to the possible development of coumarin necrosis.

The clinical indications for oral anticoagulant therapy with coumarins are presented in Tab. 16.2-14 – Indications for oral anticoagulant therapy with coumarins, while the corresponding contra indications are summarized in Tab. 16.2-15 – Contraindications of oral anticoagulant therapy.

Therapy is usually monitored by determining the PT. The therapeutic range for the PT was defined as 15–30% of normal. The standardization of the PT and, thus, the comparability of the available PT is achieved by mathematical conversion into a so-called International Normalized Ratio (INR).

The therapeutic ranges are shown in Tab. 16.2-16 – Indication-related therapeutic reference intervals, defined based on various INR intervals.

The PT is not suited to control the efficacy of low-dose oral anticoagulant therapy. The advantage of low-dose oral anticoagulant therapy lies in the fact that the bleeding tendency is lower than at higher dose. Indications, for which low-dose oral anticoagulant therapy is recommended, include:

• Secondary prophylaxis of deep venous thrombosis
• Peri operative thrombosis
• Peri operative thromboprophylaxis as part of surgical procedures
• Permanent central venous catheters
• Non-rheumatic atrial fibrillation, especially in elderly patients, possibly also certain types of heart valve implants.

Orally administered thrombin inhibitors

Contrary to vitamin-K antagonist therapy with orally administered coumarins, orally administered, direct thrombin antagonists have a much more specific site of action regarding the inhibition of thrombin-mediated clot formation and thromboembolism. For further information. See Section 16.28 – Monitoring of antithrombotic therapy.

16.2.8 Heparin therapy monitoring

Unfractionated heparin is a mixture of mucopolysaccharides with different chain lengths; these mucopolysaccharides are mainly collected from the intestinal mucosa of pigs or the lungs of cattle. The molecular weight of unfractionated heparin ranges from 3 to 30 kDa while that of low molecular weight heparin ranges from 1.5 to 12 kDa. Unfractionated heparin and low molecular weight heparin differ with regard to half-life, bio availability and their effects exerted on platelets and lipolysis (Tab. 16.2-17 – Characteristics of heparins).

Low molecular weight heparin has the following advantages:

• Higher t-PA release from vascular endothelium
• No increase in platelet aggregation
• No release of platelet factor 4
• Low probability of osteoporosis in long-term treatment
• Less frequent occurrence of heparin-induced thrombocytopenia.

Unfractionated heparin exerts its effect by significantly accelerating the inactivation of activated coagulation factors, especially of F Xa and thrombin; it does so by forming a complex with the naturally occurring coagulation inhibitor antithrombin (heparin-antithrombin complex). Significantly lower heparin doses can inhibit F Xa in comparison to those required for the inhibition of thrombin. This is the mechanism on which the effect of low-dose heparin is based in thromboprophylaxis.

Distinction is made between high-dose heparin therapy (30,000–50,000 IU/24 h) and low-dose heparin therapy or heparin prophylaxis using approximately 15,000 IU/24 h (up to 20,000 or 25,000 IU/24 h).

Indications for high-dose heparin therapy are listed in Tab. 16.2-18 – Indication for high-dose heparin therapy.

Indications for thromboprophylaxis using low-dose heparin are presented in Tab. 16.2-19 – Indication for thromboprophylaxis with low-dose heparin.

Monitoring of high-dose heparin therapy

The aPTT is used for monitoring high-dose heparin therapy. In the treatment of thromboses, a 1.5–2.5-fold prolongation of the aPTT is considered to be the therapeutic range. Alternatively, the heparin effect can be monitored by determining the thrombin time. Some laboratories measure two thrombin times parallel to each other i.e., one with a low and one with a high thrombin concentration, thus allowing the determination of lower and higher heparin activity levels.

Monitoring of low-dose heparin prophylaxis with fractionated heparin

Low-dose heparin therapy usually does not require monitoring as long as hemorrhagic diathesis or a bleeding tendency have been excluded in the patients prior to start of therapy. If monitoring is required (e.g., in patients with chronic renal insufficiency) an anti-F Xa test is used for determination. See Section 16.28 – Monitoring of antithrombotic therapy.

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15. Barthels M. Gerinnungsdiagnostik. Hämostaseologie 2004; 24: 123–34.

16. Takada A, Takada Y, Urano T. The physiological aspects of fibrinolysis. Thrombos Res 1994; 76: 1–31.

17. Hirsch DR, Goldhaber Z. Laboratory parameters to monitor safety and efficacy during thrombolytic therapy. Chest 1991; 99: 113S–20S.

18. Kremer Hovinga JA, George JN. Hereditary thrombotic thrombocytopenic purpura. N Engl J Med 2019; 381: 1653–62.

19. Botero JP, Lee K, Branchford B, Bray BF, Freson K, Lambert MP, et al. Glanzmann thrombasthenia: genetic basis and clinical correlates. Haematologica 2020; 105 (4): 888–94.

20. Leebeek FWG, Miesbach W. Gene therapy for hemophilia: a review on clinical benefit, limitations, and remaining issues. Blood 2021; 138 (11): 923–31.

16.3 Hemostasis in childhood

Lothar Thomas

16.3.1 Plasma coagulation factors

The maternal coagulation factors do not cross the placental barrier. Independent fibrinogen synthesis starts at embryonic week 5–6 and coagulability of the blood starts at gestational week 11. Fetal plasma concentrations which are present from gestational week 19 are markedly lower than those of the full-term newborn. The concentration of the following plasma coagulation factors in full-term newborns is only approximately 50% of that in adults /1/:

• Vitamin K-dependent factors II, VII, IX and X
• Contact factors XI, XII and pre kallikrein.

During the first 6 months of life, the concentration of vitamin K-dependent factors and contact factors increases to 80% and remains approximately at this level throughout childhood.

The levels of fibrinogen, factors V, VIII, XIII and von Willebrand factor (VWF) in newborns and children are not decreased and correspond to those in adults.

The VWF and its high-molecular-weight multimers are elevated during the first two months of life and then gradually decline to adulthood concentrations.

The neonatal form of fibrinogen in newborns differs from the form present in adulthood by higher sialylation. The fibrinogen concentration at birth is similar to that in adults, increases in the first weeks of life and then decreases again to adulthood levels.

The physiologically decreased plasma concentration of coagulation factors during the first 6 months of life has the following consequences /2/:

• Mild prolongation of the prothombin time (PT) due to a decrease in vitamin K dependent factors
• Prolongation of the activated partial thromboplastin time (aPTT) due to the decreased concentration of contact factors.

16.3.2 Plasma inhibitors

During the first weeks of life, antithrombin, protein C, protein S and heparin cofactor II are decreased to levels corresponding to those of heterozygous deficiency in adults. The concentration of protein C during childhood and adolescence is lower than that in adults.

C1-esterase inhibitor and α₂-macroglobulin have roughly adult concentrations at birth and increase to 150% and 200%, respectively, at the age of 6 months. α₂-macroglobulin is a more potent thrombin inhibitor in neonates and children than in adults and compensates antithrombin deficiency /2/.

16.3.3 Thrombin formation

Thrombin is decreased in a similar way as the coagulation factors and inhibitors. Based on adult concentrations representing 100%, the thrombin levels are /1/:

• 80% in childhood
• 35% in full-term neonates
• 25% in pre term infants

16.3.4 Fibrinolytic system

In the neonate, plasminogen occurs in a fetal form with increased sialylation and in an adult form. The neonatal levels are 50% of adult ones /1/. Plasminogen activator inhibitor is elevated and α₂-antiplasmin concentration at birth corresponds to 80% of the adult value.

16.3.5 Reference interval

Reference intervals for coagulation factors are listed

• of the fetus in Tab. 16.3-1 – Reference intervals for coagulation factors and coagulation inhibitors
• of children in Tab. 16.3-2 – Reference values for coagulation testing in children in comparison to adults.

16.3.6 Diagnostic work-up

The medical history is especially important for investigating a predisposition to bleeding in children because their hemostatic system has not yet been exposed to major challenges.

The type of bleeding is of differential diagnostic significance:

• Mucosal bleeding (gingiva, menorrhagia), petechiae and nosebleed point to thrombocytopenia or von Willebrand syndrome
• Spontaneous deep muscular hemorrhage and hemarthrosis, widespread suffusions and hematomas rather point to factor deficiency (e.g., hemophilia).

The onset of symptoms is also significant /1/:

• An acute onset and persistence for several days suggests an acquired disorder. This applies, for example, vitamin K deficiency, liver disease and disseminated intravascular coagulation. Common causes of liver disease include viral hepatitis, hypoxic liver damage, biliary atresia and total parenteral nutrition. In many cases, renal diseases are associated with platelet dysfunction, and a malabsorption syndrome can cause bleeding due to vitamin K deficiency.
• Although acquired hemostatic disorders occur in great excess over congenital problems, the latter need to be considered because they frequently present initially during the first weeks or months of life. The most congenital hemorrhagic disorders to present with bleeding at birth or during early infancy are deficiencies of F VIII and F IX. In contrast, von Willebrand disease, which is the most common congenital hemorrhagic disorder to present during childhood and in adults, rarely presents with bleeding during the first weeks of life.

16.3.7 Laboratory diagnostic investigations

The following laboratory investigations should be performed in children with bleeding disorders depending on the anamnesis and clinical findings /3/.

Complete blood count

The complete blood count is significant in determining thrombocytopenia and anemia. Anemia in combination with bleeding (e.g., frequent nosebleed) points to primary hemostatic disorders, whereas microcytic anemia points to long-term loss of blood. The combination of anemia and thrombocytopenia can indicate impaired megakaryopoiesis in leukemia, lymphoma or toxic bone marrow damage.

Blood smear

A blood smear can be used for estimating the platelet count and platelet quality in suspected thrombocytopenia or thrombocytopathy. Using a 100x objective, one platelet/field will give an estimated platelet count of 15 × 10⁹/L. Platelet clumps, which are not taken into account by the analyzer, are visible under the microscope. Giant platelets are seen in the Bernard-Soulier syndrome, May-Hegglin syndrome and after major bleeding.

PT and aPTT

The clotting time in one of the two tests is prolonged if the activity of the relevant coagulation factor analyzed in the test is decreased to below 40%.

Plasma mixing test

If PT or aPTT are prolonged, a plasma mixing study should be performed using a mixture of patient plasma and the same amount of pool plasma of healthy individuals. Thus, any factor deficiency is compensated. Normalization of the clotting time indicated by aPTT or PT points to factor deficiency. If the clotting time does not normalize, the presence of an inhibitor to a coagulation factor must be assumed. In most children, coagulation factor inhibitors will not cause hemorrhagic diathesis.

Factor analysis

Factor analysis is required depending on the information from family history or the results of the plasma mixing test and is performed using a modified PT or aPTT test. If the aPTT plasma mixing test points to factor deficiency, it is important:

• To analyze the sample for a deficiency in factors VIII, IX and XI because a deficiency in these factors may be associated with bleeding
• To determine the factors XII, pre kallikrein and high molecular weight kininogen. A deficiency in these factors can also lead to prolonged aPTT but is not associated with bleeding.

Fibrinogen

The functional activity of fibrinogen is determined by measuring clot formation. Clot formation is decreased in the rare disorder dysfibrinogenemia, but the immunologically determined fibrinogen concentration is normal.

Laboratory tests for primary hemostasis

The following tests are required to determine platelet dysfunction and the von Willebrand syndrome:

• Bleeding time: used as a screening test; however, the diagnostic reliability of this test is low
• Platelet aggregation test: the test should primarily be focused on determining von Willebrand syndrome. If the result is negative, further investigation of the platelet function is necessary.

16.3.8 Clinical significance

Hemorrhage

Severe congenital hemostatic disorders can already manifest in the peri-/postnatal period. For example, umbilical cord bleeding, suffusions, gastrointestinal and more rarely intracerebral bleeding are typical symptoms of a deficiency in factors II, V, VII, X and XIII and afibrinogenemia /4/. Hemophilia A and B will be diagnosed no later than after injury in toddlers. Inborn disturbances of platelet function e.g., Bernard-Soulier syndrome (VWF receptor defect) and Morbus Glanzmann-Naegeli (deficiency of GPIIb/IIIa) can be conspicuous with petechial bleeding after birth.

Acquired coagulatory bleeding, mostly results from organic disease of the child in the setting of hepatic disease, hematopoiesis, lymphatic system disorder, severe infection, or after medication with valproic acid or asparaginase.

Tab. 16.3-3 – Differential diagnosis in children with a history of bleeding shows combinations of pathological results and their significance in differential diagnosis.

Prolonged PT and/or aPTT occasionally measured in asymptomatic children can be caused by:

• Presence of an inhibitor not associated with bleeding
• Deficiency in contact factors (e.g., high molecular weight kininogen, pre kallikrein or F XII): the aPTT is prolonged
• Vitamin K deficiency. The National Institute for Health and Excellence recommends a dose of 1 mg of konakion postnatally administered by intramuscular injection. According to a study /6/, 11 in 3.15 million newborns in the United Kingdom had vitamin K deficiency bleeding (VKDB). Six babies with early bleeding received no VK prophylaxis, two babies with late VKDB received incomplete oral prophylaxis and three babies with late VKDB had liver disease

Neonatal major bleeding occurs in about 5–15% of pre term neonates admitted to a neonatal intensive care unit. A dynamic prediction of bleeding risk model was developed that included the variables thrombocyte count, gestational age, postnatal age, intrauterine growth retardation, necrotizing enterocolitis, sepsis and mechanical ventilation. The median cross-validated c-index was 0.74. A c-Index of 1.0 indicates perfect discrimination between newborns with and without major bleeding /7/.

Thrombosis

The incidence of symptomatic thromboembolism in children is lower than in adults /4/. It is 5.1 per 100,000 births, but 5% of sick newborns develop a thrombosis /5/. The congenital risk factors in children are the same as in adults. Most of the pre term neonates have a thrombocyte count below 50 × 10⁹/L

References

1. Male C, Johnston M, Sparling C, Brooker LA, Andrew M, Massicotte P. The influence of developmental hemostasis on the laboratory diagnosis and management of hemostatic disorders during infancy and childhood. Clin Lab Med 1999; 19: 39–69.

2. Kuhnle S, Male C, Mitchell S. Developmental hemostasis: Pro- and anticoagulant systems during childhood. Semin Thrombos Hemostas 2003; 29: 329–37.

3. Allen GA, Glader B. Approach to the bleeding child. Pediatr Clin N Am 2002; 49: 1239–56.

4. Kurnik K. Hämostaseologie in der Pädiatrie. Hämostaseologie 2004; 24: 116–22.

5. Nowak-Göttl U, von Kries R, Göbel U. Neonatal symptomatic thromboembolism in Germany. Arch Dis Childh 1997; 76: F163–7.

6. Busfield A, Samuel R, McNinch A, Tripp JH. Vitamin K deficiency after NICE guidance and withdrawal of konakion neonatal: British pediatric surveillance unit study, 2006–2008. Arch Dis Child 2013; 98: 41–7.

7. Fustolo-Gunnink SF, Fijnvandraat K, Putter H, Ree IM, Deelder CC, Andriessen P, et al Dynamic prediction of bleeding risk in throbocaytopenic preterm neonates. Haematologica 2019; 104 (11) 2300–6.

16.4 Hemostasis in pregnancy

Lothar Thomas

During normal pregnancy hormonal and physiological modifications cause changes in thrombocyte count, coagulation and fibrinolytic activity leading to a pro coagulant state Tab. 16.4-1 – Pro coagulants, inhibitors, and fibrinolysis activators /1/. The advantage is a decreased probability of bleeding during delivery, but the drawback is an increased risk of venous thromboembolism /2/.

16.4.1 Thrombocyte count

Thrombocytopenia is observed in 6–15% of pregnant women at the end of pregnancy and is defined as a platelet count below 150 × 10⁹/L. In mild thrombocytopenia, the number lies within a range of down to 75 × 10⁹/L. Criteria for gestational thrombocytopenia are /3/:

• Asymptomatic thrombocytopenia > 75 × 10⁹/L
• No history of thrombocytopenia in the past except during a previous pregnancy
• Thrombocytopenia occurred during the last trimester
• No fetal or neonatal thrombocythemia
• Spontaneous resolution postpartum.

Thrombocytopenia based on pathogenic events is determined in /3/:

• Preeclampsia and HELLP syndrome (approximately 20% of the cases)
• Idiopathic thrombocytopenic purpura (ITP) (5% of the cases). Approximately 5–40% of the neonates from mothers with ITP have a platelet count below 150 × 10⁹/L.

16.4.2 Determination of coagulation activity

Serial measurements of coagulation activity are determined in serious pregnancy complications e.g., postpartum hemorrhage, disseminated intravascular coagulation, amniotic fluid embolism, preeclampsia.

Prothrombin time (PT): normal clotting time with a tendency to become shorter in the last trimester /4/.

International normalized ratio (INR): normal with a tendency to become decreased in the last trimester /4/.

Activated thromboplastin time (aPTT): normal clotting time with a tendency to become shorter in the last trimester /4/.

Fibrinogen: the concentration increases about the factor 1.5 e.g., from 3.1 g/L in the gestational weeks 5–9 to 4.5 g/L in the gestational weeks 37–40 /4/. Normalization 6 weeks post partum.

Indicators of fibrinolysis activation: tissue plasminogen activator (t-PA) and urinary plasminogen activator (u-PA) are increased. D-dimer concentration increases continuously and finally markedly in the early postpartal period /5/.

In preeclampsia, increased amounts of F VIII are consumed and the levels of markers indicating increased thrombin activity (thrombin-antithrombin complex and prothrombin fragments F₁₊₂) are increased /6/.

16.4.3 Tests in suspicion of hereditary bleeding disorders

Factor VIII and von Willebrand factor are determined if a hereditary bleeding or a carrier status is suspected.

Factor VIII:C: the concentration increases about the factor 2,3 e.g., from 138% in the gestational weeks 5–9 to 320% in the gestational weeks 37–40 /4/. Normalization 6 weeks post partum. Reported symptomatic cases of hemophilia A in pregnant women are not based on homozygously mutated alleles, but on extensive lyonization. Acquired inhibitors against F VIII occur at a prevalence of 1 in 1 million and cause severe bleeding. In most cases, the affected patients are older than 50 years (i.e., pregnant women are affected even more rarely). The plasma mixing test is used for diagnosing.

Von Wllebrand factor antigen (vWF:Ag): the level increases about the factor 2.6 e.g., from 98% in the gestational weeks 5–9 to 257% in the gestational weeks 37–40 /4/. Normalization 6 weeks post partum. The clinical presentation of the von Willebrand syndrome varies greatly in pregnant women. Most cases are asymptomatic, except in extreme situations such as surgery. Occurring bleeding is of the platelet type i.e., mucosal bleeding, susceptibility to bruises, increased bleeding postpartum. See also Section 16.18 – von Willebrand factor).

Very rare coagulation factor deficiencies: hereditary F XIII deficiency (prevalence 1 in 2 million pregnancies) and fibrinogen deficiency (afibrinogenemia, hypo fibrinogenemia, dysfibrinogenemia) are associated with the inability to carry pregnancies to term /7/.

16.4.4 Venous thromboembolism (VTE)

VTE is a leading cause of morbidity and mortality in pregnancy and puerperium. The risk of VTE events in pregnancy is approximately 5-fold higher than in non-pregnant women. The incidence of pregnancy associated VTE ranges from 1 in 1,000 to 1 in 2,000 deliveries. In absolute numbers per 1,000 pregnancies, the prevalence is /8/:

• 0.57 prepartum (venous thrombosis 0.50, pulmonary embolism 0.07)
• 0.29 postpartum (venous thrombosis 0.21, pulmonary embolism 0.08).

Maternal deep venous thrombosis is more common in the left leg. Pulmonary embolism occurs in about 16% of pregnant women with untreated deep venous thrombosis and remains the most frequent cause of maternal death.

Due to the shorter period postpartum, the temporal risk of VTE is 3-fold higher and that of pulmonary embolism is 8-fold higher than during pregnancy /8/. Risk determinants of VTE include:

• Age above 35 years
• Cesarean section
• Obesity (above 80 kg)
• Multiparity (more than 4 pregnancies)
• Deep vein thrombosis in medical history.

In women with a prior idiopathic VTE who carry an additional hereditary risk factor or who have a positive family history of thrombosis, a high risk of VTE in pregnancy can be expected (> 10%); approximately 70% of the women with VTE in pregnancy have such history. Previous thromboembolism, in particular, represents an increased risk of recurrent VTE during pregnancy.

Congenital and acquired risk factors are listed in Tab. 16.4-2 – Acquired and congenital risk factors for thromboembolic disease in pregnancy. However, the majority of pregnant women heterozygous for a risk factor (e.g., factor V Leiden) do not suffer from a thromboembolic event. The occurrence of such an event is enhanced by the presence of several genetic or acquired risk factors /8, 9/.

Causes of predisposition to bleeding and thrombophilia in women suffering recurrent miscarriages are listed in Tab. 16.4-3– Hemorrhagic disorder and thrombophilia in women suffering recurrent miscarriages.

The risk of VTE depending on the hereditary disorder and antiphospholipid syndrome is shown in Tab. 16.4-4 – Risk of venous thromboembolism during pregnancy depending on the disorder and the odds ratios in the presence of hereditary thrombophilia are listed in Tab. 16.4-5 – Odds ratios for complications in pregnancy in the presence of hereditary thrombophilia.

16.4.5 Pathophysiology

Successful pregnancy outcome is dependent on the development and maintenance of adequate placental circulation. The inability of the vascular system, abnormalities of placental vasculature and of the hemostatic system to ensure adequate hemostasis may result in a number of gestational pathologies, including first and second trimesters miscarriages, intrauterine growth retardations, intrauterine fetal death, pre-eclampsia and detachment of the placenta.

Recurrent miscarriage is caused by chromosomal aberration (7%), approximately 15% in hormonal defects (progesterone, estrogen), diabetes mellitus, thyroid disorders, unknown reasons (about 6%) and blood coagulation or platelet defects (55–62%) /10/.

During pregnancy the plasma concentrations and activities of proteins involved in blood coagulation and fibrinolysis change. These changes promote /11/:

• Increase in coagulation (elevation in fibrinogen, F VIII:C and von Willebrand factor)
• Decrease in anticoagulation (reduction in antithrombin and protein S)
• Inhibition of fibrinolysis (increase in plasminogen activator inhibitors PAI-1 and PAI-2).

In total the hemostatic equilibrium is shifted toward hyper coagulability, presumably to prevent hemorrhagic complications during childbirth. Therefore, the pregnant women has a higher potential of pro coagulant factors and an increased fibrinogen concentration /11/. Activation of the blood coagulation system increases continuously with gestational age, while fibrinolytic activity is decreased.

During labor, the blood flow is initially stopped by myometrial contraction and vessels are then blocked with a thrombus by the coagulation system. There is a significant consumption of platelets, coagulation factors and fibrinogen during delivery.

Fibrinolytic activity increases again after the child has been born and the placenta expelled and all hemostatic processes that had changed during pregnancy return to normal 4–6 weeks postpartum.

References

1. Franchini M. Haemostasis in pregnancy. Thromb Haemost 2006 95: 401–13.

2. Jacobsen AF, Skjeldestad FE, Sandset PM. Incidence and risk patterns of venous thromboembolism in pregnancy and puerperium- a register-based case control study. Am J Obstet Gynecol 2008; 198: 233 e1–7.

3. Boehlen F. Thrombocytopenia during pregnancy. Hämostaseologie 2006; 26: 72–4.

4. Kristoffersen AH, Petersen PH, Bjorge L, Roraas T, Sandberg S. Within-subject biological variation of activated partial thromboplastin time, prothrombin time, fibrinogen, factor VII and von Willebrand factor in pregnant women. Clin Chem Lab Med 2018; 56: 1297–1308.

5. O’Riordan MN, Higgins JR. Hemostasis in normal and abnormal pregnancy. Best Practice & Research Clinical Obstetrics & Gynecology 2003; 17: 385–96.

6. Bremme K, Blomback M. Hemostatic abnormalities may predict chronic hypertension after preeclampsia. Gynecologic and Obstetric Investigation 1996; 41: 20–6.

7. Inbal A, Muszbek L. Coagulation factor deficiencies and pregnancy loss. Semin Thrombos Hämostas 2003; 29: 171–4.

8. Zotz RB, Gerhardt A, Scharf RE. Venöse Thrombose in der Schwangerschaft. Hämostaseologie 2006; 26: 63–71.

9. Luxembourg B, Lindhoff-Last E. Genomische Diagnostik thrombophiler Gerinnungsstörungen bei Frauen. Hämostaseologie 2007; 27: 22–31.

10. Bick RL, Hoppenstaedt D. Recurrent miscarriage syndrome and infertility due to blood coagulation protein/platelet defects: a review and update. Clin Appl Thrombosis/Hemostasis: 2005; 11: 1–13.

11. Cerneca F, Ricci G, Simeone R, et al. Coagulation and fibrinolysis changes in normal pregnancy, increased levels of procoagulants and reduced levels of inhibitors during pregnancy induce a hypercoagulable state, combined with a reactive fibrinolysis. Eur J Obstet Gynecol Reprod Biol 1997; 73: 31–6.

12. Hellgren M. Hemostasis during normal pregnancy and puerperium. Semin Thrombos Hemostas 2003; 29: 125–30.

13. Nizzi FA, Mues G. Hemorrhagic problems in obstetrics, exclusive of disseminated intravascular coagulation. Hematol Oncol Clin North Am 2000; 14: 1171–82.

14. Robertson L, Wu O, Langhorne P, et al. For the Thrombosis Risk and Economic Assessment of Thrombophilia Screening (TREATS) study. Thrombophilia in pregnancy: a systemic review. Br J Haematol 2005; 132: 171–96.

16.5 Hemostasis in trauma, inflammation and perioperative setting

Lothar Thomas

Coagulopathy is a condition in which the blood’s ability to clot is impaired. Coagulopathies resulting from injured sites leading to major bleeding are often triggered by massive transfusion after major surgery, severe trauma, gastrointestinal or obstetric hemorrhage. Four major risk factors for coagulopathy and their relative risk (RR) are identified:

• pH below 7.1 (RR 12.3)
• Core temperature below 34 °C (RR 8.7)
• Injury severity score higher than 25 (RR 7.7)
• Systolic blood pressure below 70 mmHg (RR 5.8).

The coagulopathy of trauma is a syndrome of non-surgical bleeding from mucosal lesions, serosal surfaces, and wound and vascular access sites, the tissue oozing that continues after identifiable vascular bleeding has been controlled. It occurs in the presence of profoundly decreased platelet count and coagulation factors /1/.

The differential diagnosis of excessive bleeding from non-injured sites includes /1/:

• Congenital and acquired causes of bleeding
• Bleeding due to anticoagulants or inhibitors of platelet aggregation
• Bleeding due to hypothermia, acidosis or massive transfusion used to treat hypovolemia
• Disseminated intravascular coagulation (DIC) due to initial tissue injury or treatment thereof. An early and a late type of DIC are distinguished. The early type is associated with initial tissue injury and the late one results from organ failure.

The outcome of the interaction between cellular components and coagulation factors of the hemostatic pathways in massive tissue injury is decisive as to whether the body responds with adequate hemostasis, bleeding or thrombosis. Massive bleeding is the second most common cause of death following severe trauma. Patients die in the course of a bloody vicious cycle of bleeding, emergency therapy, hemodilution, coagulation disorder and continuous bleeding.

Coagulation disorder in severe trauma is mainly triggered by the following risk factors:

• pH value ≤ 7.2. At such pH values, the activity of the F VIIa/TF complex, the F Xa/Va complex and F VIIa decreases by up to 90% and interaction with phospholipids is impaired
• Hypothermia ≤ 34 °C. The activity of the coagulation factors decreases and especially the interaction between the von Willebrand factor and the platelet glycoprotein Ib/IX complex is reduced
• Systolic blood pressure < 70 mmHg
• Massive transfusion. When the blood components, erythrocytes, thrombocytes, and fresh frozen plasma, are mixed in an equal unit ratio in massively transfused trauma patients, the resulting hematocrit, thrombocyte count, and coagulation factor activities will all be below common transfusion triggers. Thus, dilutional reductions in clotting activity are inevitable in massively transfused patients and coagulopathy is very likely /1/.

16.5.1 Preoperative diagnostics

The best approach of determining the coagulation state prior to planned surgery and identifying patients with increased risk of peri operative bleeding is by anamnesis and medical examination /2/. For laboratory tests, see Tab. 16.5-1 – Preoperative use of coagulation tests.

16.5.2 Diagnostics in trauma and peri operative setting

In trauma or a peri operative setting, bleeding may occur due to dilution resulting from a decrease in coagulation factors or due to consumption resulting from the release of tissue factor from ischemic tissue induced by shock. Hyper fibrinolysis, presumably resulting from protein C activation, may also make a major contribution to bleeding.

16.5.2.1 Coagulopathy of trauma

Coagulopathy of trauma is a syndrome of non-surgical bleeding from mucosal lesions, serosal surfaces, and wound and vascular access sites associated with serious injury, hypothermia, acidosis, hemodilution and occasionally with classic disseminated intravascular coagulation /1, 7/. Bleeding in massively injured patients increases with additional coagulopathy. Acute coagulopathy of trauma correlates independently with an 8-fold increase in mortality within 24 h and a quadrupled total mortality /7/. Tissue injury results in hypo perfusion and hyper fibrinolysis which, in turn, correlate with the severity of the trauma. Approximately 30% of the severely traumatized patients suffer from coagulation disorders on admission to the emergency department. Coagulation disorders in severe trauma may have multiple causes and can result from a combination of factor depletion and dilutional coagulopathy, hypothermia, acidosis and hyperfibrinolysis /7/. Main effects on the hemostatic system such as hypothermia ≤ 34 °C, pH ≤ 7.2, ionized calcium ≤ 0.9 mmol/L and anemia ≤ 100 g/L are not identified by laboratory coagulation tests.

The systemic damage of vascular endothelium caused by the bleeding shock and systemic inflammation is the main pathologic event of acute trauma /8/. Vessel wall injury activates the blood coagulation cascade through the release of tissue factor. Since the fibrinolytic system is also activated at the same time (above all, plasminogen is activated by the tissue plasminogen activator) in order to maintain the hemostatic equilibrium, coagulation factors and platelets will rapidly decrease in severely injured patients.

All visceral surgery should be carried out taking monotherapy using acetylsalicylic acid. Before endoscopic interventions with low risk of bleeding (below 1.5%) other inhibitors of thrombocyte aggregation (e.g., Clopidogrel, Prasurgel) also should be paused. Before visceral surgery a pause is required. Heparin bridging is reserved to high risk patients /15/.

16.5.2.2 Dilutional coagulopathy

The term dilutional coagulopathy summarizes coagulation disorders developing during surgery in the absence of a disease that might impair the coagulation pathways. This coagulopathy is caused by massive transfusion used to treat hypovolemia. The extent, severity and changes in loss of blood and hemodilution are based on hypovolemia. The loss of ≤ 750 mL of blood (up to 15% of the blood volume) does not cause major changes. A loss of blood of 15–30% results in the stimulation of the sympathetic nervous system in conjunction with tachycardia and mild hypotension, and hemorrhagic shock in conjunction with coagulation disorders occurs at a loss of > 40% of the blood volume.

Laboratory findings

Hemostaseological assays and findings in peri operative patients are presented in Tab. 16.5-2 – Perioperative patients: diagnostically significant tests and findings

16.5.2.3 Disseminated intravascular coagulation (DIC)

DIC is defined by the International Society on Thrombosis and Hemostasis (ISTH) as “an acquired syndrome characterized by the intravascular activation of coagulation with loss of localization arising from different causes”. This condition typically originates in the microvasculature and can cause damage of such severity that it leads to organ dysfunction. DIC usually presents as hemorrhage, with only 5–10% of cases presenting with micro thrombi. Sepsis is the most common cause of DIC. The up regulation of tissue factor activates coagulation, leading to the widespread deposition of fibrin and to microvascular thromboses and may contribute to organ dysfunction, such as development of renal insufficiency and ARDS, hypotension, and circulatory failure. The consumption of coagulation factors and platelets produces a bleeding tendency, with thrombocytopenia, prolonged PT and aPTT, hypo fibrinogenemia and elevated levels of fibrin degradation products, such as D-dimers. The coagulation localizing capacity is lost due to the loss of antithrombin.

Classic DIC comprises the following steps /11/:

• Activation of the coagulation system and hyper coagulability
• Secondary response of the fibrinolytic system resulting in temporarily elevated coagulation and fibrinolysis biomarkers
• Impaired fibrinolysis caused by elevated levels of plasminogen activator inhibitor.

This has the following consequences:

• Thrombotic complications due to excessive fibrin formation
• Consumption of coagulation factors (consumptive coagulopathy) and platelets. Bleeding primarily occurs at wounds and vascular accesses, but profuse hemorrhage is also possible.

The diagnostic relevance of laboratory tests in DIC is shown in Tab. 16.5-3 – Hemostaseological assays in patients with DIC and in sepsis.

The ISTH/SSC score system facilitates assessment of the diagnostic laboratory test results (Tab. 16.5-4 – Diagnostic algorithm for the diagnosis of overt DIC). A D-dimer level above the reference intervals is assigned the point value 1 and a level 5-fold higher than the upper reference interval is assigned the point value 2 /11/.

It is important for therapy to maintain a platelet count above 5 × 10⁹/L and to prevent prolongation of the PT and aPTT to more than 1.5-fold the normal level and a decrease in the fibrinogen concentration to below 1.5 g/L by the administration of fresh frozen plasma.

16.5.3 Sepsis

Hemostasis is influenced by systemic bacterial and fungal infections. This can manifest in many ways /12/, ranging

• from subclinical activation in conjunction with an increase in biomarkers of activated coagulation pathways
• to fulminant DIC culminating in intravascular fibrin formation and deposition in the microvasculature, multiple organ dysfunction syndrome (MODS), hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP) and vasculitis.

Coagulation activation in systemic inflammation can be based on /13/:

• Systemic inflammatory response syndrome (SIRS) from trauma, acute pancreatitis, snake bite. Coagulation activation is indirectly influenced by pro inflammatory cytokines such as TNF-α, IL-6 and by the complement system.
• Sepsis mediated by components of microorganisms (lipopolysaccharides, endotoxin) or by bacterial exotoxins such as the staphylococcal α-hemolysin.

The clinical picture can be dominated by hemorrhage, thrombosis or a combination of the two. None of the syndromes (DIC, MODS, HUS, TTP, vasculitis) results from a specific cause or specific microbial pathogen. Manifestation of the syndromes depends on the patient ’s condition, the location and severity of trauma and criteria such as virulence, amount and site of entry of the pathogens in sepsis and antibiotic treatment.

The pro coagulatory activity induced by sepsis is generally more severe than in severe trauma because of the systemic nature of prothrombotic diathesis. Thrombin is not only generated by vascular endothelium but also by activated macrophages. Consequently, tissue factor is available in almost unlimited quantities and the general activation of the coagulation system leads to a high consumption of coagulation factors.

DIC in sepsis has the following hallmarks /13/:

• Systemic inflammation during sepsis leads to the generation of pro inflammatory cytokines TNF-α, ΙL-1 and IL-6 that orchestrate coagulation activation as well as the down-regulation of fibrinolysis. This results in an imbalance between intravascular fibrin formation and its removal.
• Reduced anticoagulant capacity leads to excessive fibrin formation and consumption of clotting factors and anticoagulants, causing microvascular thrombosis in conjunction with multiple ischemic organ damage (MODS) and skin necrosis.

16.5.3.1 Coagulation activation in sepsis

The exogenous pathway is activated by the tissue factor (TF). TF is synthesized by activated monocytes/macrophages and endothelial cells. After binding to exposed TF, F VII is activated (F VIIa). The TF/F VIIa complex then activates F X (F Xa), by which prothrombin is converted to thrombin (see also Section 16.1.3.2 – Hemostatic function of platelets).

16.5.3.2 Anticoagulant pathways in sepsis

Anticoagulant mechanisms in sepsis are impaired by /13/:

• Inactivation of antithrombin (AT) by elastase released from granulocytes as well as rapid clearance of thrombin-antithrombin (TAT) complexes from circulation
• Decreased thrombomodulin synthesis due to pro inflammatory cytokines, leading to inadequate activation of protein C
• Relative deficiency of tissue factor pathway inhibitor (TFPI) which inhibits the activity of the FVIIa/TF complex.

The decrease in the physiological anticoagulant effect leads to a thrombin burst and, as a result, enhances fibrin formation.

16.5.3.3 Fibrinolysis in sepsis

Generation of thrombin in sepsis also initiates fibrinolysis, primarily through the release of tissue-type plasminogen activator (t-PA) and the urokinase-like plasminogen activator (u-PA). Endothelial cells are the principle source of t-PA. The initially increased fibrinolytic activity in sepsis is markedly decreased by the following processes /13/:

• Increasing PAI-1 levels. PAI-1 forms stable complexes with t-PA and u-PA thus reducing fibrinolysis
• Increased synthesis of α₂-antiplasmin which inactivates plasminogen by forming plasminogen-α₂-antiplasmin complexes.

References

1. Hunt Bj. Bleeding and coagulopathies in critical care. N Engl J Med 2014; 370: 847–59.

2. Management of severe preoperative bleeding. Guidelines from the European Society of Anaesthesiology. Eur J Anaesthesiol 2013; 30: 270–382.

3. Segal JB, Dzik WH. Paucity of studies to support the abnormal coagulation test results predecting bleeding in the setting of invasive procedures: an evidence based review. Transfusion 2005; 45: 1413–25.

4. Blome M, Isgro F, Kiessling AH, Skuras J, Haubelt H, Hellstern P, et al. Relationship between factor XIII activity, fibrinogen, haemostasis screening tests and postoperative bleeding in cardiopulmonary bypass surgery. Thromb Haemost 2005; 89: 1101–7.

5. Lloyd de L, Bovington R, Kaye A, Collis RE, Rayment R, Sanders J, et al. Standard haemostatic tests following major obstetric haemorrhage. Int J Obstet Anesthesia 2011; 20: 135–41.

6. Korte W, Gabi K, Rohner M, Gähler A, Szadkowski C, Schnider DW, et al. Preoperative fibrin monomer measurement allows risk stratification for high intraoperative blood loss in elective surgery. Thromb Haemost 2005; 94: 211–5.

7. Lier H, Böttiger BW, Hinkelbein J, Krep H, Bernhard M. Coagulation management in multiple trauma: a systemic review. Intensive Care Med 2011; 37: 572–82.

8. Maegele M. The diagnosis and treatment of acute traumatic bleeding and coagulopathy. Dtsch Arztebl 2019; 116: 799–806.

9. Fries D. Dilutionskoagulopathie. Hämostaseologie 2006; 26, suppl 1: S15–S19.

10. Singbartl K, Innerhofer P, Radvan J, et al. Hemostasis and hemodilution – a quantitative mathematical guide for clinical practice. Anesth Analg 2003; 96: 929–35.

11. Levi M. Disseminated intravascular coagulation: what’s new? Crit Care Clin 2005; 21: 449–67.

12. Zeerleder S, Hack E, Wuillemin WA. Disseminated intravascular coagulation in sepsis. Chest 2005; 128: 2864–75.

13. Dellinger RP. Inflammation and coagulation: implications for the septic patient. Clin Infect Dis 2003; 36: 1259–65.

14. Bick RL. Disseminated intravascular coagulation. Current concepts of etiology, pathophysiology, diagnosis, and treatment. Hematol Oncol Clin N Am 2003; 17: 149–76.

15. Aulinger BA, Saner FH, Stark K, Meyerle J, Lange CM. Platelet aggregation inhibitors and anticoagulants in gastroenterological and visceral surgical procedures – an update. Dtsch Arztebl 2022; 119: 851–60.

16.6 Hemostasis in liver disease

Lothar Thomas

Hemostasis is closely associated with the function of the liver /1, 2/:

• Most factors of the coagulation system are synthesized by liver parenchymal cells
• The inhibitors of the coagulation system which establish an coagulation equilibrium by counter regulation are synthesized by parenchymal cells. Inhibitors include antithrombin, protein C, protein S, plasminogen activator inhibitor (PAI-1) and α₂-anti plasmin
• The activators of fibrinolysis (e.g., tissue-plasminogen activator; t-PA), are removed from circulation. The half-life of t-PA is prolonged in liver injury, resulting in increased fibrinolysis in conjunction with bleeding tendency. The clearance not only of activated coagulation and fibrinolysis factors but also of activation complexes and end products of the fibrinogen/fibrin conversion is accomplished by the reticuloendothelial system of the liver.

The von Willebrand factor (VWF) and t-PA are produced by endothelial cells and urinary plasminogen activator (u-PA) by kidney cells.

The vitamin K dependent coagulation factors II, VII, IX and X as well as the inhibitors protein C and protein S require post translational modification in order to acquire their physiological function. All of these proteins have a number of glutamic acid residues in their amino terminal region that must be converted to γ-glutamic acids by a carboxylase that requires vitamin K. Only in their carboxylated form can these proteins bind to phospholipid surfaces via Ca²⁺ bridges, thus contributing to the formation of activation complexes such as the prothrombin complex. The unavailability of the binding site for Ca²⁺ due to vitamin K deficiency leads to reduced coagulation capacity.

Liver diseases may generally be associated with the following hemostatic problems:

• Decreased biosynthesis of coagulation factors
• Increased consumption of coagulation factors
• Synthesis of abnormal coagulation factors
• Abnormal clearance of activated components of the coagulation system.

Vitamin K deficiency may also lead to significant coagulation problems. The pattern of hemostatic proteins observed in various liver diseases and in conjunction with vitamin K deficiency are presented in Tab. 16.6-1 – Coagulopathy in liver disease and vitamin K deficiency.

Plasmatic hemostatic disorders and thrombocytopenia usually occur in different severities resulting in a balanced decrease in pro- and anti-thrombogenic factors /5/.

The INR is a diagnostic criterion of a prognostic score to predict mortality in patients with acute-on-chronic liver failure (CLIF) /6, 7/. Refer to Tab. 16.6-2 – The CLIF-organ failure score system.

References

1. Mammen EF. Coagulation abnormalities in liver disease. Hematol Oncol Clin North Am 1992; 6: 1247–57.

2. Páramo JA, Rocha E. Hemostasis in advanced liver disease. Semin Thromb Hemost 1993; 19: 184–90.

3. Mammen EF. Coagulation defects in liver disease. Med Clin North Am 1994; 78: 545–54.

4. van Wersch JWJ, Russel MGVM, Lustermans FAT. The extent of diffuse intravascular coagulation and fibrinolysis in patients with liver cirrhosis. Eur J Clin Chem Clin Biochem 1992; 30: 275–9.

5. Riess H. Acquired coagulopathies. Hämostaseologie 2004; 24: 242–51.

6. Jalan R, Saliba F, Pavesi M, Amoros A, Mroreau R, Gines P, et al. Development and validation of a prognostic score to predict mortality in patients with acute-on- chronic liver failure. J Hepatol 2014; 61: 1038–47.

7. Arroyo V, Moreau R, Jalam R. Acute-on-chronic liver failure. N Engl j Med 2020; 382: 2137–45.

16.7 Hemostasis in renal disease

Lothar Thomas

Patients with decreased renal function may display not only disorders of the plasma coagulation system and hemorrhage but also thromboembolic complications.

Thrombocyte dysfunction and increased bleeding time are due to a defect in primary hemostasis. Prolonged may be the coagulation time of the PT (extrinsic pathway) and the aPTT (intrinsic pathway) of the coagulation system because of a decline in F II, F VII and F X. The factors V and VIII are normal, fibrinogen is most often increased. Hemostatic disorders in renal disease are shown Tab. 16.7-1 – Hemostatic disorders in renal diseases.

16.7.1 Nephrotic syndrome

Thromboembolic complications are the major threat to the nephrotic patient. The incidence is about 35%. Patients with membranous glomerulopathy and severe proteinuria are at highest risk /1/. Common events include deep vein thrombosis, pulmonary embolism, renal vein thrombosis and also thrombosis involving peripheral arteries and the coronary arteries.

Renal vein thrombosis is asymptomatic in most cases, but it may present itself by flank pain, macroscopic hematuria or by unexplained rapid decline in renal function /2/.

