Primary Hemostasis

Hemostasis involves the interaction of vasoconstriction, platelet aggregation, and coagulation enzymes to stop bleeding (Table 40-1). Primary hemostasis refers to the role of blood vessels and platelets in the initial formation of a platelet plug in response to a vascular injury. Primary hemostasis mechanisms are activated either by injuries to blood vessels or by the commonplace desquamation of dying or damaged endothelial cells. In primary hemostasis, the blood vessel contracts to seal the wound or reduce the blood flow (vasoconstriction). Procoagulant substances are exposed or released by the damaged endothelium. Platelets are activated, adhere to exposed collagen, secrete the contents of their granules, and aggregate with other platelets to form a platelet plug. Vasoconstriction and platelet plug formation are the initial, rapid, short-lived response to vessel damage, but by themselves they are inadequate to control major bleeding in the long term. Eventually the plug must be reinforced by fibrin. Defects in the primary hemostasis system such as thrombocytopenia, platelet disorders, or von Willebrand disease can cause debilitating, sometimes fatal, chronic hemorrhage.

Secondary Hemostasis

Secondary hemostasis is the activation of a series of plasma proteins in the coagulation system to form a fibrin clot. Blood plasma transports at least 16 glycoproteins, mostly trypsinlike enzymes called serine proteases. These proteins circulate as inactive zymogens that become activated during the process of coagulation and, in turn, form complexes that activate other zymogens to ultimately generate thrombin, an enzyme that converts fibrinogen to a localized fibrin clot. Some coagulation proteins are cofactors that join in forming a complex and function to stabilize and enhance enzymatic action. Still others are control proteins that regulate each step of the process and prevent excessive thrombin generation. Figure 40-1 shows a simplified sequence of activation of the coagulation proteins to form fibrin. Secondary hemostasis is triggered by tissue damage that exposes TF, a protein present in the membrane of subendothelial cells—fibroblasts and smooth muscle cells—but not on endothelial cells. Exposed TF forms a complex with activated factor VII, which activates factors IX and X. Factor IX combines with its cofactor, factor VIII, also to activate factor X. The complex formed by factor X and its cofactor, factor V, converts prothrombin to thrombin. Thrombin is the key enzyme in coagulation. It splits peptides from fibrinogen to produce fibrin, activates factor XIII to cross-link and stabilize the clot, activates platelets, and feeds back to activate factors V, VIII, and XI. Fibrin formation in secondary hemostasis is necessary to control bleeding, especially from large wounds incurred through trauma, surgery, or dental procedures. The coagulation system, similar to other humoral amplification mechanisms, is complex because it translates a diminutive physical or chemical stimulus into a profound lifesaving event.¹ The absence of a single plasma procoagulant may doom the individual to lifelong anatomic hemorrhage, chronic inflammation, and transfusion dependence.

Fibrinolysis

The final event of hemostasis is fibrinolysis, the slow digestion and removal of the fibrin clot as healing occurs.² Plasminogen is bound to fibrin during coagulation and is activated by tissue plasminogen activator (TPA) into the serine protease, plasmin. Plasmin slowly and systematically degrades the fibrin clot into fragments called fibrin degradation products, designated X, Y, D, E, and D-dimer.

Although the vascular intima and platelets usually are associated with primary hemostasis, and coagulation and fibrinolysis are associated with secondary hemostasis, all systems interact in early and late hemostatic events. For example, platelets, although a key component of primary hemostasis, also secrete some coagulation factors stored in their granules (V, VIII, fibrinogen, von Willebrand factor [VWF]) and, very importantly, provide a phospholipid cell surface on which coagulation complex formation can take place. The remainder of this chapter examines vascular intima, platelets, normal coagulation, coagulation control, and fibrinolysis in detail.

