THE VITAMIN K–DEPENDENT ZYMOGENS: PROTHROMBIN, FACTOR VII, FACTOR IX, FACTOR X, AND PROTEIN C
The vitamin K-dependent zymogens circulate in an inactive state and require proteolytic activation to function as a serine protease. All share a similar domain structure of a C-terminal serine protease domain and an N-terminal γ-carboxy glutamic acid (Gla) domain, which are connected by two epidermal growth factor (EGF)-like domains or kringle domains (Fig. 113–1). Each protein domain has a well-defined function and facilitates substrate recognition, interaction with protein cofactors, or binding to a negatively charged lipid surface, such as that of activated platelets or endothelial cells, thereby restricting coagulation to the site of injury. The latter is mediated via the Gla domain, a domain that is characteristic to the vitamin K–dependent proteins.
Vitamin K–dependent schematic of the vitamin K–dependent zymogens. Each circle represents an amino acid. The prepro leader sequence contains the signal peptide as well as the propeptide that directs γ-carboxylation of glutamic acid (Gla) residues. Cleavage of the prepro sequence from the mature protein is indicated by the separation between the two. The Gla domains are indicated with the Gla residues in blue. Prothrombin has a finger loop followed by two kringle domains. Factors VII, IX, X, and protein C have epidermal growth factor (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 homologous serine protease (“catalytic”) domains (shown in light red), in which the active site His, Asp, and Ser residues are indicated in dark red. Cleavages that convert the zymogen to an active enzyme are indicated by the red arrows. In factor IX, factor X, and protein C, the released activation peptide is indicated in yellow. After proteolytic activation, all of the molecules are two-chain disulfide-linked molecules, with the cysteines forming a disulfide bridge (black line) indicated in green. All catalytic domains but that of prothrombin remain attached to the Gla domain following activation.
The high level of protein and gene homology suggests that the vitamin K–dependent zymogens originate from a common ancestral gene as a result of gene duplications.1 Exon shuffling and tandem duplication may account for the generation of the ancestral gene, in which the functional domains that are encoded by a single exon each were combined and duplicated.2 This process may also account for the presence of the kringle domains as opposed to EGF-like domains in prothrombin.
The Gla domain refers to the 42-residue region located in the N-terminus of the mature protein that comprises 9 to 12 glutamic acid residues that are posttranslationally γ-carboxylated into Gla residues by a specific γ-glutamyl carboxylase in the endoplasmatic reticulum of hepatocytes.3 This γ-carboxylase requires oxygen, carbon dioxide, and the reduced form of vitamin K for its action, hence the name vitamin K–dependent proteins. For each Glu residue that is carboxylated, one molecule of reduced vitamin K is converted to the epoxide form (Fig. 113–2). Vitamin K epoxide reductase converts the epoxide form of vitamin K back to the reduced form.4 Warfarin and related 4-hydroxycoumarin–containing molecules inhibit the activity of vitamin K epoxide reductase, thereby preventing vitamin K recycling and inhibiting γ-carboxylation. This results in a heterogeneous population of circulating undercarboxylated forms of the vitamin K–dependent proteins with reduced activity. Because warfarin blocks the reductase and not the carboxylase, the inhibitory effect of warfarin can be (temporarily) reversed by administration of vitamin K. Recognition by and interaction with γ-carboxylase is facilitated by the propeptide sequence that is located C-terminal to the signal peptide. The propeptide is highly conserved among the vitamin K–dependent proteins, and amino acids at positions –18, –17, –16, –15, and –10 are critical to recognition by the γ-carboxylase.5,6 Following γ-carboxylation, the propeptide is removed through limited proteolysis prior to secretion of the mature protein.
Vitamin K–dependent γ-carboxylation. Glutamic acid residues are converted to γ-carboxy glutamic acid (Gla) residues by a specific γ-carboxylase. This reaction requires oxygen, carbon dioxide (shown in green), and reduced vitamin K in the form of a hydroquinone. Carbon dioxide is incorporated onto the γ-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 and warfarin analogues.
