Chapter 35 Part 1 PDF

662 PART VI Hemostasis and Thrombosis CASE STUDY After studying the material in this chapter, the reader should be able to respond 1. Is this hemostatic disorder typical of an acquired or an inherited condition? to the following case study. Answers can be found in Appendix C....

662 PART VI Hemostasis and Thrombosis CASE STUDY After studying the material in this chapter, the reader should be able to respond 1. Is this hemostatic disorder typical of an acquired or an inherited condition? to the following case study. Answers can be found in Appendix C. 2. Are these symptoms most likely caused by a deciency of a procoagulant or A pregnant woman developed a blood clot in her left leg (deep vein thrombosis an inhibitor? [DVT]). Her mother reportedly had a history of thrombophlebitis. She had a brother who was diagnosed with DVT after a ight from Los Angeles to Sydney, Australia. Hemostasis is a complex physiologic process that keeps blood and collagen. ey subsequently become activated, which pro- circulating in a uid state until an injury occurs, then causes motes secretion of the contents of their granules, aggregation production of a blood clot to stop the bleeding, connes the with other platelets via brinogen, VWF, and bronectin, and blood clot to the site of injury, and nally dissolves the blood formation of a platelet plug. Vasoconstriction and platelet plug clot as the wound heals. When hemostasis systems are out of formation make up the initial, rapid, and short-lived response to balance, either hemorrhage (uncontrolled bleeding) or thrombo- vessel damage. However, to control major bleeding in the long sis (pathologic clotting) can occur, which can be life-threatening. term, the platelet plug must be reinforced by brin. Defects in e absence of a single plasma procoagulant protein may cause primary hemostasis such as collagen abnormalities, thrombo- the individual to experience lifelong anatomic hemorrhage and cytopenia, qualitative platelet disorders, or von Willebrand dis- transfusion dependence. Conversely, absence of an anticoagu- ease can cause debilitating, sometimes fatal, chronic bleeding. lant protein allows coagulation to proceed unchecked and can Secondary hemostasis (see Table 35.1) describes the activation result in thrombosis, including myocardial infarction, stroke, of a series of coagulation proteins in the plasma, mostly serine pulmonary embolism, and deep vein thrombosis. proteases, to form a brin clot. ese proteins circulate as inactive Understanding the major systems of hemostasis—blood ves- zymogens (proenzymes), becoming activated during the process of sels, platelets, and plasma proteins—is essential to interpreting coagulation and, in turn, form complexes that activate other zymo- laboratory test results, along with being able to prevent, predict, gens. is pathway ultimately leads to the generation of thrombin, diagnose, and manage hemostatic disease. an enzyme that converts brinogen to brin. is allows for forma- tion of a localized brin clot, which is stabilized by factor XIII. e OVERVIEW OF HEMOSTASIS nal event of hemostasis is brinolysis, the gradual digestion and removal of the brin clot, which occurs as the injury heals.2 Hemostasis involves the interaction of vasoconstriction, plate- Although the various components of the coagulation sys- let adhesion and aggregation, and coagulation enzyme activa- tem are divided between primary and secondary hemostasis for tion to stop bleeding. e coagulation system, similar to other improved understanding and compartmentalization, they are humoral amplication mechanisms, is complex because it associated with each other during early and late-stage hemo- translates a diminutive physical or chemical stimulus into a pro- static events. For example, platelets and vascular ECs, which found lifesaving event.1 e key cellular elements of hemostasis are key components of primary hemostasis, secrete coagula- are the endothelial cells (ECs) of the vascular intima, extravas- tion factors stored in their granules and Weibel-Palade bodies, cular tissue factor (TF)–bearing cells, and platelets. e plasma respectively. In addition, platelets provide the essential cell components include the coagulation and brinolytic proteins membrane phospholipid on which coagulation complexes and their inhibitors. form. Conversely, coagulation proteins, particularly VWF and Primary hemostasis (Table 35.1) refers to the role of blood brinogen, are involved in platelet adhesion and aggregation, vessels and platelets in the initial response to a vascular injury respectively. roughout this chapter the vascular intima, plate- or to the commonplace desquamation of dying or damaged lets, coagulation factors, brinolysis proteins, and control pro- ECs. Blood vessels contract to seal the wound or reduce the teins will be detailed, illustrating how they function to promote blood ow (vasoconstriction). Platelets adhere to the damaged normal hemostasis. vessel wall by interactions with von Willebrand factor (VWF) TABLE 35.1 Primary and Secondary VASCULAR INTIMA IN HEMOSTASIS Hemostasis e vasculature consists of the blood vessels, including arter- Stage Primary Hemostasis Secondary Hemostasis ies, veins, and capillaries, carrying blood throughout the body. Activation Desquamation and small Large injuries to blood vessels A blood vessel is structured into three layers: an inner layer injuries to blood vessels and surrounding tissues (tunica intima), a middle layer (tunica media), and an outer Components Vascular intima and platelets Platelets and coagulation system layer (tunica adventitia or tunica externa). e tunica intima provides the interface between circulating blood and body tis- Timing Rapid, short-lived response Delayed, long-term response sues. is innermost lining of blood vessels is a monolayer of Mechanism Procoagulant substances Tissue factor exposed on cell metabolically active ECs (Box 35.1 and Figure 35.1; refer also exposed or released by membranes to Figure 11.9A).3 ECs are complex and heterogeneous cells damaged or activated that are distributed throughout the body. ey display unique endothelial cells structural and functional characteristics, depending on their  CHAPTER 35 Normal Hemostasis 663 environment and physiologic requirements, not only in subsets Anticoagulant Properties of Intact Vascular Intima of blood vessels such as arteries versus veins but also in the var- Intact vascular endothelium prevents thrombosis by numerous ious tissues and organs of the body.4 5 ECs play essential roles in anticoagulant mechanisms, which are specied in Table 35.2 immune response, vascular permeability, proliferation, and, of and see Figure 35.1. e rst mechanism of intact ECs is their