The Molecular Basis Review of Blood Coagulation€¦ · The Molecular Basis of Blood Coagulation Review Bruce Furie and Barbara C. Furie Center for Hemostasis and Thrombosis Research

Cell, Vol. 53, 505-518, May 20, 1988, Copyright 0 1988 by Cell Press

The Molecular Basis of Blood Coagulation

Review

Bruce Furie and Barbara C. Furie Center for Hemostasis and Thrombosis Research Division of Hematology/Oncology Departments of Medicine and Biochemistry New England Medical Center and Tufts University School of Medicine Boston, Massachusetts 02111

Overview

Blood coagulation is a host defense system that assists in maintaining the integrity of the closed, high-pressure mammalian circulatory system after blood vessel injury. After initiation of clotting, the sequential activation of certain plasma proenzymes to their enzyme forms proceeds through either the intrinsic or extrinsic pathway of blood coagulation (Figure 1A) (Davie and Fiatnoff, 1964; Mac- Farlane, 1964). These plasma glycoproteins, including factor XII, factor XI, factor IX, factor X, factor VII, and prothrombin, are zymogens of serine proteases. As a family, these proteins bear marked structural and functional homology to the digestive proteases trypsin and chymotrypsin. They are each converted from an inactive form to an active enzyme by limited proteolysis of one or two peptide bonds. However, most of the blood clotting enzymes are effective on a physiologic time scale only when assem- bled in complexes on membrane surfaces with protein cofactors such as factor VIII and factor V. Calcium ions have a critical role in that many of the component reactions in blood coagulation are either Ca2+-dependent or require Caz+ for the interaction of proteins with membrane surfaces.

Clot formation occurs when fibrinogen is converted by thrombin to fibrin, the structural protein that assembles

into the fibrin polymer. The clot, formed after tissue injury, is composed of activated platelets and fibrin. The clot mechanically impedes the flow of blood from the injured vessel and minimizes blood loss from the wound. Once a stable clot has formed, wound healing ensues. The clot is gradually dissolved by enzymes of the fibrinolytic system.

Blood coagulation may be initiated through either the intrinsic pathway, where all of the protein components are present in blood, or the extrinsic pathway, where the cell- membrane protein tissue factor plays a critical role. Initia- tion of the intrinsic pathway of blood coagulation involves the activation of factor XII to factor Xlla (see Figure lA), a reaction that is promoted by certain surfaces such as glass or collagen. Although kallikrein is capable of factor XII activation, the particular protease involved in factor XII activation physiologically is unknown. The collagen that becomes exposed in the subendothelium after vessel damage may provide the negatively charged surface required for this reaction in vivo. Factor Xlla, with high molecular weight kininogen (HMWK) as cofactor, converts factor Xl to its activated form, factor Xla. In the presence of Ca2+, factor Xla activates factor IX to factor IXa. Factor IXa, in complex with factor Villa on membrane surfaces, catalyzes the activation of factor X to factor Xa. Factor Xa activates prothrombin to thrombin in the presence of Ca2+ and factor Va bound to membrane surfaces. Throm- bin converts fibrinogen to fibrin by cleavage of two peptide bonds, thereby releasing two small amino-terminal peptides, fibrinopeptide A and fibrinopeptide B.

Although the intrinsic pathway is clearly important in the clotting of blood in vitro, its physiologic importance has been challenged by the absence of bleeding problems in patients deficient in factor XII, prekallikrein, and HMWK. Therefore the intrinsic pathway is not likely to be of importance to normal blood coagulation. The role of factor Xl is

Table 1. PropertIes of the Genes, mRNAs, and Gene Products of the Components of the Blood Coagulation Cascade

Plasma Molecular Gene mRNA Concentration

Component Weight W Chromosome WW Exons WW Function

Prothrombin 72,000 21 llpll-q12 2.1 14 100 Protease zymogen Factor X 56,000 22 13q34 1.5 8 10 Protease zymogen Factor IX 56,000 34 Xq26-27.3 2.8 8 5 Protease zymogen Factor VII 50,000 13 13q34 2.4 8 0.5 Protease zymogen Factor VIII 330,000 185 Xq28 9.0 26 0.1 Cofactor Factor V 330,000 7.0 10 Cofactor Factor Xl 160,000 23 15 5 Protease zymogen Factor XII 80,000 12 5 24 14 30 Protease zymogen Fibrinogen 340,000 3000 Structural

Au chain 66,500 4q26-q28 5 - BB chain 52,000 4q26-q28 8 -

y chain 46,500 4q26-q28 9 - Protein C 62,000 11 1 .a a 4 Protease zymogen Protein S 80,000 2.4 25 Cofactor vWF 225,000 x na 175 1 Ppter-p12 85 >42 10 Adhesion Tissue factor 37,000 1 pter-p12 2.1 0.0 Cofactorlinitlator

Chromosome assignments are taken from Royle et al. (1987). a n denotes number of subunits, where the subunlt M, is 225,000

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Intrinsic Pathway

Figure 1. The Protems of Blood Coagulation

(A) The blood coagulation cascade. Glycoprotein components of the intrinsic pathway include factors XII, XI, IX, VIII, X, and V, prothrombin, and fibrinogen. Glycoprotein components of the extrinsic pathway, initiated by the action of tissue factor located on cell surfaces, include factors VII, X, and V, prothrombin and fibrinogen. The cascade reactions culminate in the conversion of fibrinogen to fibrin and the formation of a fibrin clot. Certain reactions, including the activation of factor X and prothrombin, take place on membrane surfaces. Where indicated, reactions are Ca2+- dependent, or Ca’+ is required for interaction of proteins with membrane surfaces. Proenzymes are drawn as diamonds, enzymes as circles, procofactors as rectangles, and macromolecular complexes on membrane surfaces as shaded rectangles, Numbered factors are abbreviated FXII, FXI, etc. Other abbreviations: HMWK, high molecular weight kininogen; TF, tissue factor; PT, prothrombin; T, thrombin; FG, fibrinogen; F, fibrin, (6) Diagrammatic representation of the structural and functional relationships among protein components of hemostasis. Protein families include the serine proteases, calcium-binding proteins containing y-carboxyglutamic acid (vitamin K-dependent), procoagulant blood clotting proteins, regulatory anticoagulant proteins, and fibrinolytic proteins. Abbreviations, in addition to those in (A): TPA, tissue plasminogen activator; UK, uro- kmase; PK, prekallikrein; T, trypsin; CT, chymotrypsin: ATIII. antithrombin Ill; HCII, heparin cofactor II; TM, thrombomodulin; PS, protein S; PC, protein C; BGP, bone Gla protein; MGP, matrix Gla protein

uncertain in that patients with factor XI deficiency have a variable bleeding tendency. Although the precise mechanisms for the initiation of blood coagulation in vivo remain uncertain, it seems probable that the extrinsic pathway, including the bypass through factor IX, plays a dominant physiologic role.

