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CHAPTER 112 MOLECULAR BIOLOGY AND BIOCHEMISTRY OF THE COAGULATION FACTORS AND PATHWAYS OF HEMOSTASIS

CHAPTER 112 MOLECULAR BIOLOGY AND BIOCHEMISTRY OF THE COAGULATION FACTORS AND PATHWAYS OF HEMOSTASIS
Williams Hematology

CHAPTER 112 MOLECULAR BIOLOGY AND BIOCHEMISTRY OF THE COAGULATION FACTORS AND PATHWAYS OF HEMOSTASIS

HAROLD R. ROBERTS
DOUGALD M. MONROE III
MAUREANE HOFFMAN

Molecular Biology, Biochemistry, and Life Span of the Coagulation Factors

Vitamin K–Dependent Zymogens (Prothrombin; Factors VII, IX, and X; and Protein C)

Soluble Cofactors (Protein S, Factor V, Factor VIII, and Von Willebrand Factor)

Factor XI and the Contact Factors

Cell-Associated Cofactors

Fibrinogen

Factor XIII

Inhibitors
Pathways of Hemostasis

Early Models of Coagulation

A Cell-Based Model of Coagulation
Chapter References

Blood coagulation is a very delicately balanced system. When it functions as it should, the blood is maintained in a fluid state in the vasculature, yet rapidly clots to seal an injury. When hemostatic functions fail, hemorrhage or thromboembolic phenomena result. This chapter addresses molecular and biochemical features of the proteins of the coagulation system and how they interact with cells and with one another to provide hemostasis in the living organism. We have grouped the coagulation factors as (1) the vitamin K–dependent zymogens (prothrombin; factors VII; IX, and X; and protein C); (2) the soluble cofactors [protein S, factor V, factor VIII, and von Willebrand factor (vWf)]; (3) factor XI and the other “contact” factors; (4) cell-associated cofactors (tissue factor and thrombomodulin); (5) fibrinogen; (6) factor XIII; and (7) the plasma coagulation protease inhibitors. Major features of the coagulation factors addressed in this chapter are shown in Table 112-1. A model of the coagulation pathway is presented that is based on current understanding of cell–cell and cell–protein interactions that regulate hemostasis. This scheme emphasizes the importance of cellular localization and plasma protease inhibitors in confining the coagulation reactions to a specific site of injury.

Acronyms and abbreviations that appear in this chapter include: ATIII, antithrombin III; BiP, immunoglobulin-binding protein; C/EB, CCAAT/enhancer-binding protein; EGF, epidermal growth factor; Gla, g-carboxyglutamic acid; HK, high-molecular-weight kininogen; HNF, hepatic nuclear factor; IL, interleukin; PK, prekallikrein; PS, phosphatidylserine; SCR, short consensus repeat; TAFI, thrombin-activatable fibrinolytic inhibitor; TF, tissue factor; TFPI, tissue factor pathway inhibitor; TM, thrombomodulin; vWf, von Willebrand factor.

TABLE 112-1 CHARACTERISTICS OF COAGULATION PROTEINS

MOLECULAR BIOLOGY, BIOCHEMISTRY, AND LIFE SPAN OF THE COAGULATION FACTORS
VITAMIN K–DEPENDENT ZYMOGENS (PROTHROMBIN; FACTORS VII, IX, AND X; AND PROTEIN C)
COMMON STRUCTURAL AND FUNCTIONAL FEATURES
The vitamin K–dependent coagulation zymogens are inert precursors of serine proteases that must be proteolytically activated to express their enzymatic activity. They all share a similar protein domain structure (Fig. 112-1). Each of the mature vitamin K–dependent coagulation zymogen proteins has an amino-terminal g-carboxyglutamic acid (Gla) domain with 9 to 12 Gla residues. This is followed by a hydrophobic region. All except prothrombin have two epidermal growth factor (EGF)–like domains, and all have a serine protease domain in their carboxy-terminal regions. Prothrombin has two kringle domains instead of EGF-like domains. Specific functions are associated, at least in part, with specific domains.

FIGURE 112-1 Comparison of the Gla-containing zymogens showing their basic structural elements. Each circle is an amino acid. The pre-pro leader sequence contains the signal peptide as well as elements that direct carboxylation of glutamyl residues. Cleavage of the leader sequence is indicated by a slight separation from the mature protein. All have a Gla domain, with the Gla residues indicated by filled blue circles. Prothrombin has a finger loop followed by two kringle domains. Factors VII, IX, and X and protein C have EGF-like domains. Prothrombin, factor VII, and factor IX circulate as single-chain molecules. Factor X and protein C circulate as two chains that are disulfide linked. All have a catalytic domain that is homologous among the Gla-containing zymogens. The active-site His, Asp, and Ser residues are indicated by the black circles in the catalytic domain. Cleavages that convert the zymogen to an active enzyme are indicated by the arrows. In factor IX, factor X, and protein C, the released activation peptide is indicated by the gray circles. After cleavage, all of the factors are two-chain disulfide-linked molecules. The disulfide connecting the catalytic domain with the rest of the molecule is shown by the heavy bond. All catalytic domains except that of prothrombin remain attached to the Gla domain following activation.

In addition to the functional modules found in the mature protein, each vitamin K–dependent factor is synthesized with an amino-terminal signal sequence directing it to the endoplasmic reticulum, followed by a 19– to 25–amino acid propeptide that is recognized by the g-glutamylcarboxylase, which catalyzes carboxylation of glutamic acid residues in the amino-terminal portion of the molecule. Following translocation into the endoplasmic reticulum, the signal sequence is removed by a microsomal signal peptidase. The propeptide is cleaved following carboxylation before the mature protein is secreted.
Not only are the proteins homologous, but their gene structures are highly similar as well. The coding regions of the vitamin K–dependent factors are quite similar in size. However, the intron lengths vary substantially and account for the differences in the overall size of the genes (20 kb for prothrombin, 13 kb for factor VII, 33 kb for factor IX, 25 kb for factor X, and 10 kb for protein C). Although the cDNA of all of the vitamin K–dependent factors has been sequenced, the noncoding regions have only been characterized to varying degrees of detail. The vitamin K–dependent coagulation zymogens are synthesized primarily by the liver, and thus they all have regulatory elements that direct liver-specific expression. The regulatory elements vary, however, among the proteins.
In factors VII, IX, and X and protein C, the introns occur in identical positions in the genes,1,2 suggesting that these enzymes evolved by duplication of a common ancestral precursor gene. The regions of the molecules that are thought of as functional domains tend to be encoded in their entirety by a single exon; that is, the signal peptide by one exon, the propeptide and Gla region by the next exon, and so on. This “modular” design suggests how “exon shuffling” could splice together intact functional units of different proteins to give rise to new proteins with novel properties.
The Gla domain that is characteristic of these proteins mediates interaction of the protein with lipid membranes. The Gla domain is named for the modified amino acids found among the first 42 residues of the mature protein. Gla residues are produced by the posttranslational modification of glutamic acid residues carried out by a specific g-glutamylcarboxylase in the endoplasmic reticulum (Fig. 112-2). The propeptide sequence is required for g-carboxylation to take place and is highly conserved among the vitamin K–dependent factors. Amino acids at positions –18, –17, –16, –15, and –10 are critical for recognition by the carboxylase.3,4 This carboxylase requires oxygen, carbon dioxide, and the reduced form of vitamin K for its action. For each glutamyl residue that is carboxylated, one molecule of reduced vitamin K is converted to the epoxide form. A separate enzyme complex, vitamin K epoxide reductase, is required to convert the epoxide form of vitamin K back to the reduced form. Warfarin inhibits the activity of vitamin K epoxide reductase and prevents recycling of vitamin K back to the reduced form. The effect of warfarin is, therefore, to inhibit g-glutamylcarboxylation, with the result that a heterogeneous population of undercarboxylated forms of the Gla-containing factors appears in circulation. These undercarboxylated forms have reduced activity. Since warfarin blocks the epoxide reductase (rather than blocking the carboxylase) and prevents recycling of vitamin K, the effects of warfarin poisoning can be reversed by administration of vitamin K. Mutations of the carboxylase can lead to low levels of all of the Gla-containing factors.5

FIGURE 112-2 Vitamin K carboxylase activity. Glutamyl residues are converted to g-carboxyglutamyl residues by a specific carboxylase. This reaction requires oxygen, carbon dioxide (shown in blue), and reduced vitamin K in the form of a hydroquinone. Carbon dioxide is incorporated into the g-carbon, providing a second carboxylate group on that residue. In the process of this reaction, reduced vitamin K is converted to an epoxide. Reduced vitamin K is recycled by a specific epoxide reductase, a reaction that can be blocked by warfarin.

The calcium-bound form of the Gla domain is responsible for mediating association with phospholipid membranes. Lipids with negatively charged head groups, primarily phosphatidylserine (PS), are required for this binding. Even in the absence of the appropriate protein cofactor, binding to phospholipids increases the proteolytic activity of Gla-containing proteases. Phosphatidylserine is required for activity on synthetic phospholipid membranes. The role of PS in mediating coagulation reactions on cellular membranes is more complex. PS is not normally exposed on the outer membrane leaflet of cells in contact with flowing blood. Further, activation of cells (particularly platelets) is often accompanied by exposure of PS on the outer leaflet of cell membranes. Since this activation enhances the ability to support coagulation reactions, it has often been assumed that exposure of PS on the outer surface of cells is sufficient to account for the ability of a cell to support coagulation reactions. However, other studies have shown that the level of coagulant activity on cells does not directly correlate with the amount of PS exposure. This result is in direct contrast to studies with phospholipid membranes in which the level of coagulant activity is directly related to the amount of PS expressed. From these studies, we conclude that PS exposure is necessary for cells to support coagulation reactions, and that other features, such as cell receptors and/or binding proteins, are also necessary.
There is very high homology in the amino acid sequence in the first 42 residues of the Gla-containing proteins. This implies that the three-dimensional structure is highly conserved and that few specific interactions are determined by this region. It was once thought that the binding of Gla-containing proteins to phospholipids was mediated by calcium ion “bridging” between the Gla residues and the negatively charged phospholipid. This mechanism provided a good explanation for why both calcium and negatively charged phospholipid were required for binding. It is currently believed, however, that binding of Gla-containing factors to lipid surfaces is mediated by membrane insertion of hydrophobic residues in the first ten amino acids of the Gla domain. Calcium is essential for this to occur because calcium binding to the Gla residues induces a dramatic conformational change that exposes the hydrophobic amino acid residues in a “patch” on the surface of the protein. This patch allows the protein to insert into the phospholipid membrane (Fig. 112-3).