course, hemostasis. physical presence. ECs are rhomboid and contiguous, provid- ECs form a smooth, unbroken surface allowing for nontur- ing a smooth inner surface of the blood vessel promoting non- bulent blood ow. e ECs are supported by a basement mem- turbulent blood ow, which prevents activation of platelets and brane and an elastin-rich internal elastic lamina. Arteries have coagulation enzymes. ECs form a physical barrier, separating an additional elastin-rich external lamina component. In all blood vessels, broblasts (important for maintenance, tissue TABLE 35.2 Anticoagulant Properties of metabolism, and structural framework) occupy the connective Intact Vascular Endothelium tissue layer, where they produce collagen. Smooth muscle cells present in the walls of all blood vessels, in much larger numbers Endothelial Cell Structure/ Substance Anticoagulant Property in arteries than veins and only occasionally in capillaries, pro- mote contraction when an injury occurs and primary hemosta- Composed of rhomboid cells Present a smooth, contiguous surface sis is initiated. Secrete prostacyclin The eicosanoid platelet inhibitor Secrete nitric oxide A vascular “relaxing” factor Secrete the glycosaminoglycan An anticoagulant that regulates BOX 35.1 Vascular Intima of the Blood heparan sulfate thrombin generation Vessel Secrete TFPI A regulator of the extrinsic pathway Innermost Vascular Lining of coagulation Endothelial cells (endothelium) Express the protein C receptor An integral component of the protein EPCR C control system Supporting the Endothelial Cells Internal elastic lamina composed of elastin and collagen Express cell membrane A protein C coagulation control thrombomodulin system activator Subendothelial Connective Tissue Secrete TPA Activates brinolysis Collagen and broblasts in veins Collagen, broblasts, and smooth muscle cells in arteries EPCR, Endothelial protein C receptor; TFPI, tissue factor pathway inhibitor; TPA, tissue plasminogen activator. Anticoagulant Functions of Intact Endothelial Cells Smooth, Nitric Heparan Thrombo- TPA EC lining oxide sulfate modulin PGI2 TFPI EPCR ECs Platelets Coagulation Fibrinolysis VWF P-Selectin PAI-1 Vasocon- Collagen TF striction ADAMTS13 exposed exposed TAFI Procoagulant Functions of Damaged Endothelial Cells Figure 35.1 Hemostatic Properties of Endothelial Cells that Line the Inner Surface of All Blood Vessels. Depicted in this gure are the anticoagulant properties associated with normal intact endothelial cells (top) and the procoagulant properties associated with damaged endothelial cells (bottom) as they relate to the functions of the hemostatic system listed in the center. ADAMTS13, A disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13; ECs, endothelial cells; EPCR, endothelial cell protein C recep- tor; PAI-1, plasminogen activator inhibitor-1; PGI2 prostacyclin or prostaglandin I2; TAFI, thrombin activatable brinolysis inhibitor; TF, tissue factor; TFPI, tissue factor pathway inhibitor; TPA, tissue plasminogen activator; VWF, von Willebrand factor. 664 PART VI Hemostasis and Thrombosis platelets in blood from the collagen in the vascular intima that TABLE 35.3 Procoagulant Properties of promotes platelet adhesion. e EC barrier also separates cir- Damaged Vascular Intima culating procoagulant proteins from TF, present on underlying Structure/Substance Procoagulant Property broblasts and smooth muscle cells, that activates coagulation. Smooth muscle cells in arterioles Induce vasoconstriction e ECs are covered with carbohydrates, known as a glycocalyx, and arteries consisting of proteoglycans and glycoproteins that have a neg- ative charge. e negative charge repels cellular components, Exposed subendothelial collagen Binds VWF; binds to and activates platelets preventing binding to the adhesion molecules present on ECs.6 Damaged or activated ECs secrete Important for platelet binding to collagen e second anticoagulant mechanism relates to the variety VWF at site of injury: platelet adhesion as a of substances synthesized and secreted by ECs. Prostacyclin rst line of defense against bleeding (PGI2) is synthesized through the eicosanoid pathway (Chapter Damaged or activated ECs secrete Promote platelet and leukocyte binding 11) and prevents unnecessary or undesirable platelet activa- adhesion molecules: P-selectin, and activation at site of injury tion in intact vessels by acting as an antagonist of thrombox- ICAMs, PECAMs ane A2 (TXA2).7 8 In addition, PGI2 promotes vasodilation Exposed smooth muscle cells and Tissue factor exposed on cell membranes through a protein kinase A mechanism, leading to inhibition broblasts of the myosin light chain kinase with subsequent smooth mus- ECs in inammation Tissue factor is induced by inammation cle cell relaxation.8 Nitric oxide is synthesized in ECs, vascu- lar smooth muscle cells, neutrophils, and macrophages. Nitric ECs, Endothelial cells; ICAMs, intercellular adhesion molecules; PECAMs, platelet endothelial cell adhesion molecules; VWF, von oxide induces smooth muscle relaxation with subsequent vaso- Willebrand factor. dilation and inhibits platelet activation through a guanylate cyclase-dependent mechanism, while also being able to pro- to binding of circulating VWF, which causes binding and mote angiogenesis in healthy arterioles through vascular endo- activation of platelets (Figure 11.9B and 11.9C). Platelets sub- thelial growth factor (VEGF) and basic broblast growth factor sequently bind to the collagen through their GPVI and α2β1 (bFGF).8-10 An important EC-produced anticoagulant is tissue receptors and adhere to the damaged area until new ECs grow. factor pathway inhibitor (TFPI), which controls activation of roughout an individual’s life span, connective tissue nat- the TF pathway, also called the extrinsic coagulation pathway. urally degenerates, which leads to an increased tendency to TFPI limits the activation of the TF:VIIa:Xa complex, thereby bruise in the elderly. limiting thrombin generation. ird, ECs secrete VWF from storage sites called Weibel- Finally, ECs synthesize and express on their surfaces two Palade bodies when activated by vasoactive agents such as known inhibitors of thrombin function, thrombomodulin and thrombin. VWF is a large multimeric glycoprotein that acts heparan sulfate. rombomodulin promotes activation of the as the necessary bridge that binds platelets to exposed suben- coagulation inhibitor protein C and subsequent anticoagula- dothelial collagen in arterioles and arteries where blood ows tion. Heparan sulfate is a glycosaminoglycan that enhances the rapidly (Figure 11.9C and 11.9D).12 VWF has been described as activity of antithrombin, a blood plasma serine protease inhib- a “carpet” on which activated platelets