Anticoagulant Properties of Intact Vascular Intima

Normally, the intact vascular endothelium prevents bleeding and thrombosis by inhibiting platelet and coagulation activation and by promoting fibrinolysis. Intravascular thrombosis is prevented by several mechanisms (Box 40-2). First, endothelial cells are rhomboid and contiguous, providing a smooth inner surface that evens the blood flow and prevents harmful turbulence. Endothelial cells form a physical barrier between blood and the substances in the basement membrane that activate clotting, such as collagen, which promotes platelet activation and adhesion, and TF, which activates coagulation and fibrin formation. Endothelial cells synthesize and secrete a variety of substances that maintain normal blood flow. Prostacyclin, a platelet inhibitor, is synthesized through the eicosanoid pathway (see Chapter 13) and prevents unnecessary or undesirable platelet activation in intact vessels.⁵ Nitric oxide is synthesized in endothelial cells, vascular smooth muscle cells, neutrophils, and macrophages. Nitric oxide counteracts vasoconstriction and maintains healthy arterioles.⁶ Another important endothelial cell anticoagulant is tissue factor pathway inhibitor (TFPI), which controls the TF or extrinsic coagulation pathway. Finally, endothelial cells synthesize and secrete onto their cell surfaces two inhibitors of thrombin formation, thrombomodulin and heparan sulfate. Thrombomodulin is a membrane protein that activates the protein C pathway. The protein C pathway downregulates coagulation by digesting activated factors V and VIII, thereby inhibiting fibrin formation. Heparan sulfate is a glycosaminoglycan that retards coagulation by activating antithrombin, a coagulation regulatory protein.⁷ The pharmaceutical heparin, manufactured from porcine gut tissues, resembles heparan sulfate in its antithrombin activity. Heparin is used extensively as a therapeutic agent to prevent propagation of the thrombi that cause coronary thrombosis, strokes, deep vein thromboses, and pulmonary emboli.⁸

Procoagulant Properties of Damaged Vascular Intima

Although the intact endothelium has anticoagulant properties, when damaged, the vascular intima promotes coagulation. First, any harmful local stimulus, be it mechanical or chemical, induces vasoconstriction in arteries and arterioles (Table 40-2). Smooth muscle cells contract, the vascular lumen narrows or closes, and blood flow to the injured site is minimized. Although veins and capillaries do not have smooth muscle cells, bleeding into tissues creates some extravascular pressure on the blood vessel as well. Second, the subendothelial connective tissues of arteries and veins are rich in collagen, a flexible, elastic structural protein that binds and activates platelets. Some connective tissue degeneration occurs naturally in aging, which leads to an increased tendency toward bruising. Third, endothelial cells secrete VWF, a 600,000- to 20,000,000-D glycoprotein that is necessary for platelets to adhere to exposed subendothelial collagen in arterioles.⁹ Fourth, on activation, endothelial cells secrete and coat themselves with P-selectin, an adhesion molecule that promotes platelet and leukocyte binding.¹⁰ Endothelial cells also secrete immunoglobulin-like adhesion molecules called intercellular adhesion molecules (ICAMs) and platelet endothelial cell adhesion molecules (PECAMs) that promote leukocyte binding.¹¹ Finally, subendothelial cells (i.e., smooth muscle cells and fibroblasts) support the constitutive surface protein TF.¹² Exposed TF activates the coagulation system through factor VII. TF also appears on the surface of endothelial cells and on bloodborne monocytes during inflammation.¹³

Fibrinolytic Properties of Vascular Intima

Endothelial cells support fibrinolysis with the secretion of TPA. During thrombus formation, both TPA and plasminogen bind to polymerized fibrin. TPA activates fibrinolysis by converting plasminogen to plasmin, which slowly digests fibrin and restores blood flow. Endothelial cells, as well as other cells, may secrete plasminogen activator inhibitor 1 (PAI-1), a TPA control protein that inhibits fibrinolysis.¹⁴ Thrombin bound to thrombomodulin activates thrombin-activatable fibrinolysis inhibitor (TAFI), which increases the tendency for thrombus formation.