FIGURE 112-3 Calcium-ion binding to the Gla domain alters its conformation. The figure shows molecular models of the Gla domain of prothrombin. The calcium-bound form is taken from the x-ray crystal structure.6 The noncalcium form is modeled from the NMR structure of factor X.7 Gla residues are shown in light blue. Hydrophobic residues believed to be important in membrane insertion are shown in dark blue (residues 5, 6, and 9). Calcium ions are shown in black. In the absence of calcium, the negatively charged Gla residues are exposed to the solution, and the hydrophobic residues are buried. Calcium-ion binding to the Gla residues provides sufficient energy to alter the overall conformation of the Gla domain and expose the hydrophobic residues. In this view, only four of the seven bound calcium ions can be seen. Insertion of the hydrophobic residues into a membrane is illustrated schematically.

The striking degree of homology among the Gla domains of the vitamin K–dependent clotting factors would suggest that the affinity of the calcium–Gla complexes for phospholipids would also be very similar. However, this turns out not to be the case. Factor IX and factor X bind much more strongly (Kd » 0.25 µM) to phosphatidylcholine- and PS-containing vesicles than does factor VII (Kd = 17 µM).8 The reasons for these marked differences are not clear.
The first EGF domain of the vitamin K–dependent proteins has a calcium ion–binding site that does not involve Gla residues but does involve a b-hydroxyaspartic acid. This conserved aspartic acid residue is modified posttranslationally by a b-hydroxylase about which little is known. Binding of calcium to this EGF-1 site appears to be important in activity and probably serves to orient the Gla domain relative to the rest of the molecule. The EGF-1 and EGF-2 domains serve, at least in part, to space the serine protease domain above the lipid membrane surface. Factor VIIa interaction with its cofactor, tissue factor, is mediated to some degree by direct interaction between tissue factor and both EGF domains of factor VIIa.
All of the zymogen Gla-containing factors undergo activation by cleavage of at least one peptide bond (see Fig. 112-1). Activation is indicated by appending the letter “a” to the name of the factor, except for protein C, which is often abbreviated aPC. The cleavage that leads to activation generates a new amino terminal that folds back and interacts with specific residues in the serine protease domain. This interaction changes the conformation of the protein such that the active site residues (His, Ser, and Asp) are aligned and the protease activity of the factor is expressed.
The serine protease domains of all the Gla-containing factors show a high homology to each other and to chymotrypsin and trypsin; all have trypsinlike activity, with an almost absolute specificity for cleaving at arginyl residues. However, unlike trypsin, which shows little specificity beyond cleaving after an arginyl or lysyl residue, the activated coagulation factors have extended substrate specificity pockets such that only a small number of amino acid sequences are recognized by each activated factor. Despite the high degree of homology between the protease domains of protein C, prothrombin, and factors VII, IX, and X, each of these factors has a highly specific function in coagulation that is mediated by surface loops that are not highly homologous.
The activated forms of factors VII, IX, and X each associate with a specific cofactor. Tissue factor is the cofactor for factor VIIa; factor VIIIa is the cofactor for factor IXa; and factor Va is the cofactor for factor Xa. The factors and cofactors associate on cell membranes to form proteolytically active complexes. Thrombin does not require a cofactor for its procoagulant activity. However, upon association with the cofactor thrombomodulin (TM), its specificity is changed from procoagulant (clotting fibrinogen) to anticoagulant (cleaving and activating protein C). While each of the proteases has some activity in the absence of its cofactor, association with cofactor dramatically enhances its activity, as illustrated in Table 112-2, which shows the enhancement of factor IXa activity by calcium, activated platelets, and cofactor.9,10 Thus, the physiological coagulant activity of factors VIIa, IXa, and Xa is only expressed as a part of a complete procoagulant complex. The complexes are sometimes named for their physiological substrate: the factor IXa/VIIIa complex is termed the tenase or intrinsic tenase complex; the factor VIIa/tissue factor complex, the extrinsic tenase complex; and the factor Xa/Va complex, the prothrombinase complex (Table 112-3). The cofactors enhance proteolytic activity by two basic mechanisms: (1) They have binding sites for both substrate and enzyme and bring the two into close proximity; and (2) they associate with the protease and induce a conformational change that enhances enzymatic activity. The structure of the factor VIIa/TF complex has been determined by x-ray crystallography.11 Figure 112-4 illustrates the projected change in conformation of the factor VIIa molecule when it binds to its cofactor, tissue factor. The factor IXa/VIIIa and Xa/Va complexes have not been crystallized, but it is likely that similar conformational changes occur during formation of these complexes.

TABLE 112-2 COFACTOR ENHANCEMENT OF FACTOR IXa ACTIVITY

TABLE 112-3 PROTEASE/COFACTOR COMPLEXES

FIGURE 112-4 Complex of factors VIIa and TF. The crystal structure of TF12 and the TF complex13 are shown, along with a model of the free structure of factor VIIa (based on the crystal structure of factor IXa14). The Gla domain, EGF domains, and catalytic domain of factor VIIa are indicated. Calcium ions are shown in black. Binding to TF alters the overall structure of factor VIIa. The orientation of the EGF1 domain is identical in factor VIIa in the modeled free structure and in complex with TF. The crystal structure of the complex shows multiple close contacts between TF and multiple domains of factor VIIa.

PROTHROMBIN (FACTOR II)
Protein Structure Like the other vitamin K–dependent zymogens, plasma prothrombin is primarily synthesized in the liver. It circulates as a single-chain zymogen with a Mr of approximately 72,000 and a plasma half-life of about 60 h. A schematic representation is shown in Fig. 112-5. Prothrombin has 10 Gla residues, and instead of the EGF region present in most vitamin K–dependent zymogens, it has two kringle domains. Kringle domains are structures held together by three disulfide bonds that schematically resemble a Danish pastry called a kringle. The primary function of kringle structures appears to be to bind other proteins, such as activators, substrates, cofactors, or receptors.15

FIGURE 112-5 Domains of prothrombin. Each amino acid in prothrombin is shown. Gla residues are indicated by g. The cleavage site to remove the pre-pro leader sequence is indicated by an arrow. The active-site His, Asp, and Ser are shown by black circles. Cleavage sites for factor Xa/factor Va are shown by arrows. Cleavage removes the Gla domain and kringles, leaving thrombin composed of a small A-chain disulfide linked to the B chain (catalytic domain).

Molecular Biology The human prothrombin gene has been localized to chromosome 11, near the centromere.16 It has been completely sequenced and is composed of 14 exons separated by 13 introns (Fig. 112-6). The 5′-flanking region of the prothrombin gene contains the promoter region and two or more cis-acting enhancer sequences. Cis-acting sequences are portions of the DNA that act as promoters, enhancers, or silencers. Unlike many other promoters, the promoter region of the prothrombin gene does not contain a TATA box. It has multiple potential sites of transcription initiation extending from 3 to 38 bp upstream from the initial methionine. The site at –31 is the most likely start site. The region between –887 and –875 is likely to be a binding site for hepatic nuclear factor-1 (HNF-1), a DNA-binding protein that plays a role in the liver-specific expression of a number of genes.17 HNF-1 is an example of a trans-acting factor, a molecule that binds to a DNA sequence and affects expression of the associated gene. An additional site in the prothrombin promoter region with non-tissue-specific enhancer activity lies just upstream to the HNF-1 site.

FIGURE 112-6 Relationship between gene structure and protein structure in prothrombin. The exons, introns, mRNA, and protein structure are as indicated. Promoter elements upstream from exon 1 are not shown but are discussed in the text. The mRNA is 2 kb, with small 5′- and 3′-untranslated regions. In the protein, Pro indicates the pre-pro leader sequence. Kringle 1 and 2 are shown. LC, light chain or A chain.

One unusual feature of the prothrombin gene is the presence of many repetitive sequences in its 5′-flanking region.18 About 41 percent of the gene and upstream sequence consists of Alu repeats. The function of these repetitive sequences, if any, is not known.
Several polymorphisms of the prothrombin gene have been described, and one of these is now recognized to have important functional consequences. This G-to-A transition in the 3′-untranslated region (20210 G®A) of the prothrombin gene is associated with higher than normal levels of plasma prothrombin.19 Increased prothrombin levels have been associated with an increased risk of thromboembolic phenomena (see Chap. 127).
Activation and Activity Prothrombin is cleaved by the factor Xa/Va complex in two places (Arg271 and Arg320), as shown in Fig. 112-7.6,20,21 and 22 The catalytic domain (thrombin), Mr 36,600, is released from the remainder of the molecule (prothrombin fragment 1.2). Since one molecule of prothrombin fragment 1.2 is released for each molecule of thrombin, assays for fragment 1.2 reflect the level of prothrombin activation.

FIGURE 112-7 Activation of prothrombin. A model of prothrombin constructed from four crystal structures is shown.6,20,21 and 22 The Gla domain, both kringle domains, and the catalytic domain are indicated. Calcium ions are shown in black. Cleavage by factor Xa/factor Va releases thrombin from the rest of the molecule, fragment 1.2.

Thrombin cleaves a number of biologically important substrates. It removes fibrinopeptides A and B from fibrinogen to form fibrin monomers, which then spontaneously polymerize to form a fibrin clot (see Chap. 124). The anion-binding exosite spans residues 387 through 398 and is involved in binding to fibrinogen, thrombomodulin, hirudin, heparin cofactor II, and the proteolytically activated thrombin receptors. It is interesting to note that this region of thrombin is identical in human, bovine, rat, and mouse.23 In addition to directly clotting fibrinogen, thrombin has a procoagulant effect by participating in positive feedback loops by activating platelets and coagulation factors V, VIII, XI, and XIII.
Thrombin is a potent platelet activator through at least two types of receptors. These include the G-protein-linked, proteolytically activated receptors PAR-1 and PAR-4, as well as platelet glycoprotein Iba (see Chap. 111).
Another function of thrombin is to activate a procarboxypeptidase-B–like enzyme to its active state, a reaction enhanced by thrombomodulin. The active carboxypeptidase inhibits plasmin-mediated fibrinolysis by removing carboxyl-terminal lysine residues, which facilitate plasminogen binding, from partially degraded fibrin. Thus, the carboxypeptidase has been termed thrombin-activatable fibrinolytic inhibitor (TAFI).24
In addition to its procoagulant activity, thrombin also has an anticoagulant function. Thus, thrombin binds to the cofactor thrombomodulin on endothelial cells, which allows it to activate protein C.25 Thrombin also has growth factor and cytokinelike activities that may play a role in atherosclerosis, wound healing, and inflammation.26
The primary plasma inhibitor of thrombin in coagulation is antithrombin III (ATIII). Heparin cofactor II also inhibits thrombin and may serve as an extravascular thrombin inhibitor that regulates the growth factor and cytokinelike activities of thrombin.26
FACTOR VII
Protein Structure Factor VII circulates as a single-chain zymogen of Mr 50,000. It has the shortest half-life of the procoagulant factors, about 3.5 h (see Table 112-1) and has 10 Gla residues.
Molecular Biology The human factor VII gene is located on chromosome 13, very close to the gene for factor X. The gene consists of eight exons and seven introns, with an overall size of about 13 kb and an organization similar to that of the other vitamin K–dependent factors (Fig. 112-8).1,2

FIGURE 112-8 Relationship between gene structure and protein structure in factor VII. The exons, introns, mRNA, and protein structure are as indicated. Promoter elements upstream from exon 1 are not shown but are discussed in the text. The mRNA is 2.7 kb, with a small 5′-untranslated region and a relatively large 3′-untranslated region. In the protein, Pro indicates the pre-pro leader sequence. CR, connecting region; EGF, epidermal growth factor–like domains.