16.7.2 Chronic renal failure

Patients with chronic renal failure often suffer from ecchymoses, purpura, epistaxis and bleeding from tapping sites due to impaired primary hemostasis because of platelet dysfunction. Heavy bleeding may occur following trauma and blood loss during surgery /2/. Bleeding time is prolonged, caused by dysfunctional von Willebrand factor, reduced thromboxane synthesis, the presence of uremic toxins and modified platelet granules. The probability of hemostatic disorder is correlated with the extent of urea increase /3/. Bleeding can be induced by taking platelet function-inhibiting drugs (steroidal antiphlogistics, antibiotics) /4/.

On the other hand, uremic patients also have an increased incidence of thromboembolic events such as cardiovascular disease, ischemic stroke and thromboses of arteriovenous fistulae.

16.7.3 Kidney transplantation

Deep vein thrombosis occurs with higher frequency in renal transplant recipients as compared to age-matched patients after other major surgery /2/. The prevalence of renal vein thrombosis is 0.5–4%, especially during the first months following transplantation. At later stages, when graft function has stabilized thromboembolism may be associated with the use of immunosuppressive therapy and is thought to be caused by cyclosporine. In severe acute vascular rejection thrombosis is widespread and gives rise to a large number of small infarcts in the renal cortex. The thrombosis may involve the main vessels, with eventual development of total graft necrosis.

References

1. Sagripanti A, Barsotti G. Hypercoagulability, intraglomerular coagulation, and thromboembolism in nephrotic syndrome. Nephron 1995; 70: 271–81.

2. Rabelink TJ, Zwaginga JJ, Koomans HA, Sixma JJ. Thrombosis and hemostasis in renal disease. Kidney Int 1994; 46: 287–96.

3. Mezzano D, Tagle R, Panes O, et al. Hemostatic disorder of uremia: the platelet defect, main determinant of the prolonged bleeding time, is correlated with indices of activation of coagulation and fibrinolysis. Thromb Haemost 1996; 76: 312–21.

4. Riess H. Acquired coagulopathies. Hämostaseologie 2004; 24: 242–51.

5. Molino D, De lucia D, De Santo NG. Coagulation disorders in uremia. Semin Nephrol 2006; 26: 46–51.

6. Karpman D, Manea M, Vaziri-Sani F, Stahl A, Kristoffersson AC. Platelet activation in hemolytic uremic syndrome. Semin Thrombos Hemostas 2006; 32: 128–45.

16.8 Hemostasis in tumor patients

Lothar Thomas

The pathogenesis of malignancies is tightly linked to the vascular system. Two processes stand out as particularly ubiquitous and important /1/:

• The formation of new blood vessels (tumor angiogenesis)
• The activation of the coagulation system (coagulopathy).

Tumor activated blood vessels exhibit compromised ability to contain plasma proteins (are leaky), poorly sustain blood flow (are prone to stasis) and provide inadequate anti thrombotic luminal surfaces (promote intravascular thrombosis) /1/.

The tissue factor (TF), frequently over expressed in malignancies, plays a major role in tumor progression, metastasis and angiogenesis through signalling via its intracellular domain. TF may involve three different compartments: cancer cells, their adjacent stroma (blood vessels, fibroblastic and inflammatory cells) and the circulating blood.

Thus, tumor diseases may exhibit /1/:

• Venous thromboembolism (VTE), inclusive deep vein thrombosis
• Pulmonary embolism
• Syndromes comparable to low disseminated intravascular coagulation.

Up to 90% of patients with metastatic tumors are affected by one of these coagulopathies in the course of their disease. Besides the spontaneously occurring hemostatic disorders during the course of malignancy enhancement of the para neoplastic effects must be expected during tumor therapy (surgery, chemotherapy or irradiation), possibly resulting in clinical manifestations. Whereas solid cancers lead predominantly to VTE, myeloproliferative diseases are associated with thrombotic and hemorrhagic complications at about the same rate and in some cases even feature both at the same time /2/.

16.8.1 Pro coagulatory mechanisms of tumor cells

Tumor tissues per se activate blood coagulation, and mechanisms that are involved in the malignant transformation may also be involved in the regulation of tumor cell pro coagulant factors. This applies, for example, to the JAK2V617F mutation expression in patients with myeloproliferative disorders and for PML-RARα hybrid gene expression in patients with acute pro myelocytic leukemia. In both cases, the gene expression is associated with a pro coagulatory phenotype /3/.

Tumor cells express various pro coagulant proteins and micro particles /3/:

• Tissue factor (TF), a transmembrane glycoprotein that is the primary activator of normal blood coagulation (see Fig. 16.1-7 – Cascade of plasma coagulation). Tf forms a complex with F VIIa, activates F X and starts the extrinsic pathway (Fig. 16.8-1 – Coagulation activation in malignant tumors by the tissue factor). The TF concentration in tumor patients can be up to 1,000-fold as high as in healthy controls.
• Cancer pro coagulant, a cysteine protease which can activate F X
• TF-bearing micro particles, small membrane vesicles released from tumor cells, monocytes, platelets and endothelial cells after activation of apoptosis play an important role in tumor progression, metastasis and angiogenesis
• Fibrinolysis proteins, for example urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA), as well as plasminogen activator inhibitor 1 and 2 (PAI-1 and PAI-2)
• Cytokines and vascular endothelial growth factors (VEGF). Cytokines stimulate the synthesis of the fibrinolysis inhibitor PAI-1 and down regulate the expression of thrombomodulin (T M). T M has a potent anticoagulant function. It forms a complex with thrombin to activate the anticoagulant protein C that causes the inhibition of F Va and F VIIa.
• Expression of adhesion molecules on the tumor cell membrane allows interaction with host cells (endothel cells, platelets) and promotes localized clotting activation to the vessel wall and to start thrombus formation.

16.8.2 Cancer-related venous thromboembolism (VTE)

Approximately 20% of all new cases of VTE are associated with cancer whereas 26% of incident cases have idiopathic VTE /4, 5/. The relative risk of developing VTE is 7-fold higher in patients with active cancer. The incidence of VTE is higher in the first few months (30–60 days) after cancer diagnosis and thereafter the incidence decreases with time. The VTE incidence rate is higher in metastatic cancer and a faster growing cancer, as evidenced by early recurrence and death. The metastasis stage at the time of diagnosis is an independent risk factor for the development of VTE within the first year after tumor diagnosis. Mucinous adenocarcinomas (lung, colon, ovarian cancer) are associated with a higher VTE incidence than other solid tumors. Patients with hematological malignancies (leukemia, lymphoma, myeloma) have relative high rates of VTE.

Tab. 16.8-1 – Incidences of venous thromboembolism in different types and stages of cancer shows the cumulative incidence of VTE based on initial cancer stage. The data of the California Cancer Registry are shown and were taken from Ref. /4/.

The incidence of cancer associated VTE is strongly increased in the presence of chronic co morbid conditions such as renal insufficiency, liver disease, hypertension, cardiac disease or psychiatric diseases.

Conventional cytotoxic chemotherapy (thalidomide, lenalidomide) are associated with increased risk of VTE, especially when combined with high dose dexamethasone for the treatment of multiple myeloma. Anti-angiogenic therapy is associated with a higher risk of both arterial and venous thrombosis.

Nonspecific activation of the coagulation cascade can be caused by stasis, immobilization of the patient, necrosis of tumor tissue, in combination with inflammation or infection, due to irradiation or surgery and by foreign objects (e.g., venous catheters and ports).

16.8.3 Hemorrhagic diathesis

Tumor-related hemorrhagic diathesis is less common than VTE and primarily occurs in myeloproliferative diseases and metastatic advanced solid tumors as well as under chemotherapy and irradiation therapy. The causes are disseminated intravascular coagulation and thrombocytopenia.

16.8.3.1 Leukemia

Hemorrhage may proceed the overt clinical diagnosis of leukemia by several months /6/. The most common pre diagnostic manifestations are petechiae, purpura and ecchymoses and are present is 40–70% at the time of leukemia diagnosis. Hemorrhage is most common in acute promyelocytic leukemia. Hemorrhage is less commonly a problem in the chronic myeloid or lymphoid leukemias.

16.8.3.2 Multiple Myeloma (MM)

Thrombosis is a significant cause of morbidity and mortality in MM and optimal thromboprophylaxis is far from being reached /18/. Refer to:

• Tab. 16.8-3 – Hemostasis in malignant disease
• Tab. 16.8-4 – Recommendations regarding primary antithrombotic prophylaxis in multiple myeloma (MM).

16.8.3.3 Solid tumors

Hemorrhage may be a significant problem in solid tumors as well /5/. Intravascular coagulation in solid tumors may manifest as low-grade disseminated intravascular coagulation (DIC) or as acute fulminant DIC with massive hemorrhage and thrombosis. The acute fulminant DIC in conjunction with thrombocytopenia occurs with bleeding usually being from three unrelated sites simultaneously. The most commonly cancers are pancreatic cancer and mucinous adenocarcinoma of the bronchus, prostate, ovaries and gastrointestinal tract.

The low-grade DIC is manifested clinically by mild to moderate bleeding, usually of the integument or mucous membranes. In many cases, these events are associated with thrombosis triggered by chemotherapy /6/.

16.8.4 Thrombocytopenia

Thrombocytopenia in cancer patients may be caused by suppression of marrow megakaryopoiesis, DIC, autoimmune disease, in the course of chemotherapy or due to splenomegaly /7/.

Suppression of the bone marrow

Megakaryopoiesis is ultimately suppressed in almost all types of leukemia, advanced lymphoma, multiple myeloma and solid tumors with bone marrow metastases.

Hypersplenism

Hodgkin’s lymphoma and other lymphoproliferative diseases in conjunction with hypersplenism may cause thrombocytopenia by platelet sequestration in the spleen.

Autoantibodies

A small portion of tumor patients, especially those suffering from lymphoproliferative disease, may develop antibodies to platelets. Alloimmunization to donor HLA determinants is seen in up to 50% of patients after platelet transfusion.

Chemotherapy

The thrombocyte count increases again within 1–3 weeks after termination of therapy. However, several substances (e.g., mitomycin and nitrosoureas) have a prolonging effect on the suppression of megakaryopoiesis.

16.8.5 Laboratory tests

Laboratory tests that are associated with an increased risk of hemostasis disorders in tumor patients may identify high-risk cancer patients. A number of hemostatic tests are suggested for detecting the activation of the coagulation cascade, including D-dimers, thrombin-antithrombin complex, prothrombin fragments, thrombin generation test, PAI-1 and thrombocyte count /1/.

Risk factors for VTE comprise:

• Pre chemotherapy platelet count ≥ 350 × 10⁹/L. For example, 21.9% of tumor outpatients had these levels and presented a 3-fold higher rate of VTE as compared to patients with counts below 200 × 10⁹/L who had a rate of 1.25% /8/
• A leukocyte count ≥ 10 × 10⁹/L
• Elevated CRP and increased D-dimer concentrations (significant indicators)
• A risk model for chemotherapy-associated VTE is depicted in Tab. 16.8-2 – Predictive model for chemotherapy-associated venous thromboembolism. The different pre chemotherapy patient characteristics were assigned a risk score. A score ≥ 3 points to a VTE risk under chemotherapy.

Clinical and laboratory findings of hemostasis in malignant diseases are listed in Tab. 16.8-3 – Hemostasis in malignant disease.

References

1. Rak J, Milsom C, Magnus N, Yu J. Tissue factor in tumour progression. Best Practice & Research Clin Haematol 2009; 22: 71–83.

2. Deitcher SR. Cancer-related deep venous thrombosis: clinical importance, treatment challenges, and management strategies. Semin Thrombos Hemostas 2003; 29: 247–58.

3. Falanga A, Paova-Neova M, Russo L. Procoagulant mechanisms in tumour cells. Best Practice & Research Clin Haematol 2009; 22: 49–60.

4. Wun T, White R. Epidemiology of cancer-related venous thromboembolism. Best Practice & Research Clin Haematol 2009; 22: 9–23.

5. Sorensen HT, Mellemkjaer L, Olsen JH, Baron JA. Prognosis of cancers associated with venous thromboembolism. N Engl J Med 2000; 343: 1846–50.

6. Bick RL, Strauss JF, Frenkel EP. Thrombosis and hemorrhage in oncology patients. Hematol Oncol Clin North Am 1996; 10: 875–906.

7. Kaushansky K. The thrombocytopenia of cancer. Hematol Oncol North Am 1996; 10: 431–55.

8. Connolly GC, Khorana AA. Risk stratification for cancer-associated venous thromboembolism. Best Practice & Research Clin Haematol 2009; 22: 35–47.

9. Khorana AA, Kuderer NM, Culakova E, et al. Development and validation of a predictive model for chemotherapy-associated thrombosis. Blood 2008; 111: 4902–7.

10. Stein E, McMahon B, Kwaan H, Altman JK, Frankfurt O, Tallman S. The coagulopathy of acute promyelocytic leukaemia revisited. Best Practice & Research Clin Haematol 2009; 22: 153–63.

11. Levi M. Disseminated intravascular coagulation in cancer patients. Best Practice & Research Clin Haematol 2009; 22: 129–36.

12. Zangari M, Saghafifar F, Mehta P, Barlogie B, Fink L, Tricot G. The blood coagulation mechanism in multiple myeloma. Thrombos Hemostas 2003; 29: 275–81.

13. Kohli M, Kaushal V, Mehta P. Role of coagulation and fibrinolytic system in prostate cancer. Thrombos Hemostas 2003; 29: 301–8.

14. Geenen RWF, Delaere KPJ, van Wersch JWJ. Coagulation and fibrinolysis activation markers in prostatic carcinoma patients. Eur J Clin Chem Clin Biochem 1997; 35: 69–72.

15. Zacharaski LR. Malignancy as a solid-phase coagulopathy: implications for the etiology, pathogenesis, and treatment of cancer. Semin Thrombos Hemostas 2003; 29: 239–46.

16. Kwaan HC, Parmar S, Wang J. Pathogenesis of increased risk of thrombosis in cancer. Thrombos Hemostas 2003; 29: 283–90.

17. Tsakiris DA, Tichelli A. Thrombotic complications after haematopoetic stem cell transplantation: early and late effects. Best Practice & Research Clin Haematol 2009; 22: 137–45.

18. De Stefano V, Larocca A, Carpenedo M, Cavo M, Di Raimondo F, Falanga A, et al. Thrombosis in multiple myeloma: risk stratification, antithrombotic prophylaxis, and management of acute events. A consensus-based position paper from an ad hoc expert panel. Haematologica 2022; 107: 2536–57.

16.9 Preanalytics and methodology of plasma-based hemostasis tests

Lothar Thomas

Preanalytics covers the procedures for the collection, transport, and processing of specimens for plasma-based coagulation. Many pre analytical variables may affect test results of hemostasis assays and may influence important diagnostic and therapeutic decisions. This is especially important in institutions where coagulation analysis is centralized and collection of the blood specimens is decentralized. Such circumstances necessitate the standardization of the collection, transport and storage of the blood samples prior to centrifugation and analysis /1/.

Laboratory diagnostic tests of bleeding and venous thromboembolic disorders measure coagulation and are of two different types /2/:

• The traditional methods measure the clotting system in general and are based on detection of a blood clot in a test tube
• The second type determines the level of the individual plasmatic coagulation protein, inhibitory protein or fibrinolytic protein either by immunologic (antigenic) methodology or by analysis of enzyme-specific synthetic substrate release (amidolytic assay).

16.9.1 Collection of blood samples

Needle

To obtain venous blood from an antecubital vein, 19- to 21 gauge needles or butterfly needles are ideal. Smaller needles are used in adults with compromised veins or in children and newborns /1/. Using needles with a smaller diameter (gauge of 25 and smaller), whether used with an evacuated tube system or syringe may activate platelets or induce hemolysis. Needles larger than 16-gauge may cause turbulence-induced hemolysis. Winged blood collection sets used in combination with smaller-gauge needles, because of their longer path between vein and anticoagulant, may cause activation of platelets and coagulation.

Blood collection tube

Polypropylene or siliconized glass.

Venipuncture

Blood specimens for plasma-based coagulation assays should be collected by venipuncture using a system that collects the specimen directly into a glass or plastic evacuated tube containing the appropriate additive. The blood collection system typically includes a straight needle attached to an adapter used to hold and pierce the collection container /1/. The drawing of a discard tube when performing a PT, INR, or aPTT is abandoned in the recommendations of the Clinical and Laboratory Standards Institute (CLSI) /1/. This is also true for other parameters tested /3/: antithrombin, protein C, and factors II, V, VIII, IX and X.

It is important to avoid contamination with infusion solutions when blood specimens for coagulation assays are drawn from a vascular access device using a blood collection system or a syringe. The infusion should be stopped approximately 5 min. before compression. If blood is obtained from a normal saline lock (a capped-off intravenous port), two dead space volumes of the catheder and extension set should be discarded /4/.

In intensive care patients, blood specimens may be collected from catheters placed in the radial artery and in the subclavian vein. Patients with sepsis present no difference between arterial and venous blood regarding PT (INR), aPTT, fibrinogen and erythrocyte, leukocyte and thrombocyte counts. However, decreased AT activity and increased D-dimer concentrations have been determined in venous plasma. This is thought to be due to the formation of capillary micro thrombi /5/.

Improper venipuncture

Paravenous needle placement or excessively rapid blood collection causes release or aspiration of tissue thromboplastin with subsequent activation of coagulation, also as a result of bubble or foam formation.

Venous compression too long

Compression for too long a period of time causes local activation of fibrinolysis.

16.9.2 Blood/anticoagulant ratio

Citrate is the standard anticoagulant. In order to inactivate the Ca²⁺ ions, a trisodium citrate solution (0.109 mol/L or 3.2%) is mixed with the blood at a ratio of 1 : 10 (1 + 9) immediately after the sample is collected. Two citrate molecules bind 3 Ca²⁺ in the complex C₁₂H₁₀Ca₃O₁₄ × 4 H₂O.

Incorrect citrate-plasma ratio

A decreased filling of the collection device decreases the blood to anticoagulant ratio below 9 : 1, clotting time in seconds tend to increase /1/. At least a 90% fill volume is recommended. The effect of fill volume on clotting results will also be dependent on the PT and APTT reagent. INR prolongation was reported with less than 90% fill volume /6/.

For patients with hematocrits (HCTs) greater than 0.55 the citrate concentration in the collection tube must be adjusted by removing a portion of the citrate volume using the formula in Tab. 16.9-1 – Citrate volume correction in the presence of hematocrit over 0.55 L/L.

In common practice 0.10 mL of the citrate can be removed for the majority of samples with HCT values between 0.55 and 0.65 (5 mL collection device) /1/. The citrate solution contained in the blood collection device also plays a role. For example, when the PT was determined using reagents of different manufacturers, differences in the clotting time of up to 8.8% and in the INR of up to 10% were found /7/. The differences between the tubes was explained mostly by the effect of Mg²⁺ contamination in the citrate solution.

16.9.3 Centrifugation

For processing suitable specimens for plasma based coagulation assays the thrombocyte count should be /1/:

• Below 200 × 10⁹/L for determining the PT/INR, aPTT, antithrombin, protein S, protein C and von Willebrand factor. A relative centrifugal force (RCF) of (1200–1500) × g for 10 min. is recommended.
• Below 10 × 10⁹/L (platelet poor plasma) for determining anti F Xa and lupus anticoagulant. A RCF of 1500 × g for at least 15 min. is required.

After centrifugation, the analysis is performed directly from the collection tube or the sample is transferred to a secondary vessel for storage.

16.9.4 Plasma and blood storage

Citrated plasma /1/

PT: specimens for PT assay can be stored centrifuged or uncentrifuged in an unopened tube for up to 24 hours.

APTT: can be stored centrifuged or uncentrifuged in an unopened tube for up to 4 hours. Specimens containing unfractionated heparin should be kept at room temperature and centrifuged within one hour of collection.

Thrombin time, fibrinogen, AT and D-dimers, protein C, protein S, anti FXa, FV: can be stored centrifuged or uncentrifuged at room temperature and should be tested within 4 hours.

INR: under oral anticoagulation therapy with coumarins, the level of INR in citrate plasma remains stable for 24 h of storage at room temperature /8/.

Factors II, VII, IX and X: remain stable for 24 h of storage at room temperature /8/.

Long-term storage: for longer storage of plasma samples, rapid freezing by immersion of sample tubes in liquid nitrogen at –70 °C is recommended. Plasma samples transferred directly to the storage compartments and stored at -20 °C will yield different results. Depending on the type of coagulation test, frozen and thawed samples generate different results compared to fresh samples /9/. The PT in samples stored at –20 °C decreased by 15% after 2 months and 25% after 4 months; however, in snap-frozen samples stored at –70 °C it only decreased by 15% after 4 months. The aPTT is generally prolonged by up to 10% within 3 months in not snap-frozen samples and by up to 5% in snap-frozen samples stored at –70 °C. Frozen and thawed samples generate slightly higher fibrinogen levels compared to fresh samples. PT and aPTT should be measured in fresh samples, since freezing has an inconstant and unpredictable effect on the results /8/.

Citrated whole blood

PT, aPTT, thrombin time, fibrinogen, antithrobin D-dimer /10/: the mean percentage change after 8 and 24-h storage at ambient temperature was below 10%. Considering the changes in individual samples all parameters can be reliably tested after 8-h storage, since less than 15% of the samples demonstrated individual changes above 10%. Clinical relevant changes over 10% were detected after 24-h storage for aPTT (41% of samples). Antithrombin and fibrinogen demonstrated individual changes of above 15% in about 10% of samples /10/.

Platelet function: for determining the platelet function in platelet aggregation studies, the citrated blood should be analyzed within 4 h after blood withdrawal because of the decline in sensitivity to platelet agonists such as ADP. Platelet testing by light transmission aggregometry and measurement of thrombin adhesion can be conducted after storage of blood for 24 h in heparin (150 U/mL, 1 : 9 v/v) with stimulation by ADP, arachidonic acid, TRAP-6, U46619 and adrenaline without any loss of activity, but not with collagen or ristocetin stimulation /11/.

16.9.5 Hemolysis

Hemolysis is defined as an accelerated destruction of red blood cells (RBCs) releasing the components within the RBCs. The components are hemoglobin, lactate dehydrogenase, K⁺, neuron-specific enolase, and phospholipids. Manufacturers broadly recommend rejecting hemolytic samples if chromogenic subtrate method is used in clotting assays. The Clinical and Laboratory Standards Institute /1/ in its guidelines for PT and aPTT testing, states that samples with visible hemolysis (≥ 0.3 g Hb/L or ≥ 0.1% hemolyzed red blood cells) should not be used. It is speculated that /12/:

• Exposure of anionic membrane phospholipids during erythrocytolysis could provide a phospholipid-rich surface to accelerate coagulation reactions and, hence, shorten the coagulation time
• Exposure of membrane phospholipids could compete with thromboplastin for F VIIa availability and increase the coagulation time.

In a study /12/ in paired patient specimens with an Hb below 0.3 g/L and up to 7 g/L, with mechanical clot detection assays hemolysis only prolonged the PT from 15.8 ± 8.4 to 16.3 ± 8.7 seconds and the aPTT from 31.6 ± 18 to 32.5 ± 19 seconds. In a different study /13/ in samples containing a final lysate concentration of 0.5%, increase in PT was measured and a decrease in aPTT and fibrinogen in samples containing a final lysate concentration of 0.9%.

16.9.6 Hyperlipidemia and hyperbilirubinemia

Hyperbilirubinemia, hyperlipidemia and hemolysis do not have a major effect on coagulation tests using clot detection assays, but interfere with optical detection methods. Aiming to reduce interferences by optical properties of plasma samples, some analyzers enable light transmission to be measured not only at 405 nm, but also at 570 nm. Changing the wavelength should lead to more valid test results, because light transmission at 570 nm is less influenced by absorbance of triglycerides, bilirubin and hemoglobin /14/. The optical spectra of bilirubin, hemoglobin and triglycerides are shown in Fig. 16.9-1 – Absorbance spectra of triglycerides bilirubin and hemoglobin.

In a study /14/, the results of spectrophotometer measurements were compared with clot detection measurement. The results obtained with a clot detection analyzer is unaffected by the optical properties of the sample and was defined as the reference method for measuring PT, aPTT and fibrinogen.

Tab. 16.9-2 – Results of an optical peptide substrate method at 405 and 570 nm in comparison to a clot detection method shows that there are pronounced differences for triglyceride concentrations above 200 mg/dL (2.3 mmol/L), bilirubin above 5 mg/dL (85 μmol/L) and hemoglobin above 0.30 g/L. Changing the spectrophotometer wave length from 405 to 570 nm was associated with a marked increase in valid results.

16.9.7 Methodology of plasma-based hemostasis tests

The quantitative determination of coagulation factors and inhibitors is performed by functional testing and concentration measurements. Functional tests measure the activity of one or several coagulation factors and have the advantage of reflecting the functional behavior of the relevant coagulation factor. Since the enzymatic activity is measured, changes in test conditions (ionic strength, pH, temperature, surface) will have an effect on the result. The following functional tests are distinguished:

• Classical coagulation tests (clot detection assays)
• Tests using synthetic peptide substrates and spectrophotometrical detection (amidolytic assay).

16.9.7.1 General principles of plasma clotting tests

The clotting assays measure the time in which the mixture of reagents and sample starts to clot after Ca²⁺ are added. For the determination of PT the coagulation process is started by incubation of a plasma sample with thromboplastin and Ca²⁺. The time to formation of a fibrin clot is measured (clotting time). The clotting time correlates with the decrease in activity of the relevant coagulation factor(s). The measurement is mechanical, for example, the time when the coagulation process is started until the standstill of the ball rotating in the coagulometer due to the formed clot is recorded (Fig. 16.9-2 – Principle of the ball coagulometer). The results in percentage of normal are derived from a standard curve from measurement of PT in standard human plasma.

Turbidimetric testing: during the coagulation process in the reaction mixture agglutinates are formed and the preparation becomes increasingly turbid. The increase in agglutination is an indicator of clot formation and reduces the amount of light passing through the cuvette or increases the amount of scattered light. Measurement results are calculated from light transmission or scattered light.

Chromogenic (amidolytic) assays

The peptide substrates used in these assays are composed of a short peptide chain (2 to 4 amino acids) labeled with a chromophore (e.g., para-nitroaniline) behind the cleavage side. The peptide sequence can be selected to ensure high binding specificity for the relevant coagulation factor. The chromophore labeled peptide chain shows an absorbance maximum at shorter wavelengths than the free chromophore.

16.9.7.2 Plasma clotting tests

Assessment of hemostasis involves plasma based assays that measure plasma clotting time after addition of Ca²⁺ Plasma clotting time contains a series of reactions including contact reaction, thromboplastin formation, prothrombin conversion, and thrombin-fibrinogen interaction (Fig. 16.9-3 – Coagulation scheme illustrating the factors measured by PT, aPTT and thrombin time tests).

The plasma clotting tests are first line tests for diagnosis, intervention, management of hemorrhage and thromboembolic events, and include the following tests:

• PT for evaluation of the extrinsic activation of plasma clotting (see Section 16.10 – Prothrombin time (PT))
• aPTT for evaluation of the intrinsic activation of plasma clotting (see Section 16.11 – Activated partial thromboplastin time)
• Thrombin time (TT), a routine screening test to specifically test for disorders of the thrombin-fibrinogen reaction (see Section 16.12 – Thrombin time)
• Fibrinogen for quantitatively measuring fibrinogen because of the lack of sensitivity of the TT to changes in fibrinogen levels
• Tests using endogenous thrombin potential for determining the amount of thrombin that can be activated in patients with suspected hyper- or hypo coagulability (see Section 16.14 – Thrombin generation test).
• Thrombelastography.

16.9.7.3 Thromboelastography (TEG)

Global hemostasis assays like thrombelastography (TEG) measure the viscoelastic changes occurring during clot formation. In perioperative transfusion management TEG is better suited than the functional tests to estimate the complex interactions among procoagulant proteins, their natural inhibitors and blood cells /15/.

16.9.7.3.1 Indication

TEG is indicated in /16/:

• Peri operative blood product use by providing specific information about coagulation status and the nature of coagulation dysfunction
• Trauma-associated coagulopathy
• Identification of hyper fibrinolysis
• The combination of hyper coagulability and hyper fibrinolysis
• Advanced disseminated intravascular coagulation
• Thrombolytic drug therapy
• Surgery in children below 6 months.

TEG supplements conventional tests (PT, aPTT, thrombocyte count) and provides information on the coagulation status and the type of coagulation disorder, especially during the peri operative phase.

16.9.7.3.2 Method of determination /15/

Principle: the TEG consists of a heated sample cup that oscillates at ± 4° 45’ every 5 seconds, into which a pin is suspended by a torsion wire. During coagulation, the forming clot results in a physical connection between each cup and pin, transferring the torque of the cup to the pin. The rate of clot formation and its elastic strength affect the magnitude of motion of the pin and its range of oscillation. Computer software produces both quantitative parameters and a graphic representation of the phases of clot formation.

Refer to:

• Fig. 16.9-4 – Thromboelastogram with the measured parameters.

TEG is comprised of:

• parameter reaction time (R-time)
• speed of fibrin build-up and crosslinking (alpha angle)
• clot formation time (K-time)
• maximum clot amplitude (mA)
• percent clot lysis at 30 minutes (LY30).

16.9.7.3.3 Specimen

Citrated venous whole blood (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood): 3 mL

16.9.7.3.4 Reference interval

Example of a system:

• parameter reaction time (R): 100–240 sec.
• clot formation time (K): 30–110 sec.
• maximum clot amplitude (MA): 53–72 mm
• percent clot lysis at 30 min and 60 min after MA: below 15%.

16.9.7.3.5 Clinical significance

The TEG result is substantially influenced by the thrombocyte count, fibrinogen concentration and presence of hyper fibrinolysis. The latter is an important problem in trauma-associated coagulopathy which may require antifibrinolytic therapy in addition to substitution of blood and blood products. However, hyper fibrinolysis is not detected by conventional tests in the laboratory /16/.

Other biological influence factors affecting the TEG include F XIII activity and fibrin polymerization impaired, for example, by D-dimers and thrombin inhibitors.

Intrinsic system activation with kaolin is recommended for performing TEG in hemophilia /15/.

TEG is not sensitive enough to determine coagulation factor deficiency, the von Willebrand syndrome and the effect of platelet inhibitors /17/.

The clinical significance of thrombelastogram parameter are shown in Tab. 16.9-3 – Clinical significance of the thrombelastogram parameters.

16.9.7.4 Comments and problems

16.9.7.4.1 Biological variation

In infants below 6 months hemostatic system is not completely mature. As a result coagulation disorders and postoperative bleeding among younger infants occur at a higher rate after major surgery such as cardiopulmonary bypass /19/.

Many studies are published about biological variation of plasma hemophilia and thrombophilia parameters showing different data. In a study /16/ of healthy adults over 18 years the main objective was to determine analytical performance specifications for

• thrombophilia parameters: protein C, protein S, activated protein C resistance (APCR), and F VIII
• hemophilia parameters: F VIII, F IX, F XI
• and F V and F XII

The analytical coefficient of variation (CVa) varied from 1.5 to 4.6%, the within-subject CV from 1.6 to 8.9% and the between-subject CV from 3.8 to 24.1%. For all parameters except F XII and APCR the obtained CVa met the requirements for minimal analytical imprecision. The CVa was below 5% for all parameters.

References

1. Clinical and Laboratory Standards Institute. Collection, transport, and processing of blood specimens for testing plasma-based coagulation assays and molecular hemostasis assays; approved guideline – fifth edition. Wayne, Pennsylvania 2008.

2. Palmer RL. Laboratory diagnosis of bleeding disorders. Postgraduate Medicine 1984; 76: 137–48.

3. Raijmakers MTM, Menting CHF, Vader HL, van der Graaf F. Collection of blood specimens by venipuncture for plasma-based coagulation assays. Am J Clin Pathol 2010; 133: 331–5.

4. Powers JM. Obtaining blood samples for coagulation studies from a normal saline lock. Am J Crit Care 1999;8: 250–3.

5. Durila M, Kalincik T, Jurcenko S, Pelichovska M, Hadacova I, Cvachovec K. Blood Coagulation and Fibrinolysis 2010; 21: 770–4.

6. Chang J, Sadler MA, Witt DM. Impact of evacuated collection tube filling volume and mixing in routine coagulation measures using 2.5 mL pediatric tubes. Chest 2004; 126: 1262–6.

7. Van den Besselaar AMPH, Hoekstra MCL, Witteveen E, Didden JH, van der Meer FJM. Influence of blood collection systems on the prothrombin time and international sensitivity index determined with human rabbit thromboplastin reagents. Am J Clin Pathol 2007; 127: 724–9.

8. Christensen TD, Jensen C, Larsen TB, Maegaard M, Christiansen K, Sorensen B. International normalized ratio (INR), coagulation factor activities and calibrated automated thrombin generation – influence of 24 h storage at ambient temperature. Int Jnl Lab Hem 2010; 32: 206–14.

9. Alesci S, Borggrefe M, Dempfle CE. Effect of freezing method and storage at –20 °C and –70 °C on prothrombin time, aPTT and plasma fibrinogen levels. Thrombosis Research 2009; 124: 212–6.

10. Kemkes-Matthes B, Fischer R, Peetz D. Influence of 8 and 24-h storage of whole blood at ambient temperature on prothrombin time, activated partial thromboplastin time, fibrinogen, thrombin time, antithrombin and D-dimer. Coagulation and fibrinolysis 2011; 22: 215–20.

11. Truss NJ, Armstrong PCJ, Liverani E, Vojnovic I, Warner TD. Heparin but not citrate anticoagulation of blood preserves platelet function for prolonged periods. J Thromb Haemost 2009; 7: 1897–905.

12. Laga AC, Cheves TA, Sweeney JD. The effect of specimen hemolysis on coagulation test results. Am J Clin Pathol 2006; 126: 748–55.

13. Lippi G, Montagnana M, Salvagno GL, Guidi GC. Interference of blood cell lysis on routine coagulation testing. Arch Pathol Lab Med 2006; 130: 181–4.

14. Junker R, Käse M, Schulte H, Bäumer R, Langer C, Nowak-Göttl U. Interferences in coagulation tests – evaluation of the 570 nm method on the Dade Behring BCS analyser. Clin Chem Lab Med 2005; 43: 244–52.

15. Recommendations for performing thromboelastography/thromboelastometry in hemophilia: communication from the SCC of the ISTH. J Thromb Haemost 2014; 12: 103–6.

16. MacIvor D, Rebel A, Hassan ZU. How do we integrate thromboelastography with perioperative transfusion management. Transfusion 2013; 53: 1386–92.

17. Calatzis A, Spannagl M, Vorweg M. Zielgerichtete Behandlung akuter Hämostasestörungen mit Hilfe der Rotem-Analyse. Edition Pentapharm, München 2000.

18. Brochier A, Mairesse A, Saussoy P, Gavard C, Desmet S, Hermans C , et al. Short term biological variation study of plasma hemophilia and thrombophilia parameters in a population of apparently healthy Caucasian adults. Clin Chem Lab Med 2022; 60 (9): 1409–15.

19. Li Z, Chen X, Liu Q, Chen P, Ke Z, Hu H. A comparison between thrombelastography and conventional clotting tests in pediatric patients after cardiopulmonary bypass procedure. Clin Lab 2023; 69: 2076–82.

16.10 Prothrombin time (PT)

Lothar Thomas

The PT is the best screening test for the reactions of the extrinsic activation way. It measures the extrinsic activation of factor X by the tissue thromboplastin -factor VII complex and the resulting common pathway reaction. The test is sensitive for deficiencies of F VII, F X, F V, F II and for fibrinogen (see Fig. 16.9-3 – Coagulation scheme illustrating the factors measured by PT, aPTT and thrombin time tests). The PT specified as International Normalized Ratio (INR) is used to assess the effect of oral anticoagulant therapy.

16.10.1 Indication

Indications are /1/:

• Acquired or congenital deficiency of F VII, F X, F V, F II and fibrinogen
• Monitoring of oral anticoagulant therapy
• Diagnosing of vitamin K deficiency (neonates)
• Monitoring of substitution therapy with plasma (fresh frozen plasma, prothrombin complex)
• Assessment of the liver function
• Criterion of score systems.

16.10.2 Method of determination

The PT measures the clotting time of plasma in the presence of an optimal concentration of tissue factor extract.

16.10.2.1 Clotting test according to Quick or Owren

Principle

In citrated plasma the transformation of prothrombin into thrombin is started after the addition of tissue thromboplastin and Ca²⁺. Fibrinogen is converted into fibrin by the generated thrombin and the fibrin formation time (clotting time) is measured (see Section 16.9.7.1 – Principle of clotting tests). Tissue thromboplastin is produced from tissue (rabbit brain, human placenta) and contains tissue factor and phospholipids as active components. The PT is expressed in seconds or as Quick value in % of normal /2/. The percentage values are determined by producing a serial dilution of a plasma pool of healthy individuals with distilled water. A Quick value of 50% of normal means that the sample has the same clotting time as one volume part of plasma diluted with one volume part of distilled water. The lower the coagulation capacity of the sample, the longer the clotting time (PT) and the lower the Quick value in %.

A distinction is made between PT according to the Quick method and according to the Owren method /2/. The Quick method determines fibrinogen and factors II, V, VII and X. It has the advantage of diagnosing coagulation factor deficiency because it measures F V. PT according to Owren only measures factors II, VII and X. In oral anticoagulant therapy, PT according to Owren has the advantage that the F V, which is not affected by vitamin K antagonists, is not taken into account in the determination.

16.10.2.2 Chromogenic assay

See also Section 16.9.7.1 – Principle of clotting tests /3/.

Principle: Analogously to the coagulation test, the reaction in the chromogenic assay is started by adding a thromboplastin reagent which, besides Ca²⁺ contains a thrombin-specific chromogenic substrate (Fig. 16.10-1 – Test principle of a chromogenic substrate method for PT determination). Due to the higher affinity of the chromogenic substrate for thrombin in comparison with fibrinogen, the chromogenic substrate is cleaved first as soon as traces of thrombin are produced, thus releasing a chromophor. When a certain, predefined threshold value of the optical density is reached during the spectrophotometric monitoring of the chromophor (usually p-NA), this difference is specified as the “clotting time”. Only after the chromogenic substrate has been almost completely consumed, will fibrinogen be cleaved as well. Since the products thus produced will also cause additional optical density, the fibrinogen concentration (derived fibrinogen) can be calculated based on the total absorption after complete consumption of both substrates (chromogen and fibrinogen) minus the constant portion of the added chromogenic substrate.

16.10.2.3 Capillary whole blood method

This method is mainly used in outpatient care and patient self-monitoring.

Principle

A battery-powered laser photometric device is used for measuring /4/. A drop of capillary blood is applied to the application field of a pre warmed test pad and drawn by diffusion to the reagent pad, where it is mixed with rabbit brain thromboplastin bound to iron oxide particles, thus triggering the coagulation cascade. The blood continues to diffuse into the reagent pad and coagulates. The content of iron oxide particles in the agglutinates is dependent on the activity of the clotting system. An electromagnet located below the reagent pad in the measuring device initiates the iron oxide particles to regular pulsation at a frequency of 2 Hz. The regular pulsation pattern is recorded by reflectance photometry. As a fibrin matrix is formed, the iron oxide particle movement is inhibited and eventually stopped. This causes a decrease in reflectance and recorded as start of coagulation. Calibration is performed by the manufacturer and stored in the device.

16.10.2.4 INR/ISI model

Principle

The use of International Normalized Ratio (INR) is recommended to achieve international harmonization of measured PT values and therapeutic PT ranges for monitoring anticoagulant therapy with vitamin K antagonists /5/. The prothrombin time ratio is determined by the equation in Tab. 16.10-1 – Calculation of prothrombin time ratio and international normalized ratio. By using the ISI, the INR can be calculated from the prothrombin time ratio.

The International Sensitivity Index (ISI) is a value that describes the ratio of the manufacturers thromboplastin in relation to the WHO reference thromboplastin preparation (IRP 47/60) /1, 5/. Contemporary thromboplastins have usually ISI values of about 1 /6/. Less sensitive reagents have ISI values higher than 1.

Reporting of results

The clotting time of the PT tests is expressed in seconds or in % of normal. In patients undergoing oral anticoagulant therapy, the PT should be expressed as International Normalized Ratio (INR).

16.10.3 Specimen

• Venous citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood): 1 mL
• Capillary blood, obtained by using a sodium citrate containing capillary: 0.025 to 0.1 mL

16.10.4 Reference interval

Percentage of normal 80–130%

INR 0.85–1.15

Oral anticoagulation therapy: Refer to Tab 16.10-2 – Venous thromboprophylaxis: INR target values and intervals

16.10.5 Clinical significance

The PT is a measure of the integrity of the extrinsic pathway and the final step of fibrin formation (Fig. 16.9-3 – Coagulation scheme illustrating the factors measured by PT, aPTT and thrombin time tests). The PT test does not take account of factors VIII, IX, XI, XII, XIII and high-molecular-weight kininogen (HMWK).

Prolonged PT is caused by:

• Oral anticoagulants (vitamin K antagonists)
• Factor deficiency
• Dysfibrinogenemia and fibrin(ogen) degradation products
• Antibodies to coagulation factors
• New oral anticoagulants.

Except for F IX, the PT test according to Quick records 3 of 4 vitamin K dependent factors (F II, F VII and F X). If the platelet count, the bleeding time and the thrombin time are normal, the prolongation of the PT suggests a reduction in the vitamin K dependent factors or in F V, regardless of the aPTT value.

The extrinsic pathway is checked by means of F VII while the other factors X, V, II and fibrinogen represent the common final steps of both extrinsic and intrinsic pathways of the coagulation system. Since all of these factors are produced in the liver, the PT is a good criterion for assessing the protein synthesis capacity of the liver. F V deficiency is not detected by the PT test according to Owren.

Vitamin K antagonists result in the functional alteration of the synthesis of the four coagulation factors II, VII, IX and X as well as of the inhibitors protein C and protein S. Since the activity of factors II, VII and X is measured by the PT, its value represents a good tool for monitoring thromboprophylaxis (Tab. 16.10-2 – Venous thromboprophylaxis: INR target values and intervals).

Hemostatic disorders with prolonged PT are listed in Tab. 16.10-3 – Hemostatic disorders with prolonged PT.

16.10.6 Comments and problems

Method of determination

The underlying cause for the lack of comparability between the PT values of different manufacturers can be:

• Thromboplastin preparations present various sensitivities toward factors II, V, X and VII
• PIVKAs (protein-induced vitamin K absence), which possibly enter into the PT determination, interfere with the activation of the normal coagulation factors. PIVKA involves the non-γ-carboxylated precursor proteins of the coagulation factors II, VII, IX and X, as a result of therapy with vitamin K antagonists.

Influence factors and interference factors: see Section 16.9 – Pre analytics and methodology of plasma-based hemostasis tests.

New oral anticoagulants: rivaroxaban administered at normal dose prolongs the PT clotting time.

Stability: INR under anticoagulant therapy, storage time is up to 6 h at room temperature /14/.

Inadequate pre warming of the reagents: prolonged PT clotting time.

Heparin: depending on the reagent used, heparin may (at concentrations of 0.8–2 U/mL plasma) prolong the PT.

Fibrinogen degradation products: lead, usually at concentrations > 50 mg/L, to a prolongation of the PT.

Comparability of the PT measurement results: expression in INR establishes comparability even when different thromboplastins have been used.

Drugs: penicillins cause a decrease in the PT; this is especially important in children.

Hypertonic NaCl solution: contamination of the sample with hypertonic NaCl solution can lead to prolongations of the PT and aPTT > 10% /15/.

References

1. Spannagl M, Moessmer G. Hämostaseologische Globalteste. Hämostaseologie 2006; 26: 27–37.

2. Quick AJ, Stanley-Brown M, Bancroft FW. A study of the coagulation defect in hemophilia and in obstructive jaundice. Amer J Sci 1935; 190: 501–11.

3. Dati F, Barthels M, Conard J, Flückiger J, Girolami A, Hänseler E, et al. Multicenter evaluation of a chromogenic substrate method for photometric determination of prothrombin time. Thromb Haemostas 1987; 58: 856–65.

4. Burri S, Biasutti FD, Lämmle B, Wuillemin WA. Vergleich der Quick-/INR-Werte aus kapillärem Vollblut (CoaguChek Plus) und venösem Citratplasma bei Patienten mit und ohne orale Antikoagulation. Schweiz Med Wochenschr 1998; 128: 1723–9.

5. International Committee for Standardization in Haematology, International Committee on Thrombosis and Haemostasis. ICSH/ICTH recommendations for reporting prothrombin time in oral anticoagulant control. Thromb Haemostas 1985; 53: 155–6.

