CHAPTER 13: Overview of Hemostasis

Hemostasis is a vital host defense system that has evolved in man and other vertebrates to maintain the integrity of a high-pressure closed circulatory system. Following a break in the vasculature, the formation of a blood clot either prevents hemorrhage or lessens its extent. An exquisitely modulated orchestration of cells and plasma proteins enables clot formation to be rapid, self-limited, and reversible. The classic paradigm of blood coagulation begins with the formation of a platelet thrombus at the site of vessel injury. Initially, platelets adhere to collagen exposed on the denuded surface of the injured vessel. This adhesion event then triggers activation of platelets by collagen, thrombin generated by tissue factor, or both, allowing them to aggregate and form a tight platelet plug that stanches the further egress of blood from the injured vessel. The clot is then reinforced and strengthened by the activation of the coagulation cascade, which results in the deposition of insoluble polymers composed of fibrin. The recruitment of specific inhibitors prevents unwanted extension of the clot beyond the site of injury. The final step, fibrinolysis, enables the vessel to be recanalized, thereby restoring blood flow. Recent studies have shown that these phases are less clearly demarcated in vivo than this idealized scenario would suggest. Indeed, the use of specific fluorescent molecular markers clearly shows that fibrin deposition occurs as the platelet plug is being formed.

This chapter will first present the dynamic sequence of events involved in formation of the platelet thrombus and the initiation, propagation, limitation, and lysis of the fibrin clot. This conceptual framework will guide the discussion of the pathophysiology, diagnosis, and treatment of bleeding and thrombotic disorders in this and the following chapters.

Plasma contains a large, elongated protein called von Willebrand factor (vWF) that forms multimeric polymers with molecular weights as high as 15 million Daltons. As discussed later in this chapter, vWF binds to and transports factor VIII, the protein deficient in patients with classic hemophilia. In addition, vWF fulfills a vital role in the formation of the primary platelet plug. It has binding sites for collagen as well as for a glycoprotein complex on the surface of platelets, GPIb-IX-V (hereafter referred to simply as the GPIb complex). Under high shear stresses, such as occur within flowing blood, vWF "unwinds" to an extended conformation in which it can bind to both platelet GPIb complexes and to collagen exposed by damage to the blood vessel. Thus, vWF is a kind of molecular "glue" that allows platelets to adhere to injured vessel walls.

The platelet is activated by adhesion as well as by thrombin generated by tissue factor. Thrombin activates platelets by cleaving the platelet thrombin "receptor" in its extracellular domain, causing a conformational change that transduces an activating signal. As depicted in Figure 13-1A, activation entails a sudden and irreversible change in platelet shape from a smooth disc to a spiky "sea urchin" with multiple pseudopods, markedly increasing the surface area of the platelet. Activation also leads to a conformational change in another platelet membrane glycoprotein, GPIIb/IIIa, which allows it to bind fibrinogen. Fibrinogen is bivalent and can therefore form bridges between GPIIb/IIIa receptors on two different platelets. This cross-linking event is the crucial step in platelet aggregation and the propagation of the primary platelet plug.

In addition to adhesion and thrombin, platelet activation can be triggered by other physiologically relevant stimuli, such as adenosine diphosphate (ADP). ADP is one of a number of molecules released from the dense granules of activated platelets. In the local milieu of the platelet plug, ADP binds to receptors on nearby unstimulated platelets and causes their activation. This is one of several amplification loops that ramp-up the hemostatic process in the immediate vicinity of a vascular injury, enabling rapid clot formation.

Activation of platelets also results in the release of arachidonic acid, a long-chain polyunsaturated fatty acid, from phospholipids on the plasma membrane. Arachidonic acid is converted to thromboxane A₂, a potent inducer of platelet aggregation, by a series of enzymes including cyclooxygenase. Inhibition of platelet cyclooxygenase explains the ability of aspirin to suppress platelet activation.