The major transcription start site in the factor VII gene is at –51. Three other minor start sites have been described.27 A hormone-responsive element and binding sites for the trans-acting factors HF-4 and Sp-1 are present between –233 and –58 in the promoter region of the factor VII gene.
Activation and Activity Factor VII binds to tissue factor with a Kd in the subnanomolar range. Once bound to its cofactor, factor VII can be activated by a number of different proteases that cleave between Arg152 and Ile153. The physiological activator of factor VII is thought to be factor Xa. Unlike prothrombin, the catalytic domain of factor VII is linked to the rest of the molecule by a disulfide bond, so no portion is cleaved from the protein (see Fig. 112-1). The factor VIIa/TF complex activates both factors IX and X. It is inhibited by tissue factor pathway inhibitor (TFPI) in complex with factor Xa. It is also inhibited by ATIII, but only in the presence of heparin.
FACTOR IX
Protein Structure Factor IX, also synthesized in hepatocytes, circulates as a single-chain zymogen of Mr 57,000 and a plasma half-life of 18 to 24 h. It has 12 Gla residues. Only about 40 percent of factor IX molecules are hydroxylated at Asp64 in the EGF-1 domain. All the other Gla-containing zymogens have complete hydroxylation of the homologous residues (Fig. 112-9). Factor IX contains N- and O-linked carbohydrate moieties found mostly in the activation peptide. In the mature molecule the Tyr residue at position 155 is sulfated, while the Ser residue at position 158 is phosphorylated. Factor IX, unlike other vitamin–K dependent factors, has been shown to bind effectively to collagen IV in vitro.28 The molecule appears to bind to collagen IV in vivo and may account for the observation that, when factor IX is infused into hemophilia B patients, recovery is only 50 percent of that expected (see Chap. 123).29 The physiological relevance of this observation remains to be determined.

FIGURE 112-9 Domains of factor IX. Each amino acid in factor IX is shown. Gla residues are indicated by g. The cleavage site to remove the pre-pro leader sequence is indicated by an arrow. The active-site His, Asp, and Ser are shown by black circles. Cleavage sites for factor XIa and factor VIIa/TF are indicated by arrows.

Molecular Biology The gene for factor IX is located on the tip of the long arm of the X chromosome at position Xq27.1–q27.2.30 Therefore, deficiency of factor IX (hemophilia B) is sex linked. The gene contains eight exons, seven introns, and a long 1.4-kb 3′-untranslated region, with an overall size of 33 kb (Fig. 112-10).

FIGURE 112-10 Relationship between gene structure and protein structure in factor IX. The exons, introns, mRNA, and protein structure are as indicated. Promoter elements upstream from exon 1 are not shown but are discussed in the text. The mRNA is 2.8 kb, with a small 5′-untranslated region and a relatively large 3′-untranslated region. In the protein, Pro indicates the pre-pro leader sequence. AP indicates the activation peptide, which is released after cleavage of two bonds.

Eight polymorphisms have been described within or flanking the factor IX gene. These polymorphisms can be useful for antenatal diagnosis and carrier detection of hemophilia B by restriction fragment length polymorphism analysis.31
The promoter activity of the 5′-untranslated region of the factor IX gene resides 274 bp upstream of the major transcription start site.32 Binding sites for several trans-acting factors have been identified, including sites for CCAAT/enhancer-binding protein (C/EBP),33 D-site binding protein,34 HNF-4,35 and HNF-1.36
Activation and Activity Factor IX can be activated either by factor XIa or by the factor VIIa/TF complex. Full activation requires cleavage of two bonds (Arg145 and Arg180), releasing an activation peptide of Mr of approximately 10,000 (see Fig. 112-9).
In complex with its cofactor, factor VIIIa, on a phospholipid membrane surface, factor IXa activates factor X. Physiologically this activity is primarily expressed on the surface of activated platelets, and there is evidence suggesting that platelets express a receptor-binding protein for factor IXa that promotes assembly of the factor IXa/VIIIa complex.37
The primary plasma inhibitor of factor IXa appears to be ATIII. Inhibition of factor IXa by ATIII is slow compared to ATIII inhibition of thrombin. However, it is enhanced in the presence of heparin.
FACTOR X
Protein Structure Factor X circulates as a two-chain, disulfide-linked zymogen of Mr 59,000 (see Fig. 112-1) and has a plasma half-life of about 34 to 40 h. A three amino acid sequence (Arg180-Lys181-Arg182) is cleaved from the protein during intracellular processing. The light-chain Mr is approximately 17,000, and the heavy-chain Mr is approximately 40,000. The light chain contains the Gla domain with its 11 Gla residues, and the two EGF domains. The heavy chain contains the catalytic domain and the activation peptide. Like all other vitamin K–dependent factors, it is synthesized in the liver.
Molecular Biology The gene for human factor X is on chromosome 13q34–qter39 in close proximity to the factor VII gene. It is composed of eight exons and seven introns,1 with a size of approximately 25 kb (Fig. 112-11). The 3′-untranslated region is unusually short, being only 10 base pairs. A number of potentially useful polymorphisms have been identified.40

FIGURE 112-11 Relationship between gene structure and protein structure in factor X. The exons, introns, mRNA, and protein structure are as indicated. Promoter elements upstream from exon 1 are not shown but are discussed in the text. The mRNA is 1.5 kb with a relatively large 5′-untranslated region and a small 3′-untranslated region. In the protein, Pro indicates the pre-pro leader sequence. AP, activation peptide.

The factor X promoter region has been sequenced and characterized. It lacks a typical TATA box but contains a CCAAT sequence at –120 to –116. Factor X appears to have multiple start sites of transcription.41 This finding is consistent with the multiple start sites reported for other promoters lacking a TATA box. Like the factor IX gene, a binding site for HNF-4 has been identified.42 However, unlike the factor IX gene, there does not appear to be a binding site for C/EBP.
Activation and Activity Factor X can be activated by factor VIIa/TF or factor IXa/VIIIa by cleavage at the Arg194-IIe195 bond. It can be autocatalytically cleaved near the carboxyl terminus of the heavy chain to yield “b-Xa,” which is also enzymatically active.
Factor Xa in complex with factor Va on a phospholipid membrane surface activates prothrombin to thrombin by cleaving two peptide bonds. Factor Xa may also play a physiological role in activation of factors VII,43 VIII,44 and V.45 While any membrane surface that expresses anionic phospholipid can support prothrombinase complex assembly, the activated platelet surface is especially well suited for this purpose. Prothrombinase assembly on platelets is not strictly a function of phospholipid composition, but is coordinated by one or more specific binding proteins.46
Like thrombin, factor X has biological activities not directly related to coagulation. It is reported to have mitogenic activity for smooth muscle cells.47 Factor Xa also possesses receptor-mediated proinflammatory activities.48 The primary plasma inhibitor of factor Xa is ATIII, and the inhibition by ATIII is accelerated by heparin. Tissue factor pathway inhibitor is also a potent inhibitor of factor Xa, as shown in Table 112-4.

TABLE 112-4 CHARACTERIZATION OF TFPI AND ATIII INHIBITION OF COAGULATION FACTORS

PROTEIN C
Protein Structure Protein C, unlike the other vitamin K–dependent zymogens, is not a procoagulant but controls coagulation by inactivating factors Va and VIIIa. It circulates as a two-chain disulfide-linked zymogen with 9 Gla residues (see Fig. 112-1). It has an Mr of 59,000 and a short plasma half-life of about 6 h (see Chap. 113).
Molecular Biology The gene for human protein C is on chromosome 2q13–14.49 It was originally described as being composed of eight exons with a size of approximately 10 kb.50 Other workers have described it as having nine exons and eight introns,51 with the first exon corresponding to the 5′-noncoding region (Fig. 112-12). Thus, the first exon is transcribed from the gene into mRNA but is not translated into protein. The gene structure is very similar to the other vitamin K–dependent factors, with especially close homology to factor IX.

FIGURE 112-12 Relationship between gene structure and protein structure in protein C. The exons, introns, mRNA, and protein structure are as indicated. The mRNA is 1.8 kb, with a small 5′-untranslated region coded for by exon 1 and a relatively small 3′-untranslated region. In the protein, Pro indicates the pre-pro leader sequence. AP, activation peptide.

Activation and Activity Protein C is activated by the thrombin–thrombomodulin complex. A single cleavage at Arg169-Leu170 releases a 12 amino acid activation peptide, leading to activated protein C with a Mr of 56,000.
Activated protein C, in complex with its cofactor protein S, proteolytically inactivates factors Va and VIIIa. It has also been reported that factor V can act as a cofactor for the inactivation of factors Va and VIIIa by activated protein C.52 The primary inhibitor of activated protein C is protein C inhibitor, also known as plasminogen activator inhibitor-3.53
SOLUBLE COFACTORS (PROTEIN S, FACTOR V, FACTOR VIII, AND VON WILLEBRAND FACTOR)
PROTEIN S
Protein Structure Protein S is a single-chain plasma glycoprotein cofactor with a Mr of approximately 75,000 and a plasma half-life of about 42 h. It contains 11 Gla residues in the amino-terminal region. Its structure is somewhat different from that of the Gla-containing zymogens (Fig. 112-13). Protein S is organized into a Gla domain, a thrombin-sensitive finger region, four EGF domains, and a region with homology to steroid-binding proteins. Unlike the other vitamin K–dependent factors, it does not contain a serine protease domain and so does not have the potential to catalyze reactions. Each EGF domain contains a modified amino acid, either b-hydroxyaspartic acid or b-hydroxyasparagine. Protein S circulates both in the free form (»40% of the total amount) and in a form bound to C4b-binding protein. The steroid-hormone–binding globulinlike region of protein S is involved in binding to the alpha subunit of C4b-binding protein. Protein S is inactive when bound to C4b-binding protein.54 Like the Gla-containing zymogens, protein S is synthesized with a signal peptide that directs it to the endoplasmic reticulum and a propeptide that binds to the g-glutamylcarboxylase. The signal sequence and propeptide are removed before the mature protein is secreted.

FIGURE 112-13 Relationship between gene structure and protein structure in protein S. The exons, introns, mRNA, and protein structure are as indicated. The mRNA is 2.3 kb, with a small 5′- and 3′-untranslated region. E, EGF-like domains; T, thrombin-sensitive finger region.