6. Lang H, Scheer B, Moritz B, Legenstein E, Kaiser E, Fischer M. International normalized ratio (INR) – proficiency tests by ÖQASTA for the prothrombin time. Hämostaseologie 1995; 15: 41–8.

7. Antithrombotic and thrombolytic therapy, 8th edition. ACCP Guideline. Chest 2008; 133: 67S–70S.

8. Raj G, Kumar R, McKinney WP. Long-term oral anticoagulant therapy: update on indications, therapeutic ranges and monitoring. Amer J Med Sci 1994; 307: 128–32.

9. Hirsh J. Optimal intensity and monitoring warfarin. Amer J Cardiol 1995; 75: 39B–42B.

10. Garrison RN, Cryer H, Howard D, Polk jr HC. Classification of risk factors for abdominal operations in patients with hepatic cirrhosis. Ann Surg 1984; 199: 648–55.

11. Guidelines of the European Society of Anaesthesiology. Management of severe perioperative bleeding. Eur J Anaesthesiol 2013; 30: 270–382.

12. Baglin T, Hillarp A, Tripodi A, Elalamy I, Buller H, Ageno W. Measuring oral direct inhibitors of thrombin and factor Xa: a recommendation from the Subcommittee on Control of Anticoagulation of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. J Thrombos Haemostas 2013; 11: 756–60.

13. Tripodi A. Which test to use to measure the anticoagulant effect of rivaroxaban: the prothrombin time test. J Thromb Haemost 2013; 11: 576–8.

14. Gest Daalderop JHH, Mulder AB, Boonman-De Winter LJM, Hoekstra MMMCL van den Besselaar AMPH. Preanalytical variables and off-site blood collection. Influences on the results of PT/INR test and implications for oral anticoagulant therapy. Clin Chem 2005; 51: 561–8.

15. Reed RL, Johnston TD, Chen Y, Fischer RP. Hypertonic saline alters plasma clotting times and platelet aggregation. J Trauma 1991; 31: 8.

16.11 Activated partial thromboplastin time (aPTT)

Lothar Thomas

The aPTT is the best screening test for the reactions of the intrinsic activation. The test is sensitive for deficiencies of F IX, F VIII, F X, F V, F II and for fibrinogen (see Fig. 16.9-3 – Coagulation scheme illustrating the factors measured by PT, aPTT and thrombin time tests).Thus, it evaluates all coagulation factors except for F VII, albeit with a sensitivity different from that of the PT. The aPTT is not sensitive to minor coagulation factor deficiencies, and since the activities of many factors are measured together, overall prolongation may be masked by elevated levels of one or more individual factors. The aPTT is sensitive for the influence of heparin or similar anticoagulants on blood coagulation /1, 2/.

16.11.1 Indication

• Congenital deficiency of F VIII (hemophilia A) , FIX (hemophilia B) or of von Willebrand factor which sometimes is the reason for F VIII deficiency /1/
• Congenital deficiency of F IX (hemophilia B)
• Acquired deficiency of F VIII, F IX and F XI
• Monitoring of therapy with parenterally administrated anticoagulants (unfractionated heparin, hirudin, aprotinin)
• Screening test for anamnestic or clinically manifest bleeding tendency or predisposition to thrombosis
• Suspected presence of inhibitors (lupus anticoagulant, F VIII inhibitor) in the plasma mixing test.

In general, the aPTT is mainly used for preoperative screening and for heparin monitoring

16.11.2 Method of determination

Partial thromboplastins are lipid reagents that are used as substitutes for platelets in clotting tests. Activated partial thromboplastins are mixtures of contact activators (micronized silica, ellagic acid or kaolin) and partial thromboplastins and are used to perform the activated partial thromboplastin time (aPTT) /2/.

The PTT is a is a modification of the plasma recalcification time, a test performed in platelet rich plasma. In the aPTT, a platelet substitute (contact activator) is added and platelet activity is not tested for /2/.

The addition of a partial thromboplastin reagent to patient plasma gives maximum platelet activity (aPTT assay ) than that obtained with the simple addition of platelet poor plasma to partial thromboplastin (plasma recalcification time assay) /2/.

16.11.2.1 Clotting method

Principle

Activated partial thromboplastin is added to re calcified citrated plasma, followed by incubation at 37 °C for a few minutes. This results in the contact activation of F XII and F XI. The measured clotting time (seconds until formation of a coagulation clot) is expressed in seconds. The clotting time depends on the activation of the intrinsic pathway factors /3/.

Chromogenic (amidolytic) assay

Spectrophotometric determination including the use of a chromogenic peptide substrate /4/. See Section 16.9.7.1 – Principle of clotting assays.

16.11.3 Specimen

Citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood): 1 mL

16.11.4 Reference interval

Adults: approximately 26–36 sec., dependent on the reagent used Reference values in fetuses, neonates and children: see Tab. 16.3-2 – Reference intervals for coagulation testing in pediatric vs. adult individuals.

16.11.5 Clinical significance

The aPTT is prolonged /1, 5/:

• In cases of F XI, F IX and F VIII deficiency in the range of 30–40% of normal
• In cases of F II, F V and F X deficiency in the range below 30%.

Impaired fibrin polymerization (dysfibrinogenemia) and fibrin(ogen) degradation products (FDP) have only little influence on the coagulation time. The aPTT is only prolonged in high concentrations of FDP.

The detection limit of the aPTT depends on the reagents used. This applies also to the suitability for the therapeutic monitoring of heparin therapy and the lupus anticoagulant sensitivity.

Approximately 95% of congenital bleeding disorders are associated with prolonged aPTT. A factor deficiency may not cause prolonged aPTT until its activity has decreased to below 30–40%. Hence, mild factor deficiencies are often not identified /1/.

If the platelet count, the bleeding time and the screening tests of the coagulation system such as the PT and the thrombin time are normal, a prolonged aPTT suggests the presence of hemophilia A or B, especially when additional corresponding data in the medical history support this diagnosis. A reduction in F VIII:C and F IX activity to below 40% of normal may be associated with a significantly increased perioperative bleeding tendency. Therefore, the aPTT is prolonged in such cases. F VIII:C is the low-molecular portion of F VIII that mediates coagulation activity.

Hemostatic disorders associated with a prolongation of the aPTT are listed in Tab. 16.11-1 – Hemostatic disorders with prolonged aPTT.

16.11.5.1 Additional tests in cases with prolonged aPTT

The following tests are indicated for differentiation:

• Thrombin time and possibly reptilase time can identify a heparin effect and hypo- or dysfibrinogenemia
• Plasma mixing test can differentiate between factor deficiency and an antibody against a coagulation factor. Normalization of aPTT after addition of normal plasma to the patient plasma indicates factor deficiency. If the aPTT stays abnormal the presence of an antibody or lupus anticoagulant is possible.
• Since the aPTT is less influenced by fibrin(ogen) degradation products (FDP) than the thrombin time (TT), there is no need to examine the sample for the presence of FDP if the thrombin time test is normal.

16.11.5.2 Monitoring heparin-induced anticoagulation

Intravenous administration of unfractionated heparin enhances the inhibitory effect of AT on factors XII, XI, IX, X and II, thus resulting in a decreased activity of the intrinsic pathway of the coagulation system. In therapeutic anticoagulation with unfractionated heparin, plasma heparin concentrations of 0.2–0.5 IU/mL of plasma are achieved which corresponds to a mean 1.5–2.5-fold increase of the baseline aPTT value /6/. Values below 1.5 indicate an inadequate level of anticoagulation and are associated with an increased risk of thromboembolism. Prolongations over 2.5 times above the baseline value may indicate the risk of bleeding.

Monitoring unfractionated heparin therapy with the aPTT is limited for the following reasons /7/:

• Commercial aPTT reagents represent individual test systems which differ in the source of their partial thromboplastins. Therefore, the general specification of 1.5–2.5-fold prolongation as the therapeutic range is incorrect and may result in inappropriate heparin doses.
• aPTT values and heparin sensitivity are lower in patients with acute-phase response than in normal controls, possibly due to an increase in fibrinogen and F VIII. Especially F VIII is thought to compete for heparin with heparin binding protein.
• Unless the aPTT assay is performed on heparinized plasma within 30 min. after blood collection, unduly prolonged aPTT values will be recorded. Delays of 1 h result in approximately 20% and 90 min. result in approximately 60% prolongation of the aPTT baseline value.

Considering such and other elements of uncertainty, it is advisable to use an F Xa-based test for assessing the anticoagulation intensity instead of the aPTT test (see Section 16.28 – Monitoring of anti thrombotic therapy).

Since the low-molecular-weight heparins are primarily directed against F Xa, the aPTT is only slightly prolonged (low activity of low-molecular-weight heparins against thrombin).

Depending on the aPTT reagent used, recombinant hirudin prolongs the clotting time by 17–40% /8/. Above a certain value the aPTT will no longer show a linerar dose response relationship to hirudin (plateau effect), thus overdosing will not be identified.

16.11.5.3 Laboratory tests in cases with shortened aPTT

Shortened aPTT may be related to pre analytical problems such as improper procedure during blood collection (venous compression too long), improper handling of the sample (heavy shaking of the sample tube) or too long a storage time. Therefore, it is recommended to collect another sample and perform the analysis again. If the clotting time is still shortened, this may point to /9/:

• Increased activity of FVIII, an acute phase protein
• Increased risk of a thromboembolic event
• Multiple biological influence factors such as pregnancy, hyperthyroidism, diabetes mellitus, malignant tumor, myocardial infarction.

16.11.6 Comments and problems

Reference interval

The reference interval depends on the individual laboratory (aPTT reagent, method, analyzer). If possible, each laboratory should determine its own reference interval (2.5th and 97.5th percentile of the clotting times of at least 40 healthy individuals) for the aPTT /7/. The lower reference interval value should also be specified since a shortened aPTT indicates hyper coagulability as well as premature activation of the blood sample due to improper blood collection procedure.

aPTT reagents

The properties of the reagents depend on the type and concentration of the partial thromboplastin and the surface activator /7/. Phospholipids from placental tissue, brain extracts and plants are used as a substitute for platelet factor 3 while kaolin, ellag acid and silica are used as surface activators. The detection limit of a certain aPTT method therefore depends on the reagent used. This applies not only to the detection limit in regard to a factor deficiency but also to the suitability for the monitoring of a therapy using heparin or to the detection of lupus anticoagulant.

Performance of the aPTT determination

The incubation period specified for each aPTT reagent must be strictly observed. Too short an incubation period results in a non reproducible prolongation of the aPTT.

Drugs

Penicillins prolong the aPTT (especially important in children), as does valproic acid. Recombinant hirudin prolongs the aPTT clotting time /10/.

New oral anticoagulants: rivaroxaban prolongs the aPTT, as does dabigatran even more markedly.

Stability

Plasma aPTT remains stable for 4 h at 20 °C, unless the plasma is heparinized. For assessing the anticoagulatory activity of heparin: determination within 1 h after blood collection is needed /7/.

References

1. Spannagl M, Moessmer G. Hämostaseologische Globalteste. Hämostaseologie 2006; 26: 27–37.

2. Sirridge MS, Shannon R (eds). Laboratory evaluation of hemostasis and thrombosis. Philadephia, Lea and Febiger 1983.

3. Proctor RR, Rappaport SJ. The partial thromboplastin time with kaolin: a simple screening test for first stage plasma clotting factor deficiencies. AJCP 1961; 36: 212–9.

4. Dati F, Becker U, Hissung A, Keller F. New perspectives in diagnosis of hemostasis disorders. Ann Biol Clin 1988; 46: 201–10.

5. Hellstern P, Oberfrank K, Köhler M, Heinkel K, Wenzel E. Die aktivierte partielle Thromboplastinzeit als Screeningtest für leichte Gerinnungsfaktorenmängel – Untersuchungen zur Sensitivität verschiedener Reagenzien. Lab Med 1989; 13: 83–6.

6. Hirsh J, Fuster V. Guide to anticoagulant therapy, Part 1: Heparin. Circulation 1994; 98: 1449–68.

7. Strekerud FG, Abildgaard U. Activated partial thromboplastin time in heparinized plasma: influence of reagent, acute phase reaction, and interval between sampling and testing. Clin Appl Thrombosis/Hemostasis 1996; 2: 169–76.

8. Töpfer G, Lindhoff-Last E, Bauersachs R, Funke U, Schulze M, Friedel G, et al. Influence of recombinant hirudin on coagulation assays. J Lab Med 2000; 24: 407–13.

9. Lippi G, Salvagno GL, Ippolit L, Franchini M, Favaloro EJ. Shortened activated partial thromboplastin time: causes and management. Blood Coagulation and Fibrinolysis 2010; 21: 459–63.

10. Köhler M, Dati F, Kolde HJ. Activated partial thromboplastin time position finding. Standardization of the method, interpretation of results and limits of applicability. Lab Med 1995; 19: 162–6.

11. Brandt JT, Triplett DA, Alving B, Scharrer I. Criteria for the diagnosis of lupus anticoagulants: An update. Thromb Haemostas 1995; 74: 1185–90.

12. Baglin T, Hillarp A, Tripodi A, Elalamy I, Buller H, Ageno W. Measuring oral direct inhibitors of thrombin and factor Xa: a recommendation from the Subcommittee on Control of Anticoagulation of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. J Thrombos Haemostas 2013; 11: 756–60.

16.12 Thrombin time (TT)

Lothar Thomas

The endstage of both extrinsic and intrinsic activation of the plasmatic coagulation system is the conversion of fibrinogen to fibrin by the proteolytic action of thrombin. It is possible to isolate this reaction and estimate the quantity and reactivity of fibrinogen in plasma by adding a specific amount of exogenous thrombin and measuring the speed of clot formation. A standardized procedure of this kind is known as the thrombin time /2/.

The TT is a screening test to specifically test for disorders of the thrombin-fibrinogen action. Among the factors of the plasma coagulation system, the TT only determines fibrinogen and not the fibrin-stabilizing F XIII.

16.12.1 Indication

Indications of the TT are /1/:

• Testing for the presence of disseminated intravascular coagulation (DIC)
• Clarification of manifest bleeding tendencies
• Investigation of unclear pathological PT and aPTT findings (exclusion of impaired fibrin polymerization)
• Confirmed absence of heparin or hirudin in samples used for thrombophilia clarification
• Thrombolytic therapy. Evaluation of the total effect of decreased fibrinogen, fibrin(ogen) degradation products and (possibly) heparin
• Monitoring of dabigatran therapy.

16.12.2 Method of determination

The TT is the time elapsing between the addition of a standardized quantity of thrombin to patient plasma and the formation of fibrin. Thus, effects of plasma-inherent thrombin formation disorders on fibrin formation do no apply. The TT is a function of the fibrinogen concentration and the quality and presence of antithrombin /2/.

Clotting method

Principle: the clotting time is determined after the addition of a defined quantity of thrombin to citrated plasma and expressed in seconds /3/.

16.12.3 Specimen

Citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood): 1 mL

16.12.4 Reference interval

Approximately 16–24 sec., depending on the thrombin activity and the manufacturer’s specifications.

16.12.5 Clinical significance

The TT test is performed as a supplementary test in the laboratory without clinical order if /4, 5/:

• The aPTT clotting time is prolonged and the presence of thrombin inhibitors such as heparin and hirudin in the plasma is suspected
• Disorders in the fibrinogen-to-fibrin conversion are suspected. The TT is prolonged in hypo fibrinogenemia, dysfibrinogenemia, the presence of fibrin(ogen) degradation products (FDP) and monoclonal immunoglobulins.

If the TT is prolonged in the face of a normal platelet count, a normal bleeding time, a normal PT and a normal or slightly prolonged aPTT, this finding indicates the presence of heparin in the plasma or the presence of FDP (e.g., as seen in conjunction with disseminated intravascular coagulation).

The TT measures thrombin-induced fibrin formation and fibrin aggregation (i.e., the last step in the process of coagulation) but the test does not detect fibrin polymerization (i.e., the covalent cross linking between the fibrin chains by F XIII).

16.12.5.1 Disorders with prolonged thrombin time

The TT is prolonged if:

• The function of the test thrombin added to the patient plasma is inhibited (e.g., as observed in conjunction with heparin therapy or in the presence of thrombin antibodies). Under recombinant hirudin therapy, the mean TT prolongation is 195% and the maximum prolongation is 282% /6/.
• A defect in the aggregation of fibrin occurs due to the presence of FDP (e.g., as observed in thrombolytic therapy)
• Low fibrinogen concentrations (below 0.6 g/L) or dysfibrinogenemia (severe liver cell damage).

The desirable therapeutic range for heparin therapy and thrombolytic therapy using streptokinase or urokinase is 2–4-fold prolongation of the upper reference interval value.

In patients with severe infections (sepsis), hepatic disease and massive myocardial infarction, the TT is a better indicator of heparin therapy than the aPTT. The aPTT, due to prolongation independent from heparin, (e.g., as a result of impaired contact activation in conjunction with pre kallikrein deficiency, overestimates the heparin activity) /7/.

Hemostatic disorders wit prolonged TT ar shown in Tab. 16.12-1 – Hemostatic disorders with prolonged thrombin time.

16.12.5.2 Disorders with shortened thrombin time

Shortened TTs are of no clinical relevance and, at most, are indicators of increased fibrinogen concentrations.

16.12.6 Comments and problems

Using the same reagents and reaction mixtures turbidimetric and nephelometric analyzers measure shorter TT values than the clotting assays.

High fibrinogen levels may, in the absence of heparin, prolong the TT. In the presence of heparin, however, they will shorten the TT and may suppress the detection of heparin, especially after freezing of the sample.

The TT depends on the concentration and the type of thrombin used. The final concentration in the test sample usually amounts to 1 IU/mL of plasma.

16.12.7 Pathophysiology

Under the influence of thrombin on fibrinogen, fibrin monomers are formed as the fibrinopeptides A and B are cleaved off. These fibrin monomers form soluble aggregates. The effects of thrombin-activated F XIII and of Ca²⁺ finally result in polymerization, thus yielding insoluble, crosslinked fibrin (see Section 16.16 – Fibrinogen).

Heparin (antithrombin effect) or FDP (inhibition of fibrin polymerization in concentrations > 50 mg/L) cause a, concentration-dependent, prolongation of the TT. The effect of heparin is identified by performing tests using thrombin-like enzymes (e.g., by determining the reptilase time).

The TT does not allow differentiation between defects in the thrombin-fibrinogen interaction and defects in the aggregation of fibrin monomers. This differentiation requires the parallel use of tests with thrombin-like enzymes.

References

1. Spannagl M, Moessmer G. Hämostaseologische Globalteste. Hämostaseologie 2006; 26: 27–37.

2. Sirridge MS, Shannon R (eds). Laboratory evaluation of hemostasis and thrombosis. Philadephia, Lea and Febiger 1983.

3. Fletcher AP, Alkjaersig N, Sherry S. The maintenance of a sustained thrombolytic state in man. I. Induction and effect. J Clin Invest 1959; 38: 1096–1100.

4. Barth A, Furlan M, Lämmle B. Unerwartet verlängerteThrombinzeit. Schweiz Med Wschr 1993; 123: 523–9.

5. Bounameaux H, Marbet GA, Lämmle B, Eichlisberger R, Duckert F. Comparison of thrombin time, activated partial thromboplastin time and plasma heparin concentration and analysis of the behavior of antithrombin III. AJCP 1980; 74: 68–73.

6. Töpfer G, Lindhoff-Last E, Bauersachs R, Funke U, Schulze M, Friedel G, et al. Influence of recombinant hirudin on coagulation assays. J Lab Med 2000; 24: 407–13.

7. Hattersley PG, Hayse D. The effect of increased contact activation time on the activated partial thromboplastin time. AJCP 1976; 66: 479–82.

16.13 Tests using thrombin-like enzymes

Lothar Thomas

Batroxobin (reptilase) and thrombin coagulase are enzymes which are able to clot fibrinogen:

• Batroxobin cleaves fibrinopeptide A from fibrinogen
• Thrombin coagulase, a complex composed of prothrombin and coagulase from staphylococci, cleaves fibrinopeptide A and B.

Both thrombin-like enzymes are not influenced by heparin.

16.13.1 Indication

Indications of tests using thrombin-like enzymes are /1/:

• Screening for impaired fibrin polymerization
• Differentiation between thrombin inhibition and impaired fibrin polymerization in the presence of prolonged thrombin time
• Thrombolytic therapy: evaluation of the total effect of decreased fibrinogen, fibrin(ogen) degradation products and (possibly) heparin
• Diagnosis of predisposition to bleeding based on clinical manifest symptoms or the medical history of the patient.

16.13.2 Method of determination

Principle: a defined quantity of batroxobin or thrombin coagulase is added to citrated patient plasma. The time period is measured between the addition of the reagent and the clot formation /2, 3, 4/.

16.13.3 Specimen

Citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood): 1 mL

16.13.4 Reference interval

Batroxobin time: Adults 16–20 sec. Neonates 16–24 sec. Thrombin coagulase clotting time: adults 15–24 sec.

16.13.5 Clinical significance

The batroxobin time and the thrombin coagulase clotting time are only influenced by disorders in the polymerization of fibrin monomers.

16.13.5.1 Increase in clotting time

Causes of prolonged clotting time include:

• The inhibition by fibrin(ogen) degradation products (FDP) which form soluble complexes with fibrin monomers. As the inhibition of fibrin aggregration is concentration-dependent, the clotting times in tests using thrombin-like enzymes correlate with the concentration of the FDP. However, this only applies to concentrations above 50 mg/L.
• Low fibrinogen concentrations
• Dysfibrinogenemia.

Clotting times are not prolonged in the presence of:

• Thrombin inhibitors such as heparin and hirudin
• Immunoglobulins, penicillins, protamine chloride.

The inhibitory effect on polymerization of fibrin monomers only occurs at high immunoglobulin concentrations. Thrombin inhibitors that can be detected by a combination of tests using the thrombin time and thrombin-like enzymes are rare.

16.13.5.2 Clotting time in thrombolytic therapy

In combined thrombolytic and heparin therapy, assessment of the lytic activity with the batroxobin time and the thrombin coagulase time is more specific than with the heparin-dependent thrombin time because there is no interference by heparin-antithrombin complexes.

Tab. 16.13-1 – Thrombin time compared to batroxobin and thrombin coagulase time shows the behavior of the thrombin coagulase time and the batroxobin time compared to the thrombin time in disorders of hemostasis /2, 3, 4/.

16.13.6 Comments and problems

Tests using thrombin-like enzymes react more sensitive to decreases in the fibrinogen concentration than the thrombin time. Often, low or greatly elevated fibrinogen concentrations are observed in intensive-care patients without any significant rises in the fibrinogen degradation products. Under such circumstances, tests using thrombin-like enzymes produce slightly prolonged clotting times which may be falsely interpreted to reflect an increased state of fibrinolysis.

References

1. Spannagl M, Moessmer G. Hämostaseologische Globalteste. Hämostaseologie 2006; 26: 27–37.

2. Soulier JP, Prou-Wartelle O. Study of thrombinkoagulase. Thrombos Diathes Haemorrh 1967; 17: 321–34.

3. Donati MB, Vermylen J, Verstrate M. Fibrinogen degradation in vivo: effect on the reptilase time and on the thrombin time. Scand J Haemat 1970; 8 Suppl 13: 259.

4. Latallo ZS, Teisseyre E. Evaluation of reptilase^® and thrombin clotting time in the presence of fibrinogen degradation products and heparin. Scand J Haemat 1971; 8, Suppl 13: 261–6.

16.14 Thrombin generation time test

Lothar Thomas

The generation of thrombin is a fundamental part of the coagulation system. The thrombin generation time test (TGT) measures the amount of active thrombin produced in plasma or whole blood after recalcification. The original TGT is technically difficult to perform and time consuming. Methods based on sensitive chromogenic or fluorogenic peptide substrates have been developed that measure the endogenous thrombin potential (ETP) /1/. The ETP represents the total amount of thrombin that can be generated as a function of the balance between the pro coagulant drivers (thrombin formation) and anticoagulant drivers (thrombin consumption) operating in plasma. The pro coagulant drivers comprise the coagulation factors downstream from F XII, while the anticoagulant drivers are represented by antithrombin (AT) and protein C (PC) /2/.

By measuring the ETP in a TGT, it is possible to determine the amount of activated thrombin formed after recalcification of the plasma or whole blood.

16.14.1 Indication

Indications of the TGT are /2/:

• Investigation of patients with a congenital deficiency of anticoagulant factors
• Investigation of patients with an acquired deficiency of coagulation in which both pro- and anticoagulant drivers are decreased (liver cirrhosis, neonatal period)
• Management of patients undergoing therapy with thromboprophylactic drugs
• Situations where the PT and aPTT are not conclusive.

16.14.2 Method of determination

The TGT measures the speed of the reactions of the intrinsic activation by recalcification of plasma when there is minimal thrombocyte activity and surface activation. In contrast to PT and aPTT which only measure the initiation phase the TGT measures the initiation and propagation phase /3/.

In the propagation phase:

• The bulk of thrombin is generated via feedback loops on factors V, VIII and XI (see Fig. 16.1-4 – Phases of platelet-induced coagulation and Fig. 16.1-7 – Cascade of plasma coagulation activation and associated positive and negative feedback mechanisms)
• After that the thrombin formation declines by the anticoagulant pathways and all thrombin activity is inhibited by plasma protease inhibitors (termination phase).

16.14.2.1 Calibrated automated thrombography (CAT) assay

The CAT assay relies on a low-affinity fluorogenic substrate to continuously monitoring thrombin activity in plasma /3/.

Principle

Two fluorescence measurements of the patient plasma are required for each CAT assay. In the measurement tube tissue factor and synthetic phospholipid vesicles are added to the plasma to initiate coagulation and thrombin formation. In the calibration tube a known amount of substrate-converting activity (the thrombin calibrator) is added to plasma without activating coagulation. The thrombin calibrator consists of thrombin bound to α₂-macroglobulin, thus thrombin is protected from inhibition by protease inhibitors of the plasma. A mixture of CaCl₂ and fluorogenic substrate is subsequently added in both tubes and the developing fluorescence is recorded by a fluorometer. The fluorescence in the measurement tube is produced by thrombin generated in plasma following initiation of coagulation and forms the basis for the thrombin generation curve. In the calibration tube the fluorogenic substrate is converted at a constant rate by the added thrombin calibrator. A typical thrombin generation curve is shown in Fig. 16.14-1 – Measurement parameters for endogenous thrombin potential. A short lag phase (initiation) is followed by an intensive increase of thrombin (propagation) that subsequently disappears due to inhibition by mainly antithrombin (termination).

Parameters derived from the TGT curve are /3, 4/:

• T-lag; the lag time is defined as the time needed for thrombin concentration to reach 1/6 of the peak concentration and shows a good correlation with the plasma clotting time
• Peak time or T-max; the time elapsing until the peak of thrombin generation is reached
• Peak height or C-max; the maximum thrombin activity generated
• Endogenous thrombin potential (ETP), identical with or area under curve (AUC). The ETP represents the total enzymatic activity of thrombin during the time and is generally considered the most predictive parameter of bleeding/thrombosis risk.

Prolonged T-lag and decreased C-max or ETP indicate a hypo coagulable (pre hemorrhagic) state. Vice versa, short lag times and high ETP/peak heights point at a hyper coagulable (prothrombotic) state /2/. A test method for whole blood has also been published /5/.

16.14.3 Specimen

Citrated plasma, citrated whole blood, depending on the method used.

16.14.4 Reference interval

Depending on the method used.

16.14.5 Clinical significance

Thrombin is a multifunctional protease that, in addition to hemostasis, supports non hemostatic mechanisms, such as the regulation of vascular permeability, vascular tone, inflammation, and angiogenesis.

Among the functions of the coagulation system mediated by thrombin, the most important are the fibrinogen-to-fibrin conversion, the activation of factors V, VIII, XI, XIII and protein C, and the activation of platelets. Large amounts of thrombin are inactivated by antithrombin.

Thrombin enhances not only its own formation during the propagation phase of the coagulation system (see Fig. 16.1-7 – Cascade of plasma coagulation activation and associated positive and negative feedback mechanisms), but is also self-inhibiting, in the following manner: thrombin and PC bind to thrombomodulin of the vascular endothelium, activating PC to APC. With involvement of PS, APC inactivates factors Va and VIIIa, thus lowering an upregulated coagulation (see Fig. 16.21-1 – Activation of protein C and its anticoagulatory effect in combination with the cofactor protein S.

16.14.5.1 TGT compared to PT and aPTT tests

Determination of the ETP is superior to PT and aPTT for indicating the overall hemostatic function /2/. In these tests, plasma has been estimated to start to clot as soon as 5% of the total amount of thrombin has been generated, thus leaving the remaining 95% undetected. Furthermore, owing to the relatively short interval (10–30 sec.) from the initiation of coagulation to clot formation, naturally occurring anticoagulants operating in plasma (i.e., antithrombin and PC) cannot be fully activated, especially if one considers that they require heparin-like substances (antithrombin) and thrombomodulin for their activation. These substances are located on endothelial cells and not in the plasma. Therefore, the PT and aPTT tests are unlikely to account for the total amount of thrombin generated as a function of the pro coagulant drivers and for its inhibition as a function of the anticoagulant drivers. The PT and aPTT are well known to be suitable for detecting deficiencies of the pro coagulant factors, but they are much less suitable for detecting deficiencies of the anticoagulant factors /2/.

16.14.5.2 Clinical significance of the ETP

Hemorrhage

In cases with hemorrhage, the ETP, bleeding tendency and factor deficiency are well correlated if platelet-poor plasma is used /6/. In hemophiliacs with undetectable F VIII or F IX, ETP analyzed in platelet-rich plasma has been reported to have high potential for distinguishing between mild and severe bleeding tendencies /7/.

Thrombophilia

In patients with the risk of venous thromboembolism (VTE) and congenital thrombophilic defects such as deficiencies in AT, PC, in carriers of factor V Leiden or prothrombin G20210A mutation and an imbalance of pro- versus anti-coagulation the TGT is a valuable indicator for hyper coagulability. Numerous epidemiological prospective and retrospective studies describe significant correlations between the ETP and the risk of VTE /8/. This refers to both the first event and recurrent thrombosis. The determination of the ETP in acquired thrombophilia, for example in pregnancy /9/ and diabetes mellitus /10/, is also of diagnostic and prognostic significance.

Hemostatic disorders with changes in ETP are listed in Tab. 16.14-1 – Hemostatic disorders with change in endogenous thrombin potential.

References

1. Hemker HC, Al Dieri R, Beguin S. Thrombin generation assays: accruing clinical relevance. Curr Opin Hematol 2004; 11: 170–5.

2. Tripodi A. The long-awaited whole-blood thrombin generation test. Clin Chem 2012; 58: 1173–5.

3. Castoldi E, Rosing J. Thrombin generation tests. Thrombos Res 2011; 127, Suppl 3: S21–S25.

4. Duchemin J, Pan-Petesch B, Arnaud B, Blouch MT, Abgrall JF. Influence of coagulation factors and tissue factor concentration on the thrombin generation test in plasma. Thromb Haemost 2008; 99: 767–73.

5. Ninivaggi M, Apitz-Castro R, Dargaud Y, Bas de Laat, Hemker HC, Lindhout T. Whole-blood thrombin generation monitored with a calibrated automated thrombogram-based assay. Clin Chem 2012; 58: 1252–9.

6. Duckers C, Simoni P, Spiezia l, Radu C, Gavasso S, Rosing J, et al. Residual platelet factor V ensures thrombin generation in patients with severe congenital facor V deficiency and mild bleeding symptoms. Blood 2010; 115. 879–86.

7. Santagostino E, Mancuso ME, Tripodi A, Chantarangkul V, Clerici M, Garagiola I, et al. Severe hemophilia with mild bleeding phenotype: molecular characterization and global coagulation profile. J Thromb Haemost 2010: 8: 737–43.

8. Castoldi E, Simoni P, Tormene D, Thomassen MC, Spiezia L, Gavasso S, et al. Differential effects of high prothrombin levels on thrombin generation depending on the cause of hyperprothrombinemia. J Thromb Haemost 2007: 5: 971–9.

9. Rosernkranz A, Hiden M, Leschnik B, Weiss EC, Schlembach D, Lang U, et al. Calibrated automated thrombin generation in normal uncomplicated pregnancy. Thromb Haemost 2008: 99: 331–7.

10. Tripodi A, Branchi A, Chantarangkul V, Clerizi M, Merati G, Artoni A, et al. Hypercoagulability in patients with type 2 diabetes mellitus detected by a thrombin generation assay. J Thromb Thrombolysis 2011; 31: 165–72.

11. van Hylckama Vlieg A, Christiansen SC, Luddington R, Cannegetier SC, Rosendaal FR, Baglin TP. Elevated endogenous thrombin potential is associated with increased risk of a first deep venous thrombosis but not with the risk of recurrence. Br J Haematol 2007; 138: 769–74.

12. Eichinger S, Hron G, Weltermann A, Kollars M, Kyrle PA. Endogenous thrombin potential (ETP) for assessing the risk of recurrent venous thromboembolism. Blood 2005; 106: 1622A.

13. Debaugnies F, Azerad MA, Noubouossie D, Rozen L, Hemker HC, Corazza D, et al. Evaluation of the procoagulant activity in the plasma of cancer patients using a thrombin generation assay. Thrombosis Res 2010; 126: 531–5.

14. Smid A, Dielis AWJH, Winkens M, Spronk MH, van Oerle R, Hamulyak K, et al. Thrombin generation in patients with first acute myocardial infarction. J Thrombos Haemostas 2011; 9: 450–6.

15. Chantarangkul V, Clerici M, Bressi C, Giesen PLA, Tripodi A. Thrombin generation assessed as endogenous thrombin potential in patients with hyper- or hypocoagulability. Haematologica 2003; 88: 547–54.

16.15 Analysis of individual coagulation factors

Lothar Thomas

Analyses of individual coagulation factors are performed in suspected hereditary defects, acquired decreases or isolated increases of a coagulation factor. The focus of the investigations is on factors VII, VIII, IX and on the von Willebrand factor (VWF). Screening tests such as the prothrombin time (PT) and activated partial thromboplastin time (aPTT) can provide hints, but the analysis of individual factors is usually required for further clarification.

Hereditary factor defects and acquired factor deficiencies are distinguished. Acquired deficiencies are due to defective synthesis or turnover and manifest in the presence of underlying disease. Except for F XII deficiency, they usually involve several coagulation factors.

Factors VIII, IX and VWF play a special role because they may cause hemorrhage and venous thrombosis.

16.15.1 Indication

• Suspected congenital or acquired deficiencies or defects in one or several coagulation factors in the presence of hemorrhage
• Investigation of abnormal results obtained by one or several screening tests: PT, aPTT, thrombin time
• Therapy monitoring in conjunction with the use of coagulation factor concentrates
• Suspected genetic defects of factors VIII, IX and VWF in thrombotic patients.

16.15.2 Method of determination

The activities of factors are determined mainly by one-stage assays such as the aPTT or PT and the use of factor deficient plasmas. In many cases, the antigen concentrations of individual factors are also determined by immunological methods.

Molecular biological analyses may be required to determine the presence and extent of genetic changes of the coagulation factor.

16.15.2.1 Coagulometric analysis of individual factors

Principle

The activities of factors II (prothrombin), V, VII, VIII:C, IX, X, XI, XII, pre kallikrein (Fletcher factor) and HMWK (high molecular weight kininogen) are determined mainly by so-called one-stage assays. These assays are activity measurements which measure the fibrin formation time in one single reaction step; after all, they are variants of the aPTT (factors VIII:C, IX, XI, XII, pre kallikrein or HMWK) or the PT (factors II, V, VII, X). Factor deficient plasma to which diluted patient plasma is added is used for this purpose. The assays are adjusted in such a way that the factor to be examined is the exclusive determinant of the reaction speed /1/. The residual activity of factors in deficiency plasma is below 1% /2/. The measured activity is expressed as percentage of the activity in seconds by extrapolation to a reference curve.

Activity measurements can also be performed by specific chromogenic assays /3/. These tests are mainly employed for FV III and F IX. In these tests, the speed with which an activated coagulation factor such as F Xa, splits a chromogenic substrate is measured.

16.15.2.2 Determination of protein concentration

The protein concentrations of individual factors can also be determined by immunochemical methods. For principle, refer to Section 52.1.5 – Direct antigen or antibody assays.

16.15.2.3 Gene analysis and mutation spectrum

There are generally two possibilities of genomic diagnostics on a DNA level /4/:

• Direct genomic analysis; identification of mutations in the affected gene
• Indirect genomic analysis; characterization of markers, for example restriction fragment length polymorphisms (RFLPs), short tandem repeats or variable number tandem repeats (VNTRs), which are closely linked to the mutation located in the affected gene.

16.15.3 Specimen

Citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L, mixed with 9 parts of blood): 2 mL

16.15.4 Reference interval

Clotting time:	F II, F V, F VII, F IX, F X, F XI	70–120% of normal
	F VIII, F XII, HMWK; pre kallikrein	70–150% of normal

16.15.5 Clinical significance

Deficiency of clotting factors in hereditary and acquired coagulopathies are afibrinogenemia, prothrombin deficiency and deficiencies of FV, FVII, FVIII, FIX, FX, FXI, FXII, FXIII and congenital deficiency of Vitamin K dependent factors /5/.

16.15.5.1 Hereditary factor defects

Among the congenital factor defects, dysproteinemias are distinguished from aproteinemias. The former are characterized by the exchange of one single amino acid due to a point mutation (e.g., as observed in certain forms of hemophilia A) whereas the genetic information in the latter is altered to such a degree that it can no longer be read at all or the abnormal mRNA is immediately broken down. Both forms can be inherited in a homozygous or heterozygous pattern.

Homozygous factor deficiencies are associated with extremely low residual activities, while heterozygotes display levels of 20–50% of normal. For the autosomally inherited and encoded coagulation factors this implies that the genetic information of both autosomes must be present in order for the activity to be normal.

Dysproteinemias can be differentiated from aproteinemias only by immunochemical methods (e.g., immunofixation electrophoresis). The clinically observed bleeding tendency correlates strictly with the remaining residual activity. Hereditary factor deficiencies are listed in Tab. 16.15-1 – Hemostatic disorders associated with hereditary reduction in coagulation factors.

16.15.5.2 Hemophilia A

Hemophilia A is an X-linked recessive bleeding disorder, characterized by absent or low concentration or ineffective FVIII. Hemophilia A causes severe bleedings that are potentially life-threatening. The standard care consists in prophylactic replacement of FVIII. New therapy consists of neutralizing Emicizumab, a recombinant agent for hemophilia. Emicizumab is a humanized bispecific monoclonal IgG4 antibody designed to mimic activated FVIII by bridging factor IXa and factor X thus effecting hemostasis /19/.

Laboratory monitoring in Emicizumab-treated patients with hemophilia A

Laboratory methods are /20/:

• One common test based on the aPTT is the one-stage FVIII clotting assay (OSA). The OSA measures the ability of the patient plasma to shorten the aPTT after mixing with FVIII-deficient plasma
• An alternative to the FVIII OSA is the FVIII two-stage FVIII clotting assay
• Based on the similar principle to the two-stage clotting assay, the F VIII CSA measures the FVIII dependent activation of FX using purified human or bovine coagulation factors.

16.15.5.3 Hereditary increase in coagulation factors

Increased activities of coagulation factors and their genotypes are potentially associated with venous thromboembolism (VTE) and atherothrombosis. Factors VIII, IX and the von Willebrand factor (VWF) are important in this context.

VWF

The VWF is synthesized by endothelial cells and macrophages. It leads to the adhesion of platelets to vascular wall collagen released due to injury. It has been found that increased concentrations of VWF-F VIII complexes are associated with VTE and atherothrombosis. Individuals with A and B blood groups have an increased risk of VTE which is assumed to be linked to an 25–30% increased level of the VWF-F VIII complex /10/.

Factor VIII

A high plasma level of F VIII is a risk factor for VTE. In the Leiden thrombophilia study, patients with levels above 150 IU/dL had a 5-fold increased risk of VTE. The risk of recurrence within 30 months in patients with levels above the 90th percentile of control individuals (above 294 IU/dL) was 6.7-fold higher than in those with lower levels /11/. In many cases, high levels of F VIII may be responsible for acquired resistance to APC if F V Leiden mutation is excluded as the underlying cause.

Factor IX

The F IXa plays a key role in maintenance of thrombin (and hence fibrin) formation within the intrinsic coagulation pathway. In a study /12/, an A>G sequence variant encoding F IX (rs 6048, F9 Malmö) was identified that is associated with deep vein thrombosis.

16.15.5.4 Acquired reduction in coagulation factors

Acquired reductions in coagulation factors are more common than hereditary-type ones and usually involve several coagulation factors. The causes of hemostatic disorders with associated reduction in coagulation factors are listed in Tab. 16.15-2 – Hemostatic disorders associated with acquired reduction in coagulation factors.

16.15.5.5 Factor replacement therapy and course monitoring

Factor replacement therapy may be required in congenital hemostatic defects (e.g., in von Willebrand disease, hemophilia A, hemophilia B and in the more unusual cases of isolated deficiencies of factor II, VII, X, XI and XIII) /16, 17/.

Acquired hemostatic defects usually occur in the form of acute bleeding complications during the peri- or postoperative period or in conjunction with advanced liver disease as well as disturbances in acid-base and electrolyte homeostasis such as the hemolytic-uremic syndrome.

Prior to major surgery, the activities of the coagulation factors should be at least 60%, whereas for less extensive surgical procedures in which the risk of bleeding can be limited by local hemostatic measures an activity of 35% will be appropriate.

If, in the case of a known factor deficiency, preoperative factor replacement is required, the levels should be determined prior to (baseline) and, for monitoring, after replacement therapy by means of a one-stage assay. Intraoperative monitoring of the levels is necessary in surgery lasting > 3 h and in all episodes of intraoperative bleeding. Further monitoring of the levels is required immediately, postoperatively, and once or twice daily until wound healing is complete.

The dosing schedule for factor replacement therapy is based on the body weight of the patient and the half-lives of the individual coagulation factors (Tab. 16.15-3 – Required activities and monitoring in patients with factor deficiencies during peri operative periods).

A coagulation factor unit refers to the activity which is contained in 1 mL of pooled citrated plasma from healthy blood donors. WHO reference materials exist for factors II, VII, VIII:C, IX and X. The administration of 1 unit of a coagulation factor per 1 kg of body weight raises the activity by 1%. After surgical procedures, the activity level should be at least 60% for the first 8 days and 30% for the following 4–8 days.

Antithrombin must be determined prior to factor replacement (e.g., in surgical patients with liver parenchymal injury or in patients requiring massive intraoperative blood transfusions). The reason for this is that the replacement of pro coagulant substances may cause thromboses or disseminated intravascular coagulation in the presence of underlying antithrombin deficiency.

Factor replacement therapy can be accomplished by using:

• Fresh frozen plasma: after thawing, it contains all coagulation factors with a residual activity > 70% (≥ 0.7 U). In the case of more severe factor deficiencies, replacement is limited by the need to avoid volume overload; approximately 1 mL of plasma/kg of body weight is needed in order to raise the activity by 1%.
• PPSB: it contains the prothrombin complex (factors II, VII, X, IX as well as proteins C and S). Reduction in the prothrombin complex is the result of defective synthesis which is due to either liver parenchymal injury or vitamin K deficiency. Reduced prothrombin complex activity is also present in disseminated intravascular coagulation.
• Concentrates of factors VIII, IX, XIII and protein C.

16.15.6 Comments and problems

Although the plasma to be investigated is diluted to such a degree that influences other than those of the factor to be investigated should be eliminated, high concentrations of heparin, autoantibodies, fibrin(ogen) degradation products or other inhibitory substances may still cause falsely low activities.

Falsely high activities may result from the blood collection or storage of activated samples. This applies, in particular, to the determination of F VIII and other factors of the intrinsic coagulation pathway.

16.15.7 Pathophysiology

Almost all coagulation factors are synthesized in the liver. Acquired factor deficiencies are the result of an underlying primary disease and are due either to defects in the synthesis (practically all liver diseases, especially toxic liver failure, shock liver) or increased turnover recognizable by shortened coagulation factor half-lives. The causes for an increased turnover (consumption) are primarily based on:

• Disseminated intravascular coagulation due to intravascular thrombin formation and hyper fibrinolysis (consumption coagulopathy)
• Increased fibrinolysis (plasminemia)
• Release of proteolytic enzymes (e.g., lysosomal enzymes such as the granulocyte elastase, liberated with the lysis of leukocytes)
• Abnormal losses such as observed in patients with nephrotic syndrome or exudative enteropathy as well as in ascites (exudation into the ascitic fluid) and heavy blood losses
• Adsorption to abnormal surfaces (e.g., to amyloid fibrils in amyloidosis, or to tumor cells as well as to areas of endothelial injury).