Although the significance of the vascular intima in hemostasis is well recognized, there are few valid laboratory methods to assess the integrity of endothelial cells, smooth muscle cells, fibroblasts, and their collagen matrix.¹⁵ The diagnosis of blood vessel disorders is often based on clinical symptoms, family history, and laboratory tests that rule out platelet or coagulation disorders.

The plasma procoagulants may be serine proteases or cofactors.²⁴ Serine proteases are proteolytic enzymes of the trypsin family and include the procoagulants thrombin (factor IIa); factors VIIa, IXa, Xa, XIa, and XIIa; and prekallikrein (pre-K).²⁵ Each member has a reactive seryl amino acid residue in its active site and acts on its substrate by hydrolyzing peptide bonds, digesting the primary backbone and producing small polypeptide fragments. Serine proteases are synthesized as inactive zymogens consisting of a single peptide chain (Table 40-6). Activation occurs when the zymogen is cleaved at one or more specific sites by the action of another protease during the coagulation process. Activation is a localized cell-surface process, limited to the site of injury and controlled by regulatory mechanisms. If zymogen activation is uncontrolled and generalized, the condition is called disseminated intravascular coagulation (DIC), a serious, often life-threatening condition (see Chapter 42).

The coagulation cofactors are TF, factor V, factor VIII, and HMWK. Each cofactor binds its particular serine protease. When bound, serine proteases gain stability and increased reactivity (Table 40-7).²⁶

The remaining components of the coagulation pathway are fibrinogen, factor XIII, phospholipids, calcium, and VWF (Box 40-3). Fibrinogen is the ultimate substrate of the coagulation pathway. When hydrolyzed by thrombin, fibrinogen polymerizes to form the primary structural protein of the fibrin clot.²⁷ Factor XIIIa is a transglutaminase that catalyzes the transfer of amino acids among the γ chains of fibrin polymers. This reaction cross-links fibrin polymers to provide physical strength to the fibrin clot. Factor XIIIa reacts with other plasma and cellular structural proteins and is essential to wound healing and tissue integrity. Factor XIII is a heterodimer whose α subunit is produced mostly from megakaryocytes and monocytes. The β subunit is produced in the liver.²⁸

Coagulation occurs on the surface of platelet or endothelial cell membrane phospholipids and not in fluid phase. Any fluid phase reaction is inhibited by the coagulation control proteins. Serine proteases bind to negatively charged phospholipid surfaces, mostly phosphatidylserine, through positively charged calcium ions, so calcium is required for the assembly of coagulation complexes on phospholipid membranes. VWF is a large glycoprotein that participates in platelet adhesion and transports the procoagulant factor VIII. VWF is synthesized in megakaryocytes and endothelial cells.²⁹

Biochemical Nature of Several Procoagulants

Vitamin K–Dependent Prothrombin Group

Prothrombin, factors VII, IX, and X, and the regulatory proteins protein C, protein S, and protein Z are vitamin K dependent (Table 40-8). These are named the prothrombin group because of their structural resemblance to prothrombin: all seven proteins have 10 to 12 glutamic acid units near their amino termini. Vitamin K is a quinone found in green leafy vegetables, fish, and liver, and is produced by the intestinal organisms Bacteroides fragilis and Escherichia coli. Vitamin K catalyzes an essential posttranslational modification of the prothrombin group proteins: γ-carboxylation of amino-terminal glutamic acids (Figure 40-8). Glutamic acid is modified to γ-carboxyglutamic acid when a second carboxyl group is added to the γ carbon. With two ionized carboxyl groups, the γ-carboxyglutamic acids gain a net negative charge, which enables them to bind ionic calcium (Ca²⁺). The bound calcium permits the vitamin K–dependent proteins to bind negatively charged phospholipids, such as phosphatidylserine. Phospholipid binding is essential to coagulation reactions.