Initiation of blood coagulation associated with vessel injury involves the expression of tissue factor activity on the surfaces of injured cells. Tissue factor initiates the extrinsic pathway of blood coagulation by participating as a cofactor in the activation of factor VII to its active form, factor Vlla (Figure 1A). The protease required for factor VII activation in vivo is uncertain, but factor Xa can catalyze this reaction. The factor Vlla-tissue factor complex activates factor X and factor IX in the presence of Ca2+. Factor Xa activates prothrombin to thrombin, and thrombin converts fibrinogen to fibrin.

Factor VIII and factor V, cofactors in intermediate steps in the cascade, may function in regulating the rate of activation of blood coagulation. This effect is mediated by protein C and protein S, plasma anticoa‘gulants. Protein C is converted to its active form on endothelial cell membranes by a complex of two proteins, thrombomodulin and thrombin. Activated protein C, anchored to cell membranes through binding to membrane-bound protein S,

converts the factor Va and factor Villa to their inactive forms by limited proteolysis.

Platelets are anucleate cells that circulate in the blood in a resting form. Upon stimulation at the site of tissue injury, platelets adhere to the injured surface. This interaction requires von Willebrand factor (vWF), a large, multi- merit plasma protein that binds to a specific receptor, glycoprotein lb, on the platelet membrane. The activation of platelets by thrombin, ADP, thromboxane AZ, or epi- nephrine triggers characteristic morphological and biochemical alterations in the platelet. Activated platelets se- crete two types of granules: a granules, which contain thrombospondin, fibrinogen, vWF, factor V, and other proteins involved in hemostasis; and dense granules, which are rich in calcium ions and ADP. The expression of the glycoprotein Ilb-llla complex as a receptor for certain plasma proteins (e.g., fibrinogen) may be important for the formation of the platelet plug and fibrin clot.

Endothelial cells tile the walls of the blood vessels. These cells are unique in that, in their resting state, they function to inhibit the activation of blood coagulation or the stimulation of platelet adherence. The synthesis of prostacyclin, the expression of heparin-like substances on the membrane surface, and the presence of protein complexes (e.g., thrombomodulin-thrombin) that lead to the

Review: Molecular Basis of Blood Coagulation 507

PT

FIX

FX

FVII

PC

FXI

FXII

PS

tPA

PUK

MGP

BGP

Pm

T

generation of anticoagulants such as activated protein C are processes that prevent clot formation in normal blood vessels. After tissue injury, thrombogenic subendothelial components of the blood vessel are exposed. Receptors for blood clotting proteins may be expressed on the endothelial cell membrane, allowing for the sequential activation of the blood coagulation cascade.

During wound healing, the fibrin clot in the vessel is degraded by plasmin, a serine protease generated from the plasma zymogen, plasminogen. This reaction is catalyzed by several plasminogen activators, including two serine proteases, tissue plasminogen activator and urokinase. Their activity is in turn regulated by two plasma protease inhibitors, plasminogen activator inhibitor and a2-antiplasmin.

Structural Features of the Blood Clotting Proteins and Their Encoding Genes The plasma proteins involved in hemostasis can be sepa- rated into three functional groups with interrelated but dis- tinct physiologic roles (Figure 1B and Table 1). One group consists of the blood coagulation proteins, which promote clot formation; this group includes cofactors (e.g., factor VIII and factor V), structural proteins (e.g., fibrinogen), and proenzymes (e.g., factor Xl, prothrombin). A second group consists of the regulatory proteins that modulate and localize clot formation to regions of tissue injury; this cate- gory includes the plasma protease inhibitors (e.g., antithrombin Ill) and specific enzyines that inactivate procoagulant components (e.g., activated protein C). A third group comprises the fibrinolytic proteins, which dissolve fibrin clots as a late component of the healing process; these components include enzymes (e.g., tissue plasminogen activator) and protease inhibitors (e.g., as-antiplasmin).

Figure 2. Stuctural Domains of the Proteins involved in Hemostasis and of Related Proteins

Domains, identified in the key, are described in the text. Sites of proteolytic cleavage associated with synthesis of the mature protein are indicated by thin arrows. Sites of proteolytic cleavage associated with zymogen activation are indicated by the thick arrows. Shown, top to bottom, are schematic structures for prothrombin (Degen et al., 1983) factor IX (Kurachi and Davie, 1982; Choo et al., 1982) factor X (Leytus et al., 1984; Fung et al., 1985) factor VII (Hagen et al., 1986), protein C (Foster and Davie, 1984; Beckmann et al., 1985; Long et al., 1984) factor XI (Fujikawa et al., 1986) factor XII (Cool et al., 1985) protein S (Lundwall et al., 1986) tissue-type plasminogen activator (Ny et al., 1984). prourokinase (Holmes et al., 1985) matrix Gla protein (Price and Williamson, 1985) bone Gla protein (Pan and Price, 1985) plasminogen (Malinowski et al., 1984), and trypsin.

Some of the proteins in each of these groups are characterized structurally as zymogens of serine proteases. Other proteins contain y-carboxyglutamic acid, synthesized in a vitamin K-dependent reaction, and pos- sess unique calcium-dependent membrane-binding properties. As mentioned above, there are a number of other cellular components essential to hemostasis, including vWF on platelets and endothelial cells, receptor proteins on platelets (e.g., glycoproteins la, lb, Ilb-llla, and IV), tissue factor on cell membranes, regulatory membrane proteins such as thrombomodulin, and tissue plasminogen activator secreted from cells. The scope of this review is limited to the proteins of blood coagulation and the plasma proteins involved in the regulation of this process.

Common Domain Structures The structure and organization of the genes coding for the blood coagulation proteins emphasize that the evolution of new protein function occurs via gene duplication, gene modification, and exon shuffling (Gilbert, 1978; Patthy, 1985). Each of the exons may be considered a module coding for a homologous domain in each protein (Figures 2 and 3). The three-dimensional structures of the polypeptide backbones of these homologous domains are likely to be nearly identical, but substitution of amino acid side chains on the protein surfaces gives definition to unique properties of substrate recognition, cofactor binding, or membrane interaction (Furie et al., 1982). The blood coagulation proteins remain a prime example of the development of a family of proteins with diverse functional properties but common, unified structural elements.