When platelets are activated, another morphologically distinct organelle, the alpha granule, releases proteins that contribute to hemostasis, including platelet factor 4, thrombospondin, fibrinogen, and factor V. In addition, P-selectin in the alpha granule membrane is translocated to the surface plasma membrane of the activated platelet where it plays an important role in the interaction of platelets with other cells, including leukocytes and the vascular endothelium. These processes provide an important link to the soluble coagulation system. The factor Xa receptor, defined by phospholipid on the surface of the activated platelet, cooperates with factor V in the generation of thrombin, as shown in Figure 13-1B and discussed in more detail in the following section.

Just as the formation of the platelet plug is a highly cooperative process, so is the concomitant formation of the fibrin clot. Amplification of a small signal (vascular injury) is transduced by a series of molecular interactions into a fibrin clot. The protagonists in this dramatic process are a set of plasma proteins that normally circulate as inactive zymogens. Figure 13-2 depicts a prototypical or consensus pathway. When the coagulation cascade is activated, either in vivo or in the test tube, a zymogen is converted into an active proteolytic enzyme capable of specifically cleaving the next zymogen in the set, and so on. This highly controlled limited proteolysis leads to the eventual formation of thrombin and generation of the fibrin clot.

During the last century, biochemical studies of in vitro clot formation have gradually led to the identification of the specific proteins that participate in the coagulation cascade, along with in-depth understanding of how they interact in a precisely controlled and orchestrated fashion. A substantial portion of this cumulative knowledge has come from rigorous investigation of families with inherited defects of specific coagulation proteins (see Chapter 15).

Figure 13-3 depicts the coagulation proteins involved in clot formation prior to the formation of fibrin. The zymogens, shown in image A (prothrombin and factors VII, IX, X, and XI), are synthesized in the liver. As this figure demonstrates, they have some striking structural similarities. All have a signal peptide at the N-terminus, and except for factor XI, a propeptide is immediately adjacent. These N-terminal portions are cleaved within the hepatocyte prior to the release of the mature zymogen into the circulation. All five zymogens have a large catalytic domain at the C-terminus that, when activated, functions as a serine protease that specifically cleaves and activates the next protein in the coagulation cascade. Four of these zymogens, factors VII, IX, X, and prothrombin, undergo a crucial post-translational modification in the Golgi body prior to secretion into the plasma. Specific glutamic acid residues in the region adjacent to the propeptide undergo vitamin K–dependent carboxylation. This additional negative charge facilitates the binding of calcium ions, an essential cofactor for optimal function.

Protein cofactors play an equally important role in the regulation of the coagulation cascade. They bind to platelet and endothelial cell membranes, serving as docking sites for the zymogens and contributing importantly to the amplification that is essential for the rapid formation of the fibrin clot. As shown in Figure 13-3B, tissue factor is a transmembrane protein on the surface of extravascular cells, cells within the vessel wall, and circulating microparticles. Like the zymogens mentioned earlier, factors VIII and V are actually pro-cofactors—soluble plasma proteins that require proteolytic cleavage for activation.

In the test tube, the formation of the fibrin clot proceeds by the intrinsic pathway depicted in Figure 13-4. Exposure of the plasma to the glass surface of the tube triggers the activation of factor XII to XIIa (the a stands for activated), which in turn, with the participation of high molecular weight kininogen, activates factor XI to factor XIa. Factor XIa, in the presence of calcium ions, catalyzes the activation of factor IX to factor IXa. Factor IXa forms a complex with activated factor VIII (factor VIIIa) on membrane surfaces in the presence of calcium ions to trigger the activation of factor X to factor Xa.

According to conventional wisdom, the extrinsic pathway depicted in Figure 13-4 is responsible for the initiation of the coagulation cascade in vivo. Tissue factor is constitutively expressed on cells within blood vessels. Upon vessel injury, tissue factor is exposed and binds to minute amounts of activated factor VII (factor VIIa) that are normally present in plasma. The procoagulant complex factor VIIa/tissue factor then initiates the coagulation cascade by converting factor X to factor Xa. As Figure 13-4 shows, this is the point at which the extrinsic and intrinsic pathways converge.