TABLE 40-8

Vitamin K–Dependent Coagulation Factors

Procoagulants	Regulatory Proteins
Prothrombin (II)	Protein C
VII	Protein S
IX	Protein Z
X

In vitamin K deficiency or in the presence of warfarin, a vitamin K antagonist, the vitamin K–dependent procoagulants are released from the liver without the second carboxyl group added to the γ carbon. These are called des-γ-carboxyl proteins or proteins in vitamin K antagonism (PIVKAs). Because they lack the second carboxyl group, they cannot bind to Ca²⁺ and phospholipid, so they cannot participate in the coagulation reaction. Vitamin K antagonism is the basis for oral anticoagulant (warfarin) therapy (see Chapter 46).

Von Willebrand Factor: Factor VIII Complex

VWF is a multimeric glycoprotein composed of multiple subunits of 240,000 D each.³⁰ The subunits are produced by endothelial cells and megakaryocytes, where they combine to form molecules that range from 600,000 to 20,000,000 D. VWF molecules are stored in α-granules in platelets and in storage sites called Weibel-Palade bodies in endothelial cells. The molecules are released from storage into the plasma, where they circulate at a concentration of 7 to 10 mcg/mL.

VWF has receptor sites for both platelets and collagen (Figure 40-9) and helps bind platelets to exposed subendothelial collagen during platelet adhesion, especially in arteries and arterioles where the flow of blood is faster. The primary platelet surface receptor for VWF is GP Ib/IX/V.³¹ Arginine–glycine–aspartic acid (RGD) sequences in VWF also bind a second platelet integrin, GP IIb/IIIa, during platelet aggregation.

A third site binds to collagen, and a fourth site binds the plasma procoagulant cofactor, factor VIII. Factor VIII is produced by hepatocytes and other tissues. It is one of two plasma procoagulants whose production is sex linked, the other being factor IX. Factor VIII has a molecular mass of 260,000 D and circulates bound to VWF. Free factor VIII is unstable except during coagulation and cannot be detected in plasma. Factor VIII is labile and deteriorates over hours in stored blood despite being bound to VWF. Males with hemophilia A have diminished factor VIII activity but normal VWF levels.³² Because factor VIII depends on VWF for stability, individuals with von Willebrand disease who have diminished VWF also have diminished factor VIII activity levels. Typically, factor VIII levels decrease to hemorrhagic levels (less than 30%) only in severe von Willebrand disease.

Fibrinogen Structure and Fibrin Formation

Fibrinogen is the primary substrate of thrombin. It is a 340,000-D glycoprotein synthesized in the liver. The normal plasma concentration of fibrinogen ranges from 200 to 400 mg/dL, the highest concentration of all the plasma procoagulants. Platelet α-granules absorb, transport, and release abundant fibrinogen.³³

The fibrinogen molecule is a mirror-image dimer, with each half consisting of three nonidentical polypeptides, designated α, β, and γ, united by disulfide bonds (Figure 40-10). The six amino termini assemble to form a bulky central region called the E domain. The carboxy termini assemble at the two ends of the molecule to form two D domains.³⁴

Thrombin cleaves fibrinopeptides A and B from the protruding amino termini of each of the two α and β chains, reducing the overall molecular weight by 10,000 D. The cleaved fibrinogen is called fibrin monomer. The exposed fibrin monomer α and β chain ends (E domain) have an immediate affinity for portions of the D domain of neighboring monomers, spontaneously polymerizing to form fibrin polymer (Figure 40-11).

Factor XIIIa catalyzes the formation of covalent bonds between the carboxy termini of γ chains from adjacent D domains. These bonds link the ε-amino acid of lysine moieties and the γ-amide group of glutamine units. Multiple cross-links form to provide an insoluble meshwork of fibrin polymers, linked by their D domains. Cross-linking also covalently incorporates fibronectin, a plasma protein involved in cell adhesion, and α₂-antiplasmin, rendering the fibrin mesh resistant to fibrinolysis. Plasminogen, the primary serine protease of the fibrinolytic system, also becomes covalently bound via lysine moieties, as does TPA, a serine protease that ultimately hydrolyzes and activates bound plasminogen to initiate fibrinolysis.