Like trypsin, the enzymes involved in blood coagulation are ordinarily maintained in their zymogen, or proenzyme forms. Each is converted to an active enzyme by proteolysis of one or two peptide bonds. A common feature of

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Factor VII

Factor IX

Factor X

Protein C

Prothrombin

Factor XI

Factor XII

0 CATALYTIC

El REPEAT

ii FIBRONECTIN TYPE I

II FIBRONECTIN TYPE ,I

Figure 3. Exon-lntron Structures of the Genes Encoding the Blood Coagulation Serine Proteases

Exons are shown schematrcally (see key) and are drawn to scale. Introns, indicated by lines connecting the exons. are not drawn to scale. The exons code for most of the individual domams shown in Figure 2. Data for prothrombin are from Friezner Degen and Davie (1987); for factor IX, Yoshitake et al (1985); for factor X. Leytus et al. (1986); for factor VII. O’liara et al. (1987); for protein C, Foster et al. (1985); for factor XI, Asakai et al. (1987); and for factor XII, Cool et a;. (1987).

these serine proteases is the presence in the enzyme active site of the “catalytic triad”: a serine, histidine, and aspartic acid critical to the mechanism of catalysis by these enzymes. The plasma blood clotting serine proteases are about twice the size of trypsin. The domain in the blood clotting enzymes showing marked sequence homology and presumed three-dimensional structural homology (Furie et al., 1982) with trypsin is known as the catalytic domain. The blood clotting serine proteases have an active site and internal core nearly identical to those of trypsin, but different molecular surfaces surrounding the enzyme active sites are likely responsible for extended substrate binding sites and the unique substrate specifici- ties of these enzymes.

The amino termini of the vitamin K-dependent blood clotting proteins (Figure 2) contain between ten and twelve v-carboxyglutamic acid (Gla) residues within the first forty- two amino acids. Two, or possibly three, of these amino acids bind a single calcium ion (Furie et al., 1979), thus forming a noncovalent intramolecular bridge between regions of the polypeptide backbone. This bond stabilizes the tertiary structure of this region of the protein (Tai et al., 1984). Upon binding by calcium, the proteins undergo two conformational changes that expose a membrane-binding site (Nelsestuen, 1976; Prendergast and Mann, 1977; Borowski et al., 1986; Liebman et al., 1987). Although the G/a domain is clearly required for these conformational transitions, it remains uncertain whether some of the y-carboxyglutamic acids directly bind to membrane surfaces through calcium ion bridges (Mann et al., 1982) or whether metal binding to y-carboxyglutamic acid in the Gla domain alters the structure of adjacent domains, ex- posing a membrane-binding surface (Borowski et al., 1986).

Many of the proteins involved in hemostasis contain one or more regions, known as EGF domains, that are homologous with a module in the epidermal growth factor (EGF) precursor. This module, a 53 amino acid peptide containing three disulfide bonds, binds to specific cell-surface receptors. The receptor-binding region of EGF modules has been localized to residues 19 through 31, and residues 13-19 and 31-33 show the highest sequence conserva- tion (Appella et al., 1987). The EGF domains may be responsible for the high-affinity interaction of proteins with specific cell surfaces or, alternatively, with receptor domains on other proteins, such as factors VIII and V.

The kringle domain, another structure common to several proteins involved in hemostasis, is about 100 amino acids long and contains three disulfide bonds. Its three- dimensional structure resembles an eccentric oblate ellip- soid, and its folding is defined by close contacts between the sulfur atoms of two of the disulfide bridges, which form a sulfur cluster in the center of the “kringle” (Park and Tulinsky, 1986). Internal amino acids are well conserved among various proteins containing kringles; again, differ- ences in the molecular surfaces probably distinguish the functional properties of these proteins. The function of these domains remain uncertain, but, as a structural unit, the kringle likely contains recognition elements important for macromolecular assembly.

A short linking segment observed in numerous blood clotting proteins, termed the aromatic amino acid stack, contains the sequence Phe-Trp-X-X-Tyr. Although hydro- phobic, this region is oriented toward the surface in prothrombin fragment 1 (Park and Tulinsky, 1986) and may play a role in membrane binding.

Type I fibronectin homology regions, containing two internal disulfide bonds, are about 50 residues long and are

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Molecular Basis of Blood Coagulation

known as finger domains (Petersen et al., 1983). They may play a role in fibrin binding. The type II fibronectin homology regions are approximately 60 residues long and also contain two internal disulfide bonds (Petersen et al., 1983). This region in fibronectin contains the collagen binding site, and the homologous region in other proteins may also be responsible for collagen binding.

Blood Coagulation Proteins The protein domains described above are encoded by genes that show marked structural and organizational homology. The human (Friezner Degen and Davie, 1987) and bovine (Irwin et al., 1985) prothrombin genes, for example, demonstrate partial homology with genes encoding other vitamin K-dependent proteins (Figure 3). The prothrombin gene contains fourteen exons: exon I encodes the signal peptide; exon II encodes the propeptide, including the y-carboxylation recognition site (@ZRS; see below) (Jorgensen et al., 1987a; Foster et al., 1987), and the Gla domain; exon III codes the aromatic amino acid stack domain. Exons I, II, and Ill are homologous in structure and organization to corresponding exons in the factor IX gene family, which includes factor IX, factor X, factor VII, and protein C. Exons IV-VII code for the two kringle regions; these domains have no parallel with structures in the factor IX family, but are observed in other proteins involved in hemostasis, including tissue plasminogen activator, plasminogen, urokinase, and factor XII (Figure 2). Exons VIII and IX code for the sites of proteolytic cleavage by factor Xa, which converts prothrombin to thrombin. The catalytic thrombin domain, responsible for enzyme catalysis and substrate specificity, is encoded by exons X-XIV. The organization of this portion of the gene differs from that coding for the analogous domain in the factor IX gene family despite the marked structural homology of the catalytic domains in the proteins. In prothrombin, the amino acids that form the catalytic triad of serine proteases are each on a separate exon- histidine on exon X, aspartic acid on exon XI, and serine on exon XIII. In the factor IX gene family the entire catalytic domain is encoded by two exons, not five. This may suggest that the factor IX gene and related genes were derived ancestrally from a precursor of the prothrombin gene, but that reorganization of these genes by intron deletion led to consolidation of the exons encoding the serine protease domain. The introns in the prothrombin gene range in size from 84 to 9447 base pairs (Friezner Degan and Davie, 1987), and 40% of the prothrombin gene is composed of repetitive DNA of the Alu and Kpn family of repeats.