Protein S is synthesized primarily by hepatocytes55 but also by endothelial cells,56 megakaryocytes,57 Leydig cells,58 and osteoblasts.59
Molecular Biology The human protein S gene is on chromosome 3, spanning the centromere from p11.1 to q11.2. It is over 80 kb in length and contains 15 exons and 14 introns (see Fig. 112-13).60 Exons 1 through 8 encode protein domains that are homologous to the Gla-containing zymogens. The intron–exon structure is typical of the members of this family. Exons 9 through 15 encode protein segments homologous to steroid-hormone–binding globulin. There is also a pseudogene of protein S located on the same chromosome. It is about 55 kb in size and codes for regions corresponding to amino acids 46 through 635 of protein S.
Activity Protein S serves as a cofactor for the cleavage and inactivation of factors Va and VIIIa by activated protein C. In contrast to factors V and VIII, it does not require proteolytic activation for its cofactor activity. Protein S alone also has a low level of anticoagulant activity by virtue of its ability to compete with factor Xa for binding to factor Va.61 For protein S to serve as a cofactor for activated protein C, it must be in the free form, rather than bound to C4b-binding protein. While the C4b-binding protein is an acute-phase reactant, its alpha subunit, which binds protein S, is not increased in inflammatory states; thus, the free protein S concentration is not affected by the acute-phase response.
FACTOR V
Protein Structure Factors V and VIII are homologous in their gene structures, amino acid sequences, and protein domain structures. Factor V is a large glycoprotein with a Mr of approximately 330,000 and a plasma half-life of about 12 h, with some reports of a half-life of up to 36 h.62 It has the following domain organization: A1-A2-B-A3-C1-C2 (Fig. 112-14). The three A domains have significant homology to the copper-binding plasma protein ceruloplasmin. The C domains have some homology to fat globule proteins. The C2 domain of factor V mediates binding to lipid membranes.63 The A and C domains of factor V are approximately 40 percent identical to the homologous regions in factor VIII. In contrast, the B domains show little homology between the two proteins and are not known to be homologous to any other proteins. In factor V, unlike factor VIII, sequences in the B domain appear to be important in promoting its activation by thrombin.

FIGURE 112-14 Relationship between gene structure and protein structure in factor V. The exons, introns, mRNA, and protein structure are as indicated. The mRNA is 7 kb, with some 5′- and 3′-untranslated sequence. In the protein, P indicates the propeptide leader sequence.

The acidic regions of factor V have a high proportion of Asp and Glu residues. These regions are thought to be important in promoting activation, possibly by providing a site of interaction with the anion-binding exosite of thrombin.
Factor V shows five potential sites for tyrosine sulfation, at residues 696, 698, 1494, 1510, and 1565. Sulfation of factor V also plays a role in the protein’s activity by enhancing activation by thrombin and by promoting maximal factor Xa activation of prothrombin.64 Factor V contains both N-linked and O-linked carbohydrate moieties, most of which are clustered in the B domain.
Molecular Biology The gene for factor V is located on chromosome 1q21–q25. It is located very close to the genes for the selectin family of leukocyte adhesion molecules. The factor V gene spans about 70 kb and consists of 25 exons (see Fig. 112-14). The gene structure is very similar to that of the factor VIII gene, with exon–intron boundaries occurring at exactly the same location in 21 of 24 cases.65 The mechanisms governing factor V gene transcription and translation are not clear.
Activation and Activity Factor V circulates in plasma as a single-chain molecule. As much as 20 percent of the circulating factor V is found in platelet alpha granules. However, platelet factor V is heterogeneous because of proteolysis within the B domain. Platelet proteases, including calpain, are able to cleave the B domain and produce a partially activated form of factor V (see Chap. 122).
In platelets, but not in plasma, the factor V is complexed to a large multimeric protein called multimerin.66 Multimerin has a massive repeating structure, with some of the multimers having molecular weights of several million. Multimerin has structural features that suggest it may mediate adhesive interactions. While multimerin appears functionally similar to vWf, the two proteins share no structural homology.
Full factor V cofactor activity is achieved only after cleavage at several bonds (Fig. 112-15). Factor V is believed to be primarily activated by thrombin in vivo, although it can be activated by factor Xa as well.45 Thrombin cleaves factor V at Arg709 and Arg1545 to produce a two-chain heterodimeric molecule consisting of an A1-A2 heavy chain (Mr 110,000) that is associated with an A3-C1-C2 light chain (Mr 73,000). The two chains are noncovalently linked through metal ions (probably calcium). Activated factor V is inactivated by activated protein C cleavage at Arg306 and Arg506, followed by dissociation of the cleaved A2 fragments.62 A common Arg506Gln change confers activated protein C resistance that has been associated with an increased risk of venous thromboembolism.67

FIGURE 112-15 Activation and inactivation of factor V. For full cofactor activity, factor V requires cleavage by thrombin or factor Xa. The acidic domains, shown in dark blue, are believed to bind to the anion-binding exosite in thrombin and enhance thrombin activation of factor V. Cleavage of residue 1018 enhances cleavage at residue 1545. Heavy and light chains are held together by noncovalent interactions mediated by metal ions (Me). Membrane binding is mediated through a site in the C2 domain (shown in light blue). Cleavages at residues 306 and 506 by activated protein C inactivate factor V by releasing two A2 fragments (iVa).

FACTOR VIII
Protein Structure The domain organization of the factor VIII protein is A1-A2-B-A3-C1-C2, like that of factor V (Fig. 112-16). In factor VIII, the B domain does not appear to play a significant role in stability or activation. B-domainless factor VIII has been used successfully as a therapeutic agent in patients with classic hemophilia.

FIGURE 112-16 Relationship between gene structure and protein structure in factor VIII. The exons, introns, mRNA, and protein structure are as indicated. Promoter elements upstream from exon 1 are not shown but are discussed in the text. The mRNA is 9 kb, with some 5′-untranslated sequence and a large 3′-untranslated region. In the protein, P indicates the propeptide leader sequence.

Factor VIII is synthesized in the liver and is secreted into the plasma as a heterogeneous collection of partially cleaved forms resulting from different cleavages in the B domain. Factor VIII circulates in a noncovalent complex with vWf. The normal half-life of factor VIII is 8 to 12 h when associated with vWf. The half-life is markedly reduced in the absence of vWf, accounting for the reduced factor VIII levels observed in many patients with a deficiency of vWf.
Factor VIII has six tyrosine residues that are modified by sulfation (residues 346, 718, 719, 723, 1664, and 1680). Sulfation of these residues is required for optimal activation by thrombin, maximal activity in complex with factor IXa, and maximal affinity for vWf.
The acidic regions of factor VIII appear to promote activation by interacting with the anion-binding exosite of thrombin. In addition, the site for factor VIII binding to vWF is in the acidic domain in the light chain of factor VIII.68
Molecular Biology The factor VIII gene is on the X chromosome at q28. Deficiency of factor VIII results in classic sex-linked hemophilia A. The factor VIII gene contains 26 exons (see Fig. 112-16), one more than factor V. Exon 5 of factor V corresponds to exons 5 and 6 of the factor VIII gene.69 The gene for factor VIII is much larger than that for factor V, spanning about 190 kb. This is largely because six of the introns in the factor VIII gene are much larger than the corresponding introns in the factor V gene. The mRNA for factor VIII is also much larger than that for factor V because of a 1.8-kb 3′-untranslated region in the factor VIII message.
Attempts to produce factor VIII in cell culture for therapeutic use have recently led to advances in understanding its intracellular processing in the endoplasmic reticulum and Golgi apparatus. Factor VIII is not secreted very efficiently from the cell. Several molecular chaperone proteins have been identified that appear to play a role in regulating transit of the factor VIII protein through secretory and/or degradative pathways. Calnexin and calreticulin are chaperone proteins that preferentially interact with glycoproteins containing monoglycosylated N-linked oligosaccharides. These proteins bind to the heavily glycosylated B domain of factor VIII and enhance both its intracellular degradation and secretion.70 Factor V associates with calreticulin but not calnexin. Factor VIII, but not factor V, also interacts through its A1 domain with another chaperone protein, immunoglobulin-binding protein (BiP). Association with BiP appears to enhance the stability of factor VIII, but also retards its secretion.71
The mannose-binding protein ERGIC-53 is a protein found in the intermediate compartment of the Golgi apparatus that facilitates secretion of both factor V and factor VIII.72 The apparent chaperone function of ERGIC-53 was defined when it was discovered that mutations in the ERGIC-53 gene can lead to a hereditary deficiency of both factors V and VIII.73 While the complex mechanisms regulating factor VIII secretion are not fully understood, these recent results suggest that factor VIII has unique requirements for molecular chaperone protein interactions for its intracellular processing.
Activation and Activity Factor VIII is activated by thrombin or factor Xa by cleavages at arginyl residues 372, 740, and 1689 (Fig. 112-17). This produces a heterotrimeric molecule consisting of A1 and A2 domains noncovalently linked with an A3-C1-C2 light chain through calcium ions. Activation also results in the release of factor VIIIa from vWf. The factor VIIIa molecule is thermodynamically unstable, and dissociation of the A2 domain results in the spontaneous loss of activity. Factor VIIIa is also inactivated by thrombin or activated protein C (aPC) through additional cleavages at arginyl residues 336 and 562 (see Fig. 112-17).

FIGURE 112-17 Activation and inactivation of factor VIII. For full cofactor activity, factor VIII requires cleavage by thrombin or factor Xa. The acidic domains, shown in dark blue, are believed to bind to the anion-binding exosite in thrombin and enhance thrombin activation of factor VIII. The acidic region in the A3 domain mediates vWf binding. The chains of factor VIIIa are held together by noncovalent interactions mediated by metal ions (Me). Factor VIIIa is thermodynamically unstable because the A2 domain can spontaneously dissociate from the complex. Membrane binding is mediated through sites in the C2 domain (shown in light blue). Cleavages at residues 336 and 562 inactivate factor VIIIa, releasing the A2 fragments (iVIIIa).

