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Home , Fibrin, Kallikrein, Kininogen, Kringle domain, Platelet plug, Urokinase

Cell, Vol. 53, 505-518, May 20, 1988, Copyright 0 1988 by Cell Press The Molecular Basis Review of

Bruce Furie and Barbara C. Furie into the polymer. The clot, formed after tissue injury, Center for and Research is composed of activated and fibrin. The clot Division of Hematology/Oncology mechanically impedes the flow of blood from the injured Departments of Medicine and Biochemistry vessel and minimizes blood loss from the wound. Once a New England Medical Center stable clot has formed, ensues. The clot is and Tufts University School of Medicine gradually dissolved by of the fibrinolytic system. Boston, Massachusetts 02111 Blood coagulation may be initiated through either the in- trinsic pathway, where all of the components are present in blood, or the extrinsic pathway, where the cell- Overview membrane protein plays a critical role. Initia- tion of the intrinsic pathway of blood coagulation involves Blood coagulation is a host defense system that assists in the activation of factor XII to factor Xlla (see Figure lA), maintaining the integrity of the closed, high-pressure a reaction that is promoted by certain surfaces such as mammalian after blood vessel injury. glass or . Although is capable of factor After initiation of clotting, the sequential activation of cer- XII activation, the particular involved in factor XII tain plasma proenzymes to their forms proceeds activation physiologically is unknown. The collagen that through either the intrinsic or extrinsic pathway of blood becomes exposed in the subendothelium after vessel coagulation (Figure 1A) (Davie and Fiatnoff, 1964; Mac- damage may provide the negatively charged surface re- Farlane, 1964). These plasma , including quired for this reaction in vivo. Factor Xlla, with high mo- factor XII, factor XI, factor IX, , factor VII, and pro- lecular weight (HMWK) as , converts , are of serine . As a family, factor Xl to its activated form, factor Xla. In the presence these bear marked structural and functional ho- of Ca2+, factor Xla activates factor IX to factor IXa. Factor mology to the digestive proteases and chymotryp- IXa, in complex with factor Villa on membrane surfaces, sin. They are each converted from an inactive form to an catalyzes the activation of factor X to factor Xa. Factor Xa active enzyme by limited of one or two activates prothrombin to thrombin in the presence of bonds. However, most of the blood clotting enzymes are Ca2+ and factor Va bound to membrane surfaces. Throm- effective on a physiologic time scale only when assem- bin converts to fibrin by cleavage of two peptide bled in complexes on membrane surfaces with protein bonds, thereby releasing two small amino-terminal pep- cofactors such as factor VIII and . Calcium ions tides, fibrinopeptide A and fibrinopeptide B. have a critical role in that many of the component reac- Although the intrinsic pathway is clearly important in the tions in blood coagulation are either Ca2+-dependent or clotting of blood in vitro, its physiologic importance has require Caz+ for the interaction of proteins with mem- been challenged by the absence of problems in brane surfaces. patients deficient in factor XII, , and HMWK. Clot formation occurs when fibrinogen is converted by Therefore the intrinsic pathway is not likely to be of impor- thrombin to fibrin, the structural protein that assembles tance 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 WW Exons WW Function Prothrombin 72,000 21 llpll-q12 2.1 14 100 Protease 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 - 62,000 11 1 .a a 4 Protease zymogen 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 Cdl 506