assemble. ADAMTS13, itor.11 e pharmaceutical anticoagulant heparin, an important also secreted from ECs, serves an important function as it therapeutic agent used for many clinical indications, resembles cleaves large VWF multimers into shorter chains that support EC heparan sulfate in both its structure and its inhibitory activ- normal platelet adhesion. ity when bound to antithrombin. Fourth, on activation, ECs secrete and coat themselves with P-selectin, an adhesion molecule that promotes platelet and leu- Procoagulant Properties of Damaged Vascular kocyte binding.13 ECs also secrete immunoglobulin-like adhe- Intima sion molecules called intercellular adhesion molecules (ICAMs) Although the intact endothelium has anticoagulant properties, and platelet EC adhesion molecules (PECAMs), which further when damaged, the tunica intima (ECs and the subendothelial promote platelet and leukocyte binding.14 matrix) promotes coagulation through several procoagulant prop- Finally, subendothelial smooth muscle cells and broblasts erties (Table 35.3 and see Figure 35.1). First, any harmful local support the constitutive membrane protein TF.15 EC disruption stimulus, whether mechanical or chemical, induces vasocon- exposes TF to circulating blood, promoting activation of the striction in arteries and arterioles where blood pressure is higher coagulation system through contact with plasma coagulation than on the venous side (Figure 11.9B). Smooth muscle cells factor VII, which ultimately leads to brin formation (Figure contract, leading to narrowing of the vascular lumen, followed 35.2). e formed brin surrounds the platelet plug, securing it by decreased blood ow through the injured site. As veins and to the damaged area such that the blood ow does not dislodge capillaries do not have as many smooth muscle cells to contribute the platelet plug. to vasoconstriction, blood can escape into surrounding tissues, In arterioles and arteries, the larger VWF multimers form a creating extravascular pressure on the blood vessel and causing brillar carpet on which the platelets assemble because of the compression, which eectively minimizes the escape of blood. high blood ow; a white clot consisting of essentially platelets, Second, the subendothelial connective tissues of arteries brin, and VWF is produced.16 In veins, because of the slower and veins are rich in collagen, a exible, elastic structural pro- blood ow, a bulky red clot is produced, consisting of mostly red tein. Upon injury to the vessel, collagen is exposed, leading blood cells and brin, with some platelets.16  CHAPTER 35 Normal Hemostasis 665 Fibroblast Smooth muscle cell Endothelial cells Platelet HS TFPI Fib White blood cell Blood flow Red blood cell TM Collagen External elastic lamina von Willebrand factor Figure 35.2 Secondary Hemostasis. Immediately after the initial response to vessel injury of platelet activa- tion, adhesion, and aggregation (primary hemostasis), as detailed in Chapter 11 and Figure 11.9, the continued response of an injured blood vessel leads to the formation of a brin clot that stabilizes the initial platelet plug, as shown in this artist’s rendering. Primary hemostasis requires interaction of platelets with subendothelial VWF and collagen. Secondary hemostasis requires exposure of TF from damaged endothelial cells and phos- pholipids from activated platelets that promotes activation of the coagulation cascade, thrombin generation, and brin formation that polymerizes around the platelet aggregate. Fib, Fibrinogen; HS, heparan sulfate; TF, tissue factor, TFPI, tissue factor pathway inhibitor (EC bound and soluble forms exist); TM, thrombomodulin (EC bound and soluble forms exist). Fibrinolytic Properties of Vascular Intima Platelets serve as one of the body’s rst lines of defense rough the secretion of tissue plasminogen activator (TPA), against blood loss. At the time of injury, platelets adhere to the ECs support brinolysis (see Table 35.2 and see Figure 35.1), the site of injury, aggregate, and secrete the contents of their gran- breakdown of brin, leading to thrombus degradation and res- ules (Table 35.4 and Figure 11.9).22 23 Adhesion is the property toration of vessel patency. TPA activates brinolysis by convert- by which platelets bind to nonplatelet surfaces such as sub- ing plasminogen to the primary brinolytic enzyme plasmin, endothelial collagen and VWF. VWF links platelets to colla- which gradually digests brin and restores blood ow. ECs also gen through their GPIb/IX/V membrane receptors in areas of regulate brinolysis by providing inhibitors to prevent excessive high shear stress such as arteries and arterioles.24 In damaged plasmin generation. One of the brinolytic inhibitors secreted veins and capillaries, platelets may bind directly to collagen via by ECs, as well as by other cells, is plasminogen activator inhib- GPVI and α2β1 receptors. e importance of platelet adhesion itor-1 (PAI-1).17 Another inhibitor of plasmin generation is is underscored by bleeding disorders such as Bernard-Soulier thrombin activatable brinolysis inhibitor (TAFI), which is acti- syndrome (Chapter 37), in which the platelet GPIb/IX/V recep- vated by thrombin bound to EC membrane thrombomodulin.18 tor is absent, and von Willebrand disease (Chapter 36), in which Although the signicance of the tunica intima in hemosta- VWF is missing or defective. sis is well recognized, there are few valid laboratory methods Aggregation is the property by which platelets bind to one to assess the integrity of ECs, smooth muscle cells, broblasts, another. When platelets are activated, a change in the GPIIb/ and their collagen matrix.19 Invasive blood vessel biopsies are IIIa receptor allows binding of brinogen as well as VWF and not performed. us the diagnosis of blood vessel disorders is bronectin.25 Fibrinogen binds to GPIIb/IIIa receptors on adja- based on clinical symptoms, family history, and indirectly by cent platelets and joins them together in the presence of ionized laboratory tests that rule out platelet or coagulation disorders. calcium (Ca2+). Fibrinogen binding is essential for platelet aggre- gation as evidenced by bleeding and compromised aggregation in patients with abrinogenemia or in patients who lack the PLATELETS GPIIb/IIIa receptor (Glanzmann thrombasthenia; Chapter 37). Platelets are produced from the cytoplasm of bone marrow Platelets secrete the contents of their α-granules and dense megakaryocytes.20 Although platelets are only 2 to 3 μm in granules during adhesion and aggregation, with most secretion diameter on a xed, stained peripheral blood lm, they are occurring late in the platelet activation process. Platelets secrete complex, metabolically active cells that interact with their envi- procoagulants (e.g., factor V, VWF, factor VIII, brinogen) as ronment and initiate and control hemostasis.21 Chapter 11 pro- well as control proteins (e.g., protein S, TFPI, antithrombin, vides an in-depth description of platelet structure and function. C1-inhibitor), along with Ca2+, adenosine diphosphate (ADP) An overview of the platelet functions critical in the initial stages and other hemostatic molecules (Table 35.5).26 27 Although of hemostatic control is given in this chapter. these substances participate in thrombosis and hemostasis, the 666 PART VI Hemostasis and Thrombosis TABLE 35.4 Platelet Function phosphatidylserine, some procoagulant factors, and receptors. e importance of these substances in the blood clotting pro- Function Mechanism of Action Characteristics cess, as well as how they serve to integrate platelet function with Adhesion Platelets roll and cling to Reversible; seals endothelial activation of the plasma coagulation system, will be described nonplatelet surfaces gaps, some secretion of in this chapter. growth factors, in arterioles Erythrocytes, monocytes, and lymphocytes also participate VWF is necessary for adhesion in hemostasis. Erythrocytes add bulk and structural integ- Aggregation Platelets adhere to each Irreversible; platelet plugs rity to the brin clot. Interestingly, those with anemia have an other form, platelet contents are increased tendency to bleed, likely in part because of the lack secreted, requires brinogen of movement of platelets to the vessel wall (marginalization) by Secretion Platelets release the Irreversible; occurs during erythrocytes and the decreased number of erythrocytes able to contents of their granules aggregation, platelet be incorporated into the thrombus.30 In inammatory condi- contents are secreted, tions, monocytes and lymphocytes, and possibly ECs, provide essential to coagulation surface-borne TF that may trigger coagulation. Leukocytes also VWF, von Willebrand factor. have a series of membrane integrins and selectins that bind adhesion molecules on platelets and other cells and help stimu- late the production of inammatory cytokines that promote the TABLE 35.5 Platelet Granule Contents wound healing process and combat infection.31 Platelet α-Granules Platelet Dense Granules (Large Molecules) (Small Molecules) COAGULATION SYSTEM Factors V, VIII, XI, prothrombin, Adenosine diphosphate brinogen Adenosine triphosphate Nomenclature of Procoagulants VWF Ionized calcium Plasma transports at least 16 procoagulants called coagulation HMWK Serotonin factors. Nearly all are glycoproteins synthesized in the liver, Antithrombin, TFPI, protein S Magnesium although monocytes, ECs, and megakaryocytes produce a few C1-inhibitor (control protein of the Potassium (Table 35.6 and Figure 35.3). Eight are enzymes that circulate in contact system) Histamine an inactive form called zymogens. Others are cofactors that bind, Plasminogen PAI-1 stabilize, and enhance the activity of their respective enzymes. Platelet factor 4 Fibrinogen is the substrate for the enzymatic action of throm- Platelet-derived growth factor, other bin, the primary enzyme of the coagulation system. In addition, growth factors there are plasma glycoproteins that act as control proteins that serve the important function of regulating the coagulation pro- HMWK, High-molecular-weight kininogen; PAI-1, plasminogen activator inhibitor-1; TFPI, tissue factor pathway inhibitor; VWF, von Willebrand cess to avoid unnecessary thrombin generation, brin forma- factor. tion, and blood clotting (see Figure 35.3). Reference intervals can be found aer the Index at the end of the book. role of platelets in inammation, atherosclerosis, antimicrobial Fibrinogen and prothrombin (factor II) were known in host defense, wound healing, angiogenesis, and malignancy has 1935; factors V and VII were identied in the period 1945 to become increasingly appreciated as the function of platelets in 1950; and factors IX and X were described in the 1950s.32 In the pathophysiology of these processes is being better dened.27 1958 the International Committee for the Standardization e platelet membrane consists of a phospholipid bilayer. of the Nomenclature of the Blood Clotting Factors ocially During activation, ADP and Ca2+ activate phospholipase A2, named the plasma procoagulants and cofactors using Roman which converts platelet membrane phospholipids into arachi- numerals in the order of their initial description or discovery.33 donic acid (Figure 11.12). Arachidonic acid is converted into the An additional convention adopted was the use of a lowercase a endoperoxides prostaglandin G2 (PGG2) and prostaglandin H2 behind the Roman numeral to designate factors in their acti- (PGH2) by the cyclooxygenase enzyme-1 (COX-1). In the plate- vated state—for instance, activated factor VII is VIIa. let, thromboxane synthetase converts these prostaglandins into ere are multiple factors not usually identied by their TXA2, which causes Ca2+ to be released from the dense tubules, Roman numerals. Factors I and II are customarily called brin- thereby promoting platelet aggregation and vasoconstriction. ogen and prothrombin, respectively, although occasionally they Available Ca2+ (ionized calcium) is critical for normal platelet are identied by their numerals. e numeral III was given to function because it is involved in helping with subsequent phys- tissue thromboplastin, a crude mixture of TF and phospholipid. ical spreading of the platelet to cover the site of injury, along Now that the precise structure of TF has been described, the with promoting secretion of platelet granule content.26 28 numeral designation is seldom used. e numeral IV iden- e membrane of activated platelets is the key surface for tied the plasma cation calcium (Ca2+); however, calcium is coagulation enzyme-cofactor-substrate complex formation (see referred to by its name or chemical symbol, not by its numeral. Figure 35.2), which is the foundation for secondary hemostasis e numeral VI was assigned to a procoagulant that later was to occur.29 Platelets supply Ca2+, the membrane phospholipid determined to be activated factor V; VI was withdrawn from  CHAPTER 35 Normal Hemostasis 667 TABLE 35.6 Properties of the Plasma Procoagulants Molecular Weight Mean Plasma Factor Name Function (Daltons) Half-Life (hr) Concentration† I* Fibrinogen Thrombin substrate, 340,000 100–150 200–400 mg/dL polymerizes to form brin II* Prothrombin Serine protease 71,600 60 10 mg/dL III* Tissue factor Cofactor 44,000 Insoluble None IV* Ionized calcium Mineral 40 NA 8–10 mg/dL V Cofactor 330,000 24 1 mg/dL VII Serine protease 50,000 6 0.05 mg/dL VIII Antihemophilic factor Cofactor 260,000 12 0.01 mg/dL VWF von Willebrand factor Factor VIII carrier