A correctly γ-carboxylated Gla domain is essential for interaction of the vitamin K–dependent proteins with phosphatidylserine, a negatively charged phospholipid. Under normal conditions, phosphatidylserine is not exposed on the outer membrane leaflet of cells. However, in activated endothelial cells or platelets, phosphatidylserine is part of the extracellular cell surface where it supports blood coagulation reactions. The Gla domain interacts with the anionic cell surface in a calcium-dependent manner. These calcium ions are coordinated by Gla residues and induce a conformational change in the Gla domain that is characterized by the appearance of a hydrophobic surface loop (Fig. 113–3). Membrane binding by the Gla domain occurs when this hydrophobic surface loop penetrates into the hydrophobic portion of the phospholipid bilayer, which is facilitated by the interaction of the Gla-bound calcium ions with the phosphate head groups of phosphatidylserine.7,8 It has been shown that the phosphate head groups of exposed phosphatidylethanolamine are also capable of coordinating calcium ions, thereby contributing to the interaction of the Gla domains with the negatively charged membrane surface.9
Calcium-dependent association of the glutamic acid (Gla) domain with the anionic phospholipid surface. Non–calcium bound [Protein Data Bank (PDB) structure 2PF2] and calcium bound (PDB structure 1WHE) molecular models of the Gla domain of prothrombin are shown. Circles represent amino acids, with the Gla (GLA) residues indicated in red. Hydrophobic residues involved in membrane insertion are shown in blue. In the absence of calcium, the negatively charged Gla residues are exposed to the solution and the hydrophobic residues are buried. Association of calcium ions (in black) to the Gla residues provides sufficient energy to alter the overall conformation of the Gla domain and expose the hydrophobic residues. Membrane binding by the Gla domain occurs when this hydrophobic surface loop penetrates into the hydrophobic portion of the phospholipid bilayer (drawn schematically), which is facilitated by interaction of the Gla-bound calcium ions with the negatively charged phosphate head groups.
The serine protease domains of the vitamin K–dependent proteins are highly homologous, as they bear a chymotrypsin-like fold and display trypsin-like activity.10 Once activated, they cleave peptide bonds following a positively charged amino acid (Lys or Arg). Activation proceeds through proteolysis at one or more sites N-terminal to the serine protease domain (see Fig. 113–1). Subsequently, the newly formed N-terminus inserts into the serine protease domain to form a salt bridge with an Asp residue, which is associated with conformational changes in the serine protease domain. These lead to an optimal configuration of the active site through alignment of the active site residues His, Ser, and Asp, and to formation of the substrate-binding exosites, allowing for substrate conversion. The substrate-binding exosites are unique to each vitamin K–dependent protease and are responsible for their highly specific substrate recognition and associated function in coagulation.
Interaction of the vitamin K–dependent proteases with specific cofactors on a (anionic) membrane surface (Table 113–2) further enhances substrate recognition, as the cofactors interact with both the protease and the substrate, bridging the two together. This results in a dramatic enhancement of the catalytic activity (Table 113–3), thereby making the cofactor–protease complex the physiologic relevant enzyme. The increase in catalytic rate has also been attributed to a cofactor-induced conformational change in the protease.11 However, whether this molecular mechanism holds true for all cofactor–protease complexes remains to be determined. 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, while thrombin does not require a cofactor for its procoagulant activity. However, upon association with the cofactor thrombomodulin, thrombin’s specificity is changed from procoagulant to anticoagulant (cleaving and activating protein C). The complexes are also named for their physiologic substrate: the factor VIIIa–factor IXa complex is termed the “tenase” or “intrinsic tenase” complex; the tissue factor–factor VIIa complex is termed the “extrinsic tenase” complex; and the factor Va–factor Xa complex is termed the “prothrombinase” complex.
Table 113–2.Protease–Cofactor Complexes ||Download (.pdf) Table 113–2. Protease–Cofactor Complexes
|Protease ||Cofactor ||Substrate ||Cellular Location |
|Factor VIIa ||Tissue factor ||Factor IX ||Many cells* |
| || ||Factor X || |
|Factor IXa ||Factor VIIIa ||Factor X ||Platelets |
|Factor Xa ||Factor Va ||Prothrombin ||Platelets |
|Thrombin ||Thrombomodulin ||Protein C ||Endothelium |
|Activated protein C ||Protein S || |
Table 113–3.Cofactor Enhancement of Serine Protease Activity ||Download (.pdf) Table 113–3. Cofactor Enhancement of Serine Protease Activity
|Cofactor-Protease* ||Fold Increase† |
|TM-Thrombin ||11,000 |
|TF-VIIa ||31,000 |
|VIIIa-IXa ||9,000,000 |
|Va-Xa ||390,000 |
Prothrombin, or factor II, which was discovered by Pekelharing in 1894, is one of the four coagulation factors that were described by Paul Morawitz in 1905, in addition to fibrinogen (factor I), thromboplastin (thrombokinase, factor III, now tissue factor), and calcium (factor IV).12,13 The zymogen prothrombin is primarily synthesized in the liver and circulates in plasma as a single-chain protein of 579 amino acids (Mr ≈72,000) at a concentration of 1.4 μM with a plasma half-life of 60 hours (see Table 113–1).
Prothrombin is composed of fragment 1 (F1), fragment 2 (F2), and the serine protease domain. F1 consists of the Gla domain, which comprises 10 Gla residues, and the kringle 1 domain; F2 contains the kringle 2 domain (see Fig. 113–1). The two kringle domains, which replace the EGF-like domains present in most vitamin K–dependent zymogens, are conserved secondary protein structures that fold into large loops that are stabilized by three disulfide bonds and schematically resemble a Danish pastry called a “kringle.” Their primary function is to bind other proteins such as the cofactor Va and serine protease factor Xa that activate prothrombin.