Prothrombin (M, 72,000) assembles with factor Xa and factor Va on the membrane surface to form a catalytic unit known as the prothrombinase complex (Mann et al., 1982). In the presence of calcium ions, prothrombin is converted to thrombin by the cleavage of two peptide bonds and removal of the Gladomain, the aromatic amino acid stack domain, and the kringle domains. Thrombin is released from the membrane phase into solution. The formation of the activating complex on membrane surfaces is typical of many of the blood clotting reactions, and a possible model for this complex is shown in Figure 4.

Figure 4. Model for a Macromolecular Complex Associated with Zymogen Activation

A central feature of the proteins involved in blood coagulation is their assembly on membrane surfaces. The interaction of calcium ions (circles) with the ~carboxyglutamic acids (“v” structures) on the vitamin K-dependent proteins leads to the exposure of membrane-binding sites (drawn in black) on these proteins. The assembly of these proteins on membranes in a geometry organized by the protein cofactor facilitates enzyme-substrate interaction. The protein cofactor likely plays an important regulatory role in this complex. Factor Xa, enzyme (L, light chain; H, heavy chain); prothrombin, substrate; factor Va, cofactor.

FactorX (Leytus et al., 1986) factor IX (Yoshitake et al., 1986), factor VII (O’Hara et al., 1987) and protein C (Foster et al., 1985) show marked structural homology in both gene structure and gene organization. The genes encoding these proteins contain eight exons and seven introns (Figure 3). Exon I codes for the signal peptide domain. Exon II codes for the propeptide domain and the Gla domain: the coding of the propeptide, containing the +RS (Jorgensen et al., 1987b; Foster et al., 1987), and the Gla domain on the same exon emphasizes the functional in- terplay of these two regions. The small exon III codes for the aromatic amino acid stack. Exon IV and exon V each code for an EGF domain. In each case the activation of the proenzyme to an enzyme requires proteolytic cleavage, and the cleavage site and the surface features that define the substrate specificity for this reaction are encoded in exon VI. The catalytic domain is encoded by exon VII and exon VIII. The components of the catalytic triad are dispersed between these exons: histidine is encoded by exon VII, aspartic acid and serine by exon VIII. Intervening sequences occur in similar or identical positions in all of these genes. However, these introns demonstrate no homology in length or in sequence. In the case of factor VII, two alternative genetic organizations have been identified. One form of preprofactor VII arises from a gene analogous in organization to the factor X, factor IX, and protein C genes. The second form arises from a gene containing a ninth exon, exon IA. This exon codes for a polypeptide extension of the amino terminus that elongates the prepropeptide from 38 to 60 residues. The significance of these two preprofactor VII forms is unknown.

Factor IX is a single-chain plasma zymogen (M, 56,000) that is converted to its enzyme form, factor IXa, by the

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cleavage of two peptide bonds and the release of an activation peptide. Twelve y-carboxyglutamic acid residues are located near the amino terminus of the mature zymogen. A single f3-hydroxyaspartic acid at position 64 in the EGF domain has uncertain function. Factor X (M, 56,000) is synthesized as a single chain (Fung et al., 1985; Leytus et al., 1984) but is converted to a two-chain zymogen that circulates in the plasma. The light chain of human factor X includes eleven y-carboxyglutamic acid residues and the EGF domain, containing a single f3-hydroxyaspartic acid. The activation peptide is cleaved from the heavy chain during activation of factor X by the factor IXa-factor Villa complex or the factor Vlla-tissue factor complex to yield the enzyme factor Xa. The remaining portion of the heavy chain contains the catalytic region homologous to trypsin.

Factor VII, a single-chain zymogen (M, 56,000) that is a component of the extrinsic pathway of blood coagulation, differs from other blood coagulation proenzymes in that the zymogen expresses significant enzyme activity. The generation of factor Xa can lead to activation of factor VII to factor Vlla, a two-chain enzyme that, in complex with tissue factor, activates factor X. Factor VII contains ten y-carboxyglutamic acid residues, and two EGF domains containing a single 5-hydroxyaspartic acid.

Protein C is a vitamin K-dependent regulatory protein (M, 56,000) containing nine y-carboxyglutamic acid residues and a single 6-hydroxyaspartic acid residue. The activation of the zymogen to the enzyme form is catalyzed by the thrombin-thrombomodulin complex on endothelial cells (Esmon et al., 1982). Thrombomodulin is an integral membrane protein whose primary structure has recently been determined (Jackman et al., 1986; Wen et al., 1987). Activated protein C destroys both factor Villa and factor Va, in each case by cleavage of the polypeptide chain. In this manner, factor Villa and factor Va activities are turned off, and the activity of the coagulation pathway is dampened. It is interesting to note that structurally homologous proteins have been adapted to serve both coagulant (e.g., factor IX, factor X) and anticoagulant (e.g., protein C) functions.

The factor XI gene contains fifteen exons and fourteen intervening sequences (Asakai et al., 1987). Of these exons, fourteen code for translated regions of the mRNA. Exon II encodes an 18 residue signal sequence. There is no evidence for a propeptide (Fujikawa et al., 1986). Four “repeat” sequences are each encoded by two exons: repeat 1 by exons III and IV, repeat 2 by exons V and VI, repeat 3 by exons VII and VIII, and repeat 4 by exons IX and X. The catalytic domain is encoded in exons XII-XV. The organization of the exons coding for the catalytic domain is similar to that of corresponding regions of the genes for urokinase and tissue plasminogen activator.

The factor Xl (M, 160,000) zymogen contains two identical chains that are disulfide-linked. Upon activation by factor Xlla in a reaction stimulated by HMWK, two peptide bonds are cleaved to generate two- heavy chain-light chain homodimers linked through the heavy chain by a disulfide bond. The catalytic domain is located on the light chain, and the heavy chain contains the HMWK binding site and the recognition site for interaction of factor Xla with its substrate, factor IX (Sinha et al., 1987; Liebman et

al., 1987). The four”repeat”domains in the amino-terminal region of the protein are likely responsible for macromolecular assembly. The protein shows marked sequence homology with prekallikrein, and both factor Xl and prekallikrein circulate in plasma as complexes with HMWK. Accordingly, the tandem repeats in both proteins may be responsible for HMWK interaction.

The factor XII gene contains fourteen exons and thir- teen introns (Figure 3) (Cool and MacGillivray, 1987). Exon I encodes the signal peptide. Exon II encodes a region with no known structural homologies. Exons Ill and IV encode regions homologous to the type II fibronectin structure. Exon V and exon VII each encode EGF domains, exon VI encodes a region homologous to the fibrin finger domain of fibronectin, and exons VIII and IX code for the two kringle domains. The catalytic domain is encoded by exons XI-XIV. The organization of these exons, like those encoding the catalytic domain of factor XI, parallels the structure of the exons encoding the urokinase and tissue plasminogen activator catalytic domains. The factor XII gene lacks the consensus TATA and CAAT sequences im- mediately upstream of the transcription initiation site, suggesting alternative sites or alternative structures for the promoters of this gene.