and 500,000–20,000,000 24 1 mg/dL platelet adhesion IX Christmas factor Serine protease 57,000 24 0.3 mg/dL X Stuart-Prower factor Serine protease 58,800 48–52 1 mg/dL XI Serine protease 143,000 48–84 0.5 mg/dL XII Hageman factor Serine protease 84,000 48–70 3 mg/dL Prekallikrein Fletcher factor, pre-K Serine protease 85,000 35 35–50μg/mL High-molecular- Fitzgerald factor, HMWK Cofactor 120,000 156 5 mg/dL weight kininogen XIII Fibrin-stabilizing factor (FSF) Transglutaminase, 320,000 150 2 mg/dL transamidase Platelet factor 3 Phospholipids, Assembly molecule — Released by platelets — phosphatidylserine, PF3 *These factors are customarily identied by name rather than Roman numeral. †Clinically, plasma concentration of all coagulation factors, except brinogen, can be given as percentage of normal (%) or units/dL, where the numeric value remains the same. ZYMOGENS Prekallikrein FXII FXI FIX FX FVII Prothrombin FXIII CO-FACTORS CONTROL PROTEINS HMWK Antithrombin Tissue factor Heparin cofactor II FVIII TFPI FV Protein C Protein Z 2 Protein S 1 Thrombomodulin ZPI FIBRINOGEN SUBSTRATE Figure 35.3 Plasma Components of the Coagulation System. Four categories of plasma-based compo- nents of the coagulation system of blood clotting include procoagulants (zymogens), cofactors, anticoagu- lants (regulatory or control proteins), and the nal brinogen substrate. Throughout this chapter, gures will use the above-shaped symbols to aid in classifying each component: zymogens as circles; activated zymo- gens or serine proteases as hexagons (not shown here); cofactors as rectangles; control proteins as stars; other components as ovals. HMWK, High-molecular-weight kininogen; TFPI, tissue factor pathway inhibitor; ZPI, protein Z–dependent protease inhibitor. 668 PART VI Hemostasis and Thrombosis the naming system and never reassigned. Although factor VIII In the coagulation pathway, there are multiple procoagulant is normally referred to as factor VIII, it was originally known cofactors that augment the enzymatic process. ese cofactors as antihemophilic factor, which is a cofactor that circulates participate in the formation of enzymatically active complexes linked to a large carrier protein, VWF. Prekallikrein (pre-K), with the serine proteases. e cofactor TF is located on mem- also called Fletcher factor, and high-molecular-weight kinino- branes of broblasts and smooth muscle cells but also can be gen (HMWK), also called Fitzgerald factor, have never received found on monocytes during inammation and sepsis.36 TF serves Roman numerals because they belong to the kallikrein and to initiate the coagulation pathway when there is vascular injury. kinin systems, respectively, and their primary functions lie Soluble plasma FV and FVIII, the other procoagulant cofactors, within these systems. Platelet phospholipids, particularly phos- become activated and are then incorporated into procoagulant phatidylserine, are required for the coagulation process but complexes of coagulation factors and other cofactors. Factors V were given no Roman numeral; instead they were once called and VIII are important for proper functioning of the coagulation collectively platelet factor 3. system and normal thrombin generation. e cofactor HMWK functions in complex with FXIIa as a second means of activation Classification and Function of Procoagulants of the coagulation pathway through the intrinsic pathway. e coagulation factors work together in a pathway in which e remaining components of the coagulation pathway are one factor, when activated, activates the next factor in the brinogen, FXIII, phospholipids, ionized calcium, and VWF sequence. e purpose is to generate the key enzyme, thrombin, (Box 35.2). Fibrinogen is the ultimate substrate of the coagula- which converts brinogen to brin, allowing for formation of a tion pathway. When hydrolyzed by thrombin, soluble brinogen localized thrombus. e sequence of coagulation factor activa- is converted to the insoluble structural protein, brin, forming tion is shown in Figure 35.4 a more stable clot that is further stabilized by activated FXIII e coagulation factors thrombin (FIIa), FVIIa, FIXa, FXa, through cross-linking of the brin monomers.37 FXIa, FXIIa, and pre-K are enzymes called serine proteases Ionized calcium (Ca2+) is required for the previously stated (Table 35.7).34 Of note, while FXIII is also an enzyme, it is a coagulation complexes. ese complexes assemble on platelet or transglutaminase. Serine proteases are proteolytic enzymes of cell membrane phospholipids, predominantly phosphatidylser- the trypsin family.35 Each member has a reactive seryl amino ine. Activated serine proteases bind to the negatively charged acid residue in its active site and acts on its substrate by hydro- phospholipid surface through the positively charged calcium lyzing peptide bonds, thereby digesting the primary backbone. ions. is process serves to localize coagulation activation to Small polypeptide fragments from each substrate are produced the cell surface at the site of injury. from this process. e molecular weights, plasma concentrations, and plasma Serine proteases are synthesized as inactive zymogens con- half-lives of the procoagulant factors are given in Table 35.6 38 sisting of a single peptide chain. Activation occurs when the ese essential pieces of clinical information assist in the zymogen is cleaved at one or more specic sites by the action of interpretation of laboratory tests, monitoring of anticoagulant another protease during the coagulation process. TABLE 35.7 Plasma Procoagulant Serine Proteases XII Inactive Active TF:VII Pre-K Zymogen Protease Cofactor Substrate XI HMWK Prothrombin (II) Thrombin (IIa) — Fibrinogen, V, VIII, XI, XIII VII VIIa Tissue factor IX, X IX IXa VIIIa X X IX:VIII X Xa Va Prothrombin XI XIa — IX XII XIIa High-molecular- XI X:V weight kininogen Prekallikrein Kallikrein High-molecular- XI II Thrombin XIII weight kininogen Fibrinogen Fibrin Cross-linked fibrin Figure 35.4 Simplied Coagulation Pathway. Exposed tissue factor (TF) activates factor VII, which activates FIX and FX. The IXa:VIIIa com- BOX 35.2 Other Plasma Procoagulants plex also activates FX, and the Xa:Va complex activates prothrombin (FII). The resulting thrombin cleaves brinogen to form brin and acti- Fibrinogen vates FXIII to stabilize the clot. Thrombin also activates factors V, VIII, Factor XIII XI, and platelets in a positive feedback loop. Exposure to negatively Phospholipids charged surfaces (e.g., bacterial cell membranes) activates the contact Calcium factors FXII, prekallikrein (pre-K), and high-molecular-weight kininogen von Willebrand factor (HMWK), which activate FXI.  