However, in vivo coagulation is more complicated. Low concentrations of tissue factor may be concentrated in the thrombus early in its development. The model that best reflects in vivo coagulation in humans is depicted in Figure 13-5. As mentioned earlier, the primary initiating trigger is the exposure of tissue factor on the injured blood vessel. Tissue factor, along with phosphatidylserine on the plasma membrane of platelets and endothelial cells, forms a complex with activated factor VII (factor VIIa), which not only catalyzes the activation of factor X but also the activation of factor IX. The latter event is of little importance in the test tube but is critical in vivo, because individuals with deficiencies of factor IX or its cofactor, factor VIII, suffer from hemophilia (Chapter 15), a severe bleeding disorder.

At each successive step in the bottom of the cascade, the concentration of the respective zymogen increases, thus favoring amplification of clot formation. It follows then that by far the most abundant coagulation protein in the plasma is the structural protein fibrinogen, which is polymerized to form fibrin in the final step in the cascade. As shown in Figure 13-6A, fibrinogen is a heterodimer composed of a pair of three subunits, Aα, Bβ, and γ, linked by disulfide bonds. Upon cleavage by thrombin (factor IIa), small fibrinopeptides, designated fibrinopeptide A and fibrinopeptide B, are released from the Aα and Bβ subunits, respectively. Measurement of fibrinopeptide A reflects the conversion of fibrinogen to fibrin and can be useful in monitoring patients with disseminated intravascular coagulation (Chapter 16). Upon cleavage, fibrinogen undergoes a conformational change that enables it to form noncovalent polymers via end-to-end interactions as well as side-to-side interactions between the linear strands. The strength and durability of the fibrin polymer is then greatly enhanced by the action of factor XIIIa, which catalyzes the formation of covalent crosslinks between the fibrin strands.

Thus far, this chapter has emphasized the importance of rapid clot formation and propagation in the cessation of bleeding. However, it is equally important that coagulation be limited to the site of injury, because its continued extension to adjacent normal vessels could occlude blood flow, with possibly disastrous consequences. Thus, during evolution, molecules and pathways have emerged that limit the extent of clot formation (summarized in Table 13-1).

• Tissue factor pathway inhibitor (TFPI) is synthesized in endothelial cells and is present in plasma and platelets. It blocks the extrinsic coagulation pathway after its initial activation by binding first to factor Xa. This binary factor Xa/ TFPI complex then docks onto and inactivates factor VIIa/tissue factor complexes, shutting down the extrinsic pathway after a small amount of factor Xa, and subsequently thrombin, has been generated. The intrinsic pathway provides a burst of procoagulant activity once factor VIIIa and factor Va are generated by the action of thrombin.

• Antithrombin (also referred to as antithrombin III) is the most important of a number of plasma protease inhibitors in regulating hemostasis. Antithrombin is also known as heparin cofactor. Upon binding to endogenous glycoconjugates on the surface of endothelial cells or the anticoagulant heparin, antithrombin changes its conformation and becomes a serine protease inhibitor (serpin) that inactivates not only thrombin but also factors VIIa, IXa, Xa, and XIa. Antithrombin is a scavenger that inactivates coagulation factors as they are carried in the blood away from the developing thrombus.

• Protein C, in the presence of protein S, is activated by thrombin bound to thrombomodulin on the surface of normal endothelial cells. Protein C and protein S undergo vitamin K-dependent carboxylation and share other structural features with factors VII, IX, X, and prothrombin (see Fig. 13-3). Activated protein C attenuates the coagulation cascade by specifically inactivating factors Va and VIIIa.

• The fibrinolytic pathway promotes dissolution of unwanted clot in adjacent non-injured tissues as well as eventual removal of clot during and following healing of injured tissues, thereby restoring blood flow. Fibrinolysis is triggered by tissue plasminogen activator and urokinase-type plasminogen activator, enzymes that convert the zymogen plasminogen into plasmin, a proteolytic enzyme that degrades fibrin.

These mechanisms for limiting blood coagulation often go awry in hereditary thrombotic disorders, discussed in detail in Chapter 17, and are the targets of several important anticoagulant therapies described later in this chapter.

The platelet count (number of platelets per mm³) is a routine part of a complete blood count. Purpura (superficial bleeding beneath the skin), mucosal bleeding, and other types of hemorrhage can occur if the platelet count is too low. In addition, bleeding can be caused by congenital or acquired defects in platelet function. This topic is covered in the next chapter (Chapter 14).