References

1. Lutze G, Urbahn H. One-stage determinations of blood clotting factors – methodological aspects and quality criteria of deficient plasmas. Lab Med 1989; 13: 457–63.

2. Lutze G, Kunstmann S. Commercial factor VIII and factor IX depleted plasmas: analysis of clotting factors. J Lab Med 2001; 25: 172–6.

3. Witt I. Test systems with synthetic peptide substrates in haemostaseology. Eur J Clin Chem Clin Biochem 1991; 29: 355–74.

4. Herrmann FH, Wulff K. Gerinnungsfaktoren VII, VIII, IX und X. Ausgewählte Aspekte zur Molekulargenetik und Gendiagnostik. Hämostaseologie 2004; 24: 94–107.

5. Palla R, Peyvandi F, Shapiro AD. Rare bleeding disorders: diagnosis and treatment. Blood 2015; 125: 2052–61.

6. Perry DJ. Factor VII deficiency. Br J Haematology 2002; 118: 689–700.

7. Castaldo G, D’Argenio V, Nardiello P, Zarrilli F, Sanna V, Rocino A, et al. Hemophilia A: molecular insights. Clin Chem Lab Med 2007; 45: 450–61.

8. Tiede A, Collins P, Knoebl P, Teitel J, Kessler C, Shima M, et al. International recommendations on the diagnosis and treatment of acquired hemophilia A. Haematologica 2020; 105 (7): 1791-1801.

9. Repesse Y, Peyron I, Dimitrov JD, Dasgupta S, Moshai EF, Costa C, et al. Development of inhibitory antibodies to therapeutic factor VIII in severe hemophilia A is associated with microsatellite polymorphism in the HMOX1 promoter. Haematologica 2013; 98: 1650–5.

10. Wu O, Bayoumi N, Vickers MA, Clark P. AB0 (H) blood groups and vascular disease: a systematic review and meta-analysis. J Thromb Haemost 2008; 6: 62–9.

11. Kyrle PA, Minar E, Hirschl M, Bialonczyk C, Stain M, Schneider B, et al. High plasma levels of factor VIII and the risk of recurrent venous thromboembolism. N Engl J Med 2000; 343: 457–62.

12. Bezemer ID, Arellano AR, Tong C, Rowland CM, Ireland HA, Bauer KA, et al. F9 Malmö, factor IX and deep vein thrombosis. Haematologica 2009; 94: 693–9.

13. Bechthold H, Andrassy K, Koderisch J, Trenk D, Sonntag H, Jähnchen E. Cumarinartige Hemmung des Vitamin-K1-Stoffwechsels durch strukturell unterschiedliche Cephalosporine. FAC 1987; 6: 609–22.

14. Scharrer I, Grossmann R. Erworbene Hemmkörperhämophilie. Anaesthesist 2000; 49: 34–42.

15. Gilbert GE. The evolving understanding of factor VIII binding sites and implications for the treatment of hemophilia A. Blood Reviews 2019. doi: 10.1016/j.blre.2018.05.001.

16. Wissenschaftlicher Beirat der Bundesärztekammer Deutschlands. Leitlinien zur Therapie mit Blutkomponenten und Plasmaderivaten. Köln: Deutscher Ärzteverlag, 1995.

17. Tilsner V. Blutungskomplikationen in der perioperativen Phase. In: Mammen EF, ed. Intensivmedizin aktuell. Marburg; Medizinische Verlagsgesellschaft 1987.

18. Fusaro M, Gallieni M, Rizzo MA, Stucchi A, Delanaye P, Vavalier E, et al. Vitamin K plasma levels determination in human health. Clin Chem Lab Med 2017; 55: 789–99.

19. Nardi MA. Hemophilia A: Emicizumab monitoring and impact on coagulation testing. Advances in Clinical Chemistry 2023; 113: 273–315.

20. Müller J, Pekrul I, Pötzsch B, Berning B, Oldenburg J, Spannagl M. Laboratory monitoring in Emicizumab-treated persons with hemophilia A. Thromb Haemost 2019; 119: 1384–93.

16.16 Fibrinogen

Lothar Thomas

Fibrinogen is the plasma protein with the highest concentration in the coagulation system and plays a key role in the hemostatic system. Thrombin-catalyzed cleavage of fibrinopeptides A and B converts fibrinogen into fibrin, which spontaneously polymerizes and forms double stranded protofibrils that assemble into branched fibrin fibers, forming the fibrin clot /1/. Low fibrinogen concentrations are associated with bleeding and high ones are associated with thrombosis.

16.16.1 Indication

• Preoperatively in patients with existing hemorrhage, a history of bleeding disorder or in clinical indication (elevated liver enzymes, HELLP syndrome)
• Intra- and peri operatively in severe blood loss and massive volume replacement
• Exclusion of disseminated intravascular coagulation in combination with antithrombin, D-dimers, TT and aPTT
• Detection of congenital or acquired deficiencies or defects in fibrinogen (hypo fibrinogenemia, dysfibrinogenemia or afibrinogenemia)
• Monitoring of thrombolytic therapy (e.g., with ancrod or defibrase)
• Detection of elevated fibrinogen concentration as a marker for atherothrombosis
• Abnormal results in screening tests (e.g., PT, aPTT and TT).

16.16.2 Method of determination

The fibrinogen concentration can be determined using various methods:

• Thrombin clotting rate assay according to Clauss
• Kinetic turbidimetry (often used on automated platforms)
• Immunochemical method (used for the evaluation of dysfibrinogenemia)
• Derived fibrinogen (results correlate well with the thrombin clotting rate assay with the exception of samples containing high concentrations of fibrin(ogen) degradation products).

16.16.2.1 Thrombin clotting rate assay

Principle

Fibrinogen is determined by diluting the plasma sample (1 : 10) and adding an excess of thrombin (100 U/mL) to overcome the influence of inhibitors. The time required for clot formation is recorded (Clauss method). The clotting time of the plasma is inversely proportional to the fibrinogen concentration. The clotting time obtained is then compared with that of a normal plasma pool in which the fibrinogen concentration has been calibrated against an assayed reference material. Patient plasma is diluted 1 : 10 in 0.02 mol/L barbital puffer, warmed for 5 min. at 37 °C and 0.1 mL of thrombin (100 NIH U/mL) is added to start the coagulation /2, 3/.

16.16.2.2 Total clottable fibrinogen assay

Principle

In the definitive reference method thrombin is added to citrated plasma and during prolonged incubation in the absence of Ca²⁺ converts all fibrinogen to a fibrin clot. Epsilon aminocarproic acid is added to inhibit plasmin degradation of the fibrin clot. Soluble proteins trapped within the clot are removed by gentle expression of serum from the clot and by subsequent washing of the clot. In the absence of Ca²⁺, the clot is not stabilized and remains soluble in concentrated urea solution. The fibrinogen concentration in g/L is measured either by light absorption at 282 nm or by determining the tyrosine content using Folin’s reagent /4, 5/.

16.16.2.3 Kinetic turbidimetry

Principle

Batroxobin, a thrombin-like enzyme from the saliva of the snake Bothrops atrox, cleaves fibrinopeptide A from fibrinogen. The resulting fibrin monomers polymerize and lead to an increase in turbidity. This rise in turbidity is linear under the selected reaction conditions and can be measured at 340 or 405 nm. The increase in absorption per time unit is directly proportional to the fibrinogen concentration. The plasma from patients is used undiluted /6/.

16.16.2.4 Immunochemical methods

Principle

Immunochemical methods such as radial immunodiffusion, immunonephelometry or immunoturbidimetry are used. They employ specific antisera directed against fibrinogen /7/.

16.16.2.5 Derived fibrinogen

Principle

Using turbidimetric or nephelometric endpoint detection for PT determination, the total increase of turbidity is directly proportional to the clottable fibrinogen concentration. When in the PT measurement a certain, predefined threshold value of the optical density is reached this increase is specified as the “clotting time”. Only after the clotting time is reached, will fibrinogen be cleaved as well. Since the products thus produced will also cause additional optical density, the fibrinogen concentration (derived fibrinogen) can be calculated based on the difference of turbidity (endpoint turbidity minus clotting time turbidity) /8/.

16.16.3 Specimen

Citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood): 1 mL

16.16.4 Reference interval

Refer to Ref. /9/ and Tab. 16.16-1 – Reference intervals for fibrinogen.

16.16.5 Clinical significance

Fibrinogen or coagulation factor I:

• Is the substrate of thrombin, the last enzyme of the coagulation system
• Is the substrate of plasmin, the last enzyme of the fibrinolytic system
• Belongs to the group of acute phase proteins
• Is synthesized in the liver.

Hemostasis is the physiological process preventing loss of blood and involves the interaction of vessel wall, platelets and the pro- and anticoagulation systems.

Fibrinogen has two functions:

• In primary hemostasis, it plays a key role in platelet aggregation by linking activated platelets. On their membrane, the platelets express the glycoprotein receptor GPIIb/IIIa (integrin αIIbβ3). The receptor binds fibrinogen present in plasma and released by platelet granules (see Fig. 16.1-5 – Platelet adhesion and platelet aggregation)
• In secondary hemostasis as the activator of fibrinformation.

Diseases and conditions associated with decreased fibrinogen concentration are listed in Tab. 16.16-2 – Diseases and conditions associated with decreased fibrinogen

Diseases and conditions associated with increased fibrinogen concentration are shown in Tab. 16.16-3 – Diseases and conditions associated with increased fibrinogen.

Not all of the plasma fibrinogen is functional (clottable) /5/. This is because circulating fibrinogen shows considerable heterogeneity in structure and function, partly due to multiple genetic influences and partly to degradation by enzymes including thrombin, plasmin and neutrophil elastase. Therefore, assays such as the method according to Clauss which determine functional fibrinogen based on clot formation yield different results compared to immunological methods which determine the total fibrinogen, whether clottable or not. These assays are inappropriate for the assessment of bleeding risk. They are useful, however, to distinguish functional fibrinogen from dysfibrinogenemia. In low total fibrinogen (hypofibrinogenemia, afibrinogenemia) and dysfibrinogenemia clottable fibrinogen is low, however in dysfibrinogenemia the fibrinogen concentration is normal.

16.16.6 Comments and problems

Method of determination /2, 5/

In general, the fibrinogen concentrations determined with the thrombin clotting rate assay is too low and those determined with derived methods are too high as compared to the reference method.

Clottable fibrinogen assays (Clauss method): although clottable fibrinogen assays use thrombin as the enzyme to transform the fibrinogen, no remarkable, undesirable sensitivity towards heparin is present because the sample is pre diluted (1 : 10) and a large thrombin excess is employed.

Derived fibrinogen: in the presence of fibrin(ogen) degradation products (FDP), this method produces higher levels than the clottable fibrinogen assays. FDP aggregate with fibrin fibrils and thus increase turbidity. Heparin may have an impact on the derived fibrinogen measurement depending on the heparin dependence of each of the thromboplastin reagents used. Usually, derived fibrinogen methods based on PT determination are reliable only if the PT values are ≥ 25% of normal. Compared to the method according to Clauss, the concentrations determined with this method are too high in patients with liver cirrhosis, renal insufficiency, disseminated intravascular coagulation and dysfibrinogenemia. For instance, in a comparative study /10/ involving patients with dysfibrinogenemia, the median fibrinogen was 0.40 g/L (0.30–2.07 g/L) determined by the Clauss assay and 2.41 g/L (0.97–4.87 g/L) determined by the derived method. Fibrinogen measured with the PT-derived method was about 5-fold higher and an existing dysfibrinogenemia would not have been detected.

Precipitation methods: like the method according to Ratnoff-Menzie, they mimic falsely elevated levels, as a result of the co precipitation of other proteins.

Immunochemical methods: in the presence of FDP, falsely elevated levels are measured because of cross antigenicity with fibrinogen.

Stability

Mild increase in whole blood after 8 h, mean of 15% changes after 24 h with individual variability /11/. In plasma up to 24 hours at ambient temperature.

Differential diagnosis of hypofibrinogenemia and dysfibrinogenemia

The differential diagnosis measured by the PT-derived fibrinogen assay in comparison to the fibrinogen Clauss assay is made in patients with dysfibrinogenemia and patients with hypofibrinogenemia. In patients with hypofibrinogenemia, there was a correlation (r = 0.9016) between the fibrinogen Clauss assay and PT-derived fibrinogen assay. The results measured by the PT-derived fibrinogen assay were approximately four times higher compared to the fibrinogen Clauss assay in patients with dysfibrinogenemia /20/

.

16.16.7 Pathophysiology

Fibrinogen is synthesized in the liver and has a molecular weight of 340 kDa. It is a dimeric molecule, with each half containing three different 45 nm long polypeptide chains (Aα, Bβ, γ) which are linked by disulfide bridges. The fibrinogen chains are folded into distinct structural regions, comprising a central E region and two outer D regions linked by coiled-coil connections (Fig. 16.16-1 – Molecular structure of fibrinogen). The E domain consists of the N-terminal ends of all six polypeptide chains, whereas the D domains primarily comprise the C-terminal ends of the Bβ and γ-chains /12/. Thrombin cleaves the Aα and Bβ chains to release fibrinopeptides A and B. After release the resulting fibrin monomers undergo polymerization to form an insoluble fibrin clot.

The three polypeptide chains are encoded by the separate genes FGA, FGB and FGG. They are located in the region of 50 kilo bases on chromosome 4q31.3 (Fig. 16.16-2 – Fibrinogen locus on chromosome 4).

In the blood, fibrinogen exists in various forms with slightly different molecular weights /13/. For instance, a fibrinogen proportion includes a variably structured γ chain identified as heterodimer with an A-chain as γA/γ^– chain /14/. The γ^– chain differs from the γA chain by its C-terminus and, thus, has no impact on the functional behavior of fibrinogen. The presence of the γA/γ^– chain modulates the thrombin functions, the F XIII activity and the fibrin clot architecture and eliminates the platelet binding site. There are associations between γA/γ^– fibrinogen and atherothrombosis and VTE.

As an acute phase protein, fibrinogen rises, with a delay of 24–48 h, to high levels in systemic inflammation.Pronounced fibrinogen deficiency may be present in severe liver parenchymal injury as a result of defective synthesis. Usually, however, fibrinogen deficiency is based on increased consumption (e.g., due to disseminated intravascular coagulation, consumption coagulopathy and hyperfibrinolysis).

As a hemostatic component, fibrinogen is the substrate of the coagulation cascade. Following the activation of the coagulation system and the thrombin-catalyzed cleavage of fibrinopeptides A and B from fibrinogen, the remainder of the molecule polymerizes into fibrin. Subsequently, F XIII catalyzes the cross linking of the fibrin polymers, thus leading to the formation of a crosslinked fibrin clot which, together with other ongoing processes, causes the bleeding to cease by closing off the blood vessel.

Cryofibrinogen is a fibrinogen which can be subject to precipitation under cold temperatures (see Section 18.11 – Cryoglobulins and cryofibrinogen).

References

1. Weisel JW. Fibrinogen and fibrin. Adv Protein Chem 2005; 70: 247–99.

2. NCCLS. Procedure for the determination of fibrinogen in plasma; approved guideline. NCCLS Document H30A Vol 14 No2. 1994.

3. Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Haematol 1957; 17: 237–46.

4. Marbet GA, Duckert F. Fibrinogen. In: Jespersen J, Bertina RM, Haverkate F, eds. ECAT assay procedures: a manual of laboratory techniques. Dordrecht: Kluwer, 1992: 47–56.

5. Lowe GDO, Rumley A, Mackie IJ. Plasma fibrinogen. Ann Clin Biochem 2004; 41: 430–40.

6. Palareti G, Maccaferri M. Specific assays of hemostasis proteins: Fibrinogen. Res Clin Lab 1990; 20: 167–76.

7. Hoffmann JJML, Verhappen MAL. Automated nephelometry of fibrinogen: Analytical performance and observations during thrombolytic therapy. Clin Chem 1988; 34: 2135–40.

8. Halbmayer WM, Haushofer A, Schön R, Radek J, Fischer M. Comparison of a new automated kinetically determined fibrinogen assay with the 3 most used fibrinogen assays (functional, derived and nephelometric) in Austrian laboratories in several clinical populations and healthy controls. Haemostasis 1995; 25: 114–23.

9. Tarallo P, Henny J, Gueguen R, Siest G. Reference limits of plasma fibrinogen. Eur J Clin Chem Clin Biochem 1992; 30: 745–51.

10. Miesbach W, Schenk J, Alesci S, Lindhoff-Last E. Comparison of the fibrinogen Clauss assay and the fibrinogen PT derived method in patients with dysfibrinogenemia. Thrombosis Res 2010; 126: e428–e433.

11. Kemkes-Matthes B, Fischer R, Peetz D. Influence of 8 and 24-h storage of whole blood at ambient temperature on prothrombin time, activated partial thromboplastin time, fibrinogen, thrombin time, antithrombin and D-dimer. Coagulation and fibrinolysis 2011; 22: 215–20.

12. Meyer M. Molekularbiologie der Gerinnung: Fibrinogen, Faktor XIII. Hämostaseologie 2004; 34: 108–15.

13. Plendl H, Caliebe A, Grote W. Molekulare Varianten des Fibrinogens. Hämostaseologie 2002; 22: 76–81.

14. De Willige SU, Standeven KF, Philippou H, Ariens RA. The pleiotropic role of fibrinogen γ’chain in hemostasis. Blood 2009; 114: 3994–4001.

15. Cote HC, Lord St, Pratt KP. γ-chain dysfibrinogenemias: molecular structure-function relationships of naturally occuring mutations in the γ chain of human fibrinogens. Blood 1998; 92: 2195–12.

16. Morris TA, Marsh JJ, Chiles PG, Magana MM, Liang NC, Soler X, et al. High prevalence of dysfibrinogenemia among patients with chronic thromboembolic pulmonary hypertension. Blood 2009; 114: 1929–36.

17. Dempfle CE. Gerinnungsstörungen. Handbuch Labormedizin. Wiesbaden; Diagnostik Update 2011.

18. Giovannini I, Chiarla C, Giulante F, Vellone M, Nuzzo G. Modulation of plasma fibrinogen levels in acute-phase response after hepatectomy. Clin Chem Lab Med 2004; 42: 261–5.

19. Simon G, Thompson MA, Kienast J, Pyke SDM, Haverkate F, van de Loo JCW. Hemostatic factors and the risk of myocardial infarction or sudden death in patients with angina pectoris. N Engl J Med 1995; 332: 635–41.

20. Skornova I, Simurda T, Stasko J, Horvath D, Zolkova J, Holly P, et al. Use of fibrinogen determination methods in differential diagnosis of hypofibrinogenemia and dysfibrinogenemia Clin Lab 2021; 67: 1028–34.

16.17 Factor XIII

Lothar Thomas

Factor XIII (F XIII) is the fibrin stabilizing factor since clots formed in the absence of activated F XIII lack stability. F XIII plays an important role in the terminal phase of the coagulation system that promotes formation of cross-linked fibrin polymers leading to a stable hemostatic clot. F XIII is converted into an active trans glutaminase (F XIIIa) by fibrin and Ca²⁺. F XIII stabilizes fibrin mechanically and protects it from fibrinolysis /1/. Patients with congenital deficiency of the factor develop a severe but rare bleeding disorder. F XIII deficiency is not detected by PT, aPTT and TT tests, but can be identified by special assays and by thromboelastography.

16.17.1 Indication

F XIII deficiency is suspected in:

• Diagnostic investigation of bleeding episodes
• Disseminated intravascular coagulation (consumptive coagulopathy)
• Diagnostic investigation of impaired healing
• Umbilical cord bleeding and intracranial bleeding.

16.17.2 Method of determination

Kinetic UV test

Principle: F XIII is a pro transglutaminase activated by thrombin to form trans glutaminase (F XIIIa). F XIIIa then links a specific peptide substrate to glycine-ethyl ester, thus producing ammonia. The latter is determined by an enzymatic reaction which is set to run in parallel with the primary reaction. The decrease in NADH is measured kinetically at 340 nm (Fig. 16.17-1 – Principle of F XIII determination). Fibrinogen is not removed prior to the measurement since this is associated with a loss of F XIII. Instead, fibrin which is produced under the influence of thrombin is prevented from forming a clot by an aggregation-inhibiting protein (clot inhibitor) and thus remains in a soluble form /2/.

Immunochemical assay

Employment of the Laurell immunoelectrophoresis including the use of antiserum directed against the F XIII subunit A.

16.17.3 Specimen

Citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood): 1 mL

16.17.4 Reference interval

Factor XIII: 70–140% of normal

16.17.5 Clinical significance

F XIII circulates in plasma bound to fibrinogen and is essential for stable wound closure. It induces cross linking and mechanical stabilization of the fibrin polymers formed in the course of blood coagulation. Incorporation of α₂-antiplasmin into the fibrin aggregate protects the clot from excessively rapid lysis and facilitates the migration of connective tissue cells into the stabilized fibrin /3/.

16.17.5.1 Factor XIII deficiency

The following forms of deficiency are distinguished /1/:

• Congenital deficiency. Umbilical cord bleeding occurs in 80% of congenital F XIII deficiency
• Deficiency due to polymorphisms in the gene of F XIII. A risk factor for hemorrhagic shock among younger women and a risk for recurrent pregnancy loss are reported to the presence of Pro564leu and Tyr204Phe polymorphism in F XIII subunit A gene.
• Acquired F XIII deficiency (Tab. 16.17-1 – Hemostatic disorders in association with reduced factor F XIII).

Under normal conditions, a decrease in F XIII level to approximately 10% of normal is not associated with any significant bleeding tendency.

Acquired F XIII deficiency may occur due to increased consumption in impaired synthesis or can be induced by drugs or F XIII antibodies (e.g., in autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis). Except for Henoch- Schönlein purpura, an acute episode of ulcerous colitis and Crohn’s disease, F XIII deficiency is rarely observed as an isolated condition, but mostly occurs in combination with other coagulation disorders.

A decrease in F XIII after surgical procedures can be due to massive loss or dilution of blood. F XIII deficiency is also observed in pro myelocytic leukemia, gynecological tumors, disseminated intravascular coagulation and in the course of severe liver disease /4, 5/. The F XIII concentration in all of these patients should be at least 50% /6/. After major surgery, however, dangerous hemorrhages may occur at concentrations between 10–40% /7/.

The clinical symptoms of F XIII deficiency include umbilical cord bleeding, intracranial bleeding, delayed-onset bleeding episodes after injuries and major surgery, suffusions after blunt trauma, intramuscular bleeding, hemarthroses, delayed wound healing and spontaneous miscarriages /8/.

16.17.5.2 Elevated factor XIII levels

Elevated F XIII concentrations may be associated with the risk of atherothrombosis. It is assumed that elevated FXIII levels mechanically strengthen the fibrin clot and make it more resistant to shear forces and to fibrinolysis. In a study /9/, F XIII activity in the upper tertile of a cohort (above 120% by activity measurement and/or above 25.5 mg/L by immunochemical method) was associated with a more than two-fold risk of peripheral artery disease in females. This was not the case in males.

16.17.6 Comments and problems

Ammonia concentrations above 294 μg/dL (172 μmol/L) in the sample may interfere with the kinetic UV test, resulting in falsely low activity. If necessary, the ammonia concentration must be determined, followed by repeat testing after the sample has been appropriately diluted using normal saline.

In the case of very low (below 0.8 g/L) and very high (above 8 g/L) fibrinogen concentrations, falsely low F XIII activities may be measured. In the case of high fibrinogen concentrations, the sample may also be pre diluted using normal saline.

16.17.7 Biochemistry and physiology

F XIII belongs to the family of trans glutaminases (protein-glutamine γ-glutamyl-ε-lysyltransferase, EC 2.3.2.13), catalyzing the insertion of ε- (γ-glutamyl)-lysyl bonds between two peptide chains. In the presence of Ca²⁺ lysis of the fibrin clot is, thus, prevented in a slightly alkaline environment.

F XIII exists as cellular protein and as plasma protein. The plasma protein is a heterotetramer composed of two identical globular A subunits of 83 kDa (F XIII-A) non covalently bound to two long fibrillar units B (F XIII-B) of 73 kDa. F XIII-A is synthesized in platelets and monocytes/macrophages. Concentrations in platelets are around 100 times higher than in plasma. Damaged platelets release their F XIII A into the plasma where it binds to F XIII B which is released by the hepatocytes and functions as carrier and protective protein /10/. The entire F XIII-A₂B₂ has a molecular weight of 326 kDa. In plasma, the heterotetramer F XIII-A₂B₂ is converted to its active form by the thrombin-catalyzed hydrolysis of the Arg37–Gly38 peptide bond at the N-terminus of the F XIII-A subunit (Fig. 16.17-2 – F XIII is a heterotetramer). In the presence of Ca²⁺ the F XIII-A₂B₂ heterodimer complex dissociates, yielding F XIII-B₂ and activated F XIII-A₂ /11/. Ca²⁺ cause small but significant conformational changes in F XIII-A during activation, exposing potential exosites within F XIII-A._. Activated F XIII-A₂ stabilizes the forming protofibril by introducing ε-amino(γ-glutamyl)lysine cross-links between carboxyl terminal portions of adjacent fibrin γ chains, before lateral association of the protofibril. See Fig. 16.16-1 – Molecular structure of fibrinogen.

The fibrinogen residues αC 242-424 play a major regulatory role in the activation of F XIII-A₂B₂. Glu396 is the key amino acid residue involved in binding activated F XIII-A₂ /11/. Once a fibrin clot has formed, α₂-antiplasmin is incorporated preventing premature fibrinolysis of the clot.

F XIII is a multifunctional protein. In addition to its role in hemostasis, it is essential for carrying out pregnancy and its role in wound healing and angiogenesis has also been unequivocally demonstrated. It also plays an important role in maintaining vascular permeability and is involved in the stabilization and mineralization of extracellular matrix in bone and cartilage /12/.

References

1. Levy JH, Greenberg C. Biology of factor XIII and clinical manifestations of factor XIII deficiency. Transfusion 2013; 53: 1120–31.

2. Fickenscher K, Aab A, Stüber W. A photometric assay for blood coagulation factor XIII. Thromb Haemostas 1991; 65: 535–40.

3. van Giezen JJJ, Minkema J, Bouma BN, Jansen JWCM. Cross-linking of α₂-antiplasmin to fibrin is a key factor in regulating blood clot lysis: species differences. Blood Coag Fibrinol 1993; 4: 869–75.

4. van Wersch JWC, Peters C, Ubachs JMH. Coagulation factor XIII in plasma of patients with benign and malignant gynaecological tumours. Eur J Clin Biochem 1994; 32: 681–4.

5. Zuzuki R, Toda H, Takamura Y. Dynamics of blood coagulation factor XIII in ulcerative colitis and preliminary study of the factor XIII concentrate. Blood 1989; 59: 162–4.

6. Kohler HP. Role of blood coagulation factor XIII in vascular diseases. Swiss Med WKLY 2001; 131: 31–4.

7. Egbring R, Kröninger A, Seitz R. Erworbene Inhibitoren gegen Faktor XIII. Hämostaseologie 1996; 16: 174–9.

8. Thies HA, Richter H. Erworbener Faktor XIII-Mangel und klinische Chirurgie. Med Welt 1981; 32: 250–5.

9. Shemirani AH, Szomjak E, Csiki Z, Katona E, Bereczky Z, Muszbek L. Elevated factor XIII level and the risk of peripheral artery disease. Haematologica 2008; 93: 1430–2.

10. Greenberg CS, Birckbichler PJ, Rice RH. Transglutaminases: multifunctional cross-linking enzymes that stabilize tissues. FASEB J 1991; 5: 3071–7.

11. Smith KA, Adamson PJ, Pease RJ, Brown JM, Balmforth AJ, Cordell PA, et al. Interactions between factor XIII and the αC-region of fibrinogen. Blood 2011; 117: 3460–8.

12. Komaromi I, Bagoli Z, Muszbek L. Factor XIII: novel structural and functional aspects. J Thromb Haemost 2010; 9: 9–20.

13. Norgauer J, Mockenhaupt M, Herouy Y. Ulcus cruris: Gerinnungsfaktor XIII und Wundheilung. Die gelben Hefte 2001; 41: 115–24.

14. Meyer M. Molekularbiologie der Gerinnung: Fibrinogen, Faktor XIII. Hämostaseologie 2004; 24: 108–15.

15. Luo YY, Zhang GS. Acquired factor XIII inhibitor: clinical features, tratment, fibrin structure and epitope determination. Haemophilia 2011; 17: 393–8.

16. Shirahata A, Shirakawa Y. Intrakranielle Blutungen bei Frühgeborenen. Die gelben Hefte 1995; 35: 78–81.

17. Perez DL, Diamond EL, Castro CM, Diaz A, Buonanno F, Nogueira RG, et al. Factor XIII deficiency related recurrent spontaneous intracerebral hemorrhage: A case and literature review. Clinical Neurology and Neurosurgery 2011; 113: 142–5.

18. Fukui H. Die allergische Purpura und ihre Therapie mit Gerinnungsfaktor XIII. Die gelben Hefte 1988; 28: 112–5.

19. Dempfle CE, Gladisch R, Heene DL. Faktor XIII bei Colitis ulcerosa und Morbus Crohn. Die gelben Hefte 1992; 32: 64–9.

20. Saito H, Fukushima R, Kobori O, Kawano N, Muto T, Morioka Y. Marked and prolonged depression of factor XIII after esophageal resection. Japan J Surg 1992; 22: 201–6.

21. Gödje O, Lamm P, Schütz A, Reichart B. Faktor XIII in der Herzchirurgie. Die gelben Hefte 2000; 40: 19–26.

22. Muszbek L, Bereczky Z, Bagoly Z, Komaromi I, Katona E. Factor III: a coagulation factor with multiple plasmatic and cellular functions. Physiol Rev 2011; 91: 931–72.

16.18 von Willebrand syndrome

Reinhard Schneppenheim, Lothar Thomas

The most common of the inherited bleedimg disordes is von Willebrand disease (VWD). It is caused by a qualitative and/or quantitative decrease of von Willebrand factor (VWF) the carrier protein of FVIII. The VWF present in plasma and thrombocytes is synthesized in megakaryocytes and endothelial cells. The VWF antigenic determinant (VWFAg) plays a key role in primary hemostasis and is characterized by two main functions /1/:

• It mediates the thrombocyte adhesion to injured subendothelial tissue
• It serves as carrier protein for F VIII and stabilizes F VIII coagulant activity. The function of VWF in hemostasis is shown in Fig. 16.18-1 – Function of the vWF in hemostasis.

The function of VWF in primary hemostasis is mediated by the large multimeres, the size of which and consequently the risk of hyper function is controlled by a VWF-specific metalloprotease (ADAMTS-13). The absence of this protease is associated with the thrombotic thrombocytopenic purpura (TTP).

16.18.1 Indication

• Diagnosis and monitoring in congenital or acquired von Willebrand syndrome (VWS)
• Diagnosis and monitoring in hereditary and acquired thrombotic thrombocytopenic purpura
• Exclusion of hemophilia A.

16.18.2 Method of determination

There is no single diagnostic test due to the high variability of the VWS. Screening tests of VWD are a prolonged bleeding time closure time with the use of the platelet function analyzer (PFA).

16.18.2.1 Screening tests

Initial laboratory evaluation for the aetiology of a bleeding disorder include the following /4/: PTT, aPTT, TT or Fibrinogen and optionally the bleeding time or a platelet function analyzer (PFA-100).

Bleeding time

Methodology according to Ivy, modified according to Mielke /2/.

Platelet function analyzer (PFA 100)

Principle: VWF has a central role in platelet adhesion and aggregation under conditions of high shear stress. The PFA 100 is an automated device for assessing platelet function under high shear conditions /3/. In the PFA-100 instrument these conditions are created by aspirating whole blood through a capillary and an aperture on a membrane coated with equine tendon collagen type 1 and an additional platelet aggregating agent. In cartridge type this agent is ADP and in the other it is epinephrine. Under these circumstances platelets passing through the membrane adhere to the collagen surface, become activated and aggregate. Subsequently a stable platelet plug forms and occludes the aperture. The PFA-100 measures the required time for total occlusion and reports the "closure time”. The closure time is dependent upon VWF and the platelet receptors GPIb and GPIIb/III. Prolonged closure times induced in both epinephrine and ADP are typical of VWS.

Persons with severe type 1 VWD or type 3 VWD usually have abnormal PFA-100 values, whereas persons with mild or moderate type 1 VWD and some with type 2 VWD may not have abnormal results /4/.

16.18.2.2 Initial tests for VWD

The laboratory diagnosis of VWD is based on the results of the, VWF:Ag, FVIII and VWF:RCo. The three initial tests are also used for monitoring therapy. The ristocetin induced thrombocyte aggregation (TIPA) and the analysis of multimeres are used for defining and classifying VWD subtypes /1/. Refer to Tab. 16.18-1 – Synopsis of VWF designation, properties and assays.

Von Willebrand factor antigen (VWF:Ag)

Principle: The VWFAg determination is a quantitative test for the detection of VWD. It measures the amount of VWF circulating in plasma regardless of the function. The most widely used method is the ELISA. Results are expressed in IU/L or % of normal. Typically the level of VWF:Ag is decreased in type I VWD.

F VIII coagulant activity (F VIII:C)

Principle: F VIII is bound to and protected from proteolytic degradation by VWF. Decrease in the level of VWF causes a decline in the amount and activity of FVIII. The activity of F VIII is measured by an aPTT based assay. F VIII:C correlates well with the severity of the disease and also predicts the risk of hemorrhage. The result is expressed in IU/L or % of normal /5/. Typically, the level of FVIII:C is moderately decreased in type 1 and type 2 VWD, but it may be normal or extremely low in type 3 /1/.

Ristocetin cofactor activity (VWF:RCo)

Several methods are used to assess the thrombocyte agglutination and aggregation that result from the binding of VWF to thrombocyte GPIb induced by ristocetin.

Principle: The assay measures the ability of patient’s plasma to agglutinate formalin-fixed platelets in the presence of 1 g/L ristocetin. Ristocetin is a glycopeptide antibiotic that binds to VWF and induces VWF activation and thrombocyte aggregation. The intensity of aggregation correlates with the activity of the VWF /6/.

Ristocetin-induced platelet aggregation (VWF:RIPA)

Principle: RIPA is carried out as a part of platelet function testing by aggregometry and is carried out in the platelet-rich plasma of patients by different concentrations of ristocetin (usually between 0.5 to 1.5 g/L). Ristocetin binds the VWF to the GP-1b receptor of thrombocytes and induces the aggregation of thrombocytes. The impairment in thrombocyte aggregation correlates with the loss of VWF function. Results are expressed in % of normal . RIPA is only diagnostic in type 2B VWD disease where an enhancement in platelet aggregation is observed.

Multimer analysis

Principle: The multimeric analysis of plasma VWF is performed by sodium dodecyl sulfate agarose gel electrophoresis, followed by Western blot detection /10/. Various types and subtypes of VWF are detected (Fig. 16.18-2 – Multimer analysis of various vWF subtypes).Only types 2A, 2B and platelet-type VWD have abnormal multimer distributions with relative deficiency of the largest multimers /4/.

GPIbα binding capacity (VWF:GPIbB)

Principle: Binding of VWF in patient plasma to a particle-bound platelet GPIbα fragment containing two gain-of-function mutations. The two mutations mediate a constitutively reacting structure of the receptor enabling the VWF binding to the GPIbα fragment without the addition of ristocetin. The amount of bound VWF is determined using a luminescence immunoassay. Results are expressed in IU/L or % of normal /7/.

Collagen binding capacity (VWF:CB)

Principle: The assay measures the collagen binding activity of VWF by an ELISA. The results are expressed in % of normal /8/. As the binding to collagen is highly dependent upon the multimeric structure, the test mainly detects high molecular weight forms and is especially impaired in type 2A and 2B disease.

F VIII binding capacity (VWF:F8BC)

Principle: VWF is captured to the solid phase of an micro titer plate from patient plasma /3/. Endogenous F VIII is removed by using a high chloride concentration and a known amount of purified F VIII is added. The solid phase bound VWF and the VWF-bound F VIII are separately detected. A disproportionally decreased amount of VWF bound FVIII indicates type 2N vWD. Results are expressed in % of normal /9/. The test is used to detect an impaired ability of the VWF in patient plasma to bind F VIII in type 2N VWD.

16.18.2.3 ADAMTS13

FVIII regulates VWF in coagulation. FVIII stabilizes VWF multimers and renders them more susceptible to degradation by the metalloprotease ADAMTS13 a (disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13) while FVIIIa, which is not VWF-bound, does not have this effect /9/.

ADAMTS13 specifically cleaves the VWF subunit at the peptide bond Tyr1605–Met1606, generating fragments of 176 kDa and 140 kDa. A severely deficient ADAMTS13 activity (below 5% of normal) is caused either by mutations of the ADAMTS-13 gene or by antibodies blocking proteolysis of VWF. The ADAMTS13 deficiency causes the accumulation of ultra large VWF multimers in the circulation and the formation of thrombi in the microvasculature under high shear stress conditions. When left untreated thrombotic thrombocytopenia purpura develops. The micro thrombi cause multi-organ failure and can lead to death /11/.

16.18.2.3.1 ADAMTS13Ag

Detection by immunoassay: results are expressed in μg/L.

ADAMTS13: activity

Principle: Determination of the specific proteolytic destruction of VWF multimers following incubation of recombinant or plasma VWF with patient plasma and denaturation buffer (urea) by addition of BaCl₂. The decrease in VWFRCo, VWFCB or the large VWF multimers by multimer analysis are determined. Results are expressed in % of normal /12/.

Fluorescence resonance energy transfer (FRETS)

Principle: Double fluorescent-labeled VWF fragment containing the proteolytic cutting site for ADAMTS13 is used as substrate. Cleavage of the fragment by ADAMTS13 is correlated with the decrease in the quenching effect of both fluorescences. Results are expressed in % of normal.

16.18.2.3.2 Anti-ADAMTS13 antibodies

Principle: Neutralizing autoantibodies are determined based on the decrease in plasma ADAMTS13 activity in the plasma mixing test, whereas non-neutralizing autoantibodies are determined using ELISA. Results are expressed in U/mL.

16.18.2.3.3 Molecular genetic diagnostics

Principle: Detection of specific genetic VWF defects by the use of polymerase chain reaction and mutation analysis /12/, detection of heterozygous deletions and duplications using multiplex ligation dependent probe amplification (MLPA).

Detection of specific ADAMTS13 gene defects for differentiation between hereditary and acquired forms of thrombotic thrombocytopenic purpura /13/.

16.18.3 Specimen

• Citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood): 2 mL
• Platelet-rich citrated plasma for the determination of ristocetin-induced platelet aggregation (RIPA)
• Platelets from platelet-rich citrated plasma for the determination of platelet VWF
• Leukocyte DNA using EDTA whole blood, for molecular genetic diagnostics: 5 mL

16.18.4 Reference interval

Concentration and function: higher than 50 IU/dl

Low dose RIPA: aggregation (0.5 g of ristocetin/L) < 20%.

Multimers: all multimers are detectable, no aberrant electrophoretic bands.

ADAMTS13: > 45% of normal.

16.18.5 Clinical significance

Von Willebrand disease (VWD) is a well known inherited bleeding disorder, causing mucous membrane and skin bleeding symptoms, and bleeding with surgical or other hemostatic challenges. In screening populations the prevalence of VWD is 0.6–1.3%. Bleeding is of mild-to-moderate severity for most persons with VWD, reflecting the predominance of type 1 VWD. Factors that increase plasma levels of VWF include age, race (African Americans), acute-phase-response and hormones, particularly those associated with pregnancy and the menstrual cycle. Approximately 12% of women who have menstrual periods have excessive menstrual bleeding. This proportion is much higher among women with VWD /1, 4/. During pregnancy VWF is increased 3–5 fold above the baseline by the third trimester. ABO blood group types have an effect on plasma VWF and FVIII concentrations. The mean VWF level of individuals with blood type 0 is 75 IU/dl and approximately 25% lower than other blood group types. The diagnosis of VWD type 1 occurs more frequently in persons who have blood group type 0 /4/.

Life-threatening bleeding (central nervous system, gastrointestinal tract) can occur in persons either with type 3 VWD and in some patients with type 2 VWD. Women with VWD report a high prevalence of menorrhagia. The sensitivity of menorrhagia as a predictor of VWD may be estimated as 32–100% /4/.

The diagnosis of vWD depends on clinical and laboratory criteria. The clinical criteria include personal and/or family history and/or physical evidence of mucocutaneous bleeding. The Expert Panel /4/ recommends that 30 IU/dL be used as a the cutoff level for supporting the definite diagnosis of VWD for the following reasons:

• There is a high frequency of blood type 0 and it is associated with low VWF levels
• Bleeding symptoms are reported by a significant proportion of normal individuals
• No abnormality in the VWF gene has been identified in many individuals who have mildly to moderately low VWF:RCo levels.

Prototypical cases are shown in Tab. 16.18-2 – Prototypical cases of VWD.

16.18.5.1 Subtype differentiation of von Willebrand disease (VWD)

Test for the differentiation of VWD are shown in Tab. 16.18-1 – Synopsis of VWF disintegration, properties and assays.

Tab. 16.18-3 – Classification of von Willebrand factor deficiency presents an overview of the different types and subtypes of VWD /15/.

A systematic approach in the differential diagnosis of VWD is presented in Fig. 16.18-3– Systematic approach in the differential diagnosis of the VWS)

Type I VWD

These persons have partial quantitative deficiency of VWF. A concordant decrease in the concentration and activity of the VWF points to VWD type 1. The remaining VWF binds FVIII normally and mediates thrombocyte adhesion normally. The results of laboratory tests are as follows /1, 4/:

• Von Willebrand factor antigen (VWF:Ag) is reduced
• FVIII coagulant activity (FVIII:C) decreased
• Ristocetin Cofactor (VWF:CRo) pathologic
• The FVIII/VWF:Ag ratio 1.5–2.0
• Multimer analysis: normal pattern.

Individuals who have very low VWF levels (< 20 IU/dL) are likely to have VWF gene mutations, mucocutneous bleeding symptoms and a strongly positive family history.

Type 2 VWD

The results of VWF:Ag, FVIII:C and VWF:CRo are variable or may be normal, the multimer pattern is abnormal. Critical tests include the VWF:RIPA, the collagen binding assay, the F VIII binding assay and multimer analysis /1, 4/.

Type 2A VWD: This type of VWD may be caused by mutations that interfere with the assembly or secretion of large multimers or by mutations that increase the susceptibility of VWF multimers to proteolytic degradation in the circulation /4/. VWF:Ag and FVIII are low normal or decreased, VWF:RCo and RIPA are decreased, and the VWF multimer pattern is abnormal.

Type 2B VWD: Mutations occur within or adjacent to VWF domain A1, which changes conformation when it binds to thrombocyte GPIb. Patients typically have thrombocytopenia that is exacerbated by surgery, pregnancy or other stress. The diagnosis depends on finding abnormally increased ristocetin-induced thrombocyte aggregation (RIPA) at low concentrations of ristocetin.

Type 2N VWD: This type of VWD is caused by mutations in the F VIII binding site of VWF and impairs binding of FV III to VWF. The level of F VIII is low (< 10%) with a normal VWF:Ag and VWF:RCo. The type masquerades as an autosomal recessive form of hemophilia A. Discrimination may require assays of F VIII binding. In low F VIII concentrations, the recessively inherited type 2N can be associated with normal VWF:Ag, VWF:RCo and VWF:CB (homozygous or compound-heterozygous F VIII binding mutation of the VWF) or manifest with low VWF:Ag (compound-heterozygous F VIII binding defect with a VWF null allele) /9/. This type is the key differential diagnosis regarding hemophilia A. VWD type 2N must be excluded in cases of unclear inheritance, inadequate response to especially pure or recombinant F VIII concentrates and always in female hemophilia A /4/.

A sytematic approach for differentiation of vWD and hemophilia A is shown in Fig. 16.18-3 – Approach in the differentiation of the VWD and hemophilia.

Molecular investigations are a way to circumnavigate the problems encountered in phenotype analysis since the phenotype-genotype correlations are clear in many cases. Thus, it is possible in many unclear cases to obtain a diagnosis based on gene analysis (Fig. 16.18-4 – Localization of molecular defects in various von vWS phenotypes) /16/.