CHAPTER 35 Normal Hemostasis 669 therapy, and design of eective replacement therapies in de- calcium. As stated previously, it is the bound calcium that ciency-related hemorrhagic diseases. It is of interest to note enables the vitamin K–dependent coagulation factors to bind that brinogen, prothrombin, FVIII, and VWF are acute-phase to negatively charged phospholipids, localizing the coagulation reactants and their levels increase when there is inammation process, as well as providing a platform for the formation of as in trauma, pregnancy, infection, and stress. coagulation factor complexes. In vitamin K deciency or in the presence of the anticoagu- Vitamin K–Dependent Prothrombin Group lant drug warfarin, a therapeutic inhibitor of vitamin K (Chapter Prothrombin (factor II), FVII, FIX, and FX, along with the reg- 40), the vitamin K–dependent procoagulants are released from ulatory proteins protein C, protein S, and protein Z, are depen- the liver without the second carboxyl group added to the γ car- dent on vitamin K during synthesis to produce a functional bon. ese are called des-γ-carboxyl proteins or proteins induced structure (Table 35.8). ese factors are named the prothrombin by vitamin K antagonists (PIVKA factors). Because they lack group because of their structural resemblance to prothrombin. the second carboxyl group, they cannot bind to Ca2+ and sub- All 7 proteins have 10 to 12 glutamic acid units near their amino sequently to phospholipids, so they cannot participate in the terminal end. All are serine proteases when activated, except coagulation reaction. protein S and protein Z, which are cofactors. Vitamin K–dependent procoagulants are essential for the Vitamin K is a quinone found in green leafy vegetables (Box assembly of three complexes in the coagulation pathway (Figure 35.3); it is also produced by the intestinal organisms Bacteroides 35.6). ese are the same coagulation complexes discussed fragilis and Escherichia coli. Vitamin K is a coenzyme for the carboxylase that catalyzes an essential posttranslational modi- BOX 35.3 Food Sources High in Vitamin K cation of the prothrombin group proteins: γ-carboxylation of amino-terminal glutamic acids (Figure 35.5). Glutamic acid is Kale modied to γ-carboxyglutamic acid when a second carboxyl Spinach Turnip greens group is added to the γ-carbon. With two ionized carboxyl Collards groups, the γ-carboxyglutamic acids gain a net negative charge, Mustard greens which enables the binding of the positively charged ionized Swiss chard Brussels sprouts TABLE 35.8 Vitamin K–Dependent Broccoli Asparagus Coagulation Factors Cabbage Procoagulants Regulatory Proteins Green onions Prothrombin (II) Protein C Lettuce: Boston, romaine, or Bibb VII Protein S Avocado IX Protein Z Cauliower X Parsley, fresh Epoxide reductase VK hydroxyquinone VK epoxide Ca COO OOC COO CH2 Carboxylase CH CH2 CH2 O2 CO2 HN2 CH COOH HN2 CH COOH Glu Gla Nonactive Active coagulation factor coagulation factor Figure 35.5 Posttranslational γ-Carboxylation of the Vitamin K–Dependent Coagulation Proteins. For coagulation factors II (prothrombin), VII, IX, and X, and control proteins C, S, and Z, vitamin K hydroxyquinone transfers a carboxyl (COO–) group to the γ carbon of glutamic acid (Glu), creating γ-carboxyglutamic acid (Gla). The negatively charged pocket formed by the two carboxyl groups attracts ionic calcium, which enables the molecule to bind to phosphatidylserine. Vitamin K hydroxyquinone is oxidized to vitamin K epoxide by carbox- ylase in the process of transferring the carboxyl group but is subsequently reduced to the hydroxyquinone form by epoxide reductase. 670 PART VI Hemostasis and Thrombosis TABLE 35.9 Coagulation Complexes TF VIIa IXa VIIIa Complex Components Activates IX Extrinsic Intrinsic Extrinsic tenase VIIa, tissue factor, phospholipid, and Ca2+ IX and X tenase tenase Intrinsic tenase IXa, VIIIa, phospholipid, and Ca2+ X X X Prothrombinase Xa, Va, phospholipid, and Ca2+ Prothrombin TABLE 35.10 Hemostasis Cofactors Xa Va Cofactor Function Binds Tissue factor Procoagulant VIIa V Procoagulant Xa TF-expressing Prothrombinase VIII Procoagulant IXa cell II High-molecular-weight kininogen Procoagulant XIIa, prekallikrein Platelet Thrombomodulin Control (protein C) Thrombin Antibrinolytic (TAFI) Thrombin Protein S Control Protein C, TFPI Thr Protein Z Control ZPI Fibrinogen Fibrin TAFI, Thrombin activatable brinolysis inhibitor; TFPI, tissue factor Figure 35.6 Complexes Within the Coagulation Pathway. Coagula- pathway inhibitor; ZPI, protein Z-dependent protease inhibitor. tion complexes form on tissue factor (TF)-bearing cells (TF:VIIa) and on platelet phospholipid membranes (IXa:VIIIa and Xa:Va). Each complex and smooth muscle cells. Vessel injury exposes blood to the consists of a vitamin K–dependent serine protease coagulation factor, a cofactor, and Ca2+, bound to the cell membrane. Extrinsic tenase com- subendothelial TF-bearing cells and leads to activation of plex is FVIIa and TF on the membrane of a TF-bearing cell. This com- coagulation through FVII. e activation of FVII is limited by plex activates both FIX and FX. Intrinsic tenase complex, which is FIXa the injury itself through the size and resulting amount of TF and its cofactor VIIIa on platelet membranes, activates FX also. Pro- exposed. Of note, TF is expressed at high levels in cells of the thrombinase complex is FXa and its cofactor Va, bound to the surface brain, lung, placenta, heart, and kidney. ese ndings explain of platelets. Prothrombinase cleaves prothrombin to the active enzyme thrombin (Thr). the signicant tissue damage, morbidity, and mortality that can ensue aer a bleed into these organs. In inammatory condi- tions, including sepsis, monocytes and other cells express TF, earlier but described here in detail. e formation of these com- initiating coagulation.15 36 plexes is emphasized because of the critical role they play in Factors V and VIII are soluble plasma proteins. Factor V is thrombin generation and normal hemostasis. a glycoprotein circulating in plasma and also present in platelet Each complex is composed of a vitamin K–dependent α-granules. During platelet activation and secretion, platelets serine protease, its nonenzyme cofactor, and Ca2+, which is release partially activated FV at the site of injury. Factor Va is a bound to the negatively charged phospholipid membranes cofactor to FXa in the prothrombinase complex in coagulation. of activated platelets or TF-bearing cells. e initial complex, e prothrombinase complex accelerates thrombin generation extrinsic tenase, comprises TF and FVIIa; it activates FIX and more than 300,000-fold compared to FXa alone.40 e initial FX, which are components of the next two complexes (Table small amount of thrombin generated activates the rst amount 35.9). Intrinsic tenase