Factor XII, the first component of the intrinsic pathway, is converted from a single-chain zymogen (M, 80,000) into the enzyme factor Xlla, a two-chain disulfide-linked enzyme. This reaction can be accomplished by kallikrein, but, as noted in the Overview, the relevant physiologic activator has not been identified with certainty. Factor Xlla is capable of activating the fibrinolytic system, generating kinins and initiating blood coagulation through the activation of factor Xl. A region homologous to the type II region of fibronectin may be responsible for factor XII binding to anionic surfaces.

Protein Cofactors The majority of the serine proteases of blood coagulation require protein cofactors for efficient proteolytic activity. Tissue factor is a transmembrane protein (M, 37,000) that activates blood coagulation through the extrinsic pathway by forming a complex with factor Vlla that activates factor X. This protein is not present in plasma, but rather is a component of many cell surfaces with the exception of resting endothelial cells and monocytes. The amino acid sequence, predicted from isolated cDNA (Spicer et al., 1987; Morrissey et al., 1987) includes a 32 residue signal peptide, an extracellular domain of 219 amino acids, a short membrane-spanning domain, and a cytoplasmic domain. No propeptide is encoded. A key event in the initiation of blood coagulation involves the expression of tissue factor activity on the cell surface to form a complex with plasma factor VII.

Protein S is a vitamin K-dependent protein (M, 80,000) that functions as a plasma anticoagulant. This protein facilitates the action of activated protein C on factor Villa and factor Va. Protein S is a membrane-binding protein, interacting with cell membranes through a mechanism re- quiring y-carboxyglutamic acid. Protein S does not contain a trypsin-like catalytic domain, but rather has a unique carboxy-terminal structure that bears no homology

Review: Molecular Basis of Blood COagUlatiOn 511

to regions of other proteins (Dahlback et al., 1986). Protein S is synthesized as a single chain containing a signal peptide, a propeptide, a Gla domain, four EGF domains containing 8-hydroxyaspartic acid and 8-hydroxyasparagine, and an additional large domain likely responsible for recognition of activated protein C (Lundwall et al., 1986; Hoskins et al., 1987). Protein S exists in two forms in the blood: as a species bound to C4b-binding binding protein and as a free form.

The gene encoding facror V//l is one of the largest known, spanning 186 kb on the chromosome. The coding DNA is divided into twenty-six exons, including several of almost 3 kb, and twenty-five introns, some of almost 32 kb. The open reading frame of the factor VIII cDNA codes for a 19 amino acid signal peptide and a 2332 amino acid mature factor VIII molecule synthesized as a single chain (Gitschier et al., 1984; Toole et al., 1984). Including carbo- hydrate, a molecular weight of 330,000 has been esti- mated.

Factor VIII is a pro-cofactor that is critical to the full expression of the enzymatic activity of factor IXa for activation of factor X. Its large size, its very low concentration in plasma, and its instability during purification have com- promised study of its biochemistry, but isolation of factor VIII cDNA has allowed prediction of its primary structure. Two internal repeat sequences characterize factor VIII. The A domain is repeated three times, the C domain twice: A A B A C C. Factor VIII shows marked sequence homology with factor V and ceruloplasmin, the copper- binding plasma protein (Church et al., 1984). The homology with factor V extends to the functions of these proteins in blood coagulation: each protein acts as a cofactor in the assembly of vitamin K-dependent proteins on membrane surfaces. Removal of much of the large B domain, repre- senting close to 40% of the factor VIII sequence, does not alter biologic activity (Toole et al., 1986). Unlike most of the blood clotting proteins, which are synthesized in the liver, factor VIII is synthesized in the liver, spleen, lymph nodes, pancreas, kidney, and muscle, as shown by the expression of factor VIII mRNA in these tissues (Wion et al., 1985).

Proteolytic conversion of factor VIII to the active cofactor, factor Villa, occurs by thrombin cleavage of the peptide bonds defined by residues 372-373 and 1686-1689 (Pittman and Kaufman, 1988). Factor Villa, bound to membrane surfaces, allows the assembly of the enzyme factor IXa onto the complex in the presence of calcium ions. Fac- tor Villa is inactivated by the action of activated protein C.

Factor V is a plasma cofactor (M, 330,000) that participates in complex with factor Xa to activate prothrombin to thrombin (Figure 4). This protein is synthesized and circulates in the blood as an inactive single-chain species. Thrombin activates factor V proteolytically to the active cofactor, factor Va, which contains a heavy and light chain derived from the amino terminus and carboxyl terminus, respectively. The heavy chain-light chain interaction is noncovalent and is Ca2+-dependent. A central carbohy- drate-rich domain is released as an activation peptide. Factor Va is inactivated by activated protein C. The full- length cDNA coding sequence indicates a 28 amino acid signal peptide and a mature protein of 2196 amino acids

(Kane and Davie, 1986; Kane et al., 1987; Jenny et al., 1987).

Fibrinogen and von Willebrand Factor The structural protein fibrinogen is unique among the blood clotting proteins in that each of its three subunits is encoded by a separate gene. These three genes are clustered on a 50 kb region of chromosome 4 (Kant et al., 1985): the a chain gene is 10 kb upstream of the y chain gene, which in turn is 13 kb upstream of the 8 chain gene. The a chain, y chain, and 8 chain genes contain nine, five, and eight exons, respectively. The significant homology among these genes suggests that they arose by triplica- tion of a common ancestral gene (Crabtee and Kant, 1981; Chung et al., 1983a, 1983b). The regulation of these genes is coordinated at the transcriptional level, and three separate mRNA species can be identified (Crabtree and Kant, 1982); however, the synchronous regulation of synthesis of these mRNAs is largely unexplained.

Fibrinogen represents about 2%-3% of the plasma protein. It is composed of two pairs of each chain: the Aa chain, the B8 chain, and the y chain. Removal of fibrinopeptide A from the Aa chain and removal of fibrinopeptide B from the B8 chain leads to the generation of fibrin mono- mer. Fibrin is a prototypic example of protein self-assembly: it rapidly polymerizes to form long structural strands that represent the fibrin clot. The clot is stabilized by the cross- linking action of factor Xllla, a transglutaminase.