Occasionally, laboratory assessment of platelet function provides useful information. The bleeding time is determined by making a controlled superficial incision on the forearm and monitoring the time that elapses before the bleeding stops. As shown in Figure 13-7, if the platelets are functionally normal, the bleeding time becomes progressively prolonged as the platelet count falls below 100,000/mm³. In contrast, in individuals with abnormal platelet function, such as those with uremia, von Willebrand disease (Chapter 15), or in those who have taken aspirin, the bleeding time is prolonged even though the platelet count is normal. Although the bleeding time has a sound pathophysiologic rationale, the test is cumbersome to set up and is subject to considerable variability and poor reproducibility. For these reasons, it is seldom used.

In contrast, in vitro assessment of platelet aggregation offers highly reliable, albeit limited, information about platelet function and is therefore widely used in the evaluation of patients with bleeding disorders. Platelet-rich plasma is prepared from freshly drawn blood. Following the addition of an agonist, platelet GPIIb/IIIa receptors become competent to bind fibrinogen, which, in turn, promotes platelet aggregation. As platelets clump, the turbidity of the plasma decreases, allowing the process to be followed by increases in light transmission. The time course of normal platelet aggregation in response to agonists (ADP, epinephrine, collagen, and ristocetin) is shown in Figure 13-8. When used at the proper concentration, the first two of these agents trigger release of endogenous ADP from platelet-dense granules in a small subset of platelets; this ADP then activates the remaining platelets, inducing a second wave of platelet aggregation. In the presence of the antibiotic ristocetin, vWF in plasma binds to normal platelets and induces platelet agglutination. Aggregation requires calcium mobilization and the activation of the fibrinogen receptor, whereas agglutination merely depends on passive cross-linking of platelets mediated by bivalent binding of vWF to GPIb complexes and therefore occurs even with platelets that have been fixed with formalin. Ristocetin-induced agglutination is absent or attenuated in individuals with either von Willebrand disease or a deficiency of GPIb complexes, the binding site on platelets for vWF.

In contrast to the earlier-mentioned platelet function tests that are used only in special circumstances, laboratory assessment of the coagulation cascade is crucial in the diagnosis and monitoring of a broad range of medical, surgical, and obstetric patients with a variety of common and sometimes life-threatening disorders. The standard initial assessment includes the platelet count (discussed earlier), prothrombin time (PT), and partial thromboplastin time (PTT).

As shown in Figure 13-9, the PT is performed by the addition of recombinant tissue factor to plasma. Thus, the PT assesses the extrinsic pathway of coagulation. Normally, the plasma suddenly clots after about 12 seconds. A prolongation in the PT means that the patient has a functional deficiency of one or more of the clotting factors in the extrinsic pathway, specifically factors VII, X, V, prothrombin, or fibrinogen. In many laboratories and clinics, the report includes the international normalized ratio (INR), a standardization of the PT that takes into account the potency of the tissue factor employed.

In contrast, the PTT assesses the intrinsic pathway of coagulation. Kaolin, a porous diatomaceous earth, is added to plasma, providing an abundant artificial contact surface that initiates the cascade by activating factor XII. Figure 13-10 shows the clotting factors that participate in the clot formed in the PTT: factors XII, XI, IX, VIII, X, V, prothrombin, and fibrinogen.

The thrombin time (TT) is the time required for a clot to form after the addition of thrombin to plasma. Thus, it assesses the amount and functionality of fibrinogen as well as the presence of thrombin inhibitors such as the anticoagulant heparin (see page 155) or breakdown products of fibrinogen and fibrin, called fibrin degradation products, that interfere with fibrin formation.

The interpretation of the results of the PT, PTT, and TT can be viewed as a matrix, shown in Table 13-2. The combination of the PT, PTT, and TT is very useful in narrowing down what factor or factors are defective or deficient. Armed with this information, the laboratory can then perform specific factor assays, often by mixing the patient's plasma with plasma that is known to be deficient in a single clotting factor. In almost all of the inherited bleeding disorders (Chapter 15), only a single deficiency will be found.¹ In contrast, patients with acquired bleeding problems (Chapter 16) due to liver disease, vitamin K deficiency, or disseminated intravascular coagulation will have multiple deficiencies.