complex comprises FIXa and its cofactor of FV to FVa. As described in the “Protein C Regulatory System” FVIIIa; it also activates FX but much more eciently than the section, thrombomodulin-bound thrombin activates protein C, TF:FVIIa extrinsic tenase complex. Prothrombinase complex which inactivates FVa to FVi. erefore FV is both activated and comprises FXa and its cofactor FVa; it converts prothrombin to then ultimately inactivated by the generation of thrombin, as is thrombin in a multistep hydrolytic process that releases throm- FVIII. Factor VIII is a cofactor to FIX, which together form the bin and a peptide fragment called prothrombin fragment 1+2 intrinsic tenase complex. (F1+2). Prothrombin fragment 1+2 in plasma is thus a marker HMWK is produced by the liver and acts as a cofactor to for thrombin generation. FXIIa and pre-K in the contact factor complex. is complex of the coagulation pathway is a mechanism for activating coagu- Cofactors in Hemostasis lation via the intrinsic pathway in conditions in which foreign Procoagulant cofactors are TF, FV, FVIII, and HMWK. objects such as mechanical heart valves, bacterial membranes, Cofactors of the coagulation control proteins are thrombomod- or high levels of inammation are present. us coagulation is ulin, protein S, and protein Z (Table 35.10).39 rombomodulin initiated at the site of either the extrinsic tenase complex (FVIIa is also a cofactor in the control of brinolysis. Each cofactor and TF) or the contact factor complex (FXIIa, pre-K, and binds to its particular serine protease, promoting stability and HMWK). increased reactivity to the serine protease. rombomodulin, a transmembrane protein constitutively TF, a transmembrane receptor for FVII and FVIIa, is found expressed by vascular ECs, is a thrombin cofactor in the protein on extravascular cells surrounding ECs, including broblasts C pathway. Together, thrombomodulin and thrombin activate  CHAPTER 35 Normal Hemostasis 671 protein C, a coagulation regulatory protein. In one of many 20,000,000 Daltons.45 VWF molecules are stored in α-granules examples of carefully regulated processes within the hemostatic in platelets and in Weibel-Palade bodies in ECs. ECs release system, once thrombin is bound to thrombomodulin, it loses its ultralarge multimers of VWF into plasma, where they are nor- procoagulant ability to activate FV and FVIII, among other fac- mally degraded into smaller multimers by a VWF-cleaving tors and, through activation of protein C, leads to destruction protease, ADAMTS13 (a disintegrin and metalloprotease with of FV and FVIII, thus suppressing further generation of throm- a thrombospondin type 1 motif, member 13), in blood vessels bin—a negative feedback loop. rombin bound to thrombo- with high shear stress.46 modulin also activates TAFI, a brinolysis inhibitor. VWF has multiple binding sites for platelets, along with Both protein S and protein Z are cofactors in the regulation binding sites for collagen and FVIII (see Figure 35.7). rough and control of coagulation. Protein S is a cofactor to protein C its collagen binding site and primary platelet surface recep- as well as TFPI. Protein Z is a cofactor to Z-dependent protease tor, GPIb/IX/V, it is able to act as a bridge, binding platelets inhibitor (ZPI). ese cofactors will be discussed in detail in to exposed subendothelial collagen during platelet adhesion association with their hemostatic mechanisms described in this (Figure 11.9C). is is especially important in arteries and arte- chapter. rioles, where the ow of blood is faster and the collagen specic receptors on platelets are unable to bind to collagen directly. Factor VIII and von Willebrand Factor rough arginine-glycine-aspartic acid (RGD) sequences, Factor VIII and VWF are key proteins for hemostasis as they are VWF is also able to bind a second platelet receptor, GPIIb/IIIa, both critically involved in all protective mechanisms to avoid during platelet aggregation (Chapter 11). blood loss (i.e., platelets, vasculature, and blood coagulation e importance of VWF in hemostasis cannot be overstated leading to brin formation). as evidenced by the severe bleeding and symptoms associated Factor VIII is produced by ECs.41 Free FVIII is unstable in with abnormalities in VWF molecular structure and concen- plasma, resulting in a signicantly shortened half-life; however, tration. von Willebrand disease, which is the most commonly when bound to VWF in circulation, its half-life increases to inherited bleeding disorder, is associated with several described approximately 12 hours (Figure 35.7). Factor VIII deteriorates mutations (Chapter 36). Factor VIII deciency, another com- more rapidly than other coagulation factors in stored blood, mon bleeding disorder, can be linked to von Willebrand disease decreasing to approximately 50% aer 5 days in thawed compo- because VWF is the carrier for FVIII. nent plasma.42 During coagulation, FVIII is activated through Of note, levels of VWF vary by ABO blood type, with group cleavage from VWF by thrombin. Factor VIIIa subsequently O individuals having lower levels of VWF than the other blood binds to phospholipid on activated platelets, forming the intrin- group types.47 In addition, VWF and factor VIII are both acute sic tenase complex with FIXa and Ca2+. Like FVa, FVIIIa is also phase reactants with increased levels seen during pregnancy, inactivated by protein C. trauma, bleeding, infection, and stress. Factors VIII and IX are the only two plasma procoagulants whose production is governed by genes carried on the X chro- Factor XI and the Contact Factors mosome.43 Factor VIII is a cofactor, but its importance in hemo- e contact system is a group of plasma proteins that responds stasis cannot be overstated, as evidenced by the severe bleeding to the presence of foreign material. e contact factors consist and symptoms associated with hemophilia A and type 2N von of factor XII, prekallikrein (pre-K; Fletcher factor), and HMWK Willebrand disease (Chapter 36). (Fitzgerald factor). ey are so named because they are acti- VWF is a large multimeric glycoprotein that participates in vated by contact with negatively charged foreign surfaces such platelet adhesion and transports the procoagulant FVIII. VWF as stents, valve prostheses, and bacterial cell membranes. Upon is composed of multiple subunits of 240,000 Daltons each.44 activation, this system triggers blood coagulation via the intrin- e subunits are produced by ECs and megakaryocytes, where sic pathway. Additionally, it is responsible for the generation of they combine to form multimers that range from 500,000 to proinammatory products such as bradykinin, activation of the complement system, and plays a major role in the host-defense system.48 