Type 3 VWD

The complete absence of the VWF protein and activity points to type 3 VWD. FVIII levels usually are very low (1–19 IU/dL). Truncated mutations of the VWF gene (e.g., nonsense, splicing and nonsense mutations) and minor and major deletions and insertions as well as missense mutations are the cause /16/.

Platelet-type VWD

Platelet-type VWD is an inherited platelet disorder characterized by thrombocytopenia with large platelets caused by gain-of-function variants in GP1BA leading to enhanced GPIa VWF interaction. GPIa and vWF play a role in megakaryopoiesis. Thrombocytopenia in VWD is due to a combination of different pathogenic mechanisms, i.e., the formation of a reduced number of platelets by megakaryocytes, the ectopic release of platelets in the bone marrow, and the increased clearance of platelet/VWD complexes /25/.

Acquired VWD

Acquired VWD is a rare bleeding disorder with laboratory findings similar to those of inherited disease. However, unlike the inherited disease acquired VWD occurs in persons with no family history of bleeding. The disease is often associated with a variety of diseases, most frequently lymphoproliferative, myeloproliferative and cardiovascular disorders. Laboratory findings in acquired VWD measure defects in VWF concentration similar to those in VWD. The results include decreased values of VWF:Ag, VWF:RCo or FVIII. The VWF multimer pattern often shows a decrease in large multimers similar to that seen in type 2 VWF or a normal pattern /4/.

16.18.5.2 Thrombotic thrombocytopenic purpura (TTP)

Patients with thrombocytic thrombocytopenic purpura (TTP) may have a genetic deficiency (Upshaw-Schulman syndrome) in the ADAMTS13 enzyme (< 10% activity) or the enzyme is inhibited by autoantibodies (acquired form). The enzyme deficiency results in a failure to cleave ultralarge von Willebrand factor, leading to multisystem thrombotic microangiopathy.

Manfestation of the hereditary TTP in neonates includes thrombocytopenia, presence of schistocytes on a blood smear, and hyperbilirubinemia /17/. While low concentrations of ADAMTS13 are measured in various diseases, values < 10%, probably even only < 5%, are specific to TTP /18/.

In the hereditary form of the disease administration of a standard dose of fresh frozen plasma is sufficient to prevent TTP /18/. Concentrations below the detection limit (< 2–5% depending on the method) always indicate a corresponding predisposition /16/. The differentiation between the hereditary and the acquired form of the disease is crucial for therapy selection.

In the acquired form of TTP anti-ADAMTS13 autoantibodies block the proteolysis of VWF and/or induce ADAMTS13 clearance from the circulation. The mechanisms leading to the loss of tolerance of the immune system towards ADAMTS13 involve the predisposing genetic factor of the human leukocyte antigen class II locus DRB1* 11 and DQB1*03 alleles as well as the protective allele DRB1*04 and modifying factors such as ethnicity, sex and obesity /19/.

Single administrations of fresh frozen plasma are sufficient in the hereditary form (every 14 days for prophylaxis), whereas the autoantibody-mediated form can only be treated by repeated plasma exchange and immunosuppressive therapy.

16.18.6 Comments and problems

Blood sample collection

The setting of phlebotomy should be as calm as possible. Anxiety of the patient may falsely elevate the F VIII and VWF levels.

Laboratory testing

The CV of the VWF:RCo assay is high (≥ 20–30%), and the CV of the VWF:Ag is also relatively high (≥ 10–20%), as is the CV for the F VIII assay /4/.

16.18.7 Pathophysiology

VWF is an adhesive protein with binding sites for circulating proteins (F VIII), insoluble structures of the sub endothelium (collagen) as well as cellular surface structures (platelet surface glycoproteins GPIb, GPIIb/ IIIa). This accounts for the key role which VWF plays in primary hemostasis as a mediator of platelet adhesion to the injured sub endothelium and subsequent platelet aggregation. This process takes place primarily under strong shear stress typical of the arterial system and the micro circulation (Fig. 16.18-1 – Function of the vWF in hemostasis). The large VWF multimers are essential for this function /20/.

The second important function is the binding of F VIII thereby protecting this factor from premature degradation, for example by activated protein C (Fig. 16.21-1 – Activation of protein C and its anticoagulatory effect in combination with the cofactor protein S). In severe VWD (type 3), all these functions are impaired. Accordingly, beside a primary hemostatic defect, there is also a defect in secondary hemostasis due to a pronounced reduction in F VIII (below 0.05 U/L). This must be taken into account for the purpose of adequate therapy. In the mild form of VWD (type 1) and in types 2A, 2B and 2M with a VWF:Ag above 30 IU/dL, there is only little impairment in secondary hemostasis.

In various other subtypes of type 2 VWD, only some particular functions of VWF may be reduced. In particular, type 2 N VWD is of interest because this type becomes evident only because of a concomitant reduction in F VIII:C on the basis of the patient’s VWF which displays impaired F VIII binding capacity; clinically, this defect resembles hemophilia A (pseudohemophilia).

Isolated particular dysfunctions of the VWF can be attributed to defined defects that may be correlated with mutations in the different functional regions of the VWF (Fig. 16.18-4 – Localization of molecular defects in various von VWD phenotypes). The marked heterogeneity of the VWD can be explained by the multi functionality of the VWF, its domain structure and the numerous options of combination of the different defects /15, 21/.

Hyper function of the VWF leads to the clinical picture of thrombotic thrombocytopenic purpura, a propensity to micro angiopathic thrombosis caused by the persistence of ultra-large VWF multimers in the absence of the specific VWF metalloprotease ADAMTS13 and, consequently, the lack of VWF size regulation /22, 23, 24/. This disease can be autosomal recessively inherited or caused by autoantibodies /19/. Accordingly, there may be homozygous or compound-heterozygous mutations of the ADAMTS13 gene or specific ADAMTS13 autoantibodies with different consequences on therapy /13, 24/.

The von VWD and the thrombotic thrombocytopenic purpura are the two opposing manifestations of VWF disorders, thus underscoring the factor’s key role in hemostatic balance.

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18.. Vesely SK, George JN, Lammle B, Studt JD, Alberio L, El-Harake MA, Raskob GE. ADAMTS13 activity in thrombotic thrombocytopenic purpura-hemolytic uremic syndrome: relation to presenting features and clinical outcomes in a prospective cohort of 142 patients. Blood 2003; 102: 60–8

19. Hrdinova J, D’Angelo S, Graca NAG, Ercig B, Vanhoorenbeke K, Veyradier A, et al. Dissecting the pathophysiology of immune thrombotic thrombocytopenic purpura: interplay between genes and environmental triggers. Haematologica 2018; 103: 1099–1109.

20. Moake JL, Rudy CK, Troll JH, Weinstein MJ, Colannino NM, Azocar J, et al. Unusually large plasma factor VIII: von Willebrand factor multimers in chronic relapsing thrombotic thrombocytopenic purpura. N Engl J Med 1982; 307: 1432–5.

21. Mannucci PM, Canciani MT, Forza I, Lussana F, Lattu-ada A, Rossi E. Changes in health and disease of the metalloprotease that cleaves von Willebrand factor. Blood 2001; 98: 2730–5.

22. Furlan M, Robles R, Lämmle B. Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis. Blood 1996; 87: 4223–34.

23. Tsai HM. Physiologic cleavage of von Willebrand factor by a plasma protease is dependent on its conformation and requires calcium ion. Blood 1996; 87: 4235–44.

24. Levy GG, Nichols WC, Lian EC, Foroud T, McClintick JN, McGee BM, et al. Mutations in a member of the ADAMTS gene family cause thrombotic thrombocyto-penic purpura. Nature 2001; 413: 488–94.

25 Bury L, Malara A, Momi S, Petito E, Balduini A, Gresele P. Mechanisms of thrombocytopenia in platelet-type von Willebrand disease. Haematologica 2019; https://haematologica.org/article/view/8977

16.19 Venous thromboembolism

Lothar Thomas

16.19.1 Introduction

Venous thromboembolism (VTE), consisting of deep vein thrombosis and pulmonary embolism, is a multi-causal disease associated with substantial morbidity and mortality. VTE has been described as hemostasis in the wrong place and is a time-limited acute disease /1/.

VTE can occur /2/:

• Unprovoked (spontaneously). In most patients who suffer unprovoked VTE the role of thrombophilia testing in clinical decision making is important. Thrombophilia testing determines whether VTE has a genetic origin and whether there is a risk of recurrence. At first patients are anticoagulated for 3–6 months then risk stratification and decision-making is done. Patients with unprovoked VTE have a high risk of recurrence (50% over 10 years, if not treated with anticoagulation). Thrombophilia testing and family history are useful in these patients. However, a positive family history of VTE does not predict discovery of thrombophilia.
• Provoked VTE. The relative risk of thrombosis after immobilization and trauma in the absence of anticoagulation is high.
• VTE in cancer patients. The Onkopedia Medical Guideline recommends besides low molecular heparine, DOAKs (Dabigatran) or FXa-inhibitors (Endoxabane, Apixabane, Rivaroxabane) for antithrombosis in cancer patients /3/.

Refer to

• Tab. 16.19-1 – Annual age dependent risk of unprovoked VTE in female persons
• Tab. 16.19-2 – Risk factors for venous thromboembolism (VTE).

16.19.2 Indication

Thrombophilia testing can be considered in patients with VTE suggestive of inherited thrombophilia /4/:

• Thrombophile disorder in patients below 50 years (estimation of the risk of recurrence after the first VTE)
• Asymptomatic women who are considering oral conceptive use but are related to VTE patients with thrombophilic disorders
• Postmenopausal hormone therapy among women with thrombophilia and no prior VTE
• Strong family history of VTE (first-degree family members affected at young age).

16.19.3 Thrombophilia testing

Thrombophilia testing refers to laboratory testing performed to identify propensity to thrombosis. A panel of biomarkers to detect conditions associated with the first or recurrent VTEs is used.

Well defined 4 biomarker panel of VTE genetic risk factors:

• Factor V Leiden mutation
• Prothrombinmutation (FIIG20210A)
• Deficiency of natural anticoagulants (antithrombin, protein S, protein C)

Risk factors of VTE and/or arterial thrombosis:

• Persistence of elevated FVIII-activity (FVIII is an acute phase protein)
• Presence of anti-phospholipid antibodies

Tests not conclusively associated with risk of VTE or require further validation /4/:

• Elevated activity of F IX, F XI, plasminogen activator inhibitor type 1 (PAI-1), and the 4G/5G PAI-1 promoter polymorphism.

Tests not associated with an increased risk of either first VTE or recurrence:

• Methylene tetrahydrofolate reductase polymorphisms (677C-T, 1298A-C), which are present in up to 45% of the population worldwide /4/.

16.19.4 Specimen

Citrate plasma (blood collection: 1 part sodium citrate solution 0.11 mol/L mixed with 9 parts of blood): 10 mL

16.19.5 Thrombophilia testing

Thrombophilia is the propensity to develop VTE, based on unprovoked (genetic) or provoked (acquired) factors. Thrombosis occurs in the veins and arteries, whereas VTE is associated with veins.

Besides factors of the 4 biomarker panel manifestations of hereditary thrombophilia can be due to /5/:

• Dysregulation of plasmatic coagulation as a result of reduced counter regulation (e.g., reduction in inhibitors) or constitutive hyperactivity (e.g., of F VIII)
• Absolute reduction in the activity of proteins of the fibrinolytic pathway.

16.19.5.1 Unprovoked (hereditary) thrombophilia

Prothrombotic disorders combined with environmental factors increase the risk of unprovoked VTE. In approximately 15% of the population in the Western world and in up to 50% of individuals with VTE thrombophilic disposition is VTE detectable using the 4 biomarker panel. Positive familial history is especially significant in the assessment of the risk of VTE because a VTE episode in the family indicates a thrombophilic risk that may lead to thrombosis in relatives.

Refer to:

• Tab. 16.19-2 – Risk factors for venous thromboembolism
• Tab. 16.19-3 – Patients with thromboses or thrombophilia: thrombotic risk of relatives.

Genetically determined predisposition to thrombophilia can be detected partially by thrombophilia screening. Approximately 15% of the population are carriers of genetically determined thrombophilia markers. For influence factors refer to Tab. 16.19-2 – Clinical, analytical and pre-analytical influence factors, for interpretation of results of thrombophilia testing.

The presence of a genetic risk factor is relatively common in adults without VTE and is assumed to lead to VTE only in combination with one or several other risk factors. In children, VTE is usually only seen if there is concurrent evidence of 3 or more risk factors. The assessment of the finding of a genetic risk factor is problematic without positive familial history. In many cases, VTE is triggered by the combination of a hereditary defect and an acute disease. In addition, exogenous factors may act as triggers leading to a transient disruption of the balance between inhibitors of coagulation and fibrinolysis. For instance, major surgery, trauma and oral contraceptives trigger VTE in individuals with thrombophilic diathesis. Approximately 10–20% of VTE patients have more than one of the genetic risk factors. This concurrence of several risk factors partly explains the different forms of propensity to thrombosis in different individuals and families with the same genetic defect.

Refer to Tab. 16.19-4 – Prevalence of thrombophilic risk factors and risk of VTE.

The cerebral venous thrombosis refers to occlusions of veins of the surface of the cortex of the brain. Cerebral venous thrombosis encompasses both dural clots and cortical vein thrombosis /7/. The most important dural venous channel is the sinus venosus. The clinical feature of sinus venous thrombosis is acute or subacute headache in 70–90% of patients, often with normal neurologic examination. The headache usually progresses during hours or days, seizures, usually focal convulsions, may follow if cortical infarction occurs. If heparin induced thrombocytopenia (HIT) is suspected laboratory testing is indicated. Refer to Section 17.5 – Heparin induced thrombocytopenia.

Age dependency /6/

Unprovoked VTE is age-dependent: the annual incidence increases from 1 : 100,000 in small children to 1 : 10,000 at an age of ≤ 40 years, and about 1 : 1,000 to ≥ 40 years, and is almost 1% at an age ≥ 75 years. More than 70% of VTE cases are seen at an age > 60 years. On average, 1 in 1,000 individuals suffer from VTE every year, of whom 20% develop post thrombotic syndrome and 1% die of pulmonary embolism /1/. The incidence of VTE in the asymptomatic population is 0.1–0.2% per year. The relative risk of a relapse is 2–5% per year.

Refer to:

• Tab. 16.19-1 – Age dependent risk of venous thrombosis
• Tab. 16.19-5 – Relative and absolute risk of relapse of VTE.

Gender dependency

Compared to the baseline incidence of 1 : 10,000 women have an increased VTE risk in special circumstances /2/:

• Asymptomatic women with oral contraceptive use 3–8 : 10,000 for 1 year
• Pregnancy 5 : 10,000 for 1 year
• Postpartum period 20 : 10,000 for 1 year
• Asymptomatic obese women with factor V Leiden using hormonal contraceptives 10 : 100 for 10 years.

Rate of VTE in factor V Leiden /2/

• Heterozygous factor V Leiden: present in about 5% of whites. The incidence rate of VTE increases from 1 : 10,000 annually to 4–7 : 10,000.
• If a young woman with heterozygous factor V Leiden is obese the incidence rate of VTE increases from 1 : 10,000 annually to 8–14 : 10,000.
• If she is taking hormonal contraceptives the incidence rate of VTE increases to 34 : 10,000.
• Women 50–60 years with factor V Leiden have a 4 : 1,000 annual risk of VTE which rises to about 1% annually with use of postmenopausal estrogen plus progestin and about 35 if there is a family history of thrombosis.

Thrombophilia in combination with increased F VIII activity

Increased F VIII activity is a risk factor of thrombophilia. F VIII is an acute phase protein that is increased in inflammation and regulated by the genes AB0. In the Leiden Thrombophilia Study, patients with F VIII concentrations > 150 IU/dL had a 5-fold increased risk of VTE.

Refer to Section 16.15 – Analysis of individual coagulation factors.

Thrombophilia in combination with defects in protein S or protein C, or antithrombin

Refer to Tab. 16.19-4 – Prevalence of thrombopholic risk factors and risk of VTE in Europe and North America.

Thrombophilia in relatives

Refer to:

• Tab. 16.19-3 – Acute VTE risk of patients with thrompophilia or VTE in the family
• Tab. 16.19-4 – Prevalence of thrombophilic risk factors and risk of VTE in Europe and North America
• Tab. 16.19-6 – Hereditary thrombophilia
• Acute risk of VTE in patients with surgery without “bridging” anticoagulation.

16.19.5.2 Provoked (causal) thrombophilia

Beside the hereditary thrombophilic diatheses numerous other congenital and acquired disorders are associated with thrombophilia, for example deficiency in heparin cofactor and plasminogenysplasminogenemia, F XII deficiency, increase in histidin-rich glycoprotein, mutations in thrombomodulin, mutations in platelet GP IIb/IIIa receptor, increase in fibrinogen, VWF and F VII, impaired release of tissue plasminogen activator (t-PA), increase in t-PA inhibitor and others. At the present time, there is insufficient evidence to recommend the routine determination of these disorders for association with increased propensity to thrombophilia.

Associations of coagulation factors IX and XIII with the risk of VTE were investigated in a study /8/. Elevated levels of both factors were associated with increased rate of thrombosis, with the odds ratio being 1.4 for F IX and 2.0 for F XIII.

Traveling (by air and other modes) is associated with a 3-fold higher risk for VTE, and with an 18% higher risk for VTE for each 2-hour increase in duration of travel /9/.

16.19.5.2.1 Thrombosis in thrombocytopenia

Causes can be:

• Disseminated intravascular coagulation
• Thrombotic thrombocytopenic purpura
• Thrombotic micro angiopathy
• Heparin induced hemoglobinuria
• Hemolytic uremic syndrome
• Paroxysmal nocturnal hemoglobinuria
• Idiopathic thrombocytopenia e.g., lupus erythematosus, antiphospholipid syndrome.

16.19.5.2.2 Antiphospholipid syndrome (APS)

Antiphospholipid syndrome (APLS) is characterized by:

• VTE in pregnancy and further complications
• The finding of lupus anticoagulant (LA) and/or elevated titers of anti-cardiolipin antibodies (ACA) or anti-β₂-glycoprotein (anti-β₂-GP) antibodies. See Section 25.5 – Antiphospholipid syndrome).

LA, ACA and anti-β₂-GP are not necessarily, but can be associated with thrombophilia and disorders in pregnancy. Transient LA sometimes seen in children in association with viral infections is not associated with thrombophilia.

The laboratory findings by themselves do not allow a prediction of clinical consequences. Practical experience suggests that the risk of clinical APLS is low in patients who are asymptomatic at the detection of LA or ACA. In patients where APLS was associated with thrombophilia, VTE was seen in 2/3 and arterial thrombosis, especially cerebral thrombosis, was seen in 1/3 of the cases. VTE has a pronounced tendency to recur. There is no bleeding tendency despite prolonged aPTT, except in those cases where LA is present in association with severe prothrombin deficiency.

Approximately half of the APLS patients have no underlying disease (primary APLS). The other half suffer from autoimmune diseases such as systemic lupus erythematosus, monoclonal gammopathies, infections (in children) or drug reactions.

16.19.5.2.3 Perioperative prevention of VTE

Invasive procedures in patients undergoing long-term anticoagulant therapy imply the risk of VTE if the anticoagulant therapy is interrupted or no suitable bridging anticoagulant therapy is administered. For further information, see Ref. /10/.

Refer to:

• Tab. 16.19-7 – Acute risk of VTE in perioperative patients without bridging anti-coagulation therapy
• Tab. 16.19-6 – Hereditary thrombophilia.

16.19.5.2.4 Influence factors of VTE

The development of VTE is influenced by /2/:

• Dispositional risk factors (prothrombotic disorders). This refers to individuals with a genetically defined risk (F V Leiden mutation, prothrombin gene mutation G20210A, deficiency in antithrombin, protein C, protein S, increase in F VIII and fibrinogen) and a non-genetic risk such as the anti-phospholipid syndrome.
• Exposure risk factors either cause VTE by itself or increase the risk of dispositional factors (Tab. 16.19-2 – Risk factors for venous thromboses) /2/. The risk is especially high in individuals who already had VTE or in patients with a malignant tumor. The risk of provoked acute VTE is high in immobilized patients with acute disease and in intensive care unit patients who have not received prophylactic medication or other preventive care (Tab. 16.19-3 – Acute risk of thromboembolism in the absence of thromboprophylaxis) /2/. According to a metaanalysis /11/, anticoagulant prophylaxis prevents approximately half of the expected events.
• Dispositional risk factors (prothrombotic disorders) combined with environmental risk factors /2/.
• Heterozygous factor V Leiden, present in about 5% of whites.
• Invasive procedures in patients undergoing long-term anticoagulant therapy if therapy is interrupted or modified.

Comprehensive diagnostic investigation of thrombophilia is not necessary in acute VTE because it is therapeutically irrelevant whether the VTE has genetic or acquired causes. The antiphospholipid syndrome where early adequate therapy can achieve good therapeutic success is an exception /12/.

Refer to:

• Tab. 16.19-8 – Clinical, analytical and pre-analytical influence factors for interpretation of results of thrombophilia testing.

16.19.5.2.5 Course of VTE

VTE is a chronic disease with a recurrence rate of 30% within 5–8 years. Approximately 5% of affected patients die of pulmonary embolism. The rate of recurrence is high in the first weeks after a thrombosis and decreases pronouncedly afterwards /13/. Patients with pulmonary embolism have a higher recurrence rate and frequent episodes will also be pulmonary embolisms. The recurrence rate in patients with inborn disposition of VTE is higher than in those with acute VTE after major surgery. The following risk factors for recurrent VTE besides male gender are shown in Tab. 16.19-4 – Relative and absolute risk of relapse of thrombosis.

16.19.6 Comments and problems

Before testing patients should have completed anticoagulant therapy. Vitamin K antagonists should be withheld for a minimum of 2 weeks and direct oral anticoagulants for at least 5 half-lifes, generally a minimum of 2–3 days /4/.

References

1. Rosendaal FR. Venous thrombosis: a multicausal disease. Lancet 1999; 353: 1167–73.

2. Kamel H, Navi BB, Sriram N, Hovsepian DA, Devereux RB, Elkind MSV. Risk of a thrombotic event after the 6-week postpartum period. N Engl J Med 2014. doi: 10.1056/nejmoa1311485.

3. Lindhoff-Last E. Thrombophilia testing. DELAB education. Mainz 2015.

4. Cushman M. Thrombophilia testing in women with venous thrombosis. The 4 P’s approach. Clin Chem 2014; 60: 134–7.

5. Geerts WH, Bergqvist D, Pineo GF, Heit JA, Samana CM, Lassen MR, et al. Prevention of venous thromboembolism: American College of Chest Physicians evidence-based clinical practical guidelines. Chest 2008; 133: 381S–435S.

6. Själander A, Jansson JH, Bergqvist D, Eriksson H, Carlberg B, Svensson P. Efficacy and safety of anticoagulant prophylaxis to prevent venous thromboembolism in acutely ill medical patients: a meta analysis. J Intern Med 2008; 263: 52–60.

7. Kyrle PA. Idiopathic venous thrombosis. Hämostaseologie 2006; 26: 52–4.

8. Baglin T, Grey E, Greaves M, Hunt BJ, Keeling D, Machin S, et al. Clinical guidelines for testing for heritable thrombophilia. Br J Haematol 2010; 149: 209–20.

9. Cullen P, Adam S, Kaiser B, Neumaier M. Status der Thrombophilie-Diagnostik unter besonderer Berücksichtigung molekulargenetischer Aspekte. J Lab Med 2009; 33: 283–92.

10. Luxembourg B, Lindhoff-Last E. Genomische Diagnostik thrombophiler Gerinnungsstörungen bei Frauen. Hämostaseologie 2007; 27: 22–31.

11. Thornburg C, Pipe S. Neonatal thromboembolic emergencies. Seminars in Fetal & Neonatal Medicine 2006; 11: 198–206.

12. Gerotziafas GT. Risk factors for venous thromboembolism in children. International Angiology 2004; 23: 195–205.

13. Cushman M, O’Meara ES, Folsom AR, Heckbert SR. Coagulation factors IX through XIII and the risk of future venous thrombosis: the longitudinal investigation of thromboembolism etiology. Blood 2009; 114: 2878–83.

14. Chandra D, Parisini E, Mozaffarian D. Meta-analysis: Travel and risk for venous thromboembolism. Ann Intern Med 2009; 152 180–90.

15. Schlitt A, Jambor C, Spannagl M, Gogarten W, Schilling T, Zwissler B. Perioperativer Umgang mit Antikoagulantien und Thrombozytenaggregationshemmern. Dtsch Arztebl Int 2013; 110: 525–32.

16. Connors JM. Thrombophilia testing and venous thrombosis. N Engl J Med 2017; 377: 1177–87.

17. Kyrle PA, Minar E, Hirschl M, Bialonczik C, Stain M, Schneider B, et al. High plasma levels for factor VIII and the risk of recurrent vein thrombosis. N Engl J Med 2000; 343: 457–62.

18. Zotz R, Sucker C, Gerhardt A. Bedeutung thrombophiler Risikofaktoren für das Erst- und Rezidivthromboserisiko. Hämotherapie 2009; 13: 1–16.

19. Lijfering WM, Mulder R, ten Kate MK, Veeger NJGM, Mulder AB, van der Meer J. Clinical relevance of decreased free protein S levels: results from a retrospective family cohort study involving 1143 relatives. Blood 2009; 113: 1225–30.

20. Ropper AH, Klein JP. Cerebral venous thrombosis. N Engl J Med 2021 (385): 59–64.

16.20 Antithrombin (AT)

Lothar Thomas

Antithrombin (AT) belongs to the family of serine protease inhibitors (serpines) and is the most important inhibitor of the coagulation system. AT inhibits all of the proteinases of the intrinsic pathway, especially thrombin, F Xa, F IXa, and to a lesser extent the fibrinolytic enzyme plasmin /1/. In contrast, heparin cofactor II is a relatively specific inhibitor of thrombin /2/. In the presence of heparin, the reaction between AT and thrombin and F Xa is drastically accelerated and leads to an effective inhibition of coagulation. The clinically significant AT deficiency is differentiated into the hereditary and the acquired forms. AT has an importance in the pathogenesis of thromboembolism.

16.20.1 Indication

• Suspected congenital AT deficiency, especially in the presence of a thromboembolic disease
• In pre term births
• Suspected acquired AT deficiency (e.g., postoperatively, in sepsis and disseminated intravascular coagulation)
• Monitoring of AT replacement therapy
• Suspected heparin resistance.

16.20.2 Method of determination

The plasma AT activity is primarily determined by functional tests. If the activity is reduced, the AT defect is in a secondary step classified into types I (quantitative deficiency) and II type (qualitative deficiency) by immunological methods. Rare variants of hereditary deficiency are detected by molecular biological methods.

Chromogenic amidolytic assay

Principle: the determination of AT activity is carried out on automated platforms using chromogenic assays in which patient plasma is incubated with an excess of thrombin (F IIa) or F Xa /3, 4/. In the presence of heparin, a proportion of the activated thrombin or F Xa is inactivated by the patients endogenous AT of the sample. The residual amount of thrombin or F Xa cleaves a chromogenic peptide substrate, releasing a dye. The concentration of this dye is proportional to the AT activity and is spectrophotometrically measured at 405 nm.

Assays utilizing human F IIa are prone to interferences by heparin cofactor II, especially in patients with heparin therapy. Its effect is small in assays using bovine thrombin. F Xa based assays are not affected by heparin cofactor I, but are influenced by direct F Xa inhibitors such as rivaroxaban.

Progressive activity assay

Principle: this assay is a variant of the chromogenic amidolytic assay. Instead of using heparin, the incubation time is extended to 300 sec. in this assay. Thus, the AT activity is measured independently of the heparin binding site, allowing the differentiation of qualitative AT deficiency into type II RS (RS, reactive site) and type II HBS (HBS, heparin binding site).

Immunochemical assay

The immunochemical assays are designed to measure the quantity of AT protein regardless of the AT’s ability to function. Methods used are immunoelectrophoresis, immunonephelometric and immunoturbidimetric assays.

16.20.3 Specimen

Citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood): 1 mL

16.20.4 Reference interval

Refer to Ref. /6, 7, 8, 9/ and Tab. 16.20-1 – Antithrombin reference intervals.

16.20.5 Clinical significance

AT is the essential anticoagulant in plasma and is a key regulator of the coagulation system. The absence of AT is incompatible with life.

The primary actions of AT are /11/:

• Control of low-level thrombin formation by inhibiting F Xa
• Inhibition of thrombin-mediated fibrin clot formation
• Inhibition of activated clotting factors in the extrinsic pathway (F VIIa-tissue factor complex) and intrinsic pathway (F IXa, F XIa and F XIIa) pathways. The inhibition of these factors is less efficient than that of thrombin and F Xa.

16.20.5.1 Antithrombin increase

AT deficiency is clinically relevant, whereas an increase in AT in plasma is not. However, therapy with direct thrombin inhibitors such as hirudin and argatroban is an exception because these inhibitors cause falsely elevated AT activity in the thrombin-based AT activity assay, but not in the F Xa-based assay.

Oral anticoagulant therapy with vitamin K antagonists such as warfarin or coumarin may lead to an increase in AT activity.

16.20.5.2 Antithrombin deficiency

AT activity below 50–70% indicates an inhibitor deficiency which will lead to inadequate compensation should the levels of pro coagulants or the activities of the pro coagulant factors be increased. Therefore, this results in a shift of the hemostatic equilibrium and the risk of venous thromboembolism (VTE). AT deficiencies can be of hereditary or acquired origin. Hereditary AT deficiency is a hyper coagulable state associated with an increased risk of venous thromboembolism (VTE). The causes of acquired AT deficiency are much more common than hereditary deficiency. AT deficient states are depicted in Tab. 16.20-2 – Hemostatic disorders with changes in AT.

16.20.5.2.1 Inherited antithrombin deficiency

In congenital AT deficiency, differentiation is made between /10, 11/:

• Type I deficiency that is characterized by decreased AT activity and antigen concentration. Typically they both are below 70% of normal. Homozygous type I deficiency of AT has not been described. Heterozygous AT deficiency occurs in 0.02–0.17% of the general population and in 0.5–4.9% in patients with VTE.
• Type II deficiency is a qualitative defect, resulting in the production of a variant protein with decreased function. The type II is further subdivided into three subtypes:

a) Subtype IIa. Mutations in thrombin binding domain are associated with decreased activity and normal antigen concentration.

b) Subtype IIb. Mutations in heparin-binding domain are associated with decreased activity and normal antigen concentration. This type exhibits progressive activity (i.e., increased activity with prolonged incubation time). The prevalence of subtype II mutations in the general population is 0.03–0.04% and is associated with a low risk of thrombosis in heterozygous carriers.

c) Subtype IIc. Pleiotropic defects result from a genetic mutation in the s1C-s4B region and are associated with a moderate decrease in both AT activity and antigen levels. AT activity is lower than antigen concentration.

Further informations about hereditary AT deficiency are presented in:

• Fig. 16.20-1 – Algorithm for the diagnosis and differentiation of hereditary AT deficiency
• Tab. 16.20-2 – Hemostatic disorders with changes in AT
• Tab. 16.20-3 – Laboratory findings in hereditary AT deficiency.

The chromogenic assay of AT activity involves heparin and identifies all types of AT deficiency, however, the assay is not able to distinguish type II HBS from other type II defects. A variant of this assay, the progressive activity assay, measures activity independent of the HBS.

16.20.5.2.2 Acquired causes of low antithrombin

Since acquired AT deficiency is associated with other disorders of the hemostatic system, its clinical effects and causes are not clearly definable. Although the plasma AT activity is reduced in patients suffering from nephrotic syndrome, it remains unclear whether AT deficiency is involved in causing an increased incidence of thrombosis. Reduced AT synthesis does not result in increased incidence of thrombosis in patients with liver cirrhosis.

AT deficient states are depicted in Tab. 16.20-2 – Hemostatic disorders with changes in AT.

16.20.5.2.3 Antithrombin deficiency and thrombosis

Venous thrombosis is the usual mode of clinical presentation in AT deficient individuals. The common sites for thromboses are the deep leg veins, the iliac, femoral and superficial leg veins. Arterial thrombosis is uncommon, but has been reported. In the European Prospective Cohort on Thrombophilia (EPCOT) study /12/. The risk of first occurrence of VTE in individuals with hereditary deficiency of AT, PT, PS or F V Leiden was analyzed. In the 5.7-year course of the study, 4.5% of the patients developed VTE. The annual incidence was highest (1.7%) in patients with AT deficiency. The relative risk of VTE in AT deficiency was 25–50-fold higher than in individuals with normal AT.

16.20.5.2.4 Antithrombin therapy

In the case of AT therapy, an AT activity of 80% should be maintained. One unit of AT per kg of body weight increases the plasma AT activity by 1–2%. The half-life of replaced AT is 1.5–2.5 days unless heparin therapy or a major inflammatory response are present in which case the half-life is reduced to less than 1 day. The following markers should be determined beforehand to ensure the efficacy of replacement therapy: PT, aPTT, AT activity, thrombocyte count, fibrinogen, D-dimers /13/.

16.20.6 Comments and problems

Specimen

AT should always be measured in plasma and not in serum because the process of coagulation consumes approximately 30% of AT.

Method of determination

The method used for testing AT deficiency is important to ensure correct diagnosis and treatment of the patient.

The following must be noted /3/:

• Assays using human thrombin are subject to interference by heparin cofactor, resulting in a falsely high value, especially under heparin therapy. This is not the case in F Xa-based assays.
• Direct thrombin inhibitors such as hirudin and argatroban simulate falsely high AT activity if thrombin-based assays are used
• Some types of hereditary AT deficiency are not detected by thrombin-based assays (e.g., Hamilton mutation A382T) or several mutations in connection with type II HBS
• F Xa-based assays are interfered by direct F Xa inhibitors such as rivaroxaban, but not by heparin cofactor II or direct thrombin inhibitors /14/.
• F Xa based assays do not detect all AT variants, such as the Stockholm mutation (G392D), which can be measured using thrombin-based assays, or the Cambridge II mutation (A384S) /15/, which is in part not detectable by thrombin-based assays. F Xa-based chromogenic assays have a lower detection limit for AT deficiency than thrombin-based assays /3/.

Reference interval

There is no difference in reference intervals for thrombin-based and F Xa-based assays.

Stability

Changes in citrated blood at ambient temperature (20 °C): mean of –0.8% (range –14.9% to +12.0%) after 8 h and 3.1% (range –6.3% to +17.5%) after 24 h /16/.

16.20.7 Pathophysiology

AT is a single-chain glycoprotein with a molecular weight of 58 kDa and belongs to the family of serine protease inhibitors. In a two-step reaction, AT initially binds to the proteases much like a substrate. Subsequently, after proteolytic cleavage, AT binds firmly to the serine located in the active center of the protease (Fig. 16.20-2 – Catalysis of the thrombin-antithrombin reaction). AT inhibits thrombin, factors F Xa and F IXa at about the same level of efficiency. F XIa, F XIIa and kallikrein are inhibited to a lesser degree. All inhibitory reactions are accelerated by heparin. Whereas the reaction with thrombin and F Xa is accelerated 1,000–2,000-fold in the presence of heparin, the reactions of AT with the contact phase factors are only slightly faster. Under physiological conditions, the heparan sulfate proteoglycans incorporated into the surface membrane of endothelial cells exert an accelerating effect on the binding capacity of AT /17/. The half-life of AT is 65 hours and thus longer than that of replaced AT.

During states of enhanced activation of intravascular coagulation connected with an increased release of thrombin, the consumption rate of AT is more rapid than its production rate. The decrease in AT and the increase in the concentration of thrombin-antithrombin complex resulting from inhibition of thrombin are indicators of abnormal fibrin formation.

The decrease in AT and the formation of thrombin-antithrombin complex resulting from the inhibition of thrombin are indicators of abnormal thrombin formation. Parallel to the processes of disseminated intravascular coagulation (DIC), an impairment in the liver synthetic capacity often develops as well. The impaired AT synthesis thus contributes further to the enhancement of DIC.

The assessment of homeostasis requires that, besides the coagulation inhibitors (AT, protein C) and the indicators of activation and consumption (TAT, F₁₊₂, fibrin monomer, D-dimer), the pro coagulant factors are taken into account as well (e.g., by measuring PT and aPTT). Furthermore, in order to interpret the in vivo activity of AT, the actual pH of the blood must be taken into consideration, since acidosis is quantitatively associated with increasing functional deficits of the inhibitor. Accordingly, an AT activity of 60%, for example, measured in vitro under optimal conditions corresponds to an in vivo activity of only 10%, given a pH of 7.05. The AT activity will return to 60% if acidosis is eliminated.

References

1. Lane DA, Kunz G, Olds RJ, Thein SL. Molecular genetics of antithrombin deficiency. Blood Rev 1996; 10: 59–74.

2. Tollefsen DM. Laboratory diagnosis of antithrombin and heparin cofactor II deficiency. Semin Thrombos Hemostas 1990; 16: 162–8.

3. Merz M, Böhm-Weigert M, Braun S, Cooper PC, Fischer R, Hickey K, et al. Clinical multicenter evaluation of a new FXa-based antithrombin assay. Int Jnl Lab Hem 2011. doi: 10.1111/j.1751-553X.2011.01326.x.

4. Khor, B Van Cott EM. Laboratory tests for antithrombin deficiency. Am J Hematol 2010; 85: 947–50.

5. Reverdiau-Moliac P, Delahousse B, Body G, et al. Evolution of blood coagulation activators and inhibitors in the healthy human fetus. Blood 1996; 88: 900–6.

6. Male C, Johnston M, Sparling C, Brooker L, Andrew M, Massicotte P. The influence of developmental haemostasis on the laboratory diagnosis and management of haemostatic disorders during infancy and childhood. Clin Lab Med 1999; 19: 39–69.

7. Wege S. Untersuchungen zur Ermittlung von Referenzbereichen für Antithrombin III bei Erwachsenen im Alter von 18–90 Jahren im Vergleich zu Thrombophilie-Patienten – Ein Methodenvergleich – DG Klinische Chemie Mitteilungen 2003; 34: 40–1.

8. Andrew M, Paes B, Johnston M. Development of the hemostatic system in the neonate and the young infant. Am J Pediatr Hematol Oncol 1990; 12: 95–104.

9. Andrew M, Vegh P, Johnston M, et al. Maturation of the hemostatic system during childhood. Blood 1992; 80: 1998–2005.

10. Lane DA Bayston T, Olds RJ, Wechselstrom S, Cooper DN, Milar DS, et al. Antithrombin mutation data base: 2nd (1997) update. For the plasma coagulation Inhibitors subcommittee of the International Society of Thrombosis and Haemostasis. Thromb Haemost 1997; 77: 197–211.

11. Patnaik MM, Moll S. Inherited antithrombin deficiency: a review. Haeophilia 2008; 14: 1229–39.

12. Vossen CY, Conard J, Fontcuberta J, Markis M, van der Meer FJ, Pabinger I, et al. Risk of a first venous thrombotic event in carriers of familial thrombophilic defect. The European Prospective Cohort on Thrombophilia (EPCOT). J Thromb Haemost 2005; 3: 459–64.

13. Bundesärztekammer. Leitlinien zur Therapie mit Blutkomponenten und Plasmaderivaten. Köln; Deutscher Ärzteverlag, 2003: 164.

14. Samama MM, Martinoli JL, LeFlem L, Guinet C, Plu-Bureau G, Depasse F, et al. Assessment of laboratory assays to measure rivaroxaban – an oral, direct factor Xa inhibitor. Thrombosis Haemostasis 2010; 103: 815–25.

15. Corral J, Hernandez-Espinoza D, Soria JM, et al. Antithrombin Cambridge II (A384S): An underestimated genetic risk factor for venous thrombosis. Blood 2007; 109: 4258–63.

16. Kemkes-Matthes B, Fischer R, Peetz D. Influence of 8 and 24-h storage of whole blood at ambient temperature on prothrombin time, fibrinogen, thrombin time, antithrombin and D-dimer. Blood Coagulation and Fibrinolysis 2011; 22: 215–20.

17. De la Morena-Barrio B, Orlando C, De la Morena-Barrio EM, Vincente V, Jochmans K, Vorral J. Incidence and features of thrombosis in children with inherited antithrombin deficiency.Haematologica 2019; 104 (12) 2512–8.

18. Roldan V, Ordonez A, Zorio MF, Soria JM, Minano A, et al. Antithrombin Cambridge II (A384S) supports a role for antithrombin deficiency in arterial thrombosis. Thromb Haemost 2009; 101: 483–6.

19. Mammen EF. Clinical relevance of antithrombin deficiencies. Semin Hematol 1995; 32: 2–6.

20. Brangenberg R, Bodensohn M, Bürger U. Antithrombin-III substitution in preterm infants. Effect on intracranial hemorrhage and coagulation parameters. Biol Neonate 1997; 72: 76–83.

21. Minakami H, Morikawa M, Yamada T, Yamade T. Candidates for the determination of antithrombin activity in pregnant women. J Perinat Med 2011; 39. doi: 10.1515/jpm.2011.026.

22. Perry DJ. Acquired antithrombin deficiency in sepsis. Br J Haematol 2001; 112: 26–31.

23. Fourrier F, Chopin C, Goudemand J, Hendrcycx S, Caron C, Rime A, Marey A, Lestavi P. Septic shock, multiorgan failure, disseminated intravascular coagulation. Chest 1992; 101: 816–23.

24. Vossen CY, Conard J, Fontcuberta J, Markis M, van der Meer FJ, Pabinger I, et al. Risk of a first venous thrombotic event in carriers of familial thrombophilic defect. The European Prospective Cohort on Thrombophilia (EPCOT). J Thromb Haemost 2005; 3: 459–64.

25. Cushman M. Thrombophilia testing in women with venous thrombosis: The 4 P’s approach. Clin Chem 2014; 60: 134–7.

26. Muszbek L, Bereczky Z, Kovacs B, Komaromi I. Antithrombin deficiency and its laboratory diagnosis. Clin Chem Lab Med 2010; 48 (Suppl 1): S67–S78.

16.21 Protein C, protein S, APC, F V Leiden, prothrombin G20210A

Lothar Thomas

Environmental risk factors and genetic predisposition play an important role in the development of venous thromboembolic events (VTE). Genetic risk factors, primarily related to the hemostatic system, trigger venous thromboembolic events (VTE).

In general, two types of mutation-related, genetic defects are distinguished /1/:

• Loss of function mutations involving natural anticoagulants antithrombin, protein C and protein S
• Gain of function mutations in pro coagulant factor V (F V Leiden) and F II (prothrombin G20210A9).

The association between genetic risk factors and VTE can be classified as:

• Strong: deficiencies in antithrombin, protein S and protein C (risk of VTE is 5–10-fold increased)
• Moderate: presence of F V Leiden or prothrombin G20210A9 (risk of VTE is 2–5-fold increased)
• Weak: for instance, His95Arg replacement in the B-subunit of F XIII (risk of VTE is approximately 1.5-fold increased).

16.21.1 Inhibitors of coagulation

16.21.1.1 Protein C

Protein C (PC) and protein S are natural anticoagulants and play an important role in the regulation of the coagulation system /2/. PC is activated by thrombin in the presence of thrombomodulin (T M). T M is an endothelial cell surface receptor, which plays an important modulating role in the anticoagulant response after vascular injury.

After vascular injury binding of thrombin to TM enables thrombin to rapidly cleave the PC into the anticoagulant APC. The endothelial cells also express a PC receptor that binds PC through its Gla-domain and presents it to the thrombin-TM complex. Thrombin cleaves the activation peptide domain of PC at position Arg169 resulting in a 12-amino acid long activation peptide being released from the N-terminal end of the α-chain of PC. Activated PC (APC) inactivates membrane-bound F VIIIa and F Va by cleaving these factors at specific sites:

• F Va is cleaved at Arg 506, which is the preferred cleavage site, however, full inactivation also requires cleavage at Arg 306
• F VIIIa is cleaved at Arg 336 and Arg 562.

The main inhibitor of APC is protein C inhibitor, a single chain glycoprotein that belongs to the serpin superfamily /3/. The inhibitor is a major regulator of the anticoagulant protease APC.

Refer also to

• Fig. 16.21-1 – Activation of protein C and its anticoagulatory effect in combination with the cofactor protein S
• Tab. 16.19-6 – Prevalence of thrombophilic risk factors and risk of VTE.