Other than γ-carboxylation of Glu residues, prothrombin is posttranslationally modified via N-glycosylation in the kringle 1 (Asn78, Asn143) and serine protease domains (Asn373), which contributes to the stability of the prothrombin precursor during processing in the endoplasmatic reticulum.14,15
Prothrombin Activation and Thrombin Activity
Prothrombin is proteolytically activated by the prothrombinase complex (i.e., factor Va, factor Xa, calcium, and anionic phospholipids) that cleaves at Arg271 and Arg320 (see Fig. 113–1). Cleavage at Arg320 opens the active site of the protease domain, while cleavage at Arg271 removes the activation fragment F1.2 (F1.2). Both cleavages are necessary to generate procoagulant α-thrombin (IIα) (Fig. 113–4). The composition of the membrane surface directs the cleavage order in prothrombin and the formation of either the zymogen prethrombin 2 (initial cleavage at Arg271) or the proteolytically active intermediate meizothrombin (initial cleavage at Arg320).16,17 Meizothrombin has impaired procoagulant activity as compared to α-thrombin, but superior anticoagulant activity as it displays increased thrombomodulin-dependent protein C activation, which is likely facilitated by membrane binding of meizothrombin through its Gla domain.18 The snake venom protease Ecarin is capable of generating meizothrombin specifically through proteolysis at Arg323 only. However, this meizothrombin is instable as a result of autocatalysis at Arg155, thereby removing the Gla domain containing F1. The so formed meizo-des-F1 can be converted to thrombin by prothrombinase, but at a slower rate as it is incapable of membrane binding. Assessment of F1.2 levels reflects prothrombin activation and is commonly used as a marker for thrombin generation.
Prothrombin to thrombin conversion. A molecular model of prothrombin comprising the γ-carboxy glutamic acid (Gla; GLA) domain, both kringle domains, and the catalytic domain is shown (PDB structures 2PF2, 1HAG, 1A0H, 1HAI). Gla domain-bound calcium ions are indicated in black. Cleavage by the factor Va-Xa complex at Arg271 and Arg320 releases thrombin (with the A chain and catalytically active B chain) from the rest of the molecule (fragment 1.2).
Thrombin (IIα) is a two-chain serine protease (Mr ≈37,000) comprising a light chain of 49 residues (A chain; Mr ≈6000) that is covalently linked to the catalytic heavy chain of 259 residues (B chain; Mr ≈31,000). Thrombin’s main function is to induce the formation of a fibrin clot by removing fibrinopeptides A and B from fibrinogen to form fibrin monomers, which then spontaneously polymerize. In addition, thrombin is able to cleave a wide variety of substrates with high specificity, which is mediated via its negatively charged, deep active site cleft and via the anion binding exosites I and II that specifically interact with cofactors and/or substrates.19 The dynamic structural conformation of thrombin allows for binding to diverse ligands, and the subsequent ligand-induced conformational stabilization, known as thrombin allostery, regulates and controls thrombin activity.20,21
Thrombin initiates important procoagulant pathways by proteolytic activation of the cofactors V and VIII and zymogen factor XI that collectively amplify thrombin and fibrin formation, and by activating factor XIII that crosslinks and stabilizes the fibrin polymers. Another procoagulant function of thrombin is to inhibit fibrinolysis by proteolytic activation of the thrombin-activatable fibrinolysis inhibitor (TAFI), a reaction enhanced by the endothelial-bound cofactor thrombomodulin. Thrombin also has an anticoagulant function and upon binding to the cofactor thrombomodulin, it is capable of proteolytically activating protein C, which inactivates the cofactors Va and VIIIa.
Thrombin activates the seven-transmembrane domain, G-protein–coupled protease-activated receptors (PARs) PAR1, PAR3, and PAR4 that are expressed on a wide range of cell types in the vasculature by proteolytic cleavage of their N-terminal extracellular domains.22,23,24,25 Thrombin is one of the strongest platelet activators in vivo and activates platelet-expressed PAR1 and PAR4.25 The platelet glycoprotein (GP) Ib (GPIb) serves as a cofactor for thrombin in PAR1 cleavage (Chap. 112). Thrombin-mediated activation of endothelial-PAR1 triggers release of von Willebrand factor (VWF) and P-selectin, which promote rolling and adhesion of platelets and leukocytes. In addition, this stimulates the endothelial production of platelet-activating factor, a potent platelet and leukocyte activator, as well as the production of chemokines, cyclooxygenase (COX)-2, and prostaglandins.25 Thrombin-mediated PAR activation is not only critical for coagulation, but also plays an important role in inflammatory and proliferative responses associated with vascular injury, such as in atherosclerosis and cancer.26
The physiologic inhibitors of thrombin are the serine protease inhibitors (serpins) antithrombin, heparin cofactor II, protein C inhibitor, and protease nexin 1, with antithrombin being the primary plasma inhibitor. For all four serpins, the rate of thrombin inhibition can be accelerated by glycosaminoglycans, such as heparin (Table 113–4), through mutual binding to the serpin and thrombin (see Fig. 113–2), which ensures rapid inhibition of thrombin at the intact endothelial cell surface where heparin-like glycosaminoglycans are found.