The gene for van Wilebrand factor, an adhesive protein located in plasma and the blood vessel wall, is quite large, in excess of 175 kb (Collins et al., 1987). This gene includes more than forty-two exons, but final definition of the exon and intron structures is incomplete (Mancuso et al., 1987). The genomic clone includes a 25 kb sequence upstream of the vWF initiator methionine and a 5 kb sequence downstream of the vWF termination codon (Collins et al., 1987). A typical TATA box, required for transcription initiation of many genes, is approximately 30 bp upstream of the transcription start site.

VWF plays a critical role in the adhesion of platelets to the subendothelium during vascular injury. Synthesized in endothelial cells, vWF is packaged in the Weibel-Palade body prior to secretion (Wagner et al., 1982). vWF (M, 225,000) is synthesized as a single chain but dimerizes in the endoplasmic reticulum. The amino acid sequence of pro-vWF, derived from the cDNA sequence (Verweij et al., 1986; Ginsberg et al., 1985; Bonthron et al., 1986; Shel- ton-lnloes et al., 1986) predicts a signal peptide, a large, 741 amino acid propeptide, and a mature vWF protein of 2050 residues. Within the Pans Golgi and the Weibel- Palade body, vWF dimers multimerize into high molecular weight species that exhibit the highest biological activity. vWF circulates in the blood in a noncovalent complex with factor VIII.

Synthesis and Posttranslational Processing

Biosynthesis of the Vitamin K-Dependent Blood Clotting Proteins Six of the plasma proteins involved in blood coagulation require vitamin K for their complete synthesis. These

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Factor IX

Prothrombin

Factor x

Protein C

Factor VII

Protein S

Bone Cta protein

Ser Leu Vat His Ser Ctn

Leu Leu Leu Leu Gty Ctu

Thr Pro Ata Pro Leu Asp

Trp Lys Pro Cty Pro His

Vat Leu Pro Vat Leu Glu

Ser Gty Ala Gtu Ser Ser

Figure 5. Sequences of the Propeptide Domains of Vitamin K-Dependent Blood Coagulation Proteins

The size of the propeptide has been established for factor IX (Diuguid et al., 1986; Bentley et al., 1988) and protein C (Long et al., 1984; Foster et al., 1987). Homologous regions of other vitamin K-dependent proteins are aligned. Residues that demonstrate significant sequence homology are boxed and shaded; regions with conservative amino acid substitutions are boxed. These data are deduced from cDNA sequences (Kurachi and Davie, 1982; Choo et al., 1982; Degen et al., 1983; Fung et al., 1985; Hagen et al., 1986; Foster et al., 1985; Dahlback et al.,1986; Celeste et al.,1986). The protein S sequence is bovine; all others are human.

proteins-prothrombin, factors IX, X, and VII, protein C, and protein S-contain between ten and twelve residues of y-carboxyglutamic acid (Stenflo et al., 1974; Nelsestuen et al., 19i4) near their amino termini. They represent a unique class of calcium-binding proteins that assemble on membrane surfaces in the presence of calcium ions. These proteins are each synthesized in a precursor form; after translocation of the precursor to the rough endoplasmic reticulum, the glutamic acid residues in the prozy- mogen are selectively y-carboxylated. This reaction, catalyzed by a membrane-bound y-carboxylase, requires reduced vitamin K, molecular oxygen, Con, and the protein precursor substrate (Suttie, 1980).

Determination of the length of the propeptide of factor IX, the observation of incomplete carboxylation associated with a propeptide point mutation, and the marked sequence homology of this domain in y-carboxyglutamic acid-containing proteins (Diuguid et al., 1986; Pan and Price, 1985) (Figure 5) led to the proposal of a role for the propeptide in designating protein precursors for subse- quent y-carboxylation. Using site-specific mutagenesis, Jorgensen et al. (1987b) demonstrated that factor IX lacking the 18 amino acid propeptide was not carboxylated. Similarly, point mutations at position -16 (Phe-Ala) or -10 (Ala-Glu) eliminate y-carboxylation. These results indicate that the propeptide contains a recognition element, termed the y-CRS, which designates factor IX and the other vitamin K-dependent proteins for y-carboxylation. Foster et al. (1987) have demonstrated that the expression of deletion mutants of proprotein C lacking residues in the -17 to -12 portion of the propeptide is associated with impaired y-carboxylation. In a single known instance, the y-CRS is found not in a propeptide sequence but in the sequence of y-carboxylated bone matrix Gla protein (Price et al., 1987).

Through use of synthetic peptides as substrates for in vitro carboxylation with a partially purified enzyme prepa- ration (Soute et al., 1987) Ulrich et al. (1988) have shown that the vitamin K-dependent carboxylase itself, or a closely associated protein, has the ability to recognize the y-CRS. A 28 residue synthetic peptide based on residues -18 to +lO in prothrombin is efficiently carboxylated, with

a K, of 3 PM. In contrast, peptides lacking the y-CRS, the commonly used carboxylase pentapeptide substrate

FLEEL (K, 2200 PM) (Suttie and Hageman, 1976), a 20 residue peptide corresponding to residues -10 to +lO in prothrombin, and a 10 amino acid peptide analogous to residues +l to +lO in prothrombin are all poor substrates for the carboxylase. In vitro, recombinant protein C expressed in E. coli incorporated 14COz into y-carboxyglutamic acid only minimally better if the protein substrate also included a segment (residues -10 to -1) of the propeptide (Suttie et al., 1987). Given that the propeptide of protein C extends from -24 to -1 (Foster et al., 1987), these results and those of Ulrich et al. (1988) indicate the importance of the intact y-CRS for efficient carboxylation both in vivo and in vitro.

Recently, Knobloch and Suttie (1987) have shown that an 18 residue peptide based on the predicted structure of the propeptide of factor X stimulates 8-fold the carboxylation of the pentapeptide FLEEL by the partially purified carboxylase. These results raise the possibility that the propeptide plays a role as an allosteric regulator of the carboxylase in addition to its role as a recognition element. An unanswered question is whether the propeptide is suf- ficient to designate proteins for y-carboxylation.

P-Hydroxylation An unusual amino acid, erythro$-hydroxyaspartic acid, has been found in the amino-terminal EGF domains of protein C (Drakenberg et al., 1983) and factors IX, X, and VII. Recently, erythro$-hydroxyasparagine has been identified in protein S (Stenflo et al., 1987). These amino acids are formed by posttranslational hydroxylation of aspartic acid and asparagine. Their function remains unknown, although proposals have been made suggesting that they are involved in defining the metal-binding properties of these proteins.