16.21.1.2 Protein S

Protein S (PS) is present in bound and free form /2/. The free form of PS is an important cofactor of APC, enhancing its affinity to negative charged phospholipid surfaces. Free PS is able to displace F Xa from its complex with F Va, allowing APC to cleave F Va at Arg506. In the process of F VIIIa inactivation, APC activity is synergistically stimulated by PS. PS is encoded by the gene PROS 1 consisting of 15 exons. Free PS is the only cofactor of APC. Bound PS forms a complex with the complement 4b binding protein (C4bBP); this complex lacks APC cofactor activity.

16.21.1.3 Activated protein C resistance

APC resistance is defined as a poor anticoagulant response to activated PC /4/.

Hereditary disorder

Activated PC resistance is caused by a single point mutation in the F V gene, also known as F V Leiden, resulting in insufficient inactivation of F Va by APC.

Acquired disorder

In the absence of F V Leiden, an acquired activated PC resistant phenotype may be present during pregnancy, with the use of oral contraceptives, in the presence of lupus anticoagulant, in cases with increased F VIII concentration and in patients with multiple myeloma.

16.21.1.4 Factor V Leiden

Factor V functions as a synergistic cofactor with PS in the degradation of F VIII when this factor is part of the tenase complex (F Xa/F Va) /5/. The genetic background for the APC resistance phenotype is a single G to A nucleotide substitution at position 1691 in the F V gene (G1691A or F V Leiden), resulting in the replacement of Arg 506 by Gln.

16.21.1.5 Prothrombin G20210A variation

A mutation in the Prothrombin gene is the second most common cause of inherited thrombophilia /6/. This mutation involves a single base-pair substitution (guanine to adenine) at nucleotide 20210 in the 3’-untranslated region of the Prothrombin gene. Heterozygous carriers have higher prothrombin plasma levels, with a 2 to 5-fold increased risk of VTE without concomitant risk factors. There is a more than additive synergistic risk of VTE when other thrombophilic factors, particularly factor V Leiden are simultaneously present /6/.

16.21.2 Indication

Testing can be considered in patients with VTE suggestive of inherited thrombophilia:

• Thrombosis at a young age (< 50 years), especially in association with weak provoking factors (minor surgery, combination of oral contraceptives, or immobility) or unprovoked VTE
• Strong family history of VTE (first-degree family members affected at young age)
• Recurrent VTE events, especially at a young age
• VTE in unusual sites such as splanchnic or cerebral veins.

16.21.3 Method of determination

Laboratory evaluation of APC resistance includes /7/:

• Primarily a functional coagulation assay for identifying APC resistance
• Secondarily, in the case of APC resistance, molecular screening for factor V Leiden
• Tertiarily, if molecular screening is negative, the determination of PC and PS and, possibly, molecular genetic analysis of the gene PROS 1.

16.21.3.1 Activated protein C resistance

Functional coagulation assay for APC resistance. It is a screening test for determining F V Leiden and the entire potential of the PC pathway (PC and PS activity) /8/.

Principle

The protein C activity dependent clotting time (PCAT) is determined using the aPTT-based assay. The second-generation aPTT method uses patient plasma with pre dilution of F V deficient plasma (1+4). This modification is highly sensitive to F V Leiden and can also be used in patients undergoing anticoagulation therapy with vitamin K antagonists (coumarins).

The aPTT is determined in the presence of a PC activator, the snake venom Agkistrodon contortix (PCAT sample), and in the absence of this activator (PCAT 0 sample). Normal plasma coagulates in an aPTT assay in 28–35 seconds. If activated PC (APC) is added, the aPTT clotting time is prolonged 2-fold or more to 60–100 seconds. This plasma is sensitive to APC. Lower sensitivity of the patient plasma to APC (clotting time only prolonged 1.5–1.7-fold; typical of heterozygous F V Leiden carriers) indicates that the plasma is resistant to APC.

The ratios PCAT/PCAT 0 are obtained by transformation of the clotting times in normalized ratios (NR). NR below 0.80 indicates the presence of F V Leiden.

This assay also detects cooperative effects between the involved factors and, thus, also recognizes PC deficiency.

16.21.3.2 Protein C (PC)

A differentiation must be made between determination of PC activity (functional assay) and that of PC antigen concentration (immunochemical assay) /2/. There are conditions where the two assays may not provide identical results.

Coagulation assay (functional assay)

In the coagulation assay, the anticoagulant activity of PC, (i.e., the capacity to inactivate F VIIIa and/or F Va) is the object of measurement /9/.

Principle: the majority of methods are based on the aPTT which depends on F V and F VIII. The activated factors F Va and F VIIIa are inactivated by activation of PC. Prolongation of the aPTT occurs which is a measure of the anticoagulant activity of PC. After the addition of aPTT reagent, an enzyme, usually extracted from the snake venom of Agkistrodon contortrix, is added which activates PC. Since the reaction mixture contains an excess of all of the coagulation factors, the measured clotting time depends on the PC activity. The result is read off a standard curve as the clotting time and is expressed in % of normal. Lupus anticoagulant and FVIII concentrations above 150% interfere with coagulation assays.

Amidolytic assay (functional assay)

The enzyme activity of PC is determined using a chromogenic peptide substrate. Contrary to the coagulation assay, a change in the phospholipid binding domain, for example under oral anticoagulant therapy, has no effect.

Principle: PC is activated by a PC activator (snake venom); subsequently, the cleavage of the chromogenic substrate is determined kinetically using spectrophotometric measurement. The activity is expressed in % of normal.

Enzyme immunoassay

The concentration of the PC antigen is determined. There is currently no need to differentiate between type I and type II. Therefore, the enzyme immunoassay is used for confirmation of decreased activity measured in the functional assay.

Principle: antibodies to PC are bound on the surface of micro titer wells; these bind PC contained in the plasma sample. Subsequently, a peroxidase-labeled antibody to PC binds to the immune-complex-bound PC. The amount of bound peroxidase which is proportional to the quantity of PC is measured by the addition of substrate. The result is expressed in % of normal or in mg/L.

16.21.3.3 Protein S (PS)

Approximately 60% of PS in the plasma is bound to the C4b-complement-binding protein (C4bBP) while 40% are present in a free circulating form /10/. Only free PS can exert its anticoagulant effect as a cofactor of APC. A functional assay as well as an immunochemical assay should be used. The functional assay is the screening test /2/. The analysis of the gene PROS 1 includes the investigation of all 15 exons for sequence variants.

Coagulation assay

The functional activity of free PS (i.e., the capacity as an APC cofactor to degrade F Va and F VIIIa) and, thus, to prolong the clotting time, is measured by the prolongation of the aPTT or the PT or the Russell’s viper venom time (RVVT) /11/. Analogously to the PC determination by a coagulation assay, the sample is diluted and mixed with plasma deficient in PS. The clotting time is measured after the addition of a coagulation activator and already activated PC. The result is expressed in % of normal.

Enzyme immunoassay

Using an enzyme immunoassay, the total and the free PS concentrations can be determined. In order to determine the free proportion, bound PS is precipitated using polyethylene glycol, followed by the determination of the remaining portion in the supernatant. Newer methods use monoclonal antibodies which bind to the PS region covered by C4bBP. This makes the precipitation of bound PS of the sample unnecessary.

16.21.3.4 Factor V Leiden

Molecular genetic analysis

A fragment of exon 10 of the FV gene is amplified by PCR (DNA polymerase chain reaction). The mutation of the Leiden variant of F V is located in this fragment. The amplified fragment is cut at two sites by the restriction enzyme Mnl. The mutation results in the loss of one of the two interface sites for the restriction enzyme, thus making a higher-molecular-weight-DNA band visible during the gel electrophoresis of samples belonging to individuals afflicted with this defect. The differentiation between heterozygous and homozygous carriers of this defect is only possible by using this method.

16.21.3.5 Prothrombin G20210A variation

Molecular genetic analysis with differentiation between homozygous and heterozygous variations.

16.21.4 Specimen

APC resistance, PC and PS: citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood): 1 mL

Molecular genetic analysis for F V Leiden, prothrombin G20210A variation: EDTA blood: 3–5 mL

16.21.5 Reference interval

Refer to Tab. 16.21-1 – Reference intervals for PC and PS.

16.21.6 Clinical significance

Genetic defects in coagulation factors and anticoagulant proteins are the main risk factors for VTE. Refer to Section 16.19 – Thrombophilia and venous thromboembolism.

16.21.6.1 Activated protein C resistance

The diagnostic procedure in suspected APC resistance is depicted in Fig. 16.21-2 – Diagnostic approach in suspected APC resistance. Based on a APC ratio below 0.80 the assessment is as follows /7/:

• All patients are diagnosed with F V Leiden. Relevant studies report a diagnostic sensitivity of 100% with a specificity of 95%. One study reported a specificity of only 72%.
• Impaired functional activity of PC is detected with a diagnostic sensitivity of 82–100%
• PS deficiency is diagnosed with a sensitivity of 87–97% and a specificity of only 30% /10/.

The functional coagulation assay is a screening test used to determine F V Leiden and PC deficiency whereas detection of PS deficiency is problematic. The assay is clinically useful to detect defects as well as to assess the capacity of the PC pathway and, thus, to a certain extent predict a future risk of VTE. In patients with PS deficiency, the screening assay is a good tool for assessing the risk of a thrombotic event. Borderline or positive findings should undergo molecular genetic analysis for F V Leiden.

16.21.6.2 Factor V Leiden

Factor V Leiden mutation is one of the most common inherited risk factors for VTE. The prevalence of heterozygous carriers in the general population is 2–15%. This mutation is limited to populations of Caucasian origin and their descent in North and South America. The incidence of VTE is 1 in 10,000 per year in young women and is increased to 4–7 in female heterozygous carriers of F V Leiden. If a young women with F V Leiden is obese, the incidence increases to 8–14 in 10,000, and if she is taking oral contraceptives, the incidence is even 34 in 10,000 /12/. Related to both sexes, the probability of VTE is increased 3–7-fold by a heterozygous defect and 50–100-fold by a homozygous defect. About 25% of family members with F V Leiden suffer their first VTE event before the age of 45.

Perioperative risk /13/

VTE after general surgery does not appear to be increased in the presence of F V Leiden heterozygosity when patients are under prophylactic anticoagulation. There may be an increased risk of arterial thrombosis and myocardial infarction after vascular surgery. Coronary bypass graft occlusion within the first few months after surgery may be increased in the presence of F V Leiden.

Hormone substitution /12/

The annual incidence of VTE in women above 50 years of age is 4 in 10,000. This rate increases to 10 in 10,000 if these women are taking postmenopausal estrogens and progesterone. In women with a familial history of VTE, the annual incidence increases to 30 in 10,000.

16.21.6.3 Protein C deficiency

In hereditary PC deficiency, a clinically irrelevant differentiation is made between two types:

• Type I; both the protein concentration as well as the activity are decreased due to a defect in synthesis
• Type II; a dysfunctional protein is present (i.e., the protein concentration is within the reference interval whereas the activity is significantly reduced).

The majority of patients heterozygous for PC deficiency have PC activities of 30–65% /2/. Homozygous carriers of the defect have almost undetectable PC activity. Most of the about 250 mutations detected to date are causative of type I deficiency. Approximately 70% of these patients have a single mutation in the encoding region of the gene PROC, leading to amino acid changes in PC. For acquired PC deficiency, see Tab. 16.21-2 – Diseases and conditions with acquired PC deficiency.

16.21.6.4 Protein S deficiency

PS deficiency is differentiated into three subtypes /2, 7/:

• Type I; both the antigen concentration as well as the activity are decreased due to a defect in synthesis
• Type II; a dysfunctional protein is present (i.e., the antigen concentration is within the reference interval whereas the activity is significantly reduced)
• Type III; the concentration of free PS is decreased due to an elevated concentration of C4bBP (i.e., the antigen concentration is within the reference interval although the activity and the concentration of free PS antigen are reduced). A causal relation between this type and VTE has not been established to date.

Acquired PS deficiency is much more common than the hereditary type. Moreover, the PS concentrations and activities of healthy individuals overlap significantly with those of carriers of the hereditary defect. Therefore, the PS reference values are not suited for diagnosing a hereditary defect. The classification of hereditary PS deficiency is shown in Tab. 16.21-3 – Classification of PS deficiency based on activity and concentration of free and total PS.

Hereditary PS deficiency

PS deficiency types I and II are defects associated with decreased PS activity and account for 95% of the cases /14/. Investigations for PS deficiency should always include a functional assay and the determination of the PS concentration. The PS values of a study /10/ are shown in Tab. 16.21-4 – Protein S activity as a function of the type of mutation. Only PS values below 45% generally point to a disease defined by a mutation in the gene PROS 1 /10/. In these cases, a molecular genetic analysis of the 15 exons of PROS 1 should be performed. A diagnostic algorithm is recommended /10/.

Heterozygous PC and PS deficiency present with the same clinical features. Only sporadic cases of homozygous deficiency have been known to date. In patients below 45 years with venous thrombosis, heterozygous PS deficiency type I was found in approximately in 2–3% of cases. See also Section 16.19 – Thrombophilia and venous thromboembolism.

Acquired protein S deficiency

PS like PC is a vitamin K dependent protein. Therefore, under oral anticoagulation, a decrease in free PS is observed. However, this decline is slower and less pronounced than that of PC /15/.

Liver disease causes but a moderate decrease in PS, and DIC is associated with normal PS levels. The decline in PS in the presence of acute respiratory distress syndrome affects the free PS portion significantly more than the bound form /16/.

Estrogens result in a decreased release of PS, thus explaining why premenopausal women have physiologically lower levels in comparison to men (median of approximately 80%). Hormone therapy or the intake of oral contraceptives also cause a significant decrease in PS activity.

16.21.6.5 Prothrombin G20210A mutation

The Prothrombin G20210A mutation involves a single base-pair substitution (guanine to adenine) at nucleotide 20210 in the 3’ un translated region of the Prothrombin gene. Carriers of this mutation have a higher endogenous thrombin potential compared to those with the wild type genotype. Prothrombin is increased by 30% in heterozygous carriers and by 70% in homozygous carriers /17/. This mutation is associated with a hyper coagulable state and represents the second highest risk of hereditary thrombophilia /18/. Heterozygous carriers among the Caucasian population:

• Have a prevalence of 2%
• Have a 2–5-fold risk of thrombosis (10-fold in homozygous carriers)
• Account for 20% of VTE patients.

Women with the Prothrombin G20210A mutation undergoing oral anticoagulation have a 150-fold increased risk of cerebral venous sinus thrombosis and a 16–59-fold increased risk of other deep venous thromboses compared to those without oral anticoagulation /12/.

However, the Prothrombin G20210A mutation may not be the only cause of a higher endogenous thrombin potential and VTE in affected carriers. Increased thrombin generation is thought to be associated with other genetic and/or environmental factors /19/.

16.21.6.6 MTHFR-C677T mutation

The homozygous genotype of MTHFR gene polymorphism at position C677T is linked to the Caucasian population. This mutation is associated with an increase in plasma homocysteine, depending on the diet. Carriers among the Caucasian population:

• Have a prevalence of 10%
• Have a 2–3-fold risk of thrombosis
• Account for 15–20% of VTE patients.

The risk of cerebral venous sinus thrombosis is 20-fold increased in women with the MTHFR C677T mutation and hyper homocysteinemia undergoing hormonal anticonception compared to those without hormonal anticonception /20/. The association between the MTHFR-C677T mutation and venous thromboembolism has been discussed controversially.

16.21.7 Comments and problems

Coagulation assay for APC resistance /2/

Preanalytics: since the vitamin K⁺-dependent coagulation factors and protein S are decreased, patients should not be on oral anticoagulant treatment. APC resistance is simulated. Vitamin K inhibitors do not interfere with assays which use samples diluted with F V deficient plasma /21/. The investigation can be performed on patients on heparin treatment if the plasma heparin concentration is below 1 IU/mL because the reagents contain heparin-neutralizing polybrenes.

Influence factors: despite using patient plasma diluted with F V deficient plasma, the presence of lupus anticoagulant, direct thrombin and F Xa inhibitors and heparin values higher than 1 IU/mL, all of which prolong the clotting time, may result in a falsely elevated APC, leading to missed detection of APC resistance. The presence of argatroban, a direct thrombin inhibitor, should be kept in mind if the APC resistance assay yields unexpectedly prolonged clotting times in samples with and without APC /22/. The sensitivity of patient plasma to APC is lower in the presence of elevated F VIII seen, for example, in inflammatory conditions and pregnancy. This results in shorter clotting times and simulated APC resistance.

Stability: up to 8 h at room temperature. Longer-term stability is achieved if deep-frozen at –20 to –70 °C /23/.

Protein C /2/

F VIII activities higher than 150% shorten the clotting time and are connected with falsely low protein C activities in the functional assay. Anticoagulation in the patient does not interfere with the determination.

Amidolytic assays for PC determination are not sensitive to lupus anticoagulant, high F VIII concentration and F V Leiden. Chromogenic peptide substrates which have no high specificity for APC may overestimate PC in the presence of other proteolytic enzymes, such as plasmin, kallikrein and thrombin. Moreover, they are insensitive to a certain type of qualitative PC deficiency.

Protein S

Platelets cause falsely low activities. Therefore, platelet-poor plasma samples should be used for analysis.

Direct oral anticoagulant interference on thrombophilia testing

In a study /25/ the introduction of DOACs into the US marketplace has caused significant interference in thrombophilia assays. Approximately 5-10% of tests for thrombophilia risk have potential DOAC interference. False positive test results for lupus anticoagulant, falsely increase protein S and protein C activities, and interference with FV dependent prothrombin venom based, activated protein C resistance were registered. Laboratories trying to determine whether samples submitted for thrombophilia testing have potential DOAC interference, should perform a thrombin time and a test for the anti-Xa activity, if test results as described above, are present.

16.21.8 Pathophysiology

PC is a vitamin K dependent glycoprotein synthesized as proenzyme in the liver. The mature protein has a molecular weight of 62 kDa and consists of a heavy chain (41 kDa) and a light chain (21 kDa) connected by a disulfide bond between Cys 141 and Cys 265.

Circulating plasma PC is a proenzyme that needs to be activated for anticoagulatory action. It is activated by thrombin bound in a thrombomodulin-thrombin complex on the vascular endothelial cells, in the presence of Ca²⁺ (Fig. 16.21-1 – Activation of protein C and its anticoagulatory effect in combination with the cofactor protein S).

APC causes proteolysis at the phospholipid surface and, thus, inactivates F Va and F VIIIa. This process depends on the presence of Ca²⁺ and is accelerated by protein S. APC is regulated by the serine proteinase inhibitors α₁-antitrypsin, α₂-antiplasmin and the heparin-stimulated PC inhibitor as well as by α₂-macroglobulin.

PS has a molecular weight of 69 kDa and is synthesized in the liver and vascular endothelial cells. In comparison to PC with a half-life of only 8 h, the half-life of PS is 60 h. About 40% of PS are present in a free form while 60% are bound to C4bBP. Free PS circulates as cofactor of PC and is present in its active form.

The gene encoding PC (PROC) is located at position 2q13–q14 and contains 9 exons and 8 introns. The gene encoding PS (PROS1) is located at position 3q11.2 and contains 15 exons and 14 introns.

A defect in the synthesis or a functional defect of PC or PS cause a reduction in the anticoagulant potential and therefore can result in hyper coagulability which is characterized by an increased susceptibility to venous thromboembolic events.

The variant Leiden of F V is another defect which counteracts the PC pathway. Under normal conditions, F V circulates in plasma and shows no notable pro- or anticoagulant activity.

However, the pro coagulant activity of F Xa and thrombin and the anticoagulant activity of APC have an influence on F V:

• Enhanced pro coagulant activity is achieved upon activation of the coagulation system in the event of vascular injury. In this case, F Xa or thrombin cleave three arginine-associated peptide bonds in F V, thus detaching domain B in the F V molecule and causing F V to become the activated pro coagulant F Va (Fig. 16.21-3 – Activation of factor V by thrombin and/or F Xa). F Va is an important cofactor of F Xa in prothrombin activation. Both activated pro coagulators form the pro thrombokinase complex (F IXa, F VIIIa, F Xa, F IIa) on the surface of phospholipids.
• F V follows a different direction, however, if the protein C pathway is activated. APC regulates the pro coagulant activity of the pro thrombokinase complex by proteolytic degradation of F V, cleaving the F V molecule at positions Arg 306 and Arg 506. Cleavage at position 306 leads to the complete loss of F V activity, whereas cleavage at position 506 results in an intermediate retaining F Xa cofactor activity. The F V intermediate cleaved off by APC forms an anticoagulant complex with protein S which cleaves F VIII present in the tenase complex, thus inactivating the tenase complex. The tenase complex consists of F IXa and F VIIIa bound to the phospholipid surface. It is efficient in F Xa activation and cannot be down regulated by APC or PC.

References

1. Bafunno V, Margaglione M. Genetic basis of thrombosis. Clin Chem Lab Med 2010; 48 (Suppl 1): S41–S51.

2. Bereczky Z, Kovacs KB, Muszbek L. Protein C and protein S deficiencies: similarities and differences between two brothers playing in the same game. Clin Chem Lab Med 2010; 48 (Suppl 1): S53–S66.

3. Elisen MGLM, Kr von dem Borne P, Bouma BN, Meijers JCM. Protein C inhibitor acts as a procoagulant by inhibiting the thrombomodulin-induced activation of protein C in human plasma. Blood 1998; 91: 1542–7.

4. Dahlbaeck B. APC resistance: what we have learned since 1993? J Lab Med 2004; 28: 21–7.

5. Nicolaes GAF, Dahlbaeck B. Activated protein C resistance (FV Leiden) and thrombosis: factor V mutations causing hypercoagulable states. Haematol Oncol Clin N Am 2003; 17: 37–61.

6. Poort SR, Roosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3’ untranslated region of the prothrombin gene is associated with elevated prothrombin levels and an increase in venous thrombosis. Blood 1996; 88: 3698–703.

7. Margetic S. Diagnostic algorithm for thrombophilia screening. Clin Chem Lab Med 2010; 48 (Suppl 1): S27–S39.

8. Dati F, Hafner G, Erles H, Prellwitz W, Kraus M, Niemann F, Noah M, Wagner C. Pro C^® Global: the first functional screening assay for the complete protein C pathway. Clin Chem 1997; 43: 1719–23.

9. Bertina RM. Specificity of protein C and protein S assays. Res Clin Lab 1990; 20: 127–38.

10. Dübgen S, Dick A, Marschall C, Spannagl M. Gerinnungsphysiologische und genetische Diagnostik bei hereditärem Protein S-Mangel. J Lab Med 2013; 37: 79–89.

11. Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, van der Velden PA, Reitsma PH. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 64–7.

12. Cushman M. Thrombophilia testing in women with venous thrombosis: The 4 P’s approach. Clin Chem 2014; 60: 134–7.

13. Donahue BS. Factor V Leiden and perioperative risk. Anesth Analg 2004; 98: 1623–34.

14. Marlar RA, Gausman JN. Protein S abnormalities: a diagnostic nightmare. Am J Hematol 2011; 86: 418–21

15. Bick Rl. Prothrombin G20210A mutation, antithrombin, heparin cofactor II, protein C and protein S defects. Hematol Oncol 2003; 17: 9–36.

16. Gouault-Heilmann M, Leroy-Matheron C, Levent M. Inherited protein S deficiency: clinical manifestations and laboratory findings in 63 patients. Thromb Res 1994; 76: 269–79.

17. Pajic T. Factor V Leiden and FII 20210 testing in thromboembolic disorders. Clin Chem Lab Med 2010; 48 (Suppl 1): S79–S87.

18. Luxembourg B, Lindhoff-Last E. Genomische Diagnostik thrombophiler Gerinnungsstörungen bei Frauen. Hämostaseologie 2007; 27: 22–31.

19. Lavigne-Lissalde G, Sanchez C, Castelli C, Alonso S, Mazoyer E, Bal dit Sollier C, et al. Prothrombin G20210A carriers the genetic mutation and a history of venous thrombosis contributes to thrombin generation independently of factor II plasma levels. J Thrombos Haemost 2010; 8: 942–9.

20. Seitz R, Rappe N, Wolf M, Heidtmann HH, Maasberg M, Immel A, Kraus M, Egbring R, Pfab R, Havemann K. Impaired anticoagulant activity of protein C and activation of neutrophils in extensive lung cancer. Clin Appl Thromb Haemostas 1995; 1: 131–4.

21. Quincampoix JC, Legarff M, Rittling C, Andiva S, Toulon P. Modification of the Pro C Global assay using dilution of patient plasma in factor V-depleted plasma as a screening assay for factor V Leiden mutation. Blood Coagul Fibrinolysis 2001; 12: 569–76.

22. Shaikh S, van Cott EM. The effect of argatroban on activated protein C resistance. Am J Clin Pathol 2009; 131: 828–33.

23. Price DT, Ridker PM. Factor V Leiden mutation and the risks for thromboembolic disease: a clinical perspective. Ann Intern Med 1997; 127: 895–903.

24. Sheth SB, Carvalho AC. Protein S and C alterations in acutely ill patients. Amer J Haematol 1991; 36: 14–8.

25. Wong CC, Worfolk LA, Sahud M, Dlott J.S. Prevalence of suspected direct oral anticoagulant interference on thrombophilia testing at US National Reference Laboratory. Clin Chem 2020; 66 (4): 618–20.

16.22 Antiphospholipid syndrome

Lothar Thomas

The antiphospholipid antibody syndrome (APS) has a broad spectrum of thrombotic, non thrombotic and obstetrical manifestations. The APS is generally considered to confer a high risk of recurrent venous thromboembolism (VTE), arterial and microvascular thrombosis. Tests for antiphospholipid antibodies are generally included in the workup for hyper coagulable state. Obstetrical APS is characterized by fetal loss after the 10th week of gestation, recurrent early miscarriages, intrauterine growth restriction, or severe preeclampsia /1/.

The combination of warfanin and antiplatelet therapy appears to be an effective approach in preventing recurrent overall thrombosis. While dual antiplatelet therapy may as show promise in preventing recurrent arterial thrombosis /2/.

16.22.1 Indication

• Venous thromboembolism (VTE)
• Arterial thrombosis (myocardial infarction, stroke)
• Pregnancy disorders
• Autoimmune disorders, especially systemic lupus erythematosus
• APTT prolongation of unknown origin
• Thrombocytopenia of unknown origin.

16.22.2 Method of determination

Laboratory APS criteria are /3/:

• Lupus anticoagulant
• Anti cardiolipin immunoglobulin isotye G (IgG) and M (IgM ) or IgG and IgM anti-β₂ gycoprotein-I (anti-β₂GPI).

The criteria were revised in detail by a subcommittee /4/.

The laboratory tests of the official criteria comprise two categories of diagnostic procedures (Tab. 16.22-1 – Recommendations for the optimal laboratory detection of lupus anticoagulant (LA)) /5/:

• Coagulation assays; they indirectly identify interference by antibodies based on prolonged clotting time. Antibodies directed against phospholipids that prolong the clotting time are also referred to as lupus anticoagulants.
• Immunoassays (ELISA) for the direct determination of anti-β₂-glycoprotein I and anti-cardiolipin.

Results of first-line tests that are suggestive of antiphospholipid syndrome:

• Prolongation of aPTT clotting time
• Thrombocyte count below 100 × 10⁹/L.

16.22.2.1 Lupus anticoagulant assays

Lupus anticoagulants (LAs) are antibodies directed against phospholipids and/or phospholipid-protein complexes. They prolong the clotting time of phospholipid dependent coagulation assays in vitro, but are not associated with thrombophilia. The term lupus anticoagulant results from the year 1952 when a prolonged aPTT was for the first time determined in patients with systemic lupus erythematosus.

LA determination is to be performed according to the criteria of the three-step guideline of the International Society of Thrombosis and Hemostasis /6/ (Fig. 16.22-1 – Principles of lupus anticoagualant testing).

1. Demonstration of a prolonged phospholipid-dependent coagulation screening test based on the local clotting time threshold value.

2. Plasma mixing test to confirm the presence of an inhibitor and exclusion of coagulation factor deficiency.

3. Confirmatory test to demonstrate that the inhibitor is phospholipid-dependent and is not directed against an individual coagulation factor.

The guidelines for lupus anticoagulant detection /4/ recommend two different methods for screening since there is no test with sufficient sensitivity to all LA. The following tests are recommended:

• A test based on F X activation (diluted Russel viper venom time)
• A second test based on intrinsic coagulation activation (aPTT or kaolin clotting time).

LA is suspected when the 99th percentile of the clotting time of healthy individuals is exceeded. Tab. 16.22-1 – Recommendations for the optimal laboratory detection of lupus anticoagulant lists recommendations for optimal laboratory detection. For streamlining the test procedure, the Sidney criteria /4/ recommend the integration of screening and confirmation into a single assay. These tests do not require performance of the plasma mixing test.

Diluted Russel viper venom time (dRVVT) assay

Principle: the snake venom Russel’s viper venom contained in the reagent activates F X causing prothrombin activation in the presence of F V, Ca²⁺ and phospholipids. The high sensitivity of the assay is achieved by low phospholipid concentration in the reagent /7/.

Activated partial thromboplastin time assay (aPTT)

Principle: see Section 16.11 – Activated partial thromboplastin time. The different commercially available reagents differ by the type of activator and the type, composition and concentration of phospholipids. The kaolin clotting time (KCT) assay is recommended. Kaolin consists of silicic acid the microcrystalline form of which has a large negatively charged surface. Thus, the process of contact activation of the intrinsic pathway is initiated and F X is activated. The F VIII activity may be increased during acute phase response or during pregnancy, leading to a shortened clotting time. Therefore, specific lupus-sensitive reagents are provided for LA detection /8/.

Plasma mixing test

Perform testing on plasmas from healthy donors mixed with pooled normal plasma (PNP) at 1 : 1 proportion. Testing should be performed without pre incubation. The PNP should be prepared ad hoc (home-made) to ensure that the PNP contains less than 10 × 10⁹ platelets/L and to ensure approximately 100% activity for all clotting factors. The thrombin time performed on patient plasma or an anti-F Xa test will help to identify heparin or specific inhibitors to clotting factors /4/.

A prolonged clotting time in the plasma mixing test indicates the presence of LA in the patient plasma, whereas shortening of the clotting time points to factor deficiency that should be confirmed by determination of the relevant individual factor. In many cases, the concurrent decrease in several coagulation factors indicates the presence of LA.

Integrated test

Integrated tests include screening and confirmation testing in a single procedure. Such tests consist of testing the patient plasma twice by means of the dRVVT or aPTT performed in parallel at low (screen) and high (confirm) phospholipid concentrations. In the presence of LA, the clotting time should be prolonged in the screening plasma sample and normal in the confirmation plasma sample. The results should be interpreted by calculating the LA ratio (screen minus confirm) or the percentage correction.

Correction (%) = (screen – confirm)/screen × 100

The presence of LA induces normalization of the clotting time in the confirmation plasma sample. The presence of coagulation factor inhibitors and unfractionated heparin may lead to falsely positive results.

Threshold values for lupus anticoagulant detection

Results of screening tests and the plasma mixing test are potentially suggestive of LA when their clotting times are longer than the 99th percentile of the distribution of at least 40 PNP patients below 50 years of age. Alternatively, the cutoff in the plasma mixing study may be the value of the inhibitory coagulation activity (ICA) defined according to the equation:

ICA = [(b – c/a)] × 100

a, b and c are the clotting times of the patient plasma, mixture and normal plasma, respectively.

The clotting time of the confirmatory test in LA positive samples is not always shortened to within the normal interval of controls. It is, therefore, recommended to perform confirmatory tests in all normal controls and to use the mean of obtained clotting times to calculate the threshold value. Patient plasmas below this threshold are LA positive.

16.22.2.2 Antiphospholipid antibodies (APA) measured by ELISA

APA are directed against phospholipid-protein complexes and are determined using ELISA methodology. The results of the anti-β₂-glycoprotein-I test and the anti-cardiolipin test should always be interpreted in the context of the LA test. In a prospective study /9/, it was shown that the sensitivity of IgG antibody determination is sufficient to diagnose APS associated thrombosis, whereas the determination of IgM or IgA antibodies is not advantageous.

Antibodies to β₂-glycoprotein I (anti-β₂-GPI)

β₂-glycoprotein I (β₂-GPI) is a 50 kDa-glycoprotein which is composed of a polypeptide chain possessing 5 domains. It binds anionic phospholipids and is a cofactor of cardiolipin. The circulating β₂-GPI protein is not able to interact with cellular receptors until after its dimerization by autoantibodies. Receptor-bound APA complexes induce an intracellular signaling that leads to the deregulated activation of endothelial cells, monocytes and platelets, thus providing a possible explanation for the thrombotic predisposition in APS patients /5/.

Anti-β₂-GPI antibodies are primarily directed against the amino acids Gly40–Arg43 in domain I. Association with thrombosis has not been found for any APA not directed against this domain.

Determination of anti-β₂-GPI antibodies: the minimal requirements for the determination of antiphospholipid antibodies using ELISA are proposed by the European Forum on Antiphospholipid Antibodies /10/.

Anti-cardiolipin antibodies (aCL)

Cardiolipins (diphosphatidylglycerins) are a subgroup of phospholipids.

Principle: diluted serum is added to bovine or human serum in a cardiolipin-coated tube and allowed to react. The objective is to detect antibodies to cardiolipin and albumin-bound β₂-GPI. The protocol for determination of aCL by ELISA should be observed during testing /11/.

16.22.3 Specimen

Lupus anticoagulant: platelet-poor citrated plasma

Blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood: 1 mL

Preparation of platelet-poor citrated plasma: centrifugation performed twice to keep the thrombocyte count below 10 × 10⁹/L. Blood collection should be performed prior to anticoagulant therapy or after an adequate period after stopping anticoagulants.

Anti-β₂-GPI and aCL

Serum, plasma: 1 mL

16.22.4 Reference interval

Lupus anticoagulant

The local reference intervals are applied.

Anti-β₂-GPI

Below 10 GPL-U/mL: negative

10–40 GPL-U/mL: weak positive

Higher than 40 GPL-U/mL: positive

Higher than the 99th percentile of healthy controls /3/: positive

Anti-cardiolipin antibodies

Below MPL-U/mL: negative

10–40 MPL-U/mL: weak positive

Higher than 40 MPL-U/mL: positive

Higher than the 99th percentile of healthy controls /3/: positive

16.22.5 Clinical significance

Ten percent of healthy blood donors are positive for anti cardiolipin antibodies, and 1% are positive for lupus anticoagulant. However, after 1 year, less than 1% are still positive for these tests /12/. Patients who are positive for antiphospholipid antibodies may present with no related symptoms. Such patients are usually identified during an evaluation for systemic autoimmune disease, early miscarriages, an elevated aPTT, or a false positive result of a syphilis test /1/.

The diagnosis of antiphospholipid syndrome should be considered in patients with persistent, moderate-to-high-risk antiphospholipid antibody profiles and in patients with any antiphospholipid antibody-related finding /1/.

16.22.5.1 Thrombotic manifestations of antiphospholipid positivity

Antiphospholipid syndrome (APS) is a systemic autoimmune disease defined by thrombotic or obstetrical events that occur in patients with persistent antiphospholipid antibodies (APA) /1/. APA are the most common acquired inhibitors of the coagulation system.

APS can have the following clinical manifestations /12/:

• Venous thromboembolism (VTE). Among patients with unprovoked VTE, those with a lupus anticoagulant had a 40% increase in the risk of recurrence, as compared with patients who did not have lupus anticoagulant /2/. For patients with clinically significant, unprovoked thrombotic events, such as large pulmonary embolism or extensive lower-extremity deep vein thrombosis, and persistently high levels of APA, continued anticoagulant therapy is advised /2/.
• Arterial and microvascular thrombosis. Stroke and transient ischemic attack are the most common arterial events in patients with APS. Patients with catastrophic APS present with thrombosis involving multiple organs /13/.
• Obstetrical manifestations are fetal loss after the 10th week of gestation, recurrent early miscarriages, intrauterine growth restriction, or severe preeclampsia /1/. Approximately 1% of pregnant women experience spontaneous abortions and are positive for APS in 10–15% of the cases /14/. In pregnant women who are positive for LA, the annual rate of VTE is 1.46% and that of ischemic stroke is 0.32% /15/. A positive lupus anticoagulant test indicates a higher risk of thrombosis and a negative outcome of pregnancy after gestational week 12 than positive anti-β₂-GPI or aCL.

The following clinical findings may be a clue that a patient has the APS: livedo, signs or symptoms of another systemic autoimmune disease, unexplained prolongation of aPTT or mild thrombocytopenia. A thrombocyte count below 20 × 10⁹/L should the clinician consider other causes than APS /1/.

The clinical criteria of APS according to the Sydney conference are listed in Tab. 16.22-2 – Revised classification criteria for the APS and diseases associated with APS in Tab. 16.22-3 – Diseases associated with the antiphospholipid syndrome.

16.22.5.2 Non thrombotic manifestations of antiphospholipid positivity

Besides thrombotic the APS has a broad spectrum of non thrombotic clinical manifestations /1, 18/. Among patients with systemic lupus eryrhematosus, valve disease, pulmonary hypertension, livedo reticularis, thrombocytopenia, hemolytic anemia, acute or chronic renal vascular lesions, and moderate or severe cognitive impairment is higher than among patients with APA than among patients who are negative for such antibodies.

The prevalence of lupus anticoagulant positivity among patients with SLE is 30%, and the presence of lupus anticoagulant positivity in such patients is associated with an increased risk of thrombosis (odds ratio 5.6). Forty percent of patients with the APS also have SLE and 37% of patients with SLE have anti-β₂-glycoprotein I antibodies. These findings suggest that there is overlap in the pathogenesis of SLE and that of the APS /14/.

16.22.5.3 Diagnosis of antiphospholipid syndrome

The diagnosis of APS is based on clinical manifestation in combination with typical laboratory findings. APS is present if at least one of the clinical criteria and one of the laboratory APA test criteria are met.

Considering the probability of positive, non pathognomonic findings, it seems advisable to perform APS testing only on patients classified by the grading of evidence as follows /3/:

• Low; venous or arterial thrombosis, older patients
• Moderate; coincidental evidence of prolonged aPTT in asymptomatic individuals, recurrent spontaneous abortions, predictable thrombosis in young patients
• High; unexplained acute deep vein thrombosis or arterial thrombosis in patients below 50 years of age, thrombosis at unusual sites, miscarriage in late pregnancy, diseases associated with thrombosis or pregnancy in patients with autoimmune disease (systemic lupus erythematosus, rheumatoid arthritis, autoimmune thrombocytopenia, autoimmune hemolytic anemia) /3/.

APA can be only transiently positive, for example in the presence of inflammation and infection. Therefore, a positive finding should be confirmed by testing after an interval ≥ 12 weeks. A negative result of the second investigation indicates that the APA presence was transient.

Laboratory test criteria for APS are shown in Tab. 16.22-2 – Revised classification criteria for the APS. Evidence of APS-induced thrombosis is provided by positive findings for lupus anticoagulant and/or anti-β₂ GPI or aCL. The annual risk of a first thrombotic event in asymptomatic individuals who are positive for lupus anticoagulant, anti-β₂ GPI and aCL, referred to as triple positive patients, is 5.3%.

Patients who already experienced the clinical event 5 years ago should not undergo laboratory testing for APS.

16.22.5.3.1 Laboratory diagnostic significance of lupus anticoagulant (LA)

LA are detected based on their functional activity through interference with phospholipid-dependent steps in the coagulation cascade. High-activity LA assays are highly specific and show strong association with thromboembolic events and obstetric complications /5/. Since a single assay will not cover all APAs possibly present in APS, different methods must be used.

LA activity is reported with quantitative test results to enable numeric differentiation between low and high inhibitory activity. It is recommended to perform laboratory procedures on patients undergoing coumarin therapy 1–2 weeks after dis continuation of treatment or when the INR is below 1.5. Bridging coumarin dis continuation with low molecular weight heparin (LMWH) is recommended with the last dose of LMWH administered more than 12 h before the blood is drawn for LA testing. Alternatively, if the INR is between 1.5 and below 3.0, a 1 : 1 dilution of patient plasma and PNP can be considered.

Isolated LA positivity is significantly more frequent in individuals without clinical APS events or may be false-positive especially if identified as mild in potency, if it is found in elderly patients or if it is diagnosed for the first time /4/.

16.22.5.3.2 Laboratory diagnostic significance of anti-β₂GPI

Anti-β₂GPI antibodies are usually detected together with other APAs and are strictly associated with pregnancy complications. Patients with high concentrations of these antibodies are at high risk of thrombosis. Anti-β₂GPI antibodies are primarily detected in patients with autoimmune disease /19/. Patients tested positive in all three assays (LA, aCL and anti-β₂GPI) usually have a higher concentration of anti-β₂GPI than those with positive results in two assays.

16.22.5.3.3 Laboratory diagnostic significance aCL

Commercial assays for the determination of aCL antibodies have a significant discrepancy regarding their diagnostic sensitivity and specificity. Moreover, the diagnostic sensitivity of aCL assays is high, while specificity is low. Therefore, the evaluation should only take into account results above the 99th percentile of healthy controls or concentrations higher than 40 units/mL of IgG or IgM-aCL antibodies /3/. aCL antibody assays should only be interpreted in the context of clinical findings and considering the LA assay results. According to longitudinal studies, the positivity of aCL ELISA is not associated with the incidence of first event of deep vein thrombosis /18/.

A profile of anti-β₂GPI and aCL should always be determined and assessed in the context of LA testing. The presence of medium to high titers of these antibodies of the same isotype (most often IgG) if in agreement with a positive LA identifies patients at high risk for thrombosis /4/.

16.22.6 Comments and problems

Lupus anticoagulant (LA) testing

None of the individual assays for determining LA has 100% diagnostic sensitivity and specificity due to the difference in commercially available reagents. For instance, the presence of heparin and coagulation factor inhibitors and deficiency in coagulation factors lead to false-positive results. False-negative results are obtained if the plasma used is not platelet-poor.

Low LA concentrations may not be detectable in the plasma mixing test.

According to inter laboratory surveys, the rate of false-positive and false-negative LA results is approximately 20% and can even be 25% at low LA concentrations /21/.

The plasma to be analyzed should contain less than 10 × 10⁹ platelets per liter.

Prior to LA testing, a thrombin time or anti F Xa assay must be performed to determine the presence of heparin in the sample even if the reagent comprises a heparin neutralizer.

The LA assay must be performed within 4 h after blood collection or the sample must be quickly frozen at –70 °C. Frozen plasma must be thawed at 37 °C /4/.

anti-β₂-GPI testing

The analytic specificity of ELISA can be lower than possible, presumably because when β₂-GPI antibodies are transferred to ELISA tubes, neo antigens are formed which preferably bind non-pathogenic antibodies. Hence, fewer pathogenic antibodies are bound, resulting in decreased specificity of the assay. Inter laboratory variation between the assays for anti-β₂-GPI is not as high as for aCL, and the specificity for APS diagnosis is higher /3/.

aCL testing

IgM aCL tends to false-positive results, particularly in the low-positive range, especially in the presence of rheumatoid factor or cryoglobulins /3/.

16.22.7 Pathophysiology

The antiphospholipid syndrome is an autoimmune disorder characterized by persistently elevated concentrations of antiphospholipid antibodies. The antibodies represent a heterogenous group of autoantibodies that recognize various phospholipids, phospholipid-binding plasma proteins, and/or phospholipid-protein complexes /3/. The major target of antiphospholipid antibodies is directed against the plasma protein β₂-glycoprotein-I.

The risk of thrombosis correlates more strongly due to anti-β₂-glycoprotein autoantibodies than due to lupus anticoagulant. The thrombogenic properties are eliminated when the fraction of anti-β₂-glycoprotein-I antibodies is removed as shown in mice studies /22/. In patients with the risk of thrombosis autoantibodies bind the major B-cell epitope on domain I of the β₂-glycoprotein-I molecule. The autoantibodies confer lupus-anticoagulant activity.

The β₂-glycoprotein-I molecule can potentially exist in two forms /14/:

• In the circular form domain I interacts with domain V. In this form the B-cell epitope is hidden from the immune system
• A fish hook confirmation, exposing the domain I epitope and allowing domain I anti-β₂-glycoprotein-I antibodies to bind.

The fish hook confirmation with affinity to phospholipid containing surfaces forms an immunocomplex on cellular surfaces, especially when bound to an β₂-glycoprotein-I-antibody. The surface bound complex up regulates the expression of prothrombotic cellular adhesion proteins such as tissue factor and E-selectin. The binding of the immunocomplex suppresses the activity of the tissue factor inhibitor and reduces the activity of protein C in addition.