Table 113–4.Antithrombin Inhibition of Coagulation Proteases ||Download (.pdf) Table 113–4. Antithrombin Inhibition of Coagulation Proteases
|Second-Order Association Rate Constants (M–1s–1) |
|Protease ||– Heparin ||+ H5 ||+ UFH |
|Thrombin ||7.7 × 103 ||1.5 × 104 ||4.7 × 107 |
|Factor Xa ||2.6 × 103 ||7.6 × 105 ||6.6 × 106 |
|Factor IXa ||58 ||3.1 × 104 ||6.2 × 106 |
|TF-Factor VIIa ||33 ||4.9 × 103 ||1.5 × 104 |
|Factor Xia ||3.6 × 102 ||1.1 × 103 ||1.8 × 105 |
|Factor XIIa ||39 ||1.9 × 103 ||6.6 × 104 |
|APC ||0.08 ||1.9 ||2.1 |
Heparin and heparin-derivatives are clinically used as anticoagulants to inhibit thrombin via antithrombin. Hirudin, which originates from the salivary glands of medicinal leeches, and its recombinant and synthetic derivatives are potent and highly specific inhibitors that directly target the active site and exosite I of thrombin.27 The target-specific oral anticoagulant dabigatran also inhibits thrombin directly with high specificity and reversibly binds the active site of thrombin.27,28
Gene Structure and Variations
Prothrombin is encoded by a gene (F2) on chromosome 11p11.2 that is approximately 20 kb long.29 The coding sequence is divided over 14 exons that range in size from 25 to 315 bp (Fig. 113–5). The reference sequence of prothrombin mRNA comprises 2018 bases. There are no common, well characterized, splicing variants with known biology.
Relationship of gene structure to protein structure in prothrombin. The exons, introns, mRNA, and protein structure are as indicated. The mRNA is 2 kb with small 5′ and 3′ untranslated regions (shown in light blue). In the protein, Pro indicates the prepro leader sequence, GLA indicates the γ-carboxy glutamic acid (Gla) domain, Kringles 1 and 2 are indicated, LC indicates the light chain (also known as the A chain), and the serine protease (catalytic) domain is indicated.
Homozygosity or compound heterozygosity for loss of function mutations in the prothrombin gene leads to a bleeding tendency. This condition is quite rare with perhaps one case per 2,000,000 newborns.30 Heterozygous carriers of loss-of-function mutations are without a bleeding phenotype. Mutations have been characterized in a relatively small number of cases with homozygous or compound heterozygous prothrombin deficiency (consult the human gene mutation database at http://www.hgmd.org for details). The majority of mutations underlying prothrombin deficiency are missense mutations, but several small deletions/insertions have also been reported.
Gain-of-function mutations in the prothrombin gene increase thrombotic risk. The best known variation is G20210A.31 This variation of the last nucleotide preceding the poly(A)-tail of the mature mRNA has an effect on 3′-end mRNA processing and increases the level of prothrombin in plasma by approximately 10 to 20 percent in heterozygous individuals.32 This relatively small increase in the level of prothrombin results in a two- to threefold enhanced risk for venous thrombosis. Homozygotes for the G20210A variation are quite rare, and the risk associated with homozygosity has not been measured with certainty. The G20210A variation is relatively common in whites, with a strong south-north gradient in that the variation is most common in southern Europe.33
Factor VII, which was discovered around 1950,34 is synthesized in the liver and circulates in plasma as a single-chain zymogen of 406 amino acids (Mr ≈50,000) at a concentration of 10 nM with a short plasma half-life of 3 to 6 hours (see Table 113–1).
Factor VII consists of a Gla domain with 10 Gla residues, two EGF-like domains, a connecting region, and the serine protease domain (see Fig. 113–1). Calcium coordination in EGF-1 is mediated by partial hydroxylation of Asn63 and O-linked fucosylation of Ser60.35 Further posttranslational modifications of factor VII consist of O-linked (Ser52 in EGF-1) and N-linked (Asn154 in the connecting region, Asn322 in the serine protease domain) glycosylation.