Unlike posttranslational y-carboxylation, f%hydroxylation of factor IX is not directed by the propeptide, nor does this process require vitamin K or concomitant y-carboxylation. Synthesisof factor IX in the presence of vitamin K antago- nists such as sodium warfarin impairs y-carboxylation but not f3-hydroxylation (Rabiet et al., 1987). Moreover, factor IX mutants lacking the propeptide or containing point mutations that eliminate y-carboxylation are still substrates for 6-hydroxylation (Rabiet et al., 1987).

f3-Hydroxylation occurs in domains homologous to the


EGF precursor in certain vitamin K-dependent proteins (Stenflo et al., 1987) as well as in proteins outside of this family, including the complement proteins Clr and Cls, thrombomodulin, uromodulin, and the low density lipoprotein receptor (Stenflo et al., 1987, 1988; Przysiecki et al., 1987). A consensus sequence encompassing the P-hy- droxylated Asp and Asn residues within a number of EGF domains has been noted by Stenflo et al. (1987):

Cys-X-AspIAsn-X-X-X-X-PhelTyr-X-Cys-X-Cys.

EGF domains that lack the consensus sequence do not contain this posttranslational modification.

Multimerization of von Willebrand Factor vWF is initially synthesized as a large precursor consist- ing of a signal peptide, a large propeptide (M, 80,000), and the mature vWF subunit (M, 225,000). The major functional form of vWF is a large, multimeric, disulfide- bonded complex, and molecular weights of some species approach 107. vWF is synthesized and secreted by endothelial cells via two separate pathways: the constitutive pathway produces primarily dimeric vWF, which ha’s little platelet adherence activity; the regulated pathway releases vWF multimers from Weibel-Palade bodies. In a process that requires the propeptide, dimers of vWF multimerize in the tram Golgi or secretory granules. The pro-

peptide is cleaved, and the vWF and propeptide (known as VW Agll) circulate in the plasma (Wagner et al., 1987). If the propolypeptide is deleted by mutagenesis, the secreted vWF is unable to form multimers (Verweij et al., 1987). Interestingly, the propeptide can facilitate multimerization in trans; that is, multimerization activity can be res- cued if cDNAs coding for the propeptide and vWF are cotransfected into heterologous cells on separate plas- mids (Wise et al., 1988). These results support the original hypothesis of Wagner and Marder (1984) that the propeptide is required for dimer multimerization. However, the mechanism by which the propeptide participates in this posttranslational process is unknown.

Cleavage of Prosequences The propeptides in the vitamin K-dependent proteins and vWF are critical to posttranslational processing. After processing, these peptides are cleaved and the mature proteins circulate in the plasma. Two contiguous basic amino acids appear to the amino-terminal side of the site of propeptidase cleavage in most of the vitamin K-dependent proteins (Figure 5). Although the propeptidases have not been identified, their specificity for arginine suggests a serine protease in the trypsin family. An Arg+Ser mutation at -1 in a naturally occurring mutant, factor IX Cam- bridge, inhibits propeptide cleavage (Diuguid et al., 1986). The properties of a naturally occurring factor IX mutant, factor IX Oxford 3, suggest that the propeptidase substrate specificity is not limited to the basic residues at -1 and -2 but also includes residue -4 in the propeptide (Bentley et al., 1986), since an Arg-Gln substitution at this position precludes propeptide cleavage. Deletion mutations in protein C further emphasize the importance of

the carboxy-terminal four amino acids of the propeptide (Foster et al., 1987). The propeptidase responsible for cleavage of the propeptide in COS cells secreting vWF is highly specific for certain substrates. An Arg+Lys substitution at residue -1 in the propeptide of vWF precludes its cleavage, although this mutation does not inhibit multimerization (Wise et al., 1988).

Molecular Genetics of Hemophilia

Although a wide diversity of bleeding disorders associated with almost all phases of clot formation and its regulation are known, the structural basis for the defect has been identified in only a few instances. Genetic defects in the blood coagulation proteins are associated with a bleeding disorder known as hemophilia. Defects in factor VIII (hemophilia A) account for about 85% of this heredita.ry disorder, while defects in factor IX (hemophilia 9) are responsible for 100/o-12%. Other defects in coagulation proteins are rare, but deficiency states have been recognized for most of the blood clotting proteins. These “experiments of nature” allow appreciation of the physiologic import of each of these proteins. Furthermore, correlation of the structural defect in the gene and the protein contributes to our understanding of structure-function relationships in these proteins and to our knowledge of the basis of hereditary disease.

Hemophilia A is due to diminished or defective factor VIII molecules in the blood. Because of their considerable size and low plasma concentration, mutant factor VIII molecules have not been purified and have not been amena- ble to direct protein analysis. Rather, defective factor VIII

has been studied indirectly by analysis of factor VIII genomic DNA isolated from hemophilia A patients. By the nature of the analytical methods used, factor VIII genes characterized by gross structural changes, such as partial gene deletions, are more likely to be detected by the re- striction analyses employed.

As shown in Table 2, gene deletions from 2.5 to 80 kb have been described in eight mutant factor VIII genes leading to factor VIII deficiency and hemophilia A (Gits- chier et al., 1985; Antonarakis et al., 1985; Youssoufian et al., 1987). Exons encoding either the heavy chain (exons 6 and 7) or the light chain (exons 23-26) of the protein appear essential to the expression of normal factor VIII activity. In other families hemophilia has arisen as a result of nonsense mutations. The recognition sequence of the re- striction enzyme Taql includes the CpG dimer, a major site of methylation in mammalian DNA. CpG dimers are mutation “hotspots” in the factor VIII gene (Youssoufian et al., 1986). As noted by Gitschier et al. (1985), five of the seven Taql sites in exons of the factor VIII gene can be converted to nonsense mutations via C-to-T transitions and the intro- duction of in-frame stop codons. Mutations in Taql sites in exons 18, 24, and 26 have been associated with hemophilia (Table 2) (Gitschier et al., 1985; Antonarakis et al., 1985). A point mutation leading to the substitution of a glutamine for arginine 2307, encoded by exon 26, is responsible for a mild form of hemophilia A (Gitschier et al., 1986). The factor VIII molecule synthesized appears to be fully active, but its plasma level is significantly decreased.