VON WILLEBRAND FACTOR
The structure, molecular biology, and activities of vWf are discussed in greater detail in Chap. 135.
Protein Structure and Activity von Willebrand factor is a large multimeric glycoprotein that serves as a carrier for factor VIII and is required for normal platelet adhesion to components of the vessel wall. It is synthesized as a prepropolypeptide with a 22 amino acid signal sequence, a 741 amino acid precursor polypeptide called vWf antigen II, and the mature vWf polypeptide chain.74 The mature vWf protein contains three A domains, three B domains, two C domains, and four D domains. The A domains are structurally homologous to a family of proteins involved in extracellular matrix or cell adhesive functions.75 Factor VIII binds to the amino-terminal region of vWf, within the first 272 amino acids of the mature protein subunit.76
In the endoplasmic reticulum, the pro-vWf monomers form disulfide-stabilized dimers. The dimers move to the Golgi apparatus, where they assemble into high-molecular-weight multimers, which are also held together by disulfide bonds. The propeptide is essential for multimerization to occur. It is usually removed before secretion of the mature vWf multimers. The circulating vWf multimers range in size from Mr of approximately 500,000 to over 20,000,000.77 The higher-molecular-weight multimers are most effective in promoting platelet adhesion. However, all multimers can bind factor VIII and enhance its stability. The plasma half-life of vWF is about 12 h.
Molecular Biology The vWF gene is located on chromosome 12 and spans about 180 kb. It contains 52 exons.78 von Willebrand factor is synthesized only in endothelial cells and megakaryocytes, but how tissue-specific expression is regulated is not known.
FACTOR XI AND THE CONTACT FACTORS
FACTOR XI
Protein Structure Factor XI, along with factor XII, high-molecular-weight kininogen (HK), and prekallikrein (PK), are sometimes referred to as the contact factors. Factor XI is a zymogen precursor of a serine protease. Factor XI circulates in complex with the nonenzymatic cofactor HK.
Factor XI is synthesized in the liver and has a plasma mean half-life of 52 h.79 Although synthesized as a single chain, it circulates as a homodimer held together by a disulfide bond.80 Each subunit has a Mr of approximately 80,000, including about 5 percent carbohydrate. Each factor XI subunit contains four repeats of a structural motif called an apple domain, as shown in Fig. 112-18. Each apple domain contains 90 or 91 amino acids held together by three disulfide bonds. Specific functions have been assigned to the different apple domains within factor XI,81,82,83 and 84 including sites for binding to HK, prothrombin, platelets, factor IX, thrombin, and factor XIIa.

FIGURE 112-18 Domains of factor XI monomer. Each amino acid in circulating factor XI is shown. The apple 1 through 4 domains are named for their appearance in this scheme. The disulfide bond that links the factor XI homodimers is indicated by the Cys residue in the first apple domain. Cys321 in the apple 4 domain is also needed for dimerization. The active-site His, Asp, and Ser residues are circled. The cleavage site for factor XIIa is shown by the arrow.

Molecular Biology The human factor XI gene is 23 kb in length and is localized to chromosome 4q32–35.85 It consists of 15 exons and 14 introns (Fig. 112-19).86 The transcription initiation site has not yet been determined. Exon 1 encodes a 5′-untranslated region that is transcribed into mRNA but not translated into protein. The signal peptide is encoded in exon 2. Each of the four apple domains is encoded in two exons. The light chain is encoded in five exons, with an organization similar to that of the homologous proteins PK, tissue plasminogen activator, urokinase, and factor XII.

FIGURE 112-19 Relationship between gene structure and protein structure in factor XI. The exons, introns, mRNA, and protein structure are as indicated. The mRNA is 2.1 kb, with a small 5′- and 3′-untranslated region. In the protein, Pro indicates the pre-pro leader sequence. A, apple domains.

Activation and Activity Factor XI can be activated by more than one mechanism in vitro. There is some controversy as to the mechanism of factor XI activation in vivo. In vitro, factor XI can be activated by factor XIIa. In the fluid phase and on charged surfaces, thrombin can activate factor XI even in the absence of the other contact factors.87,88 Factor XI can also be activated by thrombin on the surface of activated platelets, and this pathway is perhaps the most likely mechanism of activation in vivo.89 It seems possible that more than one mechanism operates under certain circumstances, but definitive proof is lacking.
In contrast to the other contact factors, deficiencies of factor XI may lead to a bleeding tendency,90 reflecting the significant role of factor XI in hemostasis.
Activation by either factor XIIa or thrombin is due to cleavage of the Arg369-Ile370 bond in the factor XI subunit. This leads to the presence of two active sites in each factor XIa dimer. Each subunit has a heavy chain containing the apple domains and a light chain containing the catalytic domain (see Fig. 112-18). Both the heavy and light chains interact with the substrate, factor IX.91 Factor XIa activation of factor IX is calcium dependent but does not require any other cofactor. Factor XIa binds with high affinity to activated platelets and can activate factor IX with the same efficiency as unbound factor XIa.92 Binding to activated platelets could serve to localize factor XIa to the site of clot formation as well as protect it from plasma protease inhibitors.
Factor XIa is susceptible to inhibition by several plasma protease inhibitors that circulate in high concentrations. Of these, a1-trypsin inhibitor has the highest affinity for factor XIa, followed by ATIII, C1-esterase inhibitor, and a2-plasmin inhibitor.93 Platelets also contain a slow-reacting Kunitz-type inhibitor of factor XIa, protease nexin 2.94
FACTOR XII, PREKALLIKREIN, AND HIGH-MOLECULAR-WEIGHT KININOGEN
Protein Structure Factor XII and PK are zymogen precursors of proteases. Prekallikrein has four apple domains and is highly homologous to factor XI. Factor XII is homologous to plasminogen activators. High-molecular-weight kininogen (HK) is a nonenzymatic cofactor that circulates in complex with factor XI and with PK. In addition to its nonenzymatic role in contact activation, HK acts as a thiol protease inhibitor and as an anti-adhesive protein. High-molecular-weight kininogen is cleaved at two sites by kallikrein to release the bioactive nonapeptide bradykinin, a potent vasodilator. The plasma levels, plasma half-lives, and chromosomal locations of factor XII, PK, and HK are shown in Table 112-1.95 All three proteins are synthesized in the liver.
Molecular Biology The gene for factor XII is located on chromosome 5q33–qter and spans about 12 kb. It contains 14 exons. The intron–exon structure of the gene is similar to that of the plasminogen activator family of serine proteases. Portions of the gene are homologous to domains found in fibronectin and tissue-type plasminogen activator. The gene for PK is located on chromosome 4q35, close to the factor XI gene. The precise gene structure of the human PK gene has not yet been determined. The rat PK gene spans 22 kb, has 15 exons, and is homologous to the human factor XI gene. The gene for HK is located on chromosome 3, contains 11 exons, and spans 27 kb. High- and low-molecular-weight kininogen are produced from the same gene by alternative splicing. Both proteins serve as precursors to bradykinin, but low-molecular-weight kininogen has no interaction with the coagulation proteins.
Activation and Activity Factor XII, HK, and PK are responsible for the contact activation of blood coagulation as seen in the activated partial thromboplastin time test. In this clinical laboratory test, plasma is mixed with a reagent, such as glass, kaolin, celite, or ellagic acid, that provides a negatively charged surface. Contact activation involves both protein–protein and protein–surface interactions that lead to the activation of factor XII. The factor XIIa activates factor XI, which then activates factor IX. In spite of the fact that factor XII, HK, and PK are required for a normal aPTT, they do not appear to be required for normal hemostasis. Individuals who are deficient in any of these factors do not have a bleeding tendency, even after significant trauma or surgery. However, factor XII, HK, and PK do participate in inflammatory responses that involve the blood clotting system, fibrinolysis, and generation of kinins.
CELL-ASSOCIATED COFACTORS
TISSUE FACTOR
Protein Structure Tissue factor (TF) is the cellular receptor and cofactor for factors VII and VIIa (see Fig. 112-4). Tissue factor is composed of 263 amino acids and consists of a 219 amino acid extracellular domain, a 23 residue transmembrane portion, and a 21 residue intracytoplasmic domain (Fig. 112-20).96 A cysteine in the intracytoplasmic domain is linked to a palmityl or stearoyl fatty acid, the function of which is not known.97 While many of the coagulation factors share a high degree of homology, the structure of TF is unique. It is the only one of the procoagulant proteins that is an integral membrane protein, and it is homologous to the type-2 cytokine receptors.98 This family includes the receptors for interleukin-10 and interferons-a, -b, and -g. The TF molecule has been crystallized, and the extracellular domain has been found to fold in a manner typical of the cytokine receptor homology unit (see Fig. 112-4).13,99 These structural features suggest that TF could be a multifunctional protein with both signal-transducing and procoagulant functions.

FIGURE 112-20 Relationship between gene structure and protein structure in TF. The exons, introns, mRNA, and protein structure are as indicated. The mRNA is 2.3 kb, with a 5′-untranslated region and a large 3′-untranslated region. Cyto, cytoplasmic domain; Pro, pre-pro leader sequence; Tran, transmembrane region.

Molecular Biology The human TF gene is located on chromosome 1p21–p22.100 The DNA sequence of the TF gene has been determined and consists of 6 exons and 5 introns that span about 13 kb.101 The first exon codes for the signal peptide, whereas the second through fifth encode the extracellular domain. The sixth exon codes for the transmembrane and cytoplasmic domains, as well as a relatively long 3′-untranslated region.
The initiation site for transcription of the TF gene has been well defined, and the region with promoter activity has been found to be from –383 to –121 bp relative to the start site.102 The promoter contains a serum response element with a putative binding site for Sp-1, and a lipopolysaccharide-responsive element with AP-1 and NF-kB-like sites.
Tissue factor is expressed constitutively on many extravascular tissues. While TF is not normally expressed by cells in contact with flowing blood, TF expression can be induced on blood monocytes and vascular endothelial cells by bacterial products, inflammatory cytokines, and engagement of P-selectin glycoprotein ligand-1 on monocytes.103,104,105 and 106 Expression of intravascular TF may contribute to the procoagulant state associated with inflammation or infection.
Activation and Activity The factor VIIa/TF complex is thought to be the major physiologic initiator of blood coagulation. Tissue factor is normally expressed in the adventitia of blood vessels and by epidermal, stromal, and glial cells.107,108 It has also been shown that leukocytes, which normally have no TF activity, can express TF when exposed to vessel media or collagen.109 The process of coagulation is initiated when an injury ruptures a vessel and allows blood to come into contact with extravascular TF. When circulating factor VII binds to TF, it is rapidly converted to the active protease, factor VIIa.43 The factor VIIa/TF complex can activate both factor IX and factor X.110
The binding of factor VIIa to TF enhances its proteolytic activity by almost three orders of magnitude.111,112 However, unlike binding of factor IXa or Xa to their cofactors, binding of factor VIIa to TF does not strictly require calcium,113 and the affinity of the interaction is only slightly enhanced by the presence of anionic phospholipid.114,115 However, the cleavage of factor IX or X by factor VIIa/TF is enhanced by anionic phospholipid.115 This effect is due to the enhanced binding of the substrate rather than to any effect of the phospholipid on the catalytic efficiency of the VIIa/TF complex.
Reported Kd values for factor VIIa binding to TF on cells range from approximately 20 to 80 pM. This broad range of values may reflect the effects of the local cellular environment on the affinity of TF for factor VIIa. Binding of factor VIIa to TF that is reconstituted into synthetic phospholipid vesicles always results in enhanced factor VIIa proteolytic activity. However, binding of factor VIIa to cellular sources of TF does not always correlate with enhanced enzymatic activity. This suggests that cells can regulate the cofactor activity of TF in a manner that is not reproduced by synthetic phospholipid vesicles.
Tissue factor does not require proteolytic activation to express its activity. However, it appears that TF can occur in a latent, or “encrypted,” form116,117; that is, TF detected as antigen on the cell surface may not express cofactor activity. It has been hypothesized that the TF could form dimers that block access to the substrate binding site on TF. Dimerized (“encrypted”) TF could still bind factor VII but would be inactive because it could not bind factor IX or X. The physiologic regulators that control TF encryption are not clear, and it remains to be determined whether this is an important regulatory mechanism in vivo.
THROMBOMODULIN
Protein Structure Thrombomodulin is a transmembrane protein of Mr 78,000.118 It is the cellular cofactor for thrombin.119 Thrombomodulin has a leader sequence followed by lectinlike domains homologous to the asialoglycoprotein receptor (Fig. 112-21).120 However, TM has no known lectinlike activity. Following the lectinlike domain are six EGF-like domains, the fourth, fifth, and sixth of which are responsible for both thrombin-binding and protein-C–activating activities (see Fig. 112-21).121 A serine- and threonine-rich region follows the EGF domains and is the site of O-linked glycosylation. A chondroitin sulfate moiety, which enhances TM anticoagulant activity, is attached to Ser492 in this region.122 The 23 amino acid transmembrane domain follows the serine- and threonine-rich region, followed by a short cytoplasmic tail.