References

1. Garcia D, Erkan D. Diagnosis and management of the antiphospholipid syndrome. N Engl J Med 2018, 378: 2010–21.

2. Attachaipanich T, Aungsusiripong A, Piriyakhuntorn P, Hantrakool S, Rattarittamrong E, Rattanathatammethee T, et al. Antithrombotic therapy in antiphospholipid syndrome with arterial thrombosis: A systematic review and network meta-analysis. Front Med 2023. doi: 10 3389/fmed.2023.1196800.

3. Miyakis S, Lockshin MD, Atsumi T, Branch DW, Brey RL, Cervera R, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome. J Thrombos Haemost 2006; 4: 295–306.

4. Pengo V, Tripodi A, Reber G, Randt JH, Ortel TL, Galli M, et al. Update of the guidelines for lupus anticoagulant detection. J Thrombos Haemost 2009; 7: 1737–40.

5. Devreese K, Hoylaerts MF. Challenges in the diagnosis of the antiphospholipid syndrome. Clin Chem 2010; 56: 930–40.

6. Brandt JT, Barna LK, Triplett DA. Laboratory identification of lupus anticoagulants: Results of the second international workshop for identification of lupus anticoagulants. On behalf of the subcommittee on lupus anticoagulants/antiphospholipid antibodies of the ISTH. Thromb Haemost 1995; 74: 1597–1603.

7. Urbanus RT, Derksen RH, de Groot PG. Current insight into diagnostics and pathophysiology of the antiphospholipid syndrome. Blood Rev 2008; 2: 93–105.

8. Quehensberger P, Wagner O. Labordiagnostik von Lupushemmstoffen. Hämostaseologie 2005; 25: 50–4.

9. Forasteiro R, Martinuzzo M, Pombo G, Puente D, Rossi A, Celebrin L, et al. A prospective study of antibodies to beta2-glycoprotein I and prothrombin, and risk of thrombosis. J Thromb Haemost 2005; 3: 1231–8.

10. Ticani A, Allegri F, Balestrieri G, Reber G, Sanmarco M, Meroni P, et al. Minimal requirements for antiphospholipid antibodies ELISAs proposed by the European Forum on antiphospholipid antibodies. Thrombosis Res 2004; 114: 553–8.

11. Pierangeli SS, Harris EN. A protocol for determination of anticardiolipin antibodies by ELISA. Nature Protocols 2008; 3: 840–8.

12. Vila P, Hernandez MC, Lopez- Fernandez MF, Battle j. Prevalence, follow-up and clinical significance of the anticardiolipin antibodies in normal subjects. Thromb Haemost 1994; 72: 209–13.

13. Asherson RA, Cervera R, de Groot PG, Erkan D, Boffa MC, Piette JC, et al. Catastrophic antiphospholipid syndrome: international consensus statement on classification criteria and treatment guidelines. Lupus 2003; 12: 530–4.

14. Giannakopoulos B, Krikis SA. The pathogenesis of antiphospholipid syndrome. N Engl J Med 2013; 368: 1033–44.

15. Gris JC, Bouvier S, Molinari N, et al. Comparative incidence of a first thrombotic event in purely obstetric antiphospholipid syndrome with pregnancy loss: the NOH-APS observational study. Blood 2012; 119: 2624–32.

16. Ruffatti A, Tonello M, Del Ross T, Cavazzana A, Grava C, Noventa F, et al. Antibody profile and clinical course in primary antiphospholipid syndrome with pregnancy morbidity. Thromb Haemost 2006; 96: 337–41.

17. Levine SR, Brey RL, Tilley BE, et al. Antiphospholipid antibodies and subsequent thrombo-occlusive events in patients with ischemic stroke. JAMA 2004; 291: 576–84.

18. Ünlü O, Zuily S, Erkan D. The clinical significance of antiphospholipid antibodies in systemic lupus erythematosus. Eur J Rheumatol 2016; 3: 75–84.

19. Matsuura E, Igarashi Y, Fujimoto M, Ichikawa K, Koike T. Anticardiolipin cofactors and differential diagnosis of autoimmune disease. Lancet 1990; 336: 177–8.

20. Giannakopoulos B, Passam F, Ioannou Y, Krilis S. How we diagnose the antiphospholipid syndrome. Blood 2009; 113: 985–94.

21. Dembitzer FR, Ledford Kraemer MR, Meijer P, Peerschke EIB. Lupus anticoagulant testing. Performance and practices by North American clinical laboratories. Am J Clin Pathol 2010; 134: 764–73.

22. Arad A, Proulle V, Furie RA, Furie BC, Furie B. β₂-glycoprotein-1 autoantibodies from patients with antiphospholipid syndrome are sufficient to potentiate arterial thrombus formation in a mouse model. Blood 2011; 117: 3453–9.

16.23 Plasminogen

Michael Kraus, Lothar Thomas

Plasminogen is the inactive precursor of the fibrinolytic enzyme plasmin. Plasminogen is activated by activator substances such as tissue activator (t-PA) and urokinase (u-PA) or by the contact phase of coagulation (F XIIa-high-molecular weight kininogen complex). Therapeutically, exogenous activators such as streptokinase are employed as well. When a fibrin clot forms approximately 20% of the plasminogen content of plasma is trapped in the clot. Activators diffuse into the clot and activate plasminogen to plasmin. The lysis of fibrin clots by plasmin results in the formation of fibrinogen-fibrin degradation products. This keeps blood vessels and secretory ducts free.

16.23.1 Indication

• Suspected hyper fibrinolysis (increased consumption)
• Thrombolytic therapy monitoring
• Prevention of blood loss after Cesarean delivery
• Suspected plasminogen deficiency
• Thrombophilia of unknown origin
• Suspected dysplasminogenemia.

16.23.2 Method of determination

Activity measurement using a chromogenic substrate method

Principle: by the action of streptokinase, plasminogen present in the plasma sample is completely transformed into plasminogen activator (streptokinase-plasmin complex). This complex subsequently hydrolyzes a chromogenic substrate. The increase in absorption, measured spectrophotometrically, is proportional to the plasminogen activity /1/.

Immunochemical methods

Immunonephelometry, radial immunodiffusion.

16.23.3 Specimen

Citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of venous blood): 1 mL

16.23.4 Reference interval

Activity	75–150% of normal 2.4–3.8 CTA U/mL*
Concentration	0.06–0.25 g/L

* Related to a plasminogen preparation of the U.S.A. National Institute of Health

16.23.5 Clinical significance

Fibrinolysis is an efficient protective mechanism to limit the hemostatic process.

16.23.5.1 Plasminogen deficiency

Plasminogen deficiency causes a decrease in the ability to react to excess coagulation activity (i.e., the risk of thrombosis is increased). This situation is encountered, for example, in the form of an increased risk of re occlusion after thrombolytic therapy or after surgery. Causes of plasminogen deficiency may include inherited defects, impaired synthesis in the liver, increased consumption (e.g., as seen in DIC), sepsis or thrombolytic therapy (Tab. 16.23-1 – Diseases and conditions associated with altered plasminogen concentration).

Tranexamic acid has antifibrinolytic effects that are achieved at least in part by promotion of hemostasis through the blocking of lysine-binding sites on plasminogen molecules. Tranexamic acid reduces the need for transfusions among women who underwent cesarean delivery and received prophylactic uterotonic agents. Outside of obstetrics tranexamic acid is used to reduce mortality among patients with extracranial or mild-to-moderate intracranial trauma /8/.

16.23.5.2 Increased plasminogen concentration

Severe sepsis, malignant tumors, tissue hypoxia and diabetes mellitus may cause increased plasminogen activity, leading to an increased risk of bleeding, particularly in sepsis /2/.

Plasminogen activity is a less sensitive indicator of (past) hyper fibrinolysis than the inhibitor α₂-antiplasmin since its concentration is subject to more fluctuation. Both parameters, on the basis of consumption, only allow indirect conclusions to be drawn with regard to the actual fibrinolytic activity. For the latter purpose, the determination of the plasmin-α₂-antiplasmin complex (PAP) is better suited.

16.23.6 Comments and problems

The chromogenic assay is preferable to the immunochemical methods because it is easier and faster to perform. The activity and antigen determinations usually correlate very well except in the case of the rare type II deficiency.

16.23.7 Pathophysiology

The body is protected against bleeding and thrombosis by the equilibrium between coagulation and fibrinolysis. Plasmin is the key enzyme in fibrinolysis and has a molecular weight of 90 kDa. It is produced by activation of the proenzyme plasminogen initiated by the tissue (tPA) or urinary (uPA; urokinase) plasminogen activator (Fig. 16.23-1 – Activation and function of the plasminogen-plasmin system).

The plasmin thus formed in turn increases the activity of t-PA and the affinity of urokinase for plasminogen by proteolytically cleaving the single-chain plasminogen activators into two-chain structures. Both enzymes then synergistically participate in the activation of plasminogen. t-PA and urokinase are regulated by their inhibitors plasminogen activator inhibitor (PAI) 1 and PAI 2.

The essential role of plasminogen, in its active form plasmin, is to maintain the hemostatic equilibrium by proteolytically dissolving fibrin clots. The fibrin specificity of the plasminogen activators not only supports this process but also localizes it. Under abnormal conditions, a systemic enhancement of plasmin may occur causing the degradation of fibrinogen and thus an increased risk of bleeding /3/.

In the plasma, plasminogen occurs in two modifications which differ with regard to their level of glycosylation and their affinity for fibrin. The binding of plasminogen to fibrin is interfered by the presence of histidine-rich glycoprotein, whereas plasmin in the circulation is inactivated by its inhibitor α₂-antiplasmin. Older clots are protected from degradation by plasmin through the incorporation of α₂-antiplasmin mediated by F XIIIa. Lipoprotein(a) may competitively inhibit the binding of plasminogen to tPA due to structural similarities (i.e., so-called crinkle regions) (Fig. 16.23-2 – Activators and inhibitors in the plasminogen/plasmin pathway).

Plasmin is a relatively unspecific protease participating in numerous mechanisms at cell level, such as fertilization, cell migration and macrophage activation. These processes are thought to be rather mediated by uPA binding cell receptors. Plasmin especially participates in inflammatory degenerative processes by activating the complement and kallikrein/kinine pathways and metalloproteases playing a key role in tissue degradation, for example in rheumatic joint disease.

Plasmin not bound to fibrin or other extracellular matrices quickly binds to its specific inhibitor α₂-antiplasmin forming an irreversible, covalent complex, the plasmin-α₂-antiplasmin complex (PAP) which has a half-life of approximately 12 h.

Streptokinase, an activator extracted from streptococci, is employed therapeutically. In contrast to the physiological activators, streptokinase does not activate plasminogen itself but instead forms a 1 : 1 complex with plasminogen. As a result of this configurational alteration of plasminogen, an autocatalytic proteolytic activation into plasmin occurs. Active plasmin may be competitively displaced from its substrate (fibrin) by the administration of ε-aminocaproic acid (6-aminohexanoic acid), thus rendering plasmin more subject to inhibition by α₂-antiplasmin. Direct inhibition of plasmin can be achieved by the administration of aprotinin, a protein extracted from the lungs of cattle.

References

1. Witt I. Test systems with synthetic peptide substrates in haemostaseology. Eur J Clin Chem Clin Biochem 1991; 29: 355–74.

2. Pechlaner Ch. Plasminogen activators in inflammation and sepsis. Acta Medica Austria aca 2002; 29: 80–8.

3. Munkvad S. Fibrinolysis in patients with acute ischaemic heart disease. With particular reference to systemic effects of tissue-type plasminogen activator treatment on fibrinolysis, coagulation and complement pathways. Danish Med Bull 1993; 40: 383–408.

4. Vassalli JD, Sappino AP, Belin D. The plasminogen activator/plasmin system. J Clin Invest 1991; 88: 1067–72.

5. Sartori MT, Patrassi GM, Theodoridis P, Perin A, Pietrogrande F, Girolami A. Heterozygous type I plasminogen deficiency is associated with an increased risk for thrombosis: a statistical analysis in 20 kindreds. Blood Coag Fibrinol 1994; 5: 889–93.

6. Schölmerich J. Diagnostik und Therapie des Ascites. Internist 1987; 28: 448–58.

7. Wun TC. Plasminogen activation: biochemistry, physiology, and therapeutics. Crit Rev Biotechnol 1988; 6: 131–48.

8. Sentilhes MV, Le Lous S, Winer S, Rozenberg P, Kayem G, Verspyck E, Fuchs F, et al. Tranexamic acid for the prevention of blood loss after Cesarean delivery. N Engl J Med 2021; 384 (17): 1623–34.

16.24 α₂-antiplasmin

Michael Kraus, Lothar Thomas

α₂-antiplasmin is the physiologically most important inhibitor of the fibrinolytic enzyme plasmin. α₂-antiplasmin circulating freely or bound to fibrin clots very rapidly and irreversibly inhibits plasmin by converting it into an inactive complex (plasmin-α₂-antiplasmin complex; PAP). Plasmin bound to receptors or substrates such as fibrin, on the other hand, is protected from inhibition by α₂-antiplasmin.

16.24.1 Indication

• Suspected hyper fibrinolysis (e.g., in conjunction with disseminated intravascular coagulation or surgery involving organs with a high content of plasminogen activators)
• Thrombolytic therapy monitoring (increased risk of bleeding)
• Suspected synthesis defects (liver damage)
• Suspected congenital α₂-antiplasmin deficiency.

16.24.2 Method of determination

Activity measurement using a chromogenic substrate method

Principle: a defined excess of plasmin is added to the plasma sample to be tested. The α₂-antiplasmin contained in the sample neutralizes equivalent quantities by forming plasmin-α₂-antiplasmin complexes. The remaining quantity of plasmin is measured using a chromogenic substrate which is added to the test sample /1/.

Immunochemical methods

Laurell electrophoresis, immunonephelometry.

16.24.3 Specimen

Citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of venous blood): 1 mL

16.24.4 Reference interval

Activity	80–120% of normal
Concentration	0.06–0.10 g/L

16.24.5 Clinical significance

The plasma α₂-antiplasmin concentration is usually constant, approximately 1 μmol/L. Hence, a decrease in α₂-antiplasmin is a more sensitive biomarker of hyperfibrinolysis than a change in plasminogen activity /2/. A deficiency in α₂-antiplasmin causes less regulation of fibrinolytic pathway, resulting in hyperfibrinolysis which may therefore spread systemically and lead to bleeding complications. Causes of α₂-antiplasmin deficiency may include: inherited defects, impaired synthesis in the liver, increased consumption (e.g., as seen in DIC), surgery involving organs with an increased plasminogen activator potential (lung, prostate, uterus) or as a result of thrombolytic therapy (Tab. 16.24-1 – Diseases and conditions associated with reduced α₂-antiplasmin). In addition, the α₂-antiplasmin concentration may fluctuate due to hormonal influences.

In poor wound healing or at normal PT or aPTT clotting times, but abnormal bleeding times, α₂-antiplasmin deficiency should be suspected /3/.

16.24.6 Pathophysiology

α₂-antiplasmin with a molecular weight of 68 kDa is present in plasma in two forms, one bound to plasmin with high affinity and the other one with low affinity /2/. The first form accounts for 70% and the latter for 30% of the total. The half-life of α₂-antiplasmin is approximately 2.5 days /2/.

α₂-antiplasmin regulates the activity of the key enzyme of fibrinolysis by:

• Rapidly forming an irreversible complex with plasmin in the circulation (plasmin-α₂-antiplasmin complex), thus preventing systemic enhancement of active protease
• Competing with fibrin for plasminogen binding, thus decreasing the amount of plasminogen that may be activated on clots
• Forming F XIIIa mediated covalent links with older fibrin clots, thus preventing the clots from degradation by plasmin and enhancing wound healing.

References

1. Wiman B. Human α₂-antiplasmin. Methods in Enzymology 1981; 80: 395–408.

2. Saito H. α₂-plasmin inhibitor and its deficiency states. J Lab Med 1988; 112: 671–8.

3. Griffin GC, Mammen EF, Sokol RJ, Perrotta AL, Stoyanovich A, Abildgaard CF. Alpha 2-antiplasmin deficiency. An overlooked cause of hemorrhage. Am J Ped Hematol Oncol 1993; 15: 328–30.

4. Sane DC, Pizzo SV, Greenberg CS. Elevated urokinase-type plasminogen activator level and bleeding in amyloidosis: case report and literature review. Am J Hematol 1989; 31: 53–7.

5. Hayashi S, Yamada K. Role of α₂-plasmin inhibitor in the appearance of fibrinolytic activity during urokinase administration and an evaluation of the optimal urokinase dosage. Thromb Res 1979; 16: 393–400.

16.25 Plasminogen activators (PA), plasminogen activator inhibitors (PAI)

Carola Wagner, Lothar Thomas

The fibrinolytic pathway is activated through the conversion of the proenzyme plasminogen into the active fibrinolytic enzyme plasmin. Plasmin degrades fibrin into soluble fibrin degradation products. The fibrinolytic pathway is inhibited at the PA level by specific plasmin activator inhibitors (PAIs), especially PAI-1 or later by α₂-antiplasmin (Fig. 16.25-1 – Components of the fibrinolytic system).

The essential function of the fibrinolytic system is to maintain the circulatory form of the blood. Increased activity of the fibrinolytic pathway may lead to an increased risk of bleeding and decreased activity may cause thrombosis /1/.

16.25.1 Indication

• Suspected defects in the fibrinolytic system in the case of thromboembolic disease (e.g., acute myocardial infarction, pulmonary embolism, venous thrombosis or stroke)
• Risk indicator for thromboembolic complications in suspected hereditary or acquired defects (e.g., in patients with hyperinsulinism, diabetes mellitus, cardiovascular disease)
• Inefficacy of thrombolytic therapy in acute myocardial infarction or peripheral arterial occlusion, high-risk pregnancies, neoplasia, sepsis and surgery with a high thrombotic risk.

16.25.2 Method of determination

Determination of the PA and t-PA activity

Active, free PA in the sample, in plasma mainly t-PA, are measured by the conversion of plasminogen into plasmin, followed by the measurement of the resultant plasmin activity through the cleavage of a chromogenic substrate. The t-PA-mediated plasminogen activation requires the addition of an accelerator (e.g., fibrin monomer or fibrin fragments obtained with cyanobromide). In order to avoid any interference by α₂-antiplasmin which would immediately bind to the plasmin by forming a complex with it, a reduction in the plasma pH is required /2/.

t-PA antigen measurement

Although the determination by enzyme immunoassay is specific for t-PA, it detects, however, both the active, free form as well as the inactive form of t-PA which is complex-bound to PAI-1 /3/.

Measurements of the PAI and PAI-1 activity

The plasma PAI-1 activity is measured indirectly by the inhibition of added PA and the chromogenic determination of the residual PA activity via the activation of plasminogen. Both t-PA and u-PA can be used as the target enzyme. Due to the short incubation period of 5–15 min., only the most rapidly active PAI (i.e., PAI-1) is determined. The influence of the α₂-antiplasmin is eliminated by acidification of the reaction mixture or by oxidative inactivation /4/.

PAI-1 antigen measurement

Various enzyme immunoassays are available for the immunochemical measurement of the PAI-1 antigen. Depending on the monoclonal antibodies used, the different assays detect the four possible forms of PAI-1 (active, latent, bound to t-PA and bound to u-PA) to a varying extent /5/.

16.25.3 Specimen

Venous, platelet-poor citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood): 1 mL

t-PA activity: to avoid an in vitro reaction of t-PA with its inhibitors, acid citrate solution is necessary for the collection of plasma (final pH ~ 6.0).

16.25.4 Reference interval

Due to the absence of standardization, the reference interval depends on the test system employed. Therefore, every laboratory should establish its own reference interval under local conditions or take into account the reference interval specified in the specific test kit.

16.25.5 Clinical significance

The measurement of t-PA and PAI-1, the physiologically most important regulators of the fibrinolytic system in the circulation, serves to estimate the fibrinolytic potential of the blood.

Frequent abnormalities of this system result in a thrombophilic shift of the hemostatic equilibrium due to an increased PAI-1 release or a deficient release of t-PA. Although sporadic cases with an increased risk of bleeding as a result of PAI-1 deficiency or an excessive t-PA release have been described, they are, however, clinically irrelevant for all practical purposes because of their extremely low prevalence /6/.

16.25.5.1 Hereditary defects

Plasminogen activator

Congenital defects of plasminogen activators have not been identified to date /1/.

Plasminogen activator inhibitors

Ten PAI-1 gene polymorphisms have been described to date, but only a few of them seem to influence the inhibitor’s level in plasma. A functional role in PAI-1 synthesis and expression has been attributed to a single guanosine deletion/insertion polymorphism (4G or 5G) located at the promoter region of the PAI-1 gene at 675 bp upstream from the transcription starting site. Both polymorphic alleles may bind a transcription activator, whereas the 5G allele also binds a repressor protein to an overlapping binding site, which consequently interferes with activator binding by steric hindrance. The relationship between the 4G/5G polymorphism and PAI-1 levels, which are higher in 4G allele carriers exists in patients with deep vein thrombosis, cardiovascular and metabolic diseases, while a weaker association has been described in healthy populations. PAI-1 is assumed to play a role in thrombotic vascular disease /7/.

The PAI concentration in the 4G/4G genotype is 25% higher than in the 5G/5G genotype and associated with deep vein thrombosis. It is assumed that the regulation of the 4G allele by the plasma triglyceride concentration influences the involvement of PAI-1 in cardiovascular disease /8/.

Homozygosity for PAI-1 4G or F XIII 34 Leu polymorphisms as well as compound carrier status is associated with early pregnancy loss /9/.

16.25.5.2 Acquired defects

Refer to tables:

• Tab. 16.25-1 – Diseases and conditions associated with abnormal PA and PAI concentrations
• Tab. 16.25-2 – Influence factors on t-PA and PAI-1.

16.25.6 Comments and problems

Blood collection

Due to the pronounced circadian rhythms of PAI-1 release with the highest levels measured in the morning and the lowest in the afternoon, blood for the determination of both PAI-1 and t-PA should be drawn between 8:00 and 10:00 a.m. Prior to blood sampling, the patients should rest for at least 20 min. since increased physical activity can lead to an increased release of t-PA.

In order to prevent the release of latent PAI-1 from platelets during storage, immediate cooling of the sample to 4 °C is necessary after blood collection as well as the preparation of platelet-poor plasma (by centrifugation at 2000 × g for 15 min.).

Reporting results

The PAI-1 activity can be expressed either as a t-PA- or u-PA-inhibiting unit (1 u-PA-inhibiting unit ~ 7.8 t-PA-inhibiting units). The test system used should always be specified because of the common significant variations in the specificity and sensitivity of the immunochemical tests which are employed for the determination of the different forms of t-PA and PAI-1 in the circulation.

16.25.7 Pathophysiology

The main component of the fibrinolytic system is plasminogen, an inactive proenzyme which, following its conversion into the active enzyme, is responsible for the degradation of fibrin as well as of various other extracellular matrix proteins.

Two physiological PA are known: t-PA, which is found primarily in the circulation where it induces the degradation of fibrin clots, and u-PA, which, through a cellular tissue receptor, leads to the degradation of matrix proteins by means of pericellular, plasmin-mediated proteolysis as well as to the activation of latent proteases and growth factors.

The fibrinolytic activity is regulated:

• At the level of plasmin by the irreversible formation of a complex with α₂-antiplasmin
• Via the activation of plasmin as a result of the formation of complexes consisting of t-PA and u-PA, on the one hand, and PAI, on the other hand; PAI-1 plays the most important role in this context /7/ (Fig. 16.25-2 – Activators and inhibitors of the fibrinolytic pathway).

t-PA is continuously released from the vascular endothelium; as a result of stimulation due to (e.g., venous occlusion, strenuous physical activity or vasoactive drugs such as catecholamines or bradykinin). A rapid rise in the release of t-PA occurs within a few minutes. The half-life of t-PA in the circulation is only 5 min. due to its rapid hepatic clearance and its efficient inactivation by the formation of a complex with PAI-1.

In contrast to other hemostatic enzymes, t-PA is not released as an inactive proenzyme but instead directly as an active protease. The affinity of t-PA to plasminogen freely circulating in the plasma is too low, however, for its activation and is not sufficient unless fibrin is present /1, 6/.

PAI-1, a serine protease inhibitor with a molecular weight of 52 kDa, is released from vascular endothelial cells into the circulation in its active form and, due to a conformational change, transforms into an active form with a half-life of 30 min. PAI-1 which is contained in the α-granules of platelets and which represents about 90% of PAI-1 circulating in the blood, on the contrary, is present almost entirely in the inactive form. The binding of PAI-1 to protein S or vitronectin stabilizes the active phase of PAI-1 in plasma; however, this does not make it resistant to enzymatic degradation by thrombin or activated protein C.

References

1. Kwaan HC, Nabhan C. Hereditary and acquired defects in the fibrinolytic system associated with thrombosis. Hematol Oncol North Am 2003; 17: 103–14.

2. Verheijen JH. Tissue-type plasminogen activator activity assay. In: Jespersen J, Bertina RM, Haverkate F (eds). ECAT assay procedures. A manual of laboratory techniques. Lancaster UK: Kluwer Academic Publishers, 1992: 139–46.

3. Juhan-Vague I, Alessi MC. Tissue type plasminogen activator antigen (t-PA Ag). In: Jespersen J, Bertina RM, Haverkate F (eds). ECAT assay procedures. A manual of laboratory techniques. Lancaster UK: Kluwer Academic Publishers, 1992: 131–7.

4. Kruithof EKO. Plasminogen activator inhibitor activity assay. In: Jespersen J, Bertina RM, Haverkate F (eds). ECAT assay procedures. A manual of laboratory techniques. Lancaster UK: Kluwer Academic Publishers, 1992: 147–50.

5. Lee MH, Vosburgh E, Anderson K, McDonagh J. Deficiency of plasma plasminogen activator inhibitor 1 results in hyperfibrinolytic bleeding. Blood 1993; 81: 2357–62.

6. Lijnen HR. Pleiotropic functions of plasminogen activator inhibitor-1. J Thrombos Haemostas 2003; 3: 35–45.

7. Sartori MT, Danesin C, Saggiorato G, Tormene D, Simioni P, Spiezia L, et al. The PAI-1 gene 4G/5G polymorphism and deep vein thrombosis in patients with inherited thrombophilia. Clin Appl Thromb Hemost 2003; 9: 299–307.

8. Kohler HP, Grant PJ. Plasminogen activator type 1 and coronary artery disease. N Engl J Med 2000; 342: 1792–1801.

9. Dossenbach Glaninger A, van Trostenburg M, Dossenbach M, Oberkanis C, Moritz A, Krugluker W, Huber J, Hopmeier P. Plasminogen activator inhibitor I 4G/5G polymorphism and coagulation factor XIII (4G/5G) Val 34/Leu polymorphism: impaired fibrinolysis and early pregnancy loss. Clin Chem 2003; 49: 1081–6.

10. Sobel BE, Woodcock-Mitchell J, Schneider DJ, et al. Increased plasminogen activator inhibitor type 1 in coronary artery atherectomy specimens from type 2 diabetics compared with non diabetic patients: a potential factor predisposing to thrombosis and its persistance. Circulation 1998; 97: 2213–21.

11. Festa A, d’Augustino R, Mykkanen L, et al. Relative contribution of insulin and its precursors to fibrinogen and PAI-1 in a large population with different states of glucose tolerance: The Insulin Resistance Atherosclerotic Study (IRAS). Arterioscer Thromb Vasc Biol 1999; 19: 562–8.

12. Müller JE, Stone PH, Turi ZG, et al. Circadian variation in the frequency of onset of acute myocardial infarction. N Engl J Med 1985; 313: 1315–22.

13. Huber K, Christ G, Wojta J, Gulba D. Plasminogen activator inhibitor type-1 in cardiovascular disease. Status report 2001. Thromb Res 2001; 103: S7–S19.

16.26 Fibrin monomer

Michael Kraus, Lothar Thomas

Elevated fibrin monomer concentration is a sign of increased thrombin activity (hyper coagulability) and, thus, indicates an acute danger of clot formation (pre thrombotic state).

16.26.1 Indication

Detection of consumptive coagulopathy associated with:

• Disseminated intravascular coagulation
• SIRS, sepsis, severe infectious disease, complications of pregnancy
• States of shock (e.g., traumatic, cardiogenic or septic shock)
• Tissue necrosis (e.g., due to poly trauma, burns, acute pancreatitis, malignant tumor, immune hemolysis)
• Transplant rejection.

Recognition of a pre thrombotic state (i.e., the acute risk of developing deep vein thrombosis or pulmonary embolism):

• Postoperatively (with or without heparin prophylaxis)
• In patients with inherited inhibitor defects such as factor V Leiden and antithrombin deficiency
• Monitoring of the thrombotic risk in patients under anticoagulant therapy (e.g., following stent implantation).

16.26.2 Method of determination

Agglutination test

Principle: agglutination tests precipitate the soluble fibrin monomer by the addition of reagents, immobilized fibrin monomers or hydrophobic surfaces. The agglutination reaction can be measured macroscopically or turbidimetrically and depends on the concentration of the fibrin monomer present in the sample. The oldest method is based on the precipitation of soluble fibrin by ethanol /1/. In the hemagglutination assay, erythrocytes loaded with fibrin monomer are used as the agglutination partner /2/.

Furthermore, fibrin monomer can be precipitated using uncoated latex particles in the presence of a dye (blue dextran) /3/. Direct precipitation is feasible using protamine sulfate /4/, ristocetin /5/ or netropsin /6/.

Functional test

Principle: the functional tests are based on the characteristic ability of soluble fibrin monomer complexes to stimulate the tissue plasminogen activator (t-PA). t-PA activates plasminogen by converting it into plasmin which in turn leads to the transformation of a chromogenic substrate which is photometrically detected /7/.

Immunochemical assay

Principle: immunochemical assays involve the use of monoclonal antibodies which recognize specific fibrin epitopes. The epitopes are not created until fibrinogen is converted into fibrin (fibrinopeptide cleavage from fibrinogen; des-A-neoepitope) or become accessible only after configurational alterations (t-PA binding sites) /9/. In the event of des-A-neoepitope recognition, the samples must be pretreated using a chaotropic reagent (NaSCN) in order to cleave the fibrin complexes which attach via this epitope /10/.

16.26.3 Specimen

Platelet-poor citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of venous blood): 1 mL

16.26.4 Reference interval

Hemagglutination assay	< 15 mg/L /1/
Functional assay	< 15 mg/L /8, 11/
ELISA	< 2 mg/L /9/

The specifications of the test kit should be taken into account for each ay used.

16.26.5 Clinical significance

An elevated concentration of fibrin monomer (FM) suggests the presence of increased thrombin activity and, thus, is a direct indicator of ongoing increased intravascular coagulation activity (hyper coagulability) /12/. If this process was triggered locally and there is inadequate fibrinolytic activity, this may result in the formation of a localized thrombosis; if this process, on the other hand, is generalized, i.e., disseminated intravascular coagulation is present, consumptive coagulopathy may ensue /12/.

Trauma, sepsis and shock are the major causes of hyper coagulability. In a study /13/ involving intensive care patients, the extent of multiple organ failure and the mortality rate were directly correlated with the FM concentration. The course of pregnancy is also characterized by the increasing development of a hyper coagulable state.

Soluble fibrin is in a labile state. At a certain ratio of fibrin to fibrinogen (1–2%), the fibrin molecules begin to spontaneously aggregate. Therefore, the detection of FM is an early indicator of an acute thrombotic risk (e.g., of deep vein thrombosis after surgery). The diagnostic sensitivity of FM for deep vein thrombosis of the lower extremities is 91% with a negative predictive value of 94% /14/.

Preoperative FM values < 3 mg/L (cutoff value ≤ 2 mg/L), measured by ELISA, exclude intra operative blood loss with a diagnostic sensitivity of 92% and a negative predictive value of 0.95 /15/.

16.26.6 Comments and problems

Sample

In some assays, the use of frozen samples is not recommended. The allowed time period between the collection and the processing of the blood sample is also limited in some assays. The instructions of each test kit should be taken into consideration.

Method of determination

In general, the methods do not correlate with each other even if similar methodologies are employed. The specifications in each test kit concerning the reference interval and interpretation must therefore be taken into account.

The agglutination assays are relatively insensitive to fibrinogen. Very high concentrations of fibrin or fibrin(ogen) degradation products may inhibit aggregation by intercalating in-between the fibrin molecules. Overall, these assays are relatively imprecise and, in the case of manual methods, depend heavily on the experience of the person performing the assay.

The functional as well as the immunochemical assays provide reproducible and quantitative results. To a certain extent, the results usually correlate with fibrinogen. This is probably due to the fact that more fibrinogen can keep more fibrin soluble and that fibrinogen is elevated because of an acute phase response which is also associated with hyper coagulability (i.e., increased fibrin formation).

The des-A-neoepitope ELISA, according to Ref. /9/, is the only method strongly correlated with the D-dimer methods.

16.26.7 Pathophysiology

The fibrinogen molecule is a dimer of three different pairs of nonidentical polypeptide chains which are labeled Aα, Bβ and γ. Thrombin initially cleaves fibrinopeptide A and then fibrinopeptide B from the Aα and Bβ chains, resulting in exposure of new N-termini and causing conformational changes and exposure of polymerization sites. This generates fibrin monomer a molecule with strong tendency to polymerize. Because of its tendency to polymerize, fibrin monomer is present physiologically in only very low concentration because it rapidly self-associates to form an insoluble fibrin deposit /16/.

Refer also to

• Fig. 16.16-1 – The structure of fibrinogen
• Fig. 16.26-1 – Formation of soluble fibrin-fibrinogen complexes.

The physiological half-life of fibrin monomer is approximately 10 h.

When small amounts of thrombin are formed, producing minimal fibrin polymer production, fibrin monomers may complex with intact fibrinogen molecules. If some intravascular fibrin is formed and fibrinolysis occurs, complexes may also be formed between the larger fibrin fragments, fibrin monomers and fibrinogen molecules. These soluble complexes are called soluble fibrin monomer complexes and are protective in preventing the further polymerization of fibrin monomers /17/.

References

1. Godal HC, Abildgaard U. Gelation of soluble fibrin in plasma by ethanol. Scand J Haematol 1966; 3: 343–50.

2. Largo R, Heller V, Straub PW. Detection of soluble intermediates of the fibrinogen-fibrin-conversion using erythrocytes coated with fibrin monomers. Blood 1976; 47: 991–1002.

3. Müller UP, Largo R, Straub PW. A sensitive latex agglutination method for the detection of soluble fibrin in plasma. Blut 1977; 35: 465–79.

4. Wieding JU, Eisinger G, Köstering H. Determination of soluble fibrin by turbidimetry of its protamine sulphate-induced paracoagulation. J Clin Chem Clin Biochem 1989; 27: 57–63.

5. Watanabe K, Tullis JL. Precipitation of fibrin monomers and fibrin degradation products by ristocetin. Am J Med Sci 1978; 275: 337–44.

6. Funke U, Töpfer G, Schulze M, et al. Ein neues Verfahren zum Nachweis von löslichen Fibrinmonomerkomplexen (Netropsinpräzipitationstest). Z Klin Med 1990; 45: 683–6.

7. Wiman B, Ranby M. Determination of soluble fibrin in plasma by a rapid and quantitative spectrophotometric assay. Thromb Haemost 1986; 55: 189–93.

8. Dickneite G, Czech J, Keuper H. Formation of fibrin monomers in experimental disseminated intravascular coagulation and its inhibition by recombinant hirudin. Circul Shock 1994; 42: 183–9.

9. Lill H, Spannagl M, Trauner A, Schramm W, Schuller D, Ofenloch-Haehnle B, Draeger B, Naser W, Dessauer A. A new immunoassay for soluble fibrin enables a more sensitive detection of the activation state of blood coagulation in vivo. Blood Coag Fibrinol 1993; 4: 97–102.

10. Nieuwenhuizen A, Hoegee-De Nobel E, Laterveer R. A rapid monoclonal antibody-based enzyme immunoassay (EIA) for the quantitative determination of soluble fibrin in plasma. Thromb Haemost 1992; 68: 273–7.

11. Dempfle CE, Pfitzner SA, Dollmann M, Huck K, Stehle G, Heene DL. Comparison of immunological and functional assays for measurement of soluble fibrin. Thromb Haemost 1995; 74: 673–9.

12. Nieuwenhuizen W. Soluble fibrin as a molecular marker for a pre-thrombotic state: a mini-review. Blood Coag Fibrinol 1993; 4: 93–6.

13. Bredbacka S, Blombäck M, Wiman B. Soluble fibrin: a predictor for the development and outcome of multiple organ failure. Am J Hematol 1994; 46: 289–94.

14. Ginsberg JS, Siragusa S, Douketis J, Johnston M, Moffat K, Stevens P, Brill-Edwards P, Panju A, Patel A. Evaluation of a soluble fibrin assay in patients with suspected deep vein thrombosis. Thromb Haemost 1995; 74: 833–6.

15. Korte W, Gabi K, Rohner M, Gähler A, Szadkowski C, Snider DW, et al. Preoperative fibrin monomer measurement allows risk stratification for high intraoperative blood loss in elective surgery. Thromb Haemost 2005; 94: 211–5.

16. Horan JT, Francis CW. Fibrin degradation products, fibrin monomer and soluble fibrin in disseminated intravascular coagulation. Semin Thrombos Hemostas 2001; 27: 657–66.

17. Nieuwenhuizen W. Soluble fibrin as a molecular marker for a pre-thrombotic state: a minireview. Blood Coag Fibrinol 1993; 4: 93–6.

16.27 D-Dimer

Lothar Thomas

D-dimer is a fibrin degradation product and elevated in clinical situations with hyper coagulability such as venous thromboembolism, disseminated intravascular coagulation and a further spectrum of diseases. D-dimer is an important diagnostic marker because of its high negative predictive value /1/.

16.27.1 Indication

• Exclusion of venous thromboembolism (VTE), particularly deep vein thrombosis and pulmonary embolism
• Prediction of recurrent VTE and risk stratification of patients for VTE recurrence
• Diagnosis and monitoring of coagulation activation in suspected disseminated intravascular coagulation
• Suspected preeclampsia.

16.27.2 Method of determination

Principle: the D-dimer assays measure an epitope on degradation products of factor XIIIa crosslinked fibrin by particle-enhanced immunonephelometric and immunoturbidimetric methods /2/. The assays use monoclonal antibodies directed against an epitope that is present in the factor XIIIa crosslinked fragment D domain of fibrin.

16.27.3 Specimen

Citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of venous blood): 1 mL

16.27.4 Reference interval

50–500 Fibrinogen Equivalent Units/Liter (FEU/L = ug/L)

16.27.5 Clinical significance

Venous thromboembolism (VTE) consists of deep vein thrombosis (DVT) and pulmonary embolism. The diagnostic management of DVT based on signs and symptoms is non-specific. The diagnosis of DVT can be safely excluded based on the combination of a low to or intermediate clinical probability combined with a negative D-dimer blood test, in which case imaging studies can be avoided /3/. The D-dimer concentration indicates the extent to which fibrin formation is increased. An age-adjusted D-dimer cutoff (patient’s age years × 10 μg/L) is recommended to exclude deep vein thrombosis in patients above 50 years of age /3/.

16.27.5.1 Negative D-dimer result

D-dimer tests are performed as markers of coagulation activation in patients with suspected VTE and to serve as a diagnostic tool in support of clinical findings. The fact that only a small portion of circulating fibrinogen needs to be converted to crosslinked fibrin to generate a detectable D-dimer signal confers the diagnostic sensitivity required /1/. Clinical signs and symptoms (Wells score /4/) suggesting that patients are clinically unlikely to have VTE and normal results of the D-dimer test indicate that clinical significant intravascular coagulation is not increased.

Determination of D-dimer is the only large-scale laboratory test to exclude VTE. The following procedure is recommended in outpatient care /5/:

• Wells score ≤ 1 and D-dimer antigen ≤ 500 μg/L: no DVT present
• Wells score ≤ 1, D-dimer > 500 μg/L and positive compression ultrasound sonography: deep vein thrombosis and therapy
• Wells score > 1, positive compression ultrasound sonography: deep vein thrombosis and therapy
• Wells score > 1, negative compression ultrasound sonography, D-dimer > 500 μg/L: further serial compression ultrasound sonography; positive results of these examinations indicate the presence of a treatable deep vein thrombosis
• Wells score > 1, negative compression ultrasound sonography, D-dimer < 500 μg/L: no deep vein thrombosis.

In many cases, the determination of D-dimer in hospitalized patients is inadequate for VTE diagnosis due to low diagnostic specificity.

16.27.5.2 Positive D-dimer result

The detection of D-dimer in plasma is an indicator of increased presence of fibrin in the blood, degraded to fibrin fragments in the course of secondary hyper fibrinolysis. Elevated D-dimer does not allow any conclusion as to the site and cause of increased fibrin formation.

D-Dimer formation can be caused by /6/:

• Systemic coagulation activation
• Release of thrombin from clots following intravascular fibrin formation
• Release of fibrin complexes from clots
• Release of fibrin degradation products from clots or circulating fibrin complexes
• Release of fibrin formed extravascularly in wounds and hematoma.

A distinction is made between conditions with acutely and permanently elevated D-dimer antigen.

16.27.5.3 Acutely elevated D-dimer level

Acutely elevated D-dimer concentrations are significant in the differential diagnosis of symptoms listed in Tab. 16.27-1 – Differential diagnostic significance of acutely elevated D-dimer.

The behavior of the D-dimer concentration in thromboembolic events is described in Tab. 16.27-2 – Behavior of the D-dimer concentration in thromboembolic events.

Elevated D-dimer levels are detected in almost all patients with deep venous thrombosis (DVT) of the lower extremities. The diagnostic sensitivity is markedly higher for thrombosis of the thigh than for thrombosis of the lower leg. The use of abnormal D-dimer findings in the diagnosis of lower extremity DVT is controversial due to the fact that diagnostic specificity is acceptable in outpatients, but not in inpatients. For instance, in a study /7/, only 22% of hospitalized patients classified as free of DVT had a D-dimer level below the cutoff value. This is because hospitalized patients often present abnormal conditions associated with increased fibrin formation without a thrombotic event.

Such processes and conditions are:

• Sepsis, pneumonia, erysipelas
• Metastatic malignant tumors
• Liver cirrhosis, particularly in conjunction with shunts
• Surgery or trauma within the past 4 weeks
• Anticoagulant therapy for more than 24 h
• Thrombolytic therapy within the past 7 days.

16.27.5.4 Permanently elevated D-dimer levels

Permanently elevated D-dimer concentrations are common in the following conditions and disorders /2/:

• Pregnancy
• Vascular aneurysm, particularly aortic aneurysm
• Portocaval shunt
• Malignant disease (e.g., adenocarcinoma, myeloproliferative disease)
• Vascular malformation such as hemangioma and Kasabach-Merritt syndrome.

16.27.5.5 Pregnancy

Median D-dimer levels increase six-fold during pregnancy. The use of “multiples of the median” (MoM) is recommended as a reference in suspected VTE in pregnant women /8/.

16.27.6 Comments and problems

Method of determination

The commercially available tests do not yield identical results because the D-dimer antigen is present on fibrin degradation products of different size and the used antibodies have a different epitope specificity. Thus, a generally valid reference interval for excluding VTE events cannot be specified because of:

• The different specificity of the antibodies used
• The composition of the antigen used for calibration
• The differences in the method of determination. For instance, an enzyme immunoassay requires only one epitope for binding crosslinked degradation products, whereas in the agglutination test this epitope needs to be present at least twice

The D-dimer is reported as in fibrinogen-equivalent units (FEU) and expressed in μg/L. Because of the different assays and standardization, a test with a cutoff value of 150 μg FEU/L is not necessarily more sensitive than a test with a cutoff value of 500 μg FEU/L.

Point-of-care tests

The negative predictive value for the exclusion of deep vein thrombosis is > 98%. The diagnostic sensitivity for detection is 91–99%, with a specificity of 39–64% /9/.

Stability 

In citrated blood at 20 °C, the mean increase is 1.9% (range –14.5% to + 39.1%) after 8 h and 5.2% (range –9.8% to + 47.8%) after 24 h /10/.