Factor VII Activation and Factor VIIa Activity
Factor VII is proteolytically activated once it has formed a high-affinity complex with its cofactor tissue factor. A number of coagulation proteases including thrombin and factors IXa and XIIa are capable of cleaving factor VII at Arg152 to generate factor VIIa (see Fig. 113–1), with factor Xa being considered the most potent and physiologically relevant activator of factor VII.36 Autoactivation can also occur, which is initiated by minute amounts (approximately 0.1 nM) of preexisting factor VIIa.37
Factor VIIa is a two-chain serine protease composed of a light chain (Mr ≈20,000) comprising the Gla and EGF domains and the catalytic heavy chain (Mr ≈30,000), which are covalently linked via a disulfide bond. Factor VIIa activity is only expressed when bound to tissue factor, which induces an active conformation of the factor VIIa serine protease domain (Fig. 113–6).11 Factor VIIa interacts with tissue factor via its Gla and EGF domains.
The tissue factor–factor VIIa complex. A molecular model of factor VIIa (PDB structures 1QHK, 1WHF, 1RFN, 1DAN) and the crystal structures of the tissue factor–factor VIIa complex (PDB structure 1DAN) and the extracellular domain of tissue factor (PDB structure 2HFT) are shown. The γ-carboxy glutamic acid (Gla; GLA) domain, epidermal growth factor (EGF)-like domains 1 and 2, and serine protease (catalytic) domain of factor VIIa are indicated. Binding to tissue factor alters the overall structure of factor VIIa.
The tissue factor–factor VIIa complex activates both coagulation factors IX and X, which is considered to be the main initiating step of the extrinsic pathway of coagulation. In addition, the tissue factor–factor VIIa (–factor Xa) complex is not only critical to processes in coagulation, but also to wound healing, angiogenesis, tissue remodeling, and inflammation through proteolytic activation of PAR2.38,39,40
The ternary tissue factor–factor VIIa–factor Xa complex is inhibited by the tissue factor pathway inhibitor (TFPI). Tissue factor–factor VIIa is also inhibited by antithrombin, but only in the presence of heparin (see Table 113–4).
Gene Structure and Variations
The gene encoding factor VII (F7) is located on chromosome 13q34, is almost 15 kb in length, and comprises 9 exons (Fig. 113–7). The canonical mRNA encoding factor VII comprises 3000 bases.41 Alternatively spliced transcript variants encoding multiple isoforms have been observed, but their biology is not well characterized.42
Relationship of gene structure to protein structure in factor VII. The exons, introns, mRNA, and protein structure are as indicated. The mRNA is 2.7 kb with a small 5′ untranslated region and a relatively large 3′ untranslated region (light blue). In the protein, Pro indicates the prepro leader sequence, GLA indicates the γ-carboxy glutamic acid (Gla) domain, and epidermal growth factor (EGF)-1 and -2, as well as the serine protease (catalytic) domain, are indicated. CR indicates the connecting region that comprises the site of proteolytic activation.
Inherited factor VII deficiency is a rare autosomal recessive disorder that affects approximately one in 500,000 newborns.30 Factor VII deficiency is the most common of the inherited rare bleeding disorders, although the reported prevalences vary between countries. Homozygotes and compound heterozygotes develop a hemorrhagic diathesis that may vary from mild to severe.
The human gene mutation database (http://www.hgmd.org) lists 258 mutations in the factor VII gene. The majority of these are missense mutations, but splicing and regulatory mutations also occur. Small deletions account for almost 10 percent of the documented mutations. Other gross gene abnormalities appear to be uncommon.
The factor VII gene harbors many common polymorphisms of which three are notable: Arg353Gln in the catalytic domain, a 10-bp insertion in the promotor region, and a variable number of 37 bp repeats in intron 7.43 The minor alleles of these polymorphisms are associated with decreased levels of factor VII and explain up to 30 percent of the variation in activated factor VII levels. Furthermore, the minor alleles have been claimed to lower the risk of myocardial infarction. However, this finding has not led to routine genotyping in the management of this disorder. The relationship between factor VII levels, factor VII polymorphisms, and venous thrombosis has not been established with certainty.
Factor IX was originally reported in 1952 as “Christmas factor,” named after one of the first identified hemophilia B patients.34,44 Factor IX is synthesized in the liver and circulates in plasma as a single-chain zymogen of 415 amino acids (Mr ≈55,000) at a concentration of 90 nM with a half-life of 18 to 24 hours (see Table 113–1).