is system is also called the plasma kallikrein-kinin Platelet membrane Platelet membrane binding site: GPIb/IX/V binding site: GPIIb/IIIa system because it functions in inammatory conditions. Further to its contribution to the intrinsic coagulation path- way and brin clot formation, the contact system is involved VWF VIII Factor VIII with activation of brinolysis through FXIIa via a pre-K–medi- reactive site ated activation of plasminogen.48 49 ere is also suggestive evi- VWF:Ag dence that FXI plays a role in the downregulation of brinolysis, epitope Collagen binding sites Carries coagulation FVIII which would help stabilize the formed blood clot.48 Figure 35.7 von Willebrand Factor (VWF)–Factor VIII Complex. VWF Factor XII and pre-K are zymogens that are activated to molecules of various lengths from 0.5 to 20 mDaltons are in blood cir- become serine proteases; HMWK is a nonenzymatic cofac- culation. Factor VIII circulates covalently bound to VWF. VWF provides tor. rough coming into contact with a negatively charged three other active receptor sites: VWF binds to collagen; it binds to glycoprotein (GP) Ib/IX/V to support platelet adhesion; and it binds to surface, FXII becomes activated through autocatalytic con- GPIIb/IIIa to facilitate platelet aggregation. VWF:Ag epitope is the target version.50 51 Activation by FXIIa transforms pre-K, a glyco- of quantitative immunoassays. protein that circulates bound to HMWK, into its active form, 672 PART VI Hemostasis and Thrombosis kallikrein. Kallikrein is subsequently able to cleave HMWK to Fibrinogen Structure and Fibrin Formation, Factor XIII form bradykinin, which promotes inammation and can cause Fibrinogen is the primary substrate of thrombin; enzymatic vasodilation. action of thrombin leads to conversion of soluble brinogen to Factor XI is activated to FXIa in part by FXIIa but more insoluble brin to produce a clot. Fibrinogen is also essential for signicantly by thrombin during coagulation generated from platelet aggregation because it links activated platelets through TF:FVIIa activation (see Figure 35.4). Factor XIa activates FIX their GPIIb/IIIa platelet brinogen receptor. Fibrinogen is a and the reaction proceeds as described previously for the coag- 340,000 Dalton glycoprotein synthesized in the liver. e nor- ulation pathway. mal plasma concentration of brinogen ranges from 200 to 400 Factor XI–associated bleeding disorders are described in mg/dL, the most concentrated of all the plasma procoagulants. Chapter 36. Deciencies of FXII, HMWK, or pre-K do not cause Platelet α-granules absorb, transport, and release abundant clinical bleeding disorders.52 However, deciencies do prolong brinogen.53 Of note, brinogen is an acute phase reactant pro- laboratory tests and necessitate investigation. Factor XII is acti- tein, meaning its level increases during inammation, infection, vated invitro by negatively charged surfaces such as nonsilicon- and other stress conditions. ized glass, kaolin, or ellagic acid—used as test reagents for the e brinogen molecule is a mirror image dimer, each half partial thromboplastin time (PTT, activated PTT [APTT]) assay. consisting of three nonidentical polypeptides, designated Aα, Bβ, and γ, united by several disulde bonds (see Figure 35.8). Thrombin e six N-terminals assemble to form a bulky central region rombin is the main enzyme of the coagulation pathway with called the E domain. e three carboxyl terminals on each outer multiple key activities. e primary function of thrombin, how- end of the molecule assemble to form two D domains.37 ever, is to cleave brinopeptides A and B (FPA and FPB) from rombin cleaves FPA and FPB from the protruding N-termini the α and β chains of the brinogen molecule, triggering spon- of each of the two α and β chains of brinogen, reducing the over- taneous brin polymerization and the beginning of the insol- all molecular weight by 10,000 Daltons. e cleaved brinogen uble brin clot (Figure 35.8). FPA and FPB are measurable in is called brin monomer. e exposed brin monomer α and β plasma and serve as markers of thrombin activation. chain ends (E domain) have an immediate anity for portions rombin amplies the coagulation pathway by activating of the D domain of neighboring monomers, spontaneously poly- cofactors V and VIII and FXI, providing a positive feedback merizing to form brin polymers (Figure 35.9). mechanism that generates more thrombin (see Figure 35.4). Factor XIII is a heterodimer whose α subunit is produced rombin also activates FXIII, which forms covalent bonds mostly by megakaryocytes and monocytes, and whose β subunit between the D domains of the brin polymer to cross-link and is produced in the liver.54 e two subunits become joined in ultimately stabilize the brin clot. In addition, thrombin initi- the plasma to form FXIII. Aer activation by thrombin, FXIIIa ates aggregation of platelets, which increases the platelet com- covalently cross-links brin polymers to form a more stable ponent of the thrombus. insoluble brin clot. Factor XIIIa is a transglutaminase that cat- Further, thrombin plays roles in both anticoagulation and alyzes the formation of covalent bonds between the carboxyl antibrinolysis. rough binding to thrombomodulin, throm- terminals of γ chains from adjacent D domains in the brin bin activates the protein C pathway and TAFI. In the protein C polymer (see Figure 35.9). ese bonds link the ε-amino acid pathway, it suppresses coagulation through degradation of FVa of lysine moieties and the γ-amide group of glutamine units. and FVIIIa. rombin activated TAFI leads to suppression of Multiple cross-links form to provide an insoluble meshwork of brinolysis. brin polymers linked by their D domains, providing physical rombin therefore plays a role in coagulation (brin for- strength to the brin clot. Factor XIIIa reacts with other plasma mation), platelet activation, regulation of coagulation activation and cellular structural proteins and is essential to wound heal- (protein C), and controlling brinolysis (TAFI). Because of its ing and tissue integrity. multiple autocatalytic functions, thrombin is the key protease Cross-linking of brin polymers by FXIIIa covalently incor- of hemostasis. porates bronectin, a plasma protein involved in cell adhesion, D domain E domain D domain FPB FPB FPA FPA -chain -chain -chain -chain -chain -chain Figure 35.8 Structure of Fibrinogen. Fibrinogen has three domains. The central E domain and two terminal D domain nodules at the carboxyl ends of the molecule. The D and E domains are joined together by super- coiled α-helix regions. This trinodular structure comprises three pairs of disulde-bonded polypeptides, two each of the Aα, Bβ, and γ chains. FPA, Fibrinopeptide A; FPB, brinopeptide B.