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Table 2. Summary of the Molecular Defects Known to Cause Hemophika

Defect

Factor VIII (Hemophika A)

Size (4 Location Phenotype Reference

Deletion Deletion Deletion Deletion Deletion Deletion Deletion Deletion Nonsense mutation Nonsense mutation Nonsense mutation Nonsense mutation Nonsense mutation Point mutation

Factor IX (Hemophilia t3)

21.9 7 2.5 7

16 39 80

5.5

Exon 26 Severe Gitschier et al., 1985 Exon 6 Severe Youssoufian et al., 1987 Exon 14 Severe Youssoufian et al.. 1987 Exons 24, 25 Severe Youssoufian et al., 1987 Exons 23, 24 Severe Youssoufian et al., 1987 Exons 23, 24, 25 Severe Gitschier et al., 1985 Exons 7-22 Severe Antonarakis et al., 1985 Exon 22 Moderate Youssoufian et al., 1987 Exon 24 Severe Gitschier et al., 1985 Exon 26 Severe Gitschier et al., 1985 Exon 18 Severe Antonarakis et al., 1985 lntron 2 Severe Gitschier et al., 1985 lntron 25 Severe Gitschier et al., 1985 Glu 2307-Arg Moderate Gitschier et al., 1986

Deletion Deletion Deletion Deletion Point mutation Deletionlframeshift Point mutation Point mutation Point mutation Point mutation Point mutation

18 18

9 10

0.001

Exons I, Ii, III, IV-? Severe Giannelli et al., 1983 Exons I, II, Ill, IV-? Severe Giannelli et al., 1983 Exons Ill, IV-? Severe Giannelli et al., 1983 Exons V, VI Severe Chen et al., 1985 Splice 3’ of Exon VI Severe Rees et al., 1985 Exon V Severe Schach et al., 1987 Arg 145-His Moderate Noyes et al., 1983 Arg -I-Ser Severe Diuguid et al., 1986 Arg -4-Gln Severe Bentley et al., 1986 Arg -4-Gln Severe Ware et al., 1986 Asp 47-Gly Moderate Davis et al., 1987

Whether this is due to decreased synthesis or to acceler- ated clearance of an active but unstable factor VIII species is unknown.

Point mutations in the factor VIII introns are also known to cause hemophilia A. A mutated Taql site located in the intron between exon 2 and exon 3 of the factor VIII gene is associated with hemophilia A (Gitschier et al., 1985). This defect is not located near the intron-exon splice junc- tions, nor does it include any sequence resembling consensus splice donor or acceptor sites, Therefore the ac- tual cause of the defect in mRNA synthesis remains obscure. A new Taql site in an intron between exon 25 and exon 26 may be responsible for the factor VIII deficiency in another hemophiliac.

Hemophilia B is due to diminished or functionally defective factor IX. In about one-third of cases, the defective factor IX antigen circulates in the blood. Factor IX Chapel Hill (Arg 145-His) is the prototype for mutant zymogens that cannot be activated to their enzyme form because of mutation of the arginine adjacent to the sessile bond (Noyes et al., 1983). In factor IX Chapel Hill, this arginine is adjacent to the cleavage site between the activation peptide and the light chain of factor IXa. A parallel point mutation has been found in prothrombin Barcelona (Arg 273~Cys) (Rabiet et al., 1986). Factor IX Cambridge (Diuguid et al., 1986) factor IX Oxford 3 (Bentley et al., 1986) and factor IX San Dimas (Ware et al., 1986) contain point mutations in the propeptide domain of the factor IX precursor, profac- tor IX. In factor IX Cambridge (Arg -1 to Ser), the mutation prevents proteolytic removal of the propeptide by a pro-

peptidase. The presence of this point mutation in the propeptide partially inhibits y-carboxylation, thus leading to a protein that cannot bind to phospholipid vesicles or exhibit coagulant activity. Factor IX Oxford 3 (Arg -4-+Gln) (Bentley et al., 1986) and factor IX San Dimas (Arg -4*Gln) (Ware et al., 1986) have propeptide mutations that pre- clude propeptide cleavage. Factor IX San Dimas contains about 50% of the normal y-carboxyglutamic acids (Ware et al., 1986). Factor IX Alabama (Asp 47-Gly) lacks coagulant activity, probably because it fails to bind to cell membrane surfaces (Davis et al., 1987). Thus, there are now examples of factor IX mutants that interfere with posttranslational processing, factor IX-cell interaction, and factor IX zymogen activation. Examples of factor IX forms that lack enzyme activity or normal substrate binding properties are expected to be identified.

Alternatively, little or no factor IX antigen may circulate in the blood of some hemophiliacs. Hemophilia 6 can result from gross factor IX gene deletions (Table 2) (Gianelli et al., 1983; Chen et al., 1985). In the deletion described by Chen et al. (1985) the 5’ end of the gene is intact, and a truncated expression product of M, 36,000 is secreted but cleared from the blood into the urine (Bray and Thompson, 1986). In the abnormality described by Rees et al. (1985) a point mutation (G-T) is located at base 21,165 within the obligatory GT of a splice junction at the 3’site of exon VI. The change from GT to TT interferes with splicing, causing a fatal defect in transcription. In factor IX Seattle (Schach et al., 1987) an A at position 17,699 in the gene is deleted, which causes a frameshift leading to the

Revfew: Molecular Basis of Blood Coagulation 515

coding of a Val at residue 85 and a stop codon at position 86. As with hemophilia A, these examples emphasize the heterogeneity of hemophilia B. A single-base deletion and frameshift mutation, a point mutation in the splice junction, and partial or gross gene deletions are among the causes of severe hemophilia B.

Von Willebrand’s disease, the most common hereditary bleeding disorder, is likely caused by a variety of defects that effect a decrease in the activity of vWF. To date, molecular explanation for this disorder is limited to three cases of severe von Willebrand’s disease due to the deletion of the entire vWF gene (Shelton-lnloes et al., 1987).

Conclusion

Blood coagulation is a complex physiologic process that involves the initiation of blood clotting, the localization of the process to the area of vascular injury, and the formation of the fibrin clot and the platelet plug. The assembly of key components on cell membranes and the interaction of reactions in the solution phase with reactions on membrane surfaces are critical to the control of this process. Better understanding of the molecular basis of blood coagulation will continue to give insight into structure- function relationships of the components of this system, and the mechanisms by which these component reactions are regulated. Further study should add to our understanding of the molecular and cellular basis of bleeding and thrombotic disorders.

We are especially grateful to members of our laboratory, past and present, for their contributions to both the scientific effort and the conceptu- alization of our current thinking about blood coagulation. We also thank our colleagues for making manuscripts available prior to publi- cation, and Dr. Denisa Wagner and Dr. Richard Lawn for critical review of the manuscript. Our work was supported by grants (HL21543, HL18834, and HL38216) from the National Institutes of Health.

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The Molecular Basis Review of Blood Coagulation€¦ · The Molecular Basis of Blood Coagulation Review Bruce Furie and Barbara C. Furie Center for Hemostasis and Thrombosis Research