TF, a membrane receptor for factor VIIa, is present on vascular cells that are not normally in contact with blood, such as fibroblasts. Coagulation is triggered by the exposure of TF to plasma proteins on injury.³⁵ In inflammatory conditions, leukocytes and other cells can also express TF and initiate coagulation. Factor VIIa binds to TF in the presence of phospholipid and calcium and triggers the activation of both factor IX and factor X (Figures 40-1 and 40-12). Factor IXa binds factor VIIIa on platelet phospholipid surfaces; this complex also activates factor X.

Factor Xa, activated by both TF : VIIa and IXa : VIIIa, binds factor Va on phospholipid surfaces. This Xa : Va complex activates prothrombin in a multistep hydrolytic process that releases a peptide fragment from prothrombin called prothrombin 1+2. Activated prothrombin is termed thrombin. Thrombin cleaves fibrinopeptides A and B from plasma fibrinogen; this produces fibrin monomer, which then polymerizes. Thrombin also activates factor XIII, which cross-links and stabilizes fibrin (see Figure 40-11).

The tissue factor pathway is characterized by three multimolecular complex formations. The first complex is composed of TF, factor VIIa, phospholipid, and Ca²⁺ on the TF-bearing cell; this complex activates IX and X. The second is composed of factor IXa, factor VIIIa, phospholipid, and Ca²⁺ on platelets, sometimes called tenase; this complex also activates factor X. The third complex is composed of factor Xa, factor Va, phospholipid, and Ca²⁺ and is often called prothrombinase; this complex converts prothrombin to thrombin (Table 40-9).

Cell-Based (In vivo) Coagulation

A spectacular combination of cellular and biochemical events function in harmony to keep blood liquid within the veins and arteries, prevent blood loss from injuries by the formation of thrombi, and reestablish blood flow during the healing process.³⁷ As noted earlier, the series of cascading proteolytic reactions traditionally known as the extrinsic and intrinsic coagulation pathways do not fully describe how coagulation occurs in vivo. These pathways are not distinct independent alternative mechanisms for generating thrombin, but are actually very interdependent. For example, a deficiency of factor VII in the extrinsic pathway can cause significant bleeding, even when the intrinsic pathway is normal. Similarly, deficiencies of some intrinsic pathway proteins, notably factors VIII and IX, may cause severe bleeding problems, regardless of the presence of a normal extrinsic pathway.³⁸

Current thinking about the physiologic process of coagulation recognizes the contribution of specific cells in regulating hemostasis. The cell-based model of hemostasis (Figure 40-13) describes the interaction of two cell types: platelets, within the blood circulation, and extravascular cells that bear TF.³⁹ The cell-based model of coagulation is often described as occurring in three separate but overlapping phases: initiation, amplification, and propagation.

Tissue Factor Pathway Inhibitor

Factor VIIa and TF combine to activate factors IX and X in the tissue factor pathway. Factor Xa reacts with TF : VIIa and binds TFPI, an important coagulation regulatory protein (Figure 40-15). TFPI is synthesized by endothelial cells.⁴¹ About 80% is bound to the vessel wall and 20% circulates in the plasma. Most of the plasma TFPI is truncated and bound to low-density lipoproteins. The full-length free form, comprising only 2% of the total TFPI, is an effective inhibitor of the tissue factor pathway. TFPI inhibits coagulation in a two-step process by inactivating Xa and then binding to TF : VIIa to prevent it from activating additional Xa.^42,43 TFPI provides feedback inhibition, because it is not functional until X is activated. Protein S, the cofactor of activated protein C (APC), is also a cofactor of TFPI and stimulates Xa inhibition by TFPI.⁴⁴ Because of the inhibitory action of TFPI, the TF : VIIa : Xa reaction is short-lived. Coagulation pathway amplification occurs primarily through IXa,⁴⁵ because Xa activation by IXa : VIIIa is negligibly affected by TFPI.