16.27.7 Pathophysiology

The end of the coagulation cascade is characterized by the thrombin-mediated cleavage of fibrinopeptide A and B from fibrinogen. This results in the formation of fibrin monomers. These monomers associate to form protofibrils or associate with fibrin or fibrinogen. In addition fibrin monomer rapidly self-associates to form insoluble fibrin deposits on vessel walls. Thrombin also activates F XIII as fibrin is formed, and F XIIIa rapidly cross-links adjacent γ-chains in fibrin polymers. They are held together by non-covalent forces between the intermolecular D-domain and DE-domain. The protofibrils are stabilized by the action of F XIIIa. The formation of a crosslinked fibrin clot occurs.

Plasmic degradation of fibrinogen and non-cross-linked fibrin results in formation of fragments X and Y and the terminal degradation products D an E. However, the formation of γ-chain cross-links in fibrin, that are resistant to degradation leads to formation of unique degradation products that differ from those of fibrinogen. Consequently, soluble products are produced with γ-chain cross-link intact, and this results in formation of a large number of polymeric forms of defined structure. The smallest is D-dimer, which consists of the fragment D domains of two adjacent monomers joined by the γ-chain cross-link /25/. Refer to Fig. 16.27-1 – Stepwise process of fibrin polymerization and degradation.

References

1. Adam SS, Key NS, Greeberg CS. D-dimer antigen: current concepts and future prospects. Blood 2009; 113: 2878–87.

2. Dempfle CE, Hafner G, Lestin HG, Töpfer G, Adema E, Hubbuch A. Multizentrische Evaluierung von Tina-quant D-Dimer. J Lab Med 1996; 20: 31–7.

3. Douma RA, Tan M, Schutgens REG, Bates SM, Perrier A, Legnani C, et al. Using an age-dependent D-dimer cut-off value increases the number of older patients in whom deep vein thrombosis can be safely excluded. Haematologica 2012; 97: 1507–13.

4. Wells PS, Anderson DR, Rodger M, Forgie M, Kearon C, Dreyer J, et al. Evaluation of D-dimer in the diagnosis of suspected deep-vein thrombosis. N Engl J Med 2003; 349: 1227–35.

5. El Tabai L, Holtz G, Schürer-Maly C, Abholz HH. Accuracy in diagnosing deep and pelvic vein thrombosis in primary care. Dtsch Arztebl Int 2012; 109: 761–6.

6. Dempfle CE. Bestimmung des D-Dimer-Antigens in der klinischen Routine. Dtsch Ärztebl 2005; 102: A-428–32.

7. Heim SW, Schectman JM, Siadaty MS, Philbrick JT. D-dimer testing for deep venous thrombosis: a metaanalysis. Clin Chem 2004; 50: 1136–47.

8. Kristoffersen AH, Petersen PH, Sandberg S. A model for calculating the within-subject biological variation and likelihood ratios for analytes with a time-dependent change in concentrations; exemplified with the use of D-dimer in suspected venous thromboembolism in healthy pregnant women. Ann Clin Biochem 2012; 49: 561–9.

9. Geersing GJ, Toll DB, Janssen KJM, Oudega R, Blikman MJC, Wijland R, et al. Diagnostic accuracy and user-friendliness of 5 point-of-care D-dimer tests for the exclusion of deep vein thrombosis. Clin Chem 2010: 56: 1758–66.

10. Kemkes-Matthes B, Fischer R, Peetz D. Influence of 8 and 24-h storage of whole blood at ambient temperature on prothrombin time, fibrinogen, thrombin time, antithrombin and D-dimer. Blood Coagulation and Fibrinolysis 2011; 22: 215–20.

11. Brown MD, Lau J, Nelson D, Kline JA. Turbidimetric D-dimer test in the diagnosis of pulmonary embolism: a metaanalysis. Clin Chem 2003; 49: 1846–53.

12. Söderberg M, Brohult J, Jorfeldt L, Lärfars G, The use of D-dimer testing and Wells score in patients with high probability for acute pulmonary embolism. J Eval Clin Pract 2009; 15: 129–33.

13. Douma RA, Kamphuisen PW, Huisman MV, Buller HR. False normal results on multidetector-row spiral computed tomography in patients with high clinical probability of pulmonary embolism. J Thromb Haemost 2008; 6: 1978–9.

14. Bick RL. Disseminated intravascular coagulation. Objective laboratory diagnostic criteria and guidelines for management. Clin Lab Med 1994; 14: 729–68.

15. Fukutake K, Kuroso K, Isagai N, Shinozawa K. Clinical evaluation of D-dimer testing in disseminated intravascular coagulation (DIC). Fibrinolysis 1993; 7: 20–2.

16. Gabazza EC, Taguchi O, Yamakami T, Machishi M, Ibata H, Suzuki S. Evaluating prethrombotic state in lung cancer using molecular markers. Chest 1993; 103: 196–200.

17. Taylor jr FB, Toh C, Hoots WK, Wada H, Levi M. Towards definition, clinical and laboratory criteria, and scoring system for disseminated intravascular coagulation. Thromb Haemos 2001; 86: 1327–30.

18. McDermott MM, Greenland P, Green D, Guralnik JM, Criqui MH, Lui K, Chan C, et al. D-dimer, inflammatory markers, and lower extremity functioning in patients with and without peripheral arterial disease. Circulation 2003; 107: 3191–8.

19. Costantini V, Zacharski LR. Fibrin and cancer. Thromb Haemost 1993; 69: 406–14.

20. Mitter CG, Zielinski CC. Plasma levels of D-dimer: a crosslinked fibrin-degradation product in female breast cancer. J Cancer Res Clin Oncol 1991; 117: 259–62.

21. Simon M, Zimmermann R, Weber E. D-Dimer als Kontrollparameter für die fibrinolytische Therapie der tiefen Beinvenenthrombose. Hämostaseologie-Info 1990; 1: 8.

22. Mahe I, Drouet L, Chassany O, Mazoyer E, Simoneau G, Knellwolf AL, et al. D-dimer: a characteristic of the coagulation state of each patient with chronic atrial fibrillation. Thromb Res 2002; 107: 1–6.

23. Shorr AF, Trotta RF, Alkins SA, Hanzel GS, Diehl LF. D-Dimer assay predicts mortality in critically ill patients without disseminated intravascular coagulation or venous thromboembolic disease. Intensive Care Med 1999; 25: 207–10.

24. Brown MD, Lau J, Nelson D, Kline JA. Turbidimetric D-dimer test in the diagnosis of pulmonary embolism: a metaanalysis. Clin Chem 2003; 49: 1846–53.

25. Horan JT, Francis CW. Fibrin degradatiom products, fibrin monomer and soluble fibrin in disseminated intravascula coagulation. Semin Thromb Hemost 2001; Dec; 27(6): 657–66.

16.28 Monitoring of anti thrombotic therapy

Jan Kramer

16.28.1 Introduction

The pathogenesis of thrombosis is described by Virchow’s triad. According to this concept, there are mainly three general factors that contribute to thrombosis:

• Endothelial alterations (vascular injury)
• Hemodynamic changes (stasis or turbulence of blood stream)
• Increase in blood viscosity (polyglobulinemia and exsiccosis).

Hyper coagulability of the blood can be essentially promoted by:

• Deficiency in inhibitors of coagulation
• Excess of coagulation activators
• Increased platelet aggregation
• Dysfunctional fibrinolysis.

Anti thrombotic therapy plays a key role in the primary and secondary prophylaxis of cardiovascular and cerebrovascular disease and venous thromboembolism.

16.28.1.1 Anti thrombotic therapy in arterial and venous thromboembolism

Arterial blood clots form due to shear-induced platelet aggregration in the presence of small amounts of fibrin, as a rule based on atherosclerosis as the main underlying condition. The rupture of an atheromatous plaque leads to the exposure of a strongly thrombogenic lipid core. This is likely to result in subsequent platelet adhesion, activation and aggregation. Hence, anti-platelet therapy is the main focus of anti thrombotic therapy in arterial thrombosis.

However, anticoagulation under inhibition of the plasmatic coagulation is also employed for the prophylaxis of cardiac arterial clots and cardioembolic events in valvular or structural cardiac disease. Fibrinolytic substances can be administered to rapidly restore the blood flow in patients with acute myocardial infarction in cases where percutaneous coronary intervention is not immediately possible. The same approach can also be followed in acute ischemic stroke or acute pulmonary embolism in conjunction with hemodynamic instability.

Venous clots form under low shear stress. They are fibrin-rich and contain captured red blood cells and relatively small amounts of platelets. Although the local activation of platelets leads to the concentration of the entire coagulation process, for example, on the site of a vessel wall injury, the plasmatic coagulation system plays a key role in the formation of venous thromboembolism (VTE). Considering the predominance of fibrin clot formation, anticoagulants are the substances of choice in the prophylaxis and therapy of VTE, whereas anti thrombocytic strategies play almost no role. Drug-induced fibrinolysis is employed in the venous system in exceptional cases, only.

VTE is one of the leading causes of morbidity and mortality in the Western world. More than half of all hospitalized patients are at risk of VTE complications /1/. Therefore, anticoagulant thromboprophylaxis is recommended for almost every inpatient to prevent VTE /2, 3/. Prophylactic anticoagulation should also be considered for outpatients in the presence of thromboembolic risk factors /3/.

Indications for anticoagulation at therapeutic doses include deep vein thrombosis as well as absolute arrhythmia in atrial fibrillation, acute myocardial infarction, extra corporeal circulation, disseminated intravascular coagulation, arterial thrombosis and embolism, severe cardiac failure, condition following aortocoronary venous bypass and implantation of heart valve or arterial vascular prostheses /4/. The decision in favor of full anticoagulation therapy should always consider potential contra indications and reflect the balancing of the risk of bleeding against the benefit to the patient. For instance, it is possible to assess the risk of a thromboembolic event in atrial fibrillation using the CHADS score and the risk of bleeding using the HAS-BLED score.

Refer to

• Tab. 16.28-1 – CHADS2-Score and CHA2DS2VASc-Score for initial risk assessment of a thromboembolic event in atrial fibrillation
• Tab. 16.28-2 – Assessment of the bleeding risk despite the use of oral anticoagulation in patients with atrial fibrillation.

16.28.1.1.1 CHADS score

The CHADS₂ score is used to initially assess the risk of a thromboembolic event in atrial fibrillation.

In the absence of contra indications, a CHADS₂ ≥ 2 (high rate of thromboembolism (e.g., CHADS₂ score 2 = 4%/year; CHADS₂ score 4 = 8.5%/year; CHADS₂ score 8 = 18.2%/year) warrants oral anticoagulation with vitamin K antagonists at therapeutic doses with a target INR of 2.5 (2.0–3.0).

However, patients classified in the categories CHADS₂ score = 1 (moderate risk = 2.8%/year) and CHADS₂ score = 0 (low risk = 1.9%/year) can still be clinically at risk of developing thromboembolism.

Another method of clinical risk assessment uses the CHA₂DS₂-VAS_c scheme /5/: age and stroke/transitory ischemic attacks are considered to be major risk factors for an increased risk of thromboembolism /6/.

A CHA₂DS₂-VAS_c ≥ 2 (e.g., CHA₂DS₂-VAS_c score 2 = 2.2%/year; CHA₂DS₂-VAS_c score 4 = 4%/year; CHA₂DS₂-VAS_c score 7 = 9.6%/year; CHA₂DS₂-VAS_c score 9 = 15.2%/year) warrants oral anticoagulation with vitamin K antagonists at therapeutic doses with a target INR of 2.5 (2.0–3.0).

A CHA₂DS₂-VAS_c score = 1 (risk of stroke = 1.3%/year requires individual evaluation of the preference of vitamin K antagonist treatment and the concomitant use of acetylsalicylic acid (75–325 mg per day). At a score = 1, the administration of dabigatran (2 × 110 mg/day) should be considered due to the decreased rate of intracranial hemorrhage and major bleeding and similar efficiency in the prevention of embolism compared to warfarin /7/.

A CHA₂DS₂-VAS_c score = 0 requires the individual balancing of the preference of non-medication management against the administration of ASA (75–325 mg per day), which is associated with a risk of bleeding.

16.28.1.1.2 HAS-BLED score

The HAS-BLED score is used to assess the risk of bleeding in patients with atrial fibrillation undergoing oral anticoagulation /8/.

HAS-BLED ≥ 3 indicates a risk of major bleeding. The indication of anticoagulation with vitamin K antagonists and the administration of acetylsalicylic acid must be strictly verified. Close-meshed monitoring of the patient is required.

Provided the indication is confirmed, patients with HAS-BLED score = 2 (moderate risk of bleeding) or HAS-BLED score = 0 or 1 (no risk of bleeding) clearly benefit from oral anticoagulation. The application of dabigatran etexilate should be considered as an alternative for the dose-adjusted administration of vitamin K antagonists:

• High-dosed (2 × 150 mg of dabigatran per day) at a HAS-BLED score of 0–2 due to the increased efficacy in the prevention of embolism in conjunction with decreased rates of intracranial hemorrhage and similar incidence of major bleeding compared to warfarin /7/
• Low-dosed (2 × 110 mg of dabigatran per day) at a HAS-BLED score ≥ 3 due to similar efficacy in the prevention of embolism in conjunction with a comparable rate of intracranial hemorrhage and major bleeding compared to warfarin /7/.

16.28.1.2 Diseases warranting therapeutic anticoagulation

Diseases warranting therapeutic anticoagulation can be roughly classified in three groups based on the risk of thromboembolism to be expected without anticoagulation /9/:

• High (above 7% per year)
• Moderate (4–7% per year)
• Low (below 4% per year).

Refer to (Tab. 16.28-3 – Risk stratification scheme for perioperative thromboembolism without the use of therapeutic anticoagulation and list of surgical procedures with a high risk of bleeding)

In order to assist in the decision-making process as to the intensity and duration of anticoagulation, a concept of individualized anticoagulation monitored through D-dimer antigen determination has been discussed aiming at increased reliability in the prevention of bleeding complications and recurring thromboembolic events /10/. According to this concept, increased D-dimer antigen can be interpreted as a possible risk indicator for VTE if other causes (e.g., hematoma, effusion, wound healing, pregnancy or liver cirrhosis, have been excluded or are unlikely).

Continued low D-dimer concentration following the reduction or termination of anticoagulation is thought to indicate a low risk of recurrence of a thromboembolic event.

However, an increase in D-dimer level within a few days detected presumably indicates a relatively high risk of recurrent thromboembolism and, thus, seems to warrant continued anticoagulation.

The rate of recurrence within the first months following a thromboembolic event is higher than after a longer period. Different indications for therapeutic anticoagulation lead to correspondingly different assessments of the risk of thromboembolic events. Therefore, an indication- and risk-oriented approach is recommended to determine the duration of anticoagulation /11/. In addition, it is advisable to regularly monitor the individual bleeding risk under therapeutic anticoagulation.

The decision about individual risk-adapted heparin bridging against the background of an indicated perioperative interruption of anticoagulation or in manifest bleeding can only be made based on sound clinical evidence /12/. The risk of bleeding during surgical intervention is increased by patient-related factors (hemostatic disorders, also including the intake of platelet function inhibitors; previous perioperative bleeding) as well as various surgery-related factors, such as larger-scale trauma, urgency of surgical intervention, major surgery, limited practical knowledge of the surgeon, few possibilities of bleeding control. The interruption of oral anticoagulation is imperative in patients at high risk for perioperative bleeding. Perioperative bridging should be considered at high and moderate risk of thromboembolism (Tab. 16.28-3 – Risk stratification scheme for perioperative thromboembolism without the use of anticoagulation and list of surgical procedures with a high risk of bleeding).

Anticoagulation monitoring is an important aspect in the prevention of therapeutic complications for numerous anticoagulants. Both bleeding and recurrent thromboembolism can only be prevented under optimal anticoagulant adjustment. By contrast, prophylactic anticoagulation usually does not require any monitoring. The efficacy of new oral anticoagulants needs not be monitored by coagulation tests in clinical routine even under therapeutic use if the limitations of indication (e.g., in patients with renal insufficiency) are taken into account. Nevertheless, when using these substances, it is important to know the possibilities and limitations of monitoring in suspected subtherapeutic or supra therapeutic levels for assessment of the clinical situation.

16.28.1.3 Anti thrombotic drugs

Depending on the site of action, anti thrombotic drugs are classified into:

• Platelet function inhibitors
• Anticoagulants
• Fibrinolytic agents.

16.28.2 Platelet function inhibitors

In regions affected by atherosclerotic alterations, the adhesion, activation and aggregation of platelets may lead to vascular occlusion. In order to prevent this development, substances from the group of platelet function inhibitors induce platelet dysfunction by various mechanisms. An overview of the function and clinical significance of the individual platelet function inhibitors as well as information on diagnostic monitoring is provided in Tab. 16.28-4 – Mode of action, clinical significance and monitoring of platelet function inhibitors.

Sites of action and function of platelet function inhibitors

Collagen and von Willebrand factor (VWF) are presented at the site of the endothelial defect. Glycoproteins (GP) Ib/V/IX (a receptor for VWF; CD42b) and GP Ia/IIa (α₂-/β₁-integrin; a receptor for collagen) of the platelet membrane induce the zipper-like adhesion of platelets to the subendothelial matrix, thus triggering platelet activation (Fig. 16.28-1 – Sites of action of platelet function inhibitors).

Membrane flip-flop trans locates pro coagulant phospholipids of the platelets from inside to the outside, numerous mediators and Ca²⁺ are released and GP IIb/IIIa is to an increasing extent expressed on the membrane surface (inside-out signaling). The negatively charged phospholipids bind Ca²⁺. Coagulation factors can now also bind to the platelet surface (see Fig. 16.1-5 – Platelet adhesion and platelet aggregation at the site of vascular wall injury). Thus, a small number of thrombin molecules can locally trigger plasma coagulation on the surface of activated platelets. The released mediators recruit more platelets and trigger a positive feedback mechanism because released adenosine diphosphate binds to the receptor P2Y12 and/or thromboxane A2 (TXA₂) binds to the thromboxane receptor (TXR), thus enhancing platelet activation.

The increased expression of GP IIb/IIIa (alpha2b/beta3 integrin; CD41a) leads to platelet aggregation because fibrinogen and VWF act as bridging molecules linking two GP IIb/IIIa on neighboring platelets.

During platelet activation, cyclooxygenase (COX)-1 is responsible for the synthesis of prostaglandin from arachidonic acid. Subsequently, TXA₂ is formed by thromboxane synthase. Acetylsalicylic acid irreversibly inactivates COX-1, thereby indirectly inhibiting platelet aggregation. The reduction of activation of the intrinsic pathway by ADP receptor antagonists also has an indirect inhibiting effect on platelet aggregation. GP IIb/IIIa receptor antagonists directly inactivate aggregation by efficiently inhibiting cross-bridging between adjacent platelets.

16.28.2.1 Indication

Therapy as well as primary and secondary prevention of cardiovascular events such as myocardial infarction, stroke and peripheral arterial occlusive disease.

16.28.2.2 Therapy

Perioperative management

Perioperative treatment with platelet aggregation inhibitors increases the risk of bleeding /13/.

Monotherapy: acetylsalicylic acid (ASA) elevates the risk of a hemorrhagic complication during surgery by 50% but does not increase operative mortality. ASA that is taken for primary prevention can be interrupted for surgery. The European Society of Cardiology (ESC) recommends in general that ASA for secondary prevention should not be discontinued perioperatively. Nonetheless, for intraocular, intraspinal and intracranial procedures, even small hemorrhages can cause significant morbidity, so that temporarily discontinuing ASA would seem to be necessary.

Dual therapy: the simultaneous intake of ASA and P2Y12 inhibitors (e.g., ticlopidine, clopidogrel) usually by some patients who have received a coronary stent can cause major problems in the perioperative period. Patients with a coronary stent must take ASA for life and a P2Y12 inhibitor either for at least six weeks (bare metal stents) or for at least twelve months (drug eluting stents). Early termination of dual therapy is associated with a 90-fold increased risk of stent thrombosis. Elective surgery should be postponed until ASA mono therapy has been established. If an operation cannot be postponed and must be performed during the critical period, it is recommended that dual inhibition of platelet aggregation be continued perioperatively. If the dual inhibition of platelet aggregation has been interrupted for surgery, P2Y12 inhibitors must be restarted as soon as possible after the operation.

In patients with extremity fractures that had been treated operatively or with any pelvic or acetabular fracture thromboprophylaxis with aspirin was noninferior to low-molecular-weight heparin in preventing death and was associated with low incidences of deep-vein thrombosis and pulmonary embolism and 90-day mortality /51/.

Monitoring

Monitoring of platelet function in patients undergoing therapy with platelet aggregation inhibitors is increasingly recommended. It is crucial to avoid activation of the platelets, for example due to long transport times.

The aggregometry procedure according to Born which measures changes in light transmission of platelet-rich plasma during aggregation is the gold standard. Aggregation patterns are shown in Tab. 16.28-5 – Typical aggregation patterns under the action of platelet function inhibitors.

16.28.3 Anticoagulants

Anticoagulants are classified into directly and indirectly acting ones. Phenprocoumon and warfarin indirectly inhibit coagulation by decreasing the γ-carboxylation of the prothrombin complex (factors II, VII, IX and X). Other anticoagulants act by inhibiting F Xa and/or thrombin (F IIa).

Sites of anticoagulant action

The effect of the individual anticoagulants is as follows:

• Antithrombin (AT) inhibits coagulation by the proteolytic degradation of thrombin and inactivation of serine proteases. Binding to AT is only possible in the presence of a specific pentasaccharide sequence of heparin. The binding leads to an altered AT conformation, whereby inactivation of substrates by AT is 100–1,000-fold faster. Heparin is immediately released again and free to activate more AT molecules. An intermolecular cross linking mechanism is needed for the inactivation of thrombin by AT; this inactivation process is indirectly enhanced by heparin concurrently binding to the secondary binding site of thrombin (exosite 2). This tertiary complex needs a heparin chain length of at least 17 monosaccharide units (molecular weight > 5400 kDa). Consequently, unfractionated heparins usually have similar anti-F IIa and anti-F Xa activities. The anti-F Xa activity of low molecular weight heparins is higher than their anti-F IIa activity. By its highly specific binding to AT, the synthetic pentasaccharide fondaparinux indirectly and selectively enhances the AT-mediated inhibition of F Xa approximately 300-fold. Fondaparinux has no anti-F IIa activity.
• Direct F Xa inhibitors bind directly to F Xa, thus preventing thrombin formation during the clotting intensification phase
• Direct thrombin inhibitors (DTI) bind to the active center of thrombin (F IIa; fibrin-bound or free) and indirectly block their interaction with substrates. F IIa has the active binding site as well as two secondary bindings sites, exosite 1 (fibrinogen) and exosite 2 (heparin). The naturally occurring, bifunctional DTI hirudin irreversibly binds to exosite 1 and the active binding site. By contrast, the bifunctional binding of the synthetic DTI bivalirudin and the monofunctional binding of the synthetic DTIs argatroban and dabigatran to the active binding site are reversible.

The sites of action of directly and indirectly acting anticoagulants are depicted in Fig. 16.28-2 – Sites of action of directly and indirectly acting anticoagulants in the coagulation system.

16.28.4 Phenprocoumon and warfarin

The vitamin K antagonists phenprocoumon and warfarin inhibit the hepatic synthesis of vitamin K dependent coagulation factors.

16.28.4.1 Indication

Thromboprophylaxsis following deep vein thrombosis, pulmonary embolism, atrial fibrillation and cardiac valve replacement.

16.28.4.2 Therapy

The intensity of anticoagulation is reflected by the INR value (Tab. 16.28-6 – Mode of action, clinical significance and monitoring of anticoagulants). Preoperative heparin bridging in patients receiving vitamin K antagonists has been practiced for many years /13/.

16.28.5 Heparin

Unfractionated heparin (UFH) and fractionated (low molecular weight) heparin (FH) are distinguished. UFH inhibits the activity of thrombin. The anti-F Xa activity of FHs is higher than their anti-F IIa activity. The effect of UFH and FH is mediated by AT (Fig. 16.28-3 – Effect of indirectly and directly acting anticoagulants).

16.28.5.1 Indication

Unfractionated heparin: thromboprophylaxis in patients with mechanical heart valves, during cardiopulmonary bypass and in intensive care.

Fractionated heparin: thromboprophylaxis.

16.28.5.2 Therapy

The dose must be adapted at eGFR below 30 [mL × min^–1 × (1.73 m²)^–1] which is often seen in older patients. In these cases, the effect of heparin must be monitored by determining the anti-F Xa activity (Tab. 16.28-6 – Mode of action, clinical significance and monitoring of anticoagulants).

Because of the long half-life of fondaparinux (approximately 17 hours), a single subcutaneous injection per day suffices. In patients with an eGFR below 50 [mL × min^–1 × (1.73 m²)^–1] the dose must be lowered to 1.5 mg daily subcutaneously in prophylactic use. Routine monitoring of the anti-F Xa activity is only required in decreased GFR. Fondaparinux has almost no effect in the coagulation screening tests (Fig. 16.28-2 – Sites of action of directly and indirectly acting anticoagulants in the coagulation system). If the anti-F Xa activity needs to be determined, the test must be calibrated with fondaparinux.

16.28.6 New oral anticoagulants (NOACs)

The NOACs , also called non-vitamin-K oral anticoagulants are anticoagulants acting on specific factors within the coagulation cascade.

The NOACs include:

• The thrombin inhibitor dabigatran given as prodrug dabigatran etexilate (Pradaxa), which is hydrolyzed in the body to become a direct thrombin inhibitor
• Direct acting oral anticoagulants (DOACs). These drugs include the F Xa inhibitors apixiban (Eliquis), edoxaban (Lixiana), and rivaroxaban (Xarelto).

Clinical trials have shown NOACs therapeutic equivalent, or even superior, to vitamin K antagonists (VKA), with lower rates of bleeding.

Approximately 12% of hospitalization is caused by the interactions of phenprocoumon or warfarin with other drugs. Renal and hepatic (dys)function, hemorrhagic risk, age, and interactions with other drugs must be considered when NOACs are prescribed /22/. The accompanying hemorrhagic risk of NOACs is low (slight bleeding 0.8% and heavy bleeding 1.4–2.1%).

16.28.6.1 Indication

Dabigatran (Pradaxa)

• Stroke prevention in non-valvular atrial fibrillation
• Prevention of venous thromboembolism (total knee or total hip replacement)
• Treatment of deep vein thrombosis (DVT) and pulmonary embolism (PE)
• Prevention of recurrent DVT and PE.

Apixiban (Eliquis), edoxaban (Lixiana), and rivaroxaban (Xarelto)

• Prophylaxis of venous thromboembolism (total knee or total hip replacement)
• Prophylaxis of venous thromboembolism following total knee or total hip replacement
• Treatment of deep vein thrombosis (DVT) and pulmonary embolism (PE)
• Prophylaxis of deep vein thrombosis (DVT) and pulmonary embolism (PE)
• Prophylaxis of stroke and systemic embolism in non-valvular atrial fibrillation
• Prophylaxis of atherotrombotic events in acute coronary syndrome (with aspirin alone or aspirin and clopidrogel.

16.28.6.2 Therapy

NOACs are small molecules given at fixed doses. Monitoring is only required in special cases, as pharmacodynamic and pharmacokinetic responses are reliably predicted in patients with adequate renal function who are not taking other interacting drugs /23/. Several trials have evaluated the efficacy and safety of the new oral anticoagulants (NOACs) for venous thromboembolism (VTE) prevention in patients undergoing total hip arthroplasty (THA). Twenty eight to 39 days of pharmacological prophylaxis with an NOAC compared with enoxaparin reduced the relative odds of symptomatic VTE by about 60% in patients undergoing THA with no excess of major bleeding /24/. A three-year prospective study of the presentation and clinical outcomes of major bleeding episodes associated with oral anticoagulant use in the UK (ORANGE study) /25/ showed the following results: bleeding sites were intracranial (44%), gastrointestinal (33%), and other (24%). The in-hospital mortality was 21% overall, and 33% for patients with intracranial hemorrhage. Compared to warfarin-treated patients, patients treated with direct NOACs were older and had lower odds of subdural/epidural, subarachnoid and intracerebral bleeding. The mortality rate due to major bleeding was not different between patients being treated with warfarin or direct NOACs.

16.28.6.3 Shortcomings with non-vitamin K oral anticoagulants (NOACs)

In contrast to vitamin K antagonists and heparin, no routine coagulation monitoring is required in patients taking NOACs. However, potential indications and shortcomings must be noted /26/:

• Potential indications for coagulation testing include emergency situations (i.e., trauma) urgent surgery, urgent invasive procedures, major bleeding, overdose/attempted suicide, acute thrombosis, renal failure, adherence verification, and potential drug-drug interactions
• Routine clotting test are not reliable for monitoring patients
• Before starting therapy renal function monitoring prevents adverse events
• Each of the NOACs, except dabigatran, is metabolized by the liver. As such, routine liver function monitoring (every 6–12 months) should be considered in patients with, or at risk for, hepatic dysfunction
• Because of insufficient studies in pregnancy and during childbed, women with VTE should not be treated with NOACs.
• Because of insufficient studies, tumor patients with VTE should not be treated with NOACs.

16.28.7 Methods of monitoring the anticoagulant effect of heparins and NOACs

Plasma concentrations can be measured for all four NOACs using liquid chromatography-tandem mass spectrometry. Information on the clinical utility of coagulation assays largely comes from ex vivo studies using plasma from dabigatran treated patients or healthy volunteers and in vitro studies with dabigatran added to plasma samples /26/.

16.28.7.1 Activated clotting time (ACT)

Indication

Determination of the anticoagulant effect of heparin.

Principle

Whole blood is added to a tube containing a surface activator. Coagulation is activated via the intrinsic pathway and through in-vitro activation of F XII. The appearance of the first clot is timed. The type of activator affects the clotting time (celite, 100–170 sec.; glass, 110–190 sec.; kaolin, 90–150 sec.). In a non anticoagulated patient, the ACT is about 107 ± 13 seconds. For monitoring the heparin effect during interventions (e.g., cardiopulmonary bypass), heparin is titrated to maintain an ACT > 400–600 sec. to prevent clot formation (> 250 sec. in heparin-coated systems). During extra corporeal membrane oxygenation, an ACT in the region of 200 sec. is considered sufficient. The ACT is influenced by numerous factors and is prolonged, for example, in thrombocytopenia below (30–50) × 10⁹/L and factor deficiency including hypo fibrinogenemia.

16.28.7.2 Ecarin clotting time (ECT) and ecarin chromogenic assay (ECA)

Indication

Determination of the anticoagulant effect of direct thrombin inhibitors (lepirudin, desirudin, bivalirudin, argatroban, dabigatran).

Principle

Prothrombin is activated by ecarin, a snake venom from Echis carinatus.

In the ECT assay, plasma fibrinogen is converted to fibrin by the resulting prothrombin activation substances meizothrombin and meizothrombin-desF1 /27/.

In the ECA assay, the activation products cleave a chromogenic substrate (Fig. 16.28-4 – Ecarin clotting time and ecarin chromogenic assay).

In the ECT, the time elapsing until the appearance of a clot is measured. The ECT depends on the prothrombin and fibrinogen levels in the patient plasma, contrary to the concentration-independent ECA where prothrombin is added to the reaction and cleavage of a chromogenic substrate by meizothrombin is measured spectrophotometrically.

The activity of meizothrombin and meizothrombin-desF1 is inhibited in a concentration-dependent matter by direct thrombin inhibitors. Heparin does not influence the ECA and ECT assays. There is a linear correlation between the ECT and/or ECA response time and the concentration of the direct thrombin inhibitor allowing:

• The definition of substance-specific drug levels for therapy
• The allocation to regions with increased rates of complications, regarding both the occurrence of thromboembolism in anticoagulant under dose and bleeding in anticoagulant overdose.

16.28.7.3 Anti-F IIa (thrombin) activity

Indication

Determination of the anticoagulant effect of unfractionated heparin.

Principle

Heparin activates antithrombin (AT) forming a heparin/AT complex (Fig. 16.28-3 – Effect of indirectly and directly acting anticoagulants. AT inhibits coagulation by activating serine proteases and proteolytic degradation of thrombin which very rapidly inhibits thrombin. A defined excess of thrombin added to the sample leads to the inactivation of a proportion of thrombin proportional to the heparin concentration. The residual, non-inactivated thrombin cleaves p-nitroaniline (p-NA) from a chromogenic substrate /29/. The heparin concentration is calculated based on the increase in absorption determined spectrophotometrically at 405 nm and expressed in IU/mL.

16.28.7.4 Anti-F Xa activity

Indication

Evaluation of the anticoagulant effect of coagulation therapy with anticoagulant drugs acting on F Xa within the coagulation cascade e.g., fractionated heparin, and some NOACs like apixaban, endoxaban, and rivaroxaban.

Principle

A defined amount of F Xa is added to the test sample; part of it binds to AT and is inactivated, a reaction catalyzed by heparin (Fig. 16.28-5 – Chromogenic substrate assay for determination F Xa inhibitor effect). The residual, non-inactivated F Xa cleaves p-NA from a chromogenic substrate. Heparin levels below 0.1 IU/mL are no longer detectable because platelet factor 4 (PF4) binds to heparin. Therefore, dextran sulfate is added to the sample to release heparin from PF4. The heparin concentration is calculated based on the increase in absorption determined spectrophotometrically at 405 nm and expressed in IU/mL. Calibration must be performed according to the WHO standard for low molecular weight heparin. If used to determine the anti-F Xa activity of danaparoid or fondaparinux calibration is achieved with these anticoagulants.

Anti-F Xa activity is determined in risk patients (renal insufficiency, over- or underweight, children) and during pregnancy /29/. Optimal anti-F Xa activities (if collected 4 h after subcutaneous administration) are reported as 0.4–1.0 IU/mL in plasma in therapeutic application and as 0.2–0.5 IU/mL in plasma in prophylactic application. The lower detection limit for heparins of the anti-F Xa assay is 0.01–0.05 IU/mL /28/. An assay was developed and evaluated capable of quantifying heparin in the presence of apixaban /47/.

16.28.7.5 Hemoclot thrombin inhibitor assay

Indication

Suspected high anticoagulant activity of direct thrombin inhibitors (hirudin, argatroban, dabigatran).

Principle

The hemoclot thrombin inhibitor assay is based on the inhibition of a defined quantity of thrombin by thrombin inhibitors /30/. The plasma to be analyzed is diluted and pooled normal human plasma is added. Coagulation is triggered using human α-thrombin. The time elapsing until the formation of a clot is directly proportional to the concentration of the thrombin inhibitor in the analyzed plasma. The reference interval for dabigatran 2 h after intake is 50–300 μg/L.

16.28.8 Clinical monitoring of anticoagulation

Some anticoagulants must be subjected to continuous laboratory diagnostic monitoring, while others need only to be monitored in specific patients or scenarios.

16.28.8.1 Phenprocoumon and warfarin

Patients undergoing therapy with phenprocoumon and warfarin must be monitored because of the risk of bleeding and/or recurrent thromboembolism based on inter individual differences in adjustment and dosage. This is achieved by determining the PT expressed as INR. The normal INR value is 1.0, the target value is usually 2–3 under therapy and 2.5–3.5 after mechanical heart valve replacement. Refer to Tab. 16.28-6 – Mode of action, clinical significance and monitoring of anticoagulants.

Surgery associated with a low risk of bleeding (dento- surgical interventions, cardiac catheter examinations) is performed under therapeutic INR.

Major surgery requires bridging with coagulants with a short-term effect. Bridging implies a 5-fold increased risk of bleeding. As a sub therapeutic INR is reached after 4–7 days, vitamin K antagonist therapy should be temporarily discontinued, if possible. The decision about bridging with low molecular weight heparin, (e.g., in atrial fibrillation) depends on the CHA₂DS₂-VASc score. Perioperative bridging with unfractionated heparin is required in patients under intensive care and patients with mechanical heart valves. Anticoagulation using argatroban (intravenously) or danaparoid (subcutaneously and/or intravenously) is an alternative in patients at risk of heparin-induced thrombocytopenia type II.

16.28.8.2 Heparin

The anti-F IIa (antithrombin) activity assay is used to monitor unfractionated heparin therapy (Tab. 16.28-6 – Mode of action, clinical significance and monitoring of anticoagulants). As a rule, the anticoagulant effect during low molecular weight heparin therapy needs not be monitored by the anti-F Xa assay, but be determined in older patients with an eGFR < 30 [mL × min^–1 × (1.73 m²)^–1] for dose adjustment. However, the selected drug must be calibrated with the corresponding heparin.

Heparin resistance is defined as the need for high heparin doses to achieve a target level of anticoagulation. Mechanisms of resistance are nonspecific binding of heparin, elevated concentrations of coagulation factors, antithrombin deficiency, thrombocyte interactions, and the decoy factor Andexanet alfa /50/.

Definitions are /50/:

• The need for more than 35,000 Units per day
• In patients undergoing cardiopulmonary bypass the definition of heparin resistance is the need for a dose of more than 500 U per kilogram of body weight to achieve an activated thromboplastin time of 400–480 seconds.

If heparin resistance is a concern, anti-FXa concentration is used to measure heparin. If the anti-FXa concentration is low, than unfractionated heparin should be increased to achieve the standard target of 0.3 to 0.7 U/mL.

16.28.8.3 New oral anticoagulants (NOACs)

In contrast to vitamin K antagonists and heparin, routine anticoagulation takes continuously. However, monitoring is not required in patients taking NOACs. Laboratory diagnostic monitoring of the anticoagulant effect of NOACs is recommended in the following scenarios /22/.

• Hemorrhagic risk
• Before surgery or an invasive procedure when the patient has taken a NOACs during the previous 24 h and his GFR is < 30 [mL × min^–1 × (1.73 m²)^–1]
• Identification of sub therapeutic or supra therapeutic levels in patients taking other drugs that are known to significantly affect the NOAC pharmacokinetics
• Identification of sub therapeutic or supra therapeutic levels in patients at the extremes of body weight
• Patients with deteriorating renal function
• Peri operative management
• Reversal of anticoagulation
• Suspicion of overdose
• Assessment of compliance in patients suffering thrombotic events while on NOAC treatment.

The Standardization Committee of the International Society on Thrombosis and Hemostasis considers the following tests for oral anticoagulant monitoring /23/:

• Coagulation tests that are readily available in most laboratories. Their result is semi quantitative and should be considered as indicating supra therapeutic, therapeutic or sub therapeutic anticoagulation in emergency or other clinical scenarios
• Tests such as HPLC tandem mass spectrometry. NOACs are determined quantitative and specific.

16.28.8.3.1 Monitoring of anticoagulation using NOACs

Refer to Tab. 16.28-7 – Laboratory monitoring of patients using NOAcS.

16.28.9 Fibrinolysis and fibrinolytic agents

Thrombolytic therapy may be indicated in special situations in connection with thromboembolic events /33/. Contra indications of fibrinolytic therapy must be excluded and the latest technical information applied. In general, a distinction is made between systemic fibrinolytic therapy and local (catheter-controlled) fibrinolysis frequently performed during radiological interventions. Available fibrinolytic agents include streptokinase, urokinase, rt-PA (alteplase) and tenectplase. Due to the increased risk of bleeding, fibrinolytic therapy is not recommended in deep vein thrombosis. However, fibrinolysis may still be employed in individual cases because of its superiority in clot recanalization. By contrast, systemic fibrinolytic therapy is the therapy of choice in hemodynamically unstable patients with pulmonary embolism /34/. Early fibrinolysis with rt-PA (initial bolus 10 mg/1–2 min.; maintenance dose 90 mg/2 h; not more than 100 mg in total) is usually the standard therapy.

In acute coronary syndrome and/or acute myocardial infarction, fibrinolytic therapy using, for example, rt-PA (e.g., within 6 h after the onset of symptoms; initial bolus 15 mg; maintenance dose 50 mg/30 min., then 35 mg/60 min.; not more than 100 mg in total; dose adjustment required in patients with a body weight below 65 kg) should always be considered if standard percutaneous coronary intervention cannot be performed within 90 min. /35/.

In acute ischemic stroke, systemic fibrinolysis using rt-PA (0.9 mg per kg of body weight; not more than 90 mg; 10% of the total dose as initial bolus, the rest distributed over 60 min.; not more than 90 mg in total) must be started within 3 h (to not more than 4.5 h) after the onset of symptoms; the risk of bleeding increases thereafter /36, 37/. Local intraarterial, catheter-based thrombolytic therapy in acute ischemic stroke is only performed on selected patients in specialized centers. Intraarterial thrombolytic therapy can also be considered in acute ischemia of the extremities, whereas systemic thrombolysis is objected in this case /38/.

Special diagnostic monitoring of fibrinolytic therapy beyond the determination of global coagulation parameters (aPTT) is not necessary. In pulmonary embolism, rt-PA treatment should be accompanied by intravenous heparinization as soon as the aPTT values are below 2 times the upper reference interval value. The aPTT target interval is between 50 and 70 sec. (1.5–2.5-fold the reference interval).

In patients with acute coronary syndrome, platelet function inhibition therapy using acetylsalicylic acid can be started concurrently with rt-PA fibrinolysis. By contrast, the administration of acetylsalicylic acid or intravenous heparin is contraindicated in patients receiving rt-PA for treatment of ischemic stroke within the first 24 h after start of therapy.

16.28.10 Recommended duration of therapeutic anticoagulation

The recommended duration of therapeutic anticoagulation depends on the indication. Aspects to be considered include the evidence level 1/2, strong/low recommendation and quality of studies:

• A, excellent quality of study (e.g., two studies with evidence level I; randomized case-control studies)
• B, good quality of study (e.g., 1 study with evidence level I)
• C, moderate quality of study (e.g., studies with evidence level II; cohort or outcome studies).

16.28.10.1 Venous thromboembolism (VTE)

Therapeutic anticoagulation

Therapeutic anticoagulation is accomplished in VTE with vitamin K antagonists (VKA) and/or for cancer with low-molecular-weight heparin (LMWH) /11, 39/. Initial therapy of pulmonary embolism is to employ the concept of overlapping treatment with LMWH, unfractionated heparin (UFH) or fondaparinux in therapeutic doses until an INR > 2 is attained (recommendation 1A). Venous catheder application (VCA) therapy starts on the first day of treatment (1A). The target INR using VCA in venous thrombosis or embolism is in the range of 2.5 (2.0–3.0). For the possible use of dabigatran etexilate, see Tab. 16.28-6 – Mode of action, clinical significance and monitoring of anticoagulants.

A risk/benefit analysis should be performed on a regular basis in patients on unlimited-duration anticoagulant therapy (1C). Immediate fibrinolytic therapy is indicated in massive, hemodynamically relevant pulmonary embolism (1B). In most cases, the presence of laboratory-detected thrombophilia does not influence the duration of anticoagulation if the guidelines are adhered to.

16.28.10.2 Valvular or structural heart disease

In patients with valvular or structural heart disease receiving VCA therapy, the target INR is in the range of 2.5 (2.0–3.0); exception: mechanical heart valve target INR 3.0 (2.5–3.5) /40/. Evidence of the indication for anti thrombocytic therapy is to be provided, as necessary. Dabigatran etexilate is not permitted in valvular atrial fibrillation.

16.28.10.3 Therapeutic anticoagulation

Refer to:

• Tab. 16.28-8 – Recommended duration of anti thrombotic therapy for venous thromboembolism with vitamin K antagonists and/or for cancer with low-molecular-weight heparin
• Tab. 16.28-9 – Recommended duration of therapeutic anticoagulation with vitamin K antagonists in valvular or structural heart disease
• Tab. 16.28-10 – Pharmacokinetics of anticoagulants
• Tab. 16.28-11 – Parenteral anticoagulant therapy.

16.28.10.4 Severe hemorrhage associated with oral anticoagulants

As expected with all antithrombotic agents, there is an associated increased risk of bleeding complications. Prevalence of total mortality and severe hemorrhage associated with oral anticoagulants was lower in patients treated with DOCA in comparison to VCA therapy /47/.

For the majority of severe bleeding events, the widespread approach is to withdraw the DOAC, then provide supportive measures and watchful waiting with the expectation that the bleeding event will resolve with time. However urgent reversal of anticoagulation may be advantageous in patients with serious or life-threatening bleeding. Idaruczimab has been approved for reversal of anticoagulation in dabigatran-treated patients and adexanet alfa for factor Xa inhibitor treated patients /48/.

In a study /49/ patients with atrial fibrillation (AF) or venous thrombolic disease (VTE) who were treated with a DOAC received ASA without a clear indication. Compared with DOAC monotherapy, concurrent DOAC and ASA use was associated with increased bleeding and hospitalizations but similar observed thrombosis rate.

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