FIGURE 112-21 Relationship between gene structure and protein structure in TM. The TM gene has no introns. It covers 3.7 kb on chromosome 20 (p12–centromere). The mRNA is the same size, with a small 5′-untranslated region and a large 3′-untranslated region. In the protein, Pro indicates the pre-pro leader sequence. Cyt, cytoplasmic domain; E, EGF-like domains; M, transmembrane region; S/T, serine-, threonine-rich region.

Molecular Biology The human TM gene is located on chromosome 20p12–cen123 and spans about 3.5 kb. It consists of a single exon (see Fig. 112-21). Intronless genes are uncommon and include rhodopsin, angiogenin, mitochondrial genes, interferons-a and -b, and b-adrenergic receptors. The functional significance of the lack of introns is not known.
Activation and Activity Thrombin can cleave a number of substrates without a cofactor, such as fibrinogen, factors V and VIII, and the proteolytically activated thrombin receptors. However, binding to the cofactor TM localizes thrombin to endothelial cell surfaces and induces a conformational change such that its ability to activate protein C is enhanced 1000- to 2000-fold. Thrombin bound to TM no longer activates platelets, nor does it cleave fibrinogen or activate factor V or factor VIII.124 Thus, TM changes the activity of thrombin from procoagulant to anticoagulant. Thrombomodulin also enhances the ability of thrombin to activate the thrombin-activatable inhibitor of fibrinolysis (TAFI)24
Thrombomodulin is expressed on the surface of vascular endothelial cells and appears to play a major role in preventing thrombosis from occurring on intact endothelium in the microcirculation.125 Thrombomodulin has also been detected in mesothelial cells,126 mononuclear phagocytes,127 squamous epithelium,128 megakaryocytes, and malignant cells,25,129 where its function is unknown. The level of TM expression differs among endothelial cells from different sites.130 Endothelial TM and TF expression are regulated by inflammatory cytokines in a reciprocal fashion. Thus, thrombosis may be favored at sites of inflammation by a concurrent elevation of endothelial TF and depression of endothelial TM.
Protein C inhibitor has recently been shown to be an effective inhibitor of the thrombin–TM complex.131
FIBRINOGEN
Protein Structure Fibrinogen forms the structural meshwork that consolidates an initial platelet plug into a solid hemostatic clot. The physiologic importance of fibrinogen is underscored by the bleeding diathesis associated with afibrinogenemia132,133 and some dysfibrinogenemias134 (see Chap. 124). Other dysfibrinogenemias are associated with thromboembolic disease.135
Fibrinogen is a dimeric glycoprotein whose dominant form has a Mr of 340,000. It is found in plasma and in platelet a-granules. Each of the two subunits contains three disulfide-linked polypeptide chains136 that are referred to as the Aa (Mr 66,500), Bb (Mr 52,000), and g (Mr 46,500) chains. Fibrinopeptides A and B are released from the amino termini of the Aa and Bb chains by thrombin cleavage of the Argl6-Glyl7 and Argl4-Glyl5 bonds, respectively.137 The central globular domain of fibrinogen is called the E domain. It includes the disulfide-linked amino termini of all six polypeptide chains, referred to as the N-terminal disulfide knot.138 The E domain is linked by helical, coiled-coil domains to the carboxyl-terminal globular domains of the three chains, designated the D domains. A trinodular model of fibrinogen structure has been proposed based on its electron microscopic appearance (Fig. 112-22). N-linked glycosylation occurs at Asn364 of the Bb chain and Asn52 of the g chain.

FIGURE 112-22 Structure of fibrinogen. Fibrinogen is a dimer. Each dimer consists of three chains: Aa, shown in gray; Bb, shown in blue; and g, shown in black. The disulfides that link the two dimers are in the central E domain. The D domains consist primarily of the carboxyl-terminal regions of the Bb and g chains. The helical region connecting the two domains consists of all three chains intertwined.139

In normal individuals the plasma half-life of fibrinogen is 3 to 5 days,140 with only a small proportion of the catabolism due to consumption. Plasma fibrinogen is synthesized in the liver. Fibrinogen is an acute-phase reactant, and its synthesis can be increased up to 20-fold with a strong inflammatory stimulus.141,142 Interleukin-6 (IL-6) is an important mediator of increased fibrinogen synthesis during an acute-phase response,143 and IL-6 secretion can be upregulated by fibrin- or fibrinogen-degradation products.
MOLECULAR BIOLOGY
The genes for the three chains of fibrinogen are found within a 50-kb length of DNA on chromosome 4 at q23–q32 (Fig. 112-23);144 have all been sequenced. The genomic sequences show a high degree of homology, suggesting they were derived through duplication of a common ancestral gene.145,146 The homology extends to sites upstream of the gene, suggesting that common regulatory elements may reside in these areas, thus helping to coordinate synthesis of the three chains.147

FIGURE 112-23 Relationship between gene structure and protein structure in fibrinogen. The exons, introns, mRNA, and protein structure for the three chains of fibrinogen are shown. The Bb chain is translated in the opposite direction from the Aa and g chains. In the proteins, P designates the pre-pro leader sequence. D, residues in the D domain; E, residues in the E domain; H, residues in the helical connecting region; f, fibrinopeptide residues.

Studies of tissue-specific expression and acute-phase regulation of the mRNA of the fibrinogen chains have revealed some surprises. The expression of the g chain is regulated by ubiquitous factors, such as SP1, while transcription of the Aa and Bb genes requires the liver-specific factor HNF-1.148 The Bb-chain promoter contains an IL-6 responsive element that appears to be present in the upstream sequences of the other chains as well.149 Because of the differences in the promoter regions of the genes for the three chains, the tissue distribution differs. The highest levels of mRNA for all three chains are found in the liver. However, g-chain transcripts have been found in a number of organs that lack transcripts of the other chains. Messenger RNA for Aa and Bb has been found in the kidney, consistent with the presence of HNF-1 in kidney.150
Because of the presence of fibrinogen in the alpha granules of platelets, it was initially assumed that megakaryocytes synthesized fibrinogen. However, while some g-chain transcripts are present in marrow precursors, it appears that most of the fibrinogen found within platelets is taken up from the plasma by endocytosis (see Chap. 111).151,152
ACTIVATION AND ACTIVITY
Thrombin binds to the central domain of fibrinogen and proteolytically releases two fibrinopeptides A (Aa 1–16) and two fibrinopeptides B (Bb 1–14) from each fibrinogen molecule.153 Release of the fibrinopeptides exposes binding sites in the E domain that have complementary sites in the D domains of other fibrin monomers.154,155 These complementary binding sites lead to the initial formation of two-stranded protofibrils with a half-staggered overlap configuration (Fig. 112-24). Protofibrils then aggregate into thick fibers consisting of 14 to 22 protofibrils that branch into a meshwork of interconnected thick fibers.156 The half-staggered overlap of the fibrin monomers gives a characteristic cross-banded pattern on electron micrographs.157 Calcium appears to enhance lateral fiber growth by binding to sites on human fibrinogen.158,159

FIGURE 112-24 Cleavage of fibrinogen and polymerization of fibrin. The structure of fibrinogen is indicated schematically. Cleavage sites for fibrinopeptide A by thrombin are shown. Cleavage of the B peptide is not shown in this figure. Release of fibrinopeptide A exposes binding sites in the E domain that match complementary sites in the D domain. Fibrin monomers polymerize by half-staggered overlaps. Polymerization can also lead to branched structures.139

During fibrin monomer polymerization, other plasma proteins also bind to the surface of the developing meshwork. These include elements of the fibrinolytic system and a variety of adhesive proteins, including fibronectin, thrombospondin, and vWf. These surface proteins influence the generation, cross-linking, and lysis of fibrin. Fibrin or fibrinogen also has specific integrin-binding sites that are essential for platelet binding (for additional details see Chap. 111). The thrombin that initiates fibrin polymerization also activates factor XIII, which stabilizes the fibrin polymer by cross-linking. Factor XIIIa also cross-links other bound proteins, such as plasminogen activator-1, vitronectin, fibronectin, and a2-antiplasmin, to the fibrin network.
Once formed, the fibrin mesh can be degraded by the fibrinolytic system. Plasmin cleaves fibrin and fibrinogen in an ordered sequence at arginyl and lysyl bonds, giving rise to a series of soluble degradation products.160 The plasmin digestion of fibrinogen initially cleaves the Aa polar appendage and the Bb 1–42 fragment, generating fragment X (Mr 250,000), which can still form a clot, albeit slowly. Further action of plasmin releases a D fragment (Mr 100,000) from fragment X to form fragment Y (Mr 150,000). Fragment Y is further cleaved to form another fragment D and a fragment E (Mr 50,000). Similar fragments are generated during plasmin digestion of cross-linked fibrin, with two exceptions: (1) the Bb 15–42 is released from the des 1–14 Bb chain of fibrin, and (2) D-dimer and other covalently cross-linked degradation products are cleaved from the cross-linked fibrin polymer. Monoclonal antibodies recognizing the fibrin D-dimer fragments can help to discriminate fibrin degradation products from fibrinogen degradation products.161
Although the large X fragment can still polymerize into a weak clot,162 the smaller Y and D fragments inhibit normal fibrin monomer polymerization.163 The inhibition of polymerization can prolong the thrombin time and lead to spuriously low values of fibrinogen when measured in clotting assays.
FACTOR XIII
PROTEIN STRUCTURE
Factor XIII is a 320,000 Mr glycoprotein composed of A and B subunits with a plasma half-life of about 10 days. It is a protransglutaminase that is activated by thrombin in the presence of calcium.164 The A chain contains the cysteine-active site, while the B chain is not enzymatically active and functions as a carrier protein. The cDNA and protein sequences of both subunits have been determined.165,166 and 167
The factor XIII A chain is a unique member of the transglutaminase family, which is composed of calcium- and thiol-dependent enzymes found in all human tissues and fluids. Factor XIIIa cross-links proteins between the g-carbon of glutamine in one protein and the e amino group of lysine in the other. The A chain contains 731 amino acids and has a Mr of approximately 83,000 (Fig. 112-25). There is no typical signal sequence to direct secretion, and thus the mechanism of secretion of the A chain is not known.