Protein C Regulatory System

During thrombosis, thrombin propagates the clot as it cleaves fibrinogen and activates factors V, VIII, XI, and XIII. In intact normal vessels, where coagulation would be inexpedient, thrombin binds the endothelial cell membrane protein thrombomodulin and triggers an essential coagulation regulatory system called the protein C system.⁴⁶ The thrombin-thrombomodulin complex activates the zymogen protein C to a serine protease (Figure 40-16). APC binds its cofactor, free plasma protein S. The stabilized APC–protein S complex hydrolyzes and inactivates factors Va and VIIIa, slowing or blocking coagulation.

Protein S, the cofactor that binds and stabilizes APC, is synthesized in the liver and circulates in the plasma in two forms: free and covalently bound to the complement control protein C4b-binding protein (C4bBP). Bound protein S cannot participate in the protein C anticoagulant pathway; only free plasma protein S can serve as the APC cofactor. Protein S–C4bBP binding is of particular interest in inflammatory conditions, because C4bBP is an acute phase reactant. When the plasma C4bBP level increases, additional protein S is bound, and free protein S levels become proportionally decreased, which increases the risk of thrombosis. Chronic acquired or inherited protein C or protein S deficiency, or mutations of protein C, protein S, or factor V, compromise downregulation of Va and VIIIa and may be associated with recurrent venous thromboembolic disease, which underscores the importance of the protein C regulatory system (see Chapter 42).

Serine Protease Inhibitors (Serpins)

Antithrombin was the first of the coagulation regulatory proteins to be identified and the first to be assayed routinely in the clinical hemostasis laboratory.⁴⁷ Other members of the serpin family are heparin cofactor II, ZPI, protein C inhibitor, α₁-protease inhibitor (α₁-antitrypsin), α₂-macroglobulin, α₂-antiplasmin, and PAI-1.⁴⁸

Antithrombin is a serine protease inhibitor (serpin) that binds and neutralizes all serine proteases except factor VIIa, including thrombin (factor IIa) and factors IXa, Xa, XIa, XIIa, kallikrein, and plasmin. Heparin cofactor II is a serpin that inactivates primarily thrombin. Antithrombin and heparin cofactor II both require heparin for effective anticoagulant activity. In vivo, heparin is available from endothelial associated mast cell granules or as endothelial cell heparan sulfate, a natural glycosaminoglycan that activates antithrombin, although not to the same intensity as unfractionated heparin. Therapeutically, unfractionated heparin, low-molecular-weight heparin, and heparin pentasaccharide are administered as anticoagulants. Unfractionated heparin is a heterogeneous glycosaminoglycan composed of 15 to 30 saccharide units. Unfractionated heparin increases the ability of antithrombin to neutralize thrombin and factors IXa, Xa, XIa, and XIIa by 1000-fold. Any heparin molecule of 17 or more saccharide units simultaneously binds antithrombin and thrombin. The antithrombin covalently binds and inactivates a thrombin molecule, forming an inactive thrombin-antithrombin complex, which is released from the heparin molecule. Laboratory measurement of thrombin-antithrombin is used as an indicator for thrombosis. Unfractionated heparin’s catalytic effect is to approximate thrombin to antithrombin (i.e., to bring the two molecules into proximity with each other) and to induce allostery, or change in steric conformation, of the antithrombin molecule (Figure 40-17). Heparin fractions of less than 17 saccharide units, such as therapeutic low-molecular-weight heparin, are able to bind antithrombin or thrombin separately but cannot bring them into the necessary spatial configuration to bind each other. Nevertheless, the allosteric changes induced in the antithrombin still make it capable of binding and inactivating factor Xa and other serine proteases. To summarize, with unfractionated heparin therapy, antithrombin preferentially inactivates thrombin, and with low-molecular-weight heparin therapy, antithrombin preferentially inactivates factor Xa.