Factor IX consists of a Gla domain, two EGF-like domains, a 35-residue activation peptide, and the serine protease domain (see Fig. 113–1). The Gla domain contains 12 Gla residues, of which the 11th and 12th Gla (Glu36 and Glu40) are not evolutionary conserved in other vitamin K–dependent proteins and are not essential for normal factor IX function.45
Factor IX comprises several posttranslational modifications that are not only important for its structure and function, but are also involved in the plasma clearance and distribution of factor IX.35 Factor IX is sulfated at Tyr155 and phosphorylated at Ser158 in the activation peptide. Hydroxylation of Asp64 in EGF 1 mediates calcium binding, and while only approximately 40 percent of total plasma factor IX carries this modification, complete absence because of a point mutation at this position dramatically reduces factor IX activity resulting in hemophilia B.46,47 An O-linked fucose (Ser61) and glucose (Ser63) are found in the EGF 1 domain, in addition to several O-linked glycans in the activation peptide (Thr159, Thr169, Thr172, and Thr179). Further modification of the activation peptide includes N-linked glycosylation of Asn residues 157 and 167, which modulates the circulating levels of factor IX.48,49,50
Factor IX binds with high affinity to the extracellular matrix component collagen IV via residue Lys5 in the Gla domain.51,52 Although factor IX variants incapable of collagen IV binding exhibit a greater recovery, collagen IV association generates an extravascular reservoir of factor IX that enables prolonged action of factor IX at a hemostatic relevant region.
Factor IX Activation and Factor IXa Activity
Limited proteolysis of factor IX at both Arg145 and Arg180 by either the tissue factor–factor VIIa complex or factor XIa results in the release of the activation peptide and generation of factor IXa (see Fig. 113–1). Cleavage at Arg180 generates factor IXaα, which displays catalytic activity toward synthetic substrates only, whereas fully active factor IXaβ is formed following cleavage at Arg145.53,54
Factor IXa is a two-chain serine protease (Mr ≈45,000) that is composed of a light chain of 145 residues (Mr ≈17,000) and the catalytic heavy chain of 235 residues (Mr ≈28,000), which are covalently linked via a disulfide bond.
Factor IXa has a low catalytic efficiency as a result of impaired access of substrates to the active site that results from steric and electrostatic repulsion.55 Reversible interaction with the cofactor VIIIa on anionic membranes and subsequent factor X binding leads to rearrangement of the regions surrounding the active site and proteolytic factor X activation.
The primary plasma inhibitor of factor IXa is the serpin anti-thrombin, and this inhibition is enhanced by heparin (see Table 113–4), which induces a conformational change in antithrombin that is required for simultaneous active site and exosite interactions with factor IXa.56
Gene Structure and Variations
The gene encoding factor IX (F9) is located on chromosome Xq27.1 and covers nearly 25 kb.57 It is divided into eight exons from which a mature mRNA molecule is transcribed with an ultimate length of 2802 bases (Fig. 113–8).
Relationship of gene structure to protein structure in factor IX. The exons, introns, mRNA, and protein structure are as indicated. The mRNA is 2.8 kb with a small 5′ untranslated region and a relatively large 3′ untranslated region (light blue). In the protein, Pro indicates the prepro leader sequence, GLA indicates the γ-carboxy glutamic acid (Gla) domain, and epidermal growth factor (EGF)-1 and -2, as well as the serine protease (catalytic) domain are indicated. AP indicates the activation peptide that is released after cleavage of two bonds.
A defect or deficiency in factor IX leads to hemophilia B. Chapter 123 discusses the prevalence, clinical characteristics, and molecular genetics of hemophilia B in detail.
Conversely, increased levels of factor IX are a strong risk factor for venous thrombosis.58 This is in agreement with a rare gain of function mutation (Arg335Leu; factor IX Padua), which renders the protein hyperfunctional and is associated with familial early-onset thrombophilia.59
Factor X was originally reported in the late 1950s as the “Stuart-Prower factor,” named after the first two identified factor X–deficient patients.60,61,62 Factor X is primarily synthesized in the liver and circulates in plasma as a two-chain zymogen of 445 amino acids (Mr ≈59,000) at a concentration of 170 nM with a half-life of 34 to 40 hours (see Table 113–1).
Factor X is synthesized as a single-chain precursor and during intracellular processing, the three-amino acid peptide Arg140-Lys141-Arg142 is excised. The resulting two-chain zymogen consists of a light chain (Mr ≈16,000), comprising the Gla domain with 11 Gla residues and the EGF domains, which is linked via a disulfide bond to the heavy chain (Mr ≈42,000) that consists of a 52-residue activation peptide and the serine protease domain (see Fig. 113–1).
Hydroxylation of Asp63 mediates calcium binding to the EGF 1 domain and orients the Gla domain, which is essential for factor X clotting activity.35 N-linked glycosylation of the activation peptide residues Asn181 and Asn191 has been implicated in prolonging the factor X half-life.63 Further posttranslational modification of factor X consists of O-linked glycosylation at Thr159 and Thr171 in the activation peptide and Thr443 in the serine protease domain. There is some evidence that glycosylation of the human factor X activation peptide may also contribute to substrate recognition by the intrinsic or extrinsic factor X-activating complex.64,65
Factor X Activation and Factor Xa Activity
Factor X is proteolytically activated by either the factor VIIIa–factor IXa (“intrinsic tenase”) or the tissue factor–factor VIIa (“extrinsic tenase”) enzyme complexes following cleavage at Arg194 in the heavy chain (see Fig. 113–1). This results in the release of the activation peptide and generation of factor Xa, also known as factor Xaα. A snake venom protease from Russell’s viper venom (RVV-X) is capable of generating factor Xa in a similar manner.