FIGURE 112-25 Relationship between gene structure and protein structure in the factor XIII A chain. The exons, mRNA, and protein structure for the factor XIII A chain are shown. The size of the introns in the factor XIII A chain has not been published. The mRNA is 4 kb, with some 5′-untranslated sequence coded in exon 1 and a large 3′-untranslated region. In the protein, AP indicates the activation peptide.

The B chain is homologous to complement-regulatory proteins. It is synthesized as a chain of 661 amino acids starting with a signal peptide. The mature B chain is composed of 641 amino acids167 and has a Mr of appproximately 76,500, including 8.5 percent carbohydrate. The B chain contains 10 short consensus repeat units (SCR, also called GP-1 or Sushi domains; Fig. 112-26). Each SCR contains 60 to 70 amino acids, containing four conserved cysteine residues with a characteristic pattern of disulfide bonds.168 Short consensus repeat sequences are found in other complement-related proteins, including factor H; C4-b–binding protein; CR1; decay-accelerating factor; and the complement factors C1r, C1s, C2, factor B, C6, and C7. Short consensus repeat units are also present in the interleukin-2 receptor, endothelial leukocyte adhesion molecule 1, and b2-glycoprotein 1.

FIGURE 112-26 Relationship between gene structure and protein structure in the factor XIII B chain. The exons, introns, mRNA, and protein structure are as indicated. Since full-length cDNA has not been isolated, the size of the mRNA is not known. In the protein, Pro indicates the propeptide. Carb; carboxyl-terminal region; S, Sushi (SCR) domains.

In addition to plasma, factor XIII also is found in platelets, monocytes, and monocyte-derived macrophages. While the plasma factor is a heterotetramer consisting of paired A and B subunits (A2B2), its cellular counterpart lacks the B subunits and is a homodimer of A subunits (A2). Monocytes-macrophages can synthesize factor XIII,169 and the factor XIII found in platelets is probably synthesized by megakaryocytes.170 Cells of marrow origin seem to be the primary site for the synthesis of subunit A in plasma factor XIII, but hepatocytes might also contribute.164 The B subunit of plasma factor XIII is synthesized in the liver.
MOLECULAR BIOLOGY
The factor XIII A-chain gene has been localized to chromosome 6 p24–p25.171 It contains 15 exons and 14 introns and is over 160 kb in size (see Fig. 112-25).166 The fibrin-binding domain is encoded by exons 2 thorugh 12. The active site, with its reactive thiol at Cys314, is present in exon 7. The structure of the factor XIII A-chain gene is quite similar to that of other transglutaminases. The transcription initiation site for the A chain is unknown, but three potential sites have been described, at –170, –150, and –40 relative to the inital methionine.166
The factor XIII B chain has been localized to chromosome 1q31–q32.1. It has 12 exons separated by 11 introns and is about 28 kb in size (see Fig. 112-26).172 Each SCR is encoded by a single exon. The regulation of the factor XIII B-chain expression is poorly understood. A total of 30 potential start sites are located upstream of the initial methionine.
ACTIVATION AND ACTIVITY
Plasma factor XIII circulates in association with its substrate, fibrinogen. The key step in the activation of plasma factor XIII is thrombin cleavage of the Arg37-Gly38 bond in the A chain to release a Mr 4500 activation peptide. This leads to dissociation of the A and B subunits and exposure of the active site on the free A subunits. Cellular factor XIII in platelets becomes activated through a nonproteolytic process. When intracytoplasmic Ca2+ is elevated during platelet activation, the zymogen, in the absence of the B chain, becomes an active configuration.164 The main physiological function of plasma factor XIIIa is to cross-link the a and g chains of fibrin to stabilize the fibrin plug. In the absence of factor XIII, a clot forms, but it is inadequate for hemostasis. Additional protein substrates of factor XIIIa include components of the clotting and fibrinolytic system, as well as multiple adhesive and contractile proteins. Factor XIIIa also protects fibrin from fibrinolysis by cross-linking it to a2-antiplasmin. Plasma factor XIII is also involved in wound healing and tissue repair, and is essential for maintaining pregnancy.
INHIBITORS
There are many protease inhibitors in plasma, but the two most specifically involved in inhibition of coagulation factors are TFPI and ATIII (see Table 112-4). A recently identified protein-Z–dependent protease inhibitor (PZI) may also play a role in regulating coagulation by inactivating surface-bound factor Xa.173 Coagulation inhibitors are discussed in detail in Chap. 113.
TISSUE FACTOR PATHWAY INHIBITOR
Protein Structure and Activity Tissue factor pathway inhibitor (TFPI) is a single-chain polypeptide of Mr 34,000 to 40,000, depending upon the degree of proteolysis of the carboxyl-terminal region. Tissue factor pathway inhibitor contains three Kunitz-type protease inhibitor domains. The second Kunitz domain binds and inhibits factor Xa, and this interaction is required for the first Kunitz domain to bind and inhibit the factor VIIa/TF complex. The function of the third Kunitz domain is not clear, but it may be involved in binding to glycosaminoglycans. Thus, TFPI is unique among the coagulation protease inhibitors in two respects: (1) It has inhibitory sites for both factor Xa and the factor VIIa/TF complex, and (2) it cannot inhibit the factor VIIa/TF complex unless it has also bound factor Xa.174,175
The primary site of plasma TFPI synthesis is endothelial cells.176 The majority of circulating TFPI is bound to lipoproteins. A second pool of TFPI is bound to heparian sulfates on the surface of endothelial cells. Administration of heparin releases the endothelial-cell–bound TFPI and raises the plasma level severalfold.177
Tissue factor pathway inhibitor is only present in the plasma at about 2.5 nM, compared to ATIII, at about 2 µM. However, its rate of reaction with factor Xa in plasma is similar to that of ATIII. Therefore, TFPI contributes significantly to the inhibition of factor Xa in vivo.
Molecular Biology The gene for TFPI has not been completely sequenced. However, it is known to be located on chromosome 2q31–q32.1 and has nine exons that span 70 kb.178 The first two exons code for a 5′-untranslated region. No TATA box is present in the promoter region of the TFPI gene. DNA sequences that are consistent with binding sites for the transcription factors GATA-2, AP-1, and NF-1 are present in the 5′-untranslated region of the TFPI gene. It is thought that GATA-2 binding is necessary for constitutive expression of TFPI by endothelial cells.176
An alternatively spliced form of TFPI has also been described. This form, TFPI-b, lacks the third Kunitz domain and instead has a unique carboxyl terminal of unknown function. It is found in plasma and has inhibitory activity similar to that of full-length TFPI.179
ANTITHROMBIN III
Protein Structure and Activity Antithrombin III (ATIII) is a member of the large family of serine protease inhibitors (serpins). These inhibitors act as “suicide” substrates for their target proteases through a surface-exposed structure termed a reactive site loop. An amino acid sequence in the reactive site loop is recognized by the target protease; it forms a one-to-one complex that blocks the active site of the protease. The primary proteases targeted by ATIII are thrombin, factor Xa, and factor IXa, while factor VIIa is resistant to inhibition by ATIII180,181 and 182 unless it is complexed to TF in the presence of heparin. All these reactions are enhanced by heparin (see Table 112-4). While the reaction between ATIII and the serine proteases is reversible, dissociation is probably insignificant under physiological conditions. The protease–serpin complex is cleared from the circulation by receptor-mediated endocytosis in the liver.183 Antithrombin III is an important physiological inhibitor of the blood coagulation proteases, since its deficiency leads to a significantly increased risk of thrombosis.
Molecular Biology The gene for ATIII is on the long arm of chromosome 1. The gene has seven exons and spans about 13.5 kb. Little is known about transcriptional regulation of ATIII. The region from –89 to –68 has been implicated in the binding of transcription factors from rat liver,184 but the specific transcription factors involved are not clear.
PATHWAYS OF HEMOSTASIS
EARLY MODELS OF COAGULATION
In the 1960s two groups proposed a model of coagulation that envisioned a sequential series of steps in which activation of one clotting factor led to the activation of another, finally leading to a burst of thrombin generation.185,186 Each clotting factor was thought to exist as a proenzyme that could be converted to an active enzyme.
The original cascade models were subsequently modified to include the observation that some procoagulants were cofactors and did not possess enzymatic activity. In addition, the clotting sequences were divided into so-called extrinsic and intrinsic systems, as shown in Fig. 112-27. The extrinsic system consisted of factor VIIa and TF, the latter being viewed as extrinsic to the circulating blood. The factors in the so-called intrinsic system were all viewed as being intravascular. Both pathways could activate factor X, which, in complex with its cofactor Va, could convert prothrombin to thrombin. While these earlier concepts of coagulation were extremely valuable, several groups recognized that the intrinsic and extrinsic systems could not operate independently of one another and that all the clotting factors were somehow interrelated. Only in this way could hemostasis in vivo be explained.

FIGURE 112-27 Cascade model of coagulation. This model shows successive activation of coagulation factors proceeding from the top of the schematic to thrombin generation and fibrin formation at the bottom. The intrinsic and extrinsic pathways are as indicated.

REVISION OF THE COAGULATION MODELS
Key observations made by several groups have led to a revision of earlier models of coagulation. A major observation was that a complex of factor VIIa/TF activated not only factor X but also factor IX.110 Other important observations led to the conclusion that the major initiating event in hemostasis in vivo was the formation of a factor VIIa/TF complex at the site of injury.187,188 and 189 This led to the belief that factor VIII and IX deficiencies, which resulted in hemophilia A and B respectively were, in fact, abnormalities of the VIIa/TF pathway, even though factors IX and VIII were considered components of the intrinsic system. It was also recognized that in vivo coagulation was regulated by control mechanisms, one of which was the localization of the coagulation reactions to cell surfaces. In addition, earlier and more recent observations emphasized the importance of plasma inhibitors of each step of the coagulation process. These include TFPI, which inhibits the factor VIIa/TF/Xa complex175,190; proteins C and S, which inactivate factors Va and VIIIa125,191,192; and ATIII, which inhibits thrombin and other coagulation proteases.193
A CELL-BASED MODEL OF COAGULATION
ROLE OF THE TF-BEARING CELL
The goal of hemostasis is to produce a fibrin clot to seal a site of injury or rupture in the blood vessel wall. This process is initiated when TF-bearing cells are exposed to blood at a site of injury. TF is anchored to cells via a transmembrane domain and acts as a receptor for plasma factor VII. Once bound to TF, zymogen factor VII is rapidly converted to factor VIIa through mechanisms not yet completely understood. The resulting factor VIIa/TF complex, localized by cells to the site of injury, catalyzes two very important reactions: (1) activation of factor X to factor Xa, and (2) activation of factor IX to IXa. The factors Xa and IXa formed on the TF-bearing cells have very distinct and separate functions in the process of blood coagulation.
The factor Xa formed on the TF-bearing cell interacts with its cofactor Va to form a prothrombinase complex sufficient to generate a very small amount of thrombin in the vicinity of the TF cells (Fig. 112-28). Although this amount of thrombin may not be sufficient to clot fibrinogen, it is sufficient to initiate events that “prime” the clotting system for a subsequent burst of thrombin generation. Experiments using a cell-based model have shown that minute amounts of thrombin are formed in the milieu of TF-bearing cells exposed to plasma concentrations of procoagulants, even in the absence of platelets. The small amounts of factor Va required for prothrombinase assembly on the TF-bearing cells are activated by factor Xa45 or by noncoagulation proteases elaborated by the cells.194 The small amounts of thrombin generated on the TF-bearing cells are capable of accomplishing the following89,195: (1) activating platelets, (2) activating factor V, (3) activating factor VIII and dissociating factor VIII from vWf, and (4) activating factor XI (Fig. 112-29). The activity of the factor Xa formed by the factor VIIa/TF complex is restricted to the TF-bearing cell. Factor Xa that diffuses off the cell surface is rapidly inhibited by TFPI or ATIII.