ZPI, in the presence of its cofactor, protein Z, is a potent inhibitor of factor Xa.49–51 ZPI covalently binds protein Z and Xa in a complex with Ca²⁺ and phospholipid. Protein Z is a vitamin K–dependent plasma glycoprotein that is synthesized in the liver. Although protein Z has a structure similar to that of other vitamin K–dependent proteins VII, IX, X, and protein C, it lacks an activation site and, like protein S, is nonproteolytic. Protein Z increases the ability of ZPI to inhibit Xa 1000-fold. ZPI also inhibits factor XIa, in a separate reaction that does not require protein Z, phospholipid, and Ca²⁺. The inhibition of XIa is accelerated twofold by the presence of heparin.

Protein C inhibitor is a nonspecific, heparin-binding serpin that inhibits a variety of proteases, including APC, thrombin, thrombin-thrombomodulin complex, TPA, and urokinase.⁴⁸ It is found not only in plasma but in many other body fluids and organs. Depending on its target, it can function as an anticoagulant (inhibits thrombin), as a procoagulant (inhibits thrombin-thrombomodulin and APC), or as a fibrinolytic inhibitor (inhibits TPA).

The serpins α₁-protease inhibitor and α₂-macroglobulin are able to inhibit serine proteases reversibly. See Table 40-11 and the section on fibrinolysis for further information on α₂-antiplasmin and PAI-1.

Plasminogen and Plasmin

Plasminogen is a 92,000-D plasma zymogen produced by the liver (see Table 40-11).^53,54 It is a single-chain protein possessing five glycosylated loops termed kringles. Kringles enable plasminogen to bind fibrin lysine residues during polymerization. This binding is essential to fibrinolysis. Plasminogen likewise binds plasma α₂-antiplasmin, the major fibrinolysis control protein. Fibrin-bound plasminogen becomes converted into a two-chain active plasmin molecule when cleaved between arginine at position 561 and valine at position 562 by neighboring bound TPA or urokinase. Plasmin is a serine protease that systematically digests fibrin polymer by hydrolysis of arginine-related and lysine-related peptide bonds. As fibrin becomes digested, the exposed carboxy-terminal lysine residues bind additional plasmin, which incrementally accelerates clot digestion. Plasmin is capable of digesting fluid-phase fibrinogen and factors V and VIII; however, localization to fibrin through lysine binding prevents systemic activity. Plasma α₂-antiplasmin rapidly binds and inactivates free plasmin. Several aminocarboxalic acids have an affinity for plasminogen’s kringles, which accounts for the antifibrinolytic properties of therapeutic tranexamic acid and ε-aminocaproic acid.

Endothelial cells secrete TPA, which hydrolyzes fibrin-bound plasminogen and initiates fibrinolysis. TPA, with two glycosylated kringle regions, forms covalent lysine bonds with fibrin during polymerization and segregates at the surface of the thrombus with plasminogen, where it begins the digestion process by converting plasminogen to plasmin. Free TPA circulates bound to inhibitors such as PAI-1 and is cleared from plasma. Synthetic recombinant TPAs mimic intrinsic TPA and are a family of drugs used to dissolve pathologic clots that form in venous and arterial thrombotic disease.

Fibrin Degradation Products and D-Dimer

Fibrinolysis produces a series of identifiable fibrin fragments, X, Y, D, E, and D-D (see Figure 40-19).⁵⁶ Several of these fragments inhibit hemostasis and contribute to hemorrhage by preventing platelet activation and by hindering fibrin polymerization. Fragment X is described as the central E domain with the two D domains (D-E-D), minus some peptides cleaved by plasmin. Fragment Y is the E domain after cleavage of one D domain (D-E). Eventually these fragments are further digested to individual D and E domains. The D-D fragment, called D-dimer, is two D domains from separate fibrin molecules cross-linked by the action of factor XIIIa. Fragments X, Y, D, and E are produced by digestion of either fibrin or fibrinogen by plasmin, but D-dimer is a product of digestion of fibrin. The various fragments may be detected by quantitative or semiquantitative immunoassay to reveal fibrinolytic activity. D-dimer is separately detectable by a quantitative monoclonal antibody immunoassay. The D-dimer immunoassay is used to identify chronic and acute DIC and to rule out venous thromboembolism in suspected cases of deep venous thrombosis or pulmonary embolism.