In addition to single factor assays, a number of other tests are commonly used to determine the cause of bleeding disorders.

• Assessment of fibrinogen, a normally abundant plasma protein that can be quantified by immunologic assay or tested functionally by the thrombin time.

• Measurement of fibrinogen/fibrin degradation products and D-dimers is useful in documenting the fibrinolytic process that occurs in the aftermath of clotting in vivo, such as in patients with disseminated intravascular coagulation (discussed in Chapter 16) or deep venous thrombosis (Chapter 17).

• Assessment of vWF function (often referred to as a vWF panel). These include: 1) measurement of factor VIII activity; 2) immunologic quantification of vWF, which carries factor VIII in the blood; and 3) ristocetin-induced platelet agglutination assays, which assess vWF function. This panel is particularly useful in the diagnosis of von Willebrand disease (Chapter 15).

• Mixing studies are used to detect the presence of inhibitors to blood clotting factors. For example, to evaluate a patient with a prolonged PTT, the patient's plasma is mixed with an equal volume of normal plasma, and the PTT test is repeated. Clotting factor levels that are >25% of normal are sufficient to give a normal PTT; therefore, if the patient has a simple deficiency in a clotting factor, the PTT of the mixture will be normal. In contrast, the PTT will be prolonged if the patient's plasma contains an inhibitor, such as an antibody, capable of inactivating a clotting factor in both the patient's plasma and the admixed normal plasma.

The application and interpretation of these tests in different clinical settings will be covered in Chapters 14 through 17.

Patients with bleeding and thrombotic disorders are effectively treated with a variety of modalities including specific factor replacement, transfusions of red cells, platelets and plasma, and a wide range of drugs. These applications will be covered in the four chapters that follow. However, it is fitting that this chapter conclude with a consideration of anticoagulant and antifibrinolytic drugs because they are widely used in a number of commonly encountered disorders, and the mechanisms underlying their efficacy are based on the coagulation pathways described earlier.

• Vitamin K antagonists. Because carboxylation of factors VII, IX, X, and prothrombin is required for hemostatic effectiveness, blocking of this vitamin K-dependent post-translational modification has proven to be an effective way to retard blood clotting in vivo. The most commonly used vitamin K antagonist is warfarin, which is administered daily by mouth. Patients treated with warfarin must be monitored closely with regular measurements of the PT to ensure that warfarin levels are in a therapeutic range, because insufficient anticoagulation is ineffective in preventing thrombosis, whereas excessive anticoagulation increases the risk of serious bleeding.

• Heparin and heparin-like drugs. As explained in the previous section, polysaccharides, particularly heparin, activate the serine protease inhibitor antithrombin thereby inhibiting several steps in the coagulation cascade both in vivo and in vitro. Heparin must be administered parenterally, but patients do not need to be monitored as closely as those on warfarin. Unfractionated heparin occasionally causes a potentially serious complication, heparin-induced thrombocytopenia, owing to an autoantibody against a complex of the drug and platelet factor 4 released from alpha granules. This immune response activates platelets, commonly causing thrombocytopenia and, in some patients, life-threatening venous and/or arterial thrombosis (see Chapter 17).

• Direct thrombin inhibitors. Knowledge of the three-dimensional structure of thrombin and the domain responsible for its proteolytic activity has led to the design and development of drugs such as argatroban that bind with high affinity and specificity to thrombin's active site. Argatroban is particularly useful in patients who cannot be anticoagulated with heparin because of the development of heparin-induced thrombocytopenia.

• Fibrinolytic drugs. Agents that activate the fibrinolytic pathway are often remarkably effective in opening vessel lumens occluded by thrombus and re-establishing blood flow. Initially, the proteolytic enzymes streptokinase and urokinase were used. More recently, recombinant tissue plasminogen activator has been shown to have superior efficacy. These therapies carry with them the risk of hemorrhage.