FIGURE 112-28 The role of TF-bearing cells. Factor VIIa bound to TF can activate both factor X and factor IX. Factor Xa and factor IXa activated by factor VIIa/TF play distinct roles in coagulation. Factor Xa is assembled into a prothrombinase complex on the surface of the TF-bearing cell. This generates a small amount of thrombin.

FIGURE 112-29 The role of thrombin generated by TF-bearing cells. After the initial generation of factor Xa on TF-bearing cells, subsequent factor Xa generation is shut down when TFPI reacts with factor Xa to inactivate the factor VIIa/TF complex. The small amount of thrombin generated on the TF-bearing cells plays a critical role in priming platelets for subsequent coagulation steps. This thrombin activates platelets, releases factor V from alpha granules, activates factor V, activates factor VIII and releases it from vWf, and activates factor XI.

Unlike factor Xa, the primary site of activity of the factor IXa formed by factor VIIa/TF is on activated platelets in close proximity to the TF-bearing cell. Factor IXa can diffuse to adjacent cell surfaces because it is not inhibited by TFPI and is inhibited much more slowly by ATIII than is factor Xa (see Table 112-4).
ROLE OF ACTIVATED PLATELETS
Platelets also play a major role in localizing clotting reactions to the site of injury since they adhere and aggregate at the same sites where TF is exposed. Platelet localization and activation are mediated by vWf, thrombin, platelet receptors, and vessel wall components such as collagen (see Chap. 111).
Once platelets are activated, the cofactors Va and VIIIa are rapidly localized to the platelet membrane surface (see Fig. 112-29). Cofactor binding is mediated in part by the exposure of PS on the platelet membrane, a process resulting from a flip-flop mechanism whereby PS on the inner leaflet of the membrane bilayer flips to the outside.196 In addition, it appears that the cofactors bind to the platelet surface before the binding of the respective enzymes.197
The factor IXa formed by the factor VIIa/TF complex binds to the surface of activated platelets (Fig. 112-30). Specific sites on the activated platelets bind factor IXa and promote formation of active factor IXa/VIIIa complexes.198,199 Once the platelet “tenase” complex is assembled, factor X is recruited from the plasma and is activated to factor Xa on the platelet surface. Factor Xa then associates with factor Va on the surface to generate a burst of thrombin sufficient to clot fibrinogen and form a hemostatic plug (see Fig. 112-30). Factor XIII, activated by thrombin, cross-links fibrin and stabilizes the hemostatic plug, rendering it impermeable.

FIGURE 112-30 The role of platelets. Factor IXa, generated on TF-bearing cells, is only slowly inhibited by plasma inhibitors and so can make its way to the primed platelet surface, where it binds to factor VIIIa. This factor IXa activates factor X on the platelet surface. Factor Xa complexes to factor Va and activates prothrombin, leading to the burst of thrombin generation responsible for cleaving fibrinogen. Additional factor IXa is supplied by factor XIa on the platelet surface.

Factor XIa, formed by thrombin activation of the zymogen, also associates with the platelet surface, where it can activate more factor IX to IXa.89,92 Thus, it appears that factor XIa activation enhances the platelet tenase activity and serves as a “booster” mechanism to enhance thrombin generation.
The role of factor XI in hemostasis has been a point of major interest, since even severe factor XI deficiency does not result in a hemorrhagic tendency comparable to that seen in severe factor VIII or IX deficiency. This observation can be explained if factor XI is viewed as an enhancer, or booster, of thrombin generation. In factor VIII and IX deficiency, the individual has a markedly decreased ability to generate factor Xa on the platelet surface. Thus, one would expect that patients with a severe deficiency of either factor VIII or factor IX would generate insufficient thrombin for hemostasis, since the tenase, and hence prothrombinase, activity would be markedly reduced. In contrast, patients with factor XI deficiency would always possess baseline tenase activity. Such patients only lack the ability to boost platelet surface factor X activation by producing extra factor IXa. Without the boost in thrombin generation by factor XI, there may be decreased activation of thrombin activatable fibrinolysis inhibitor (TAFI) resulting in enhanced fibrinolysis that may contribute to the bleeding tendency seen in factor-XI–deficient patients.200 Even though each step of the model has been depicted as an isolated set of reactions, they should be viewed as an overlapping continuum of events, as illustrated in Fig. 112-31.

FIGURE 112-31 A cell-based model of hemostasis. The sequence of events shown in previous figures is summarized here.

ROLE OF ENDOTHELIAL CELLS
Once a fibrin–platelet clot is formed over an area of injury, the clotting process must be terminated to avoid thrombotic occlusion in adjacent normal areas of the vasculature. If the coagulation mechanism were not controlled, clotting could occur throughout the entire vascular tree after even a modest procoagulant stimulus. Endothelial cells play a major role in confining the coagulation reactions to a site of injury and preventing clot extension to areas where an intact endothelium is present (see Chap. 114). Endothelial cells have two major types of anticoagulant-antithrombotic activities as illustrated in Fig. 112-32. The protein C–protein S–TM system is activated in response to thrombin generation. Some of the thrombin formed during the coagulation process can diffuse away or be swept downstream from a site of injury. When thrombin reaches an intact endothelial cell, it binds to TM on the endothelial surface. The thrombin–TM complex then activates protein C, which binds to its cofactor protein S and inactivates any factor Va or VIIIa that finds its way to the adjacent endothelial cell membranes. This prevents the generation of additional thrombin in the vasculature. The endothelial cell also posseses other anticoagulant features. The protease inhibitors ATIII and TFPI are always present, bound to heparan sulfates expressed on the endothelial surface, where they can inactivate proteases near an intact endothelium.201 Endothelial cells also inhibit platelet activation by releasing the inhibitors prostacyclin and nitric oxide, as well as digesting ADP by their membrane ecto-ADPase, CD39.202

FIGURE 112-32 The role of endothelial cells. Activated coagulation proteins generated on platelets localized to a site of an injury are confined to the site. Activated coagulation factors that move to an endothelial cell surface are rapidly inhibited by ATIII associated with glycosaminoglycans (GAG) on the endothelial surface. Further, thrombin that reaches the endothelial cell surface binds to TM. Once bound, thrombin can no longer cleave fibrinogen. Instead, thrombin activates protein C, leading to the formation of activated protein C (aPC)/protein S (PS) complexes on the endothelial cell, which inactivate factors Va and VIIIa (iV, iVIII).

ROLE OF PLASMA PROTEASE INHIBITORS
Like cell-based coagulation, circulating protease inhibitors are also critical in localizing the coagulation reactions to specific cell surfaces by directly inhibiting proteases that escape into the fluid phase (see Fig. 112-32). Not only are the plasma protease inhibitors key players in confining a clot to the proper location, but they also impose a threshold effect on the coagulation process.203 Thus, in the presence of inhibitors, coagulation does not proceed unless procoagulant factors are generated in sufficient amounts to overcome the effects of inhibitors. If the triggering event is not sufficiently strong, the system returns to baseline rather than continuing through the coagulation process. Under pathological conditions, the trigger for clotting may be so strong as to overwhelm the control of inhibitors and lead to disseminated intravascular coagulation or thrombosis (see Chap. 126).
ROLE OF FIBRINOLYSIS
Once a hemostatic clot has been formed, some provision must be made for its eventual removal as wound healing takes place. Dissolution of clots is accomplished by the fibrinolytic system, as discussed in detail in Chap. 116.
THE CONCEPT OF BASAL COAGULATION AND ANTICOAGULATION
A low level of basal coagulation factor activation probably occurs at all times.204 It was shown over 20 years ago that fibrinopeptides are continuously cleaved from fibrinogen at low levels in normal individuals.205 It has also been shown that there are low levels of circulating factor VIIa and of the activation peptides from factors IX and X in the blood of normal individuals.206,207 and 208 This has been called basal coagulation and may occur as a result of the minor injuries to vessels that occur during normal daily activities or perhaps when the lower-molecular-weight coagulation factors percolate through the extravascular spaces. This process does not lead to clot formation under normal circumstances. The coagulation process only proceeds when enough thrombin is generated on or near the TF-bearing cell to trigger activation of platelets and cofactors. One wonders, however, whether minute hemostatic plugs are not constantly formed throughout the body to maintain the integrity of the vascular tree. This basal coagulation must be balanced by activity of the anticoagulation and fibrinolytic systems. This is evidenced by the presence of low levels of the protein C activation peptide and tissue plasminogen activator activity in normal individuals.209
BLOOD COAGULATION AS A PART OF THE HOST DEFENSE MECHANISM
The process of hemostasis is only a small part of the overall host response to injury. While different parts of the host response are presented as though they were truly separate processes, in fact coagulation, fibrinolysis, inflammation, the immune response, and wound healing are interrelated parts of the overall response to injury.
There are many ways in which coagulation factors or byproducts play important roles in the response to injury. One clear example is the multiple roles of thrombin. It acts not only as a procoagulant to clot fibrinogen, but also as a growth factor and cytokine that promotes monocyte, fibroblast, and endothelial cell influx into an area of recent injury and sets the stage for removal of damaged tissue and for wound healing.26 Platelets also have multiple roles in the response to injury. They release several growth factors and cytokines upon activation, some of which play key roles in wound healing and atherosclerosis. Factor Xa, TF, and fibrinogen fragments, similarly, seem to have roles as inflammatory mediators and cell growth regulators. In addition, the contact factors (factor XII, PK, and HK) may play a role as a bridge between the coagulation reactions and other host defense mechanisms. No doubt the list of multifunctional molecules will grow as understanding of the blood clotting mechanism increases.
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Williams Hematology

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