Factor Xa consists of the Gla and EGF domains-comprising light chain (Mr ≈16,000) and the catalytic heavy chain (Mr ≈29,000) that are covalently linked via a disulfide bond. Factor Xa reversibly associates with its cofactor factor Va on an anionic membrane surface in the presence of calcium ions to form prothrombinase, the physiologic activator of prothrombin. Factor Xa is also involved in the proteolytic activation of factors V, VII, and VIII.66,67,68
Similar to thrombin, factor Xa plays a role in other biologic and pathophysiologic processes that are not directly related to coagulation. Factor Xa triggers intracellular signaling via activation of PAR1 and/or PAR2. Factor Xa cleaves PAR2 by itself as well as in complex with tissue factor–factor VIIa. These direct cellular effects of factor Xa contribute to wound healing, tissue remodeling, inflammation, angiogenesis, and atherosclerosis, among others.26,69
Further autocatalytic cleavage at Arg429 near the C-terminus of the factor Xa heavy chain leads to release of a 19-residue peptide, yielding the enzymatically active factor Xaβ.70,71,72 Plasmin-mediated cleavage of factor Xa at adjacent C-terminal Arg or Lys residues also results in the generation of factor Xaβ and factor Xaβ derivatives.73,74 While the coagulation activity is eliminated in the factor Xaβ derivatives, they are capable of interacting with the zymogen plasminogen and enhance its tissue plasminogen activator-mediated conversion to plasmin, thereby promoting fibrinolysis.75
A primary plasma inhibitor of factor Xa is the serpin anti-thrombin, and this inhibition is enhanced by heparin (see Table 113–4), which induces a conformational change in antithrombin that is required for simultaneous active site and exosite interactions with factor Xa.76 Another potent factor Xa inhibitor is TFPI, which inhibits both the ternary tissue factor–factor VIIa–factor Xa complex as well as free factor Xa, for which protein S functions as a cofactor.77,78 Free factor Xa is also inhibited by the protein Z/protein Z–dependent protease inhibitor (ZPI) complex on membranes.79
Low-molecular-weight heparin and synthetic derivatives (e.g.fondaparinux) are clinically used as anticoagulants to enhance factor Xa inhibition by antithrombin specifically. The target-specific oral anticoagulants rivaroxaban, apixaban, edoxaban, and analogues directly inhibit both free factor Xa and prothrombinase complex-assembled factor Xa with high specificity through a high-affinity, reversible interaction with the factor Xa active site.80,81,82,83
Gene Structure and Variations
The gene encoding factor X (F10) is located on chromosome 13q34 and spans almost 27 kb.84 The 8 exons in the factor X gene give rise to a mature mRNA of 1560 bases (Fig. 113–9). There are no common alternative splice variants with known biology.
Relationship of gene structure to protein structure in factor X. The exons, introns, mRNA, and protein structure are as indicated. 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 prepro leader sequence, GLA indicates the γ-carboxy glutamic acid (Gla) domain, and epidermal growth factor (EGF)-1 and -2, as well as the serine protease (catalytic) domain, are indicated. AP indicates the activation peptide. Before secretion, cleavage in this domain processes factor X to the two-chain mature zymogen. A second cleavage releases the activation peptide and generates factor Xa activity.
Loss of function mutations in the factor X gene lead to a rare bleeding disorder with a recessive mode of inheritance. Factor X deficiency occurs in approximately one in every 1,000,000 newborns. Most cases of documented factor X deficiency experience serious bleeding problems. In fact, factor X deficiency may be the most severe of the rare congenital bleeding disorders.30 Well over 100 mutations have been documented in cases with factor X deficiency (http://www.hgmd.org). The majority of these mutations are missense and nonsense mutations.
Gain-of-function mutations in factor X could potentially increase thrombotic risk, but such mutations have not been documented. There is uncertainty about whether common gene variations influence the level of factor X in plasma.85
Protein C, which plays a central role in the anticoagulant pathway, was discovered in 1960, and being the third protein peak (“peak C”) observed in a vitamin K–dependent plasma protein purification, it was named protein C.86,87 Protein C is synthesized in the liver and circulates in plasma as a two-chain zymogen of 417 amino acids (Mr ≈62,000) at a concentration of 65 nM with a half-life of 6 to 8 hours (see Table 113–1).
Protein C is synthesized as a single-chain precursor and during intracellular processing amino acids Lys146-Arg147 are excised. The resulting two-chain zymogen consists of a light chain (Mr ≈21,000) comprising the Gla domain with nine Gla residues and the EGF domains, which is linked via a disulfide bond to the heavy chain (Mr ≈41,000) that consists of the 12-residue activation peptide and the serine protease domain (see Fig. 113–1).
In addition to γ-carboxylation, protein C is hydroxylated at Asp71 in the EGF-1 domain, which coordinates calcium binding.35 N-linked glycosylation of Asn97 in EGF-1 and Asn248, Asn313, and Asn329 in the serine protease domain are important for efficient protein secretion, proteolytic processing of Lys146-Arg147, and proteolytic activation.88,89,90 Some of the total plasma protein C is not glycosylated at either Asn329 (β-protein C) or at both Asn329 and Asn248 (γ-protein C), of which the impact on protein function remains unclear.91
Protein C Activation and Activated Protein C Activity
Protein C is proteolytically activated by α-thrombin in complex with the endothelial cell surface protein thrombomodulin following cleavage at Arg169 (see Fig. 113–1). The activation peptide is released and the mature serine protease activated protein C (APC) is formed. Activation of protein C is enhanced by its localization on the endothelial surface through association with the endothelial cell protein C receptor (EPCR).92 Several snake venom proteases (RVV-X and Protac) are also capable of activating protein C.
APC consists of the disulfide-linked light chain comprising the Gla and EGF domains (Mr ≈21,000) and the catalytic heavy chain (Mr ≈32,000). In complex with its cofactor protein S, APC proteolytically inactivates factors Va and VIIIa in a calcium- and membrane-dependent manner. Intact factor V has been reported to function as a cofactor for the inactivation of factor VIIIa in the presence of protein S.93
Downregulation of thrombin formation through inactivation of these cofactors seems to occur preferentially on the endothelial cell surface as opposed to that of platelets,94 where it prevents coagulation and potential thrombosis. However, protein C activation is also accelerated by platelet factor 4 (PF4), which is secreted by activated platelets. Upon interaction with the Gla domain of protein C, PF4 modifies the conformation of protein C, thereby enhancing its affinity for the thrombomodulin-thrombin complex.95 This ensures APC generation in close proximity of the injury site where platelets are activated, which serves to impede dissemination of coagulation.
APC also plays a major role in the cytoprotective pathway to prevent vascular damage and stress.96 These activities include antiapoptotic activity, antiinflammatory activity, alterations of gene-expression profiles, and endothelial barrier stabilization. Most of these functions require binding to EPCR and PAR1 cleavage.
APC is primarily inhibited by the heparin-dependent serpin protein C inhibitor and by plasminogen activator inhibitor-1 (PAI-1). Because PAI-1 is the major inhibitor of tissue plasminogen activator, inhibition through complex formation with APC contributes to enhanced fibrinolysis. Chapter 114 discusses these and other factors that attenuate the anticoagulant activity of APC.
Gene Structure and Variations
The protein C gene (PROC) is located on chromosome 2q14.3 and spans almost 11 kb.97 The gene is divided into nine exons and the mature mRNA has a length of 1790 bases (Fig. 113–10). There are no alternative mRNA species with known biology.
Relationship of gene structure to 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 (light blue). In the protein, Pro indicates the prepro leader sequence, GLA indicates the γ-carboxy glutamic acid (Gla) domain, and epidermal growth factor (EGF)-1 and -2, as well as the serine protease (catalytic) domain, are indicated. AP indicates the activation peptide. Before secretion, cleavage in this domain processes protein C to the two-chain mature zymogen. A second cleavage releases the activation peptide and generates activated protein C.
Loss-of-function mutations cause protein C deficiency. In homozygous or compound heterozygous form this leads to life-threatening purpura fulminans at birth which, if left untreated, is fatal.98 In cases where there is still some protein C activity detectable, symptoms may be much milder.
Heterozygous protein C deficiency increases the risk of venous thrombosis. This is true for most deficiencies of natural anticoagulants and sets them apart from rare bleeding disorders where heterozygosity for loss of function mutations is mostly asymptomatic. The risk for venous thrombosis is increased approximately 10-fold in heterozygotes for protein C deficiency, albeit that the risk estimates vary considerably between studies.99 Family studies in particular suggest a high risk, whereas case-control studies may show markedly lower estimates.100
Heterozygous protein C deficiency can be categorized as type I or type II. In type I deficiency, antigen levels are approximately 50 percent of normal, whereas in type II deficiency, antigen levels are (near) normal but activity levels are decreased by 50 percent.
The genetic basis of protein C deficiency, consistent with what is observed in general for congenital loss of function disorders, is heterogeneous. In line with this, more than 300 mutations have been documented and are tracked in the human gene mutation database (http://www.hgmd.org). Two-thirds of these documented mutations are missense or nonsense.
Several common polymorphisms, in particular in the promotor region of the protein C gene, are known to have a small but measurable effect on plasma protein C levels. Alleles of these polymorphisms that are associated with lower protein C levels are also associated with an increased thrombotic risk, albeit that the effect is small.101 Therefore, it is not surprising that measurement of these polymorphisms have not found any clinical application.