Intrinsic Pathway

Figure 1. The Protems of Blood Coagulation (A) The blood coagulation cascade. 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 proteins, and fibrinolytic proteins. Abbreviations, in addition to those in (A): TPA, tissue ; UK, uro- kmase; PK, prekallikrein; T, trypsin; CT, : ATIII. Ill; HCII, 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 converts the factor Va and factor Villa to their inactive variable bleeding tendency. Although the precise mecha- forms by limited proteolysis. nisms for the initiation of blood coagulation in vivo remain Platelets are anucleate cells that circulate in the blood uncertain, it seems probable that the extrinsic pathway, in- in a resting form. Upon stimulation at the site of tissue in- cluding the bypass through factor IX, plays a dominant jury, platelets adhere to the injured surface. This interac- physiologic role. tion requires (vWF), a large, multi- Initiation of blood coagulation associated with vessel in- merit plasma protein that binds to a specific receptor, jury involves the expression of tissue factor activity on the glycoprotein lb, on the membrane. The activation surfaces of injured cells. Tissue factor initiates the extrin- of platelets by thrombin, ADP, AZ, or epi- sic pathway of blood coagulation by participating as a nephrine triggers characteristic morphological and bio- cofactor in the activation of factor VII to its active form, fac- chemical alterations in the platelet. Activated platelets se- tor Vlla (Figure 1A). The protease required for factor VII ac- crete two types of granules: a granules, which contain tivation in vivo is uncertain, but factor Xa can catalyze this thrombospondin, fibrinogen, vWF, factor V, and other pro- reaction. The factor Vlla-tissue factor complex activates teins involved in hemostasis; and dense granules, which factor X and factor IX in the presence of Ca2+. Factor Xa are rich in calcium ions and ADP. The expression of the activates prothrombin to thrombin, and thrombin converts glycoprotein Ilb-llla complex as a receptor for certain fibrinogen to fibrin. plasma proteins (e.g., fibrinogen) may be important for the Factor VIII and factor V, cofactors in intermediate steps formation of the and fibrin clot. in the cascade, may function in regulating the rate of acti- Endothelial cells tile the walls of the blood vessels. vation of blood coagulation. This effect is mediated by pro- These cells are unique in that, in their resting state, they tein C and protein S, plasma anticoa‘gulants. Protein C is function to inhibit the activation of blood coagulation or converted to its active form on endothelial cell mem- the stimulation of platelet adherence. The synthesis of branes by a complex of two proteins, thrombomodulin and , the expression of heparin-like substances on thrombin. Activated protein C, anchored to cell mem- the membrane surface, and the presence of protein com- branes through binding to membrane-bound protein S, plexes (e.g., thrombomodulin-thrombin) that lead to the Review: Molecular Basis of Blood Coagulation 507

PT FIX FX FVII PC FXI Figure 2. Stuctural Domains of the Proteins involved in Hemostasis FXII and of Related Proteins Domains, identified in the key, are described in the text. Sites of proteo- PS lytic cleavage associated with synthesis of the mature protein are indi- cated by thin arrows. Sites of proteolytic cleavage associated with tPA zymogen activation are indicated by the thick arrows. Shown, top to bottom, are schematic structures for prothrombin (Degen et al., 1983) PUK 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 MGP 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 BGP S (Lundwall et al., 1986) tissue-type plasminogen activator (Ny et al., 1984). prourokinase (Holmes et al., 1985) matrix Gla protein (Price Pm and Williamson, 1985) bone Gla protein (Pan and Price, 1985) plas- T minogen (Malinowski et al., 1984), and trypsin.

generation of such as activated protein C Some of the proteins in each of these groups are are processes that prevent clot formation in normal blood characterized structurally as zymogens of serine pro- vessels. After tissue injury, thrombogenic subendothelial teases. Other proteins contain y-carboxyglutamic acid, components of the blood vessel are exposed. Receptors synthesized in a vitamin K-dependent reaction, and pos- for blood clotting proteins may be expressed on the en- sess unique calcium-dependent membrane-binding prop- dothelial , allowing for the sequential acti- erties. As mentioned above, there are a number of other vation of the blood coagulation cascade. cellular components essential to hemostasis, including During wound healing, the fibrin clot in the vessel is vWF on platelets and endothelial cells, receptor proteins degraded by , a generated from on platelets (e.g., glycoproteins la, lb, Ilb-llla, and IV), tis- the plasma zymogen, plasminogen. This reaction is cata- sue factor on cell membranes, regulatory membrane pro- lyzed by several plasminogen activators, including two teins such as thrombomodulin, and tissue plasminogen serine proteases, tissue plasminogen activator and uro- activator secreted from cells. The scope of this review is . Their activity is in turn regulated by two plasma limited to the proteins of blood coagulation and the protease inhibitors, plasminogen activator inhibitor and plasma proteins involved in the regulation of this process. a2-antiplasmin. Common Domain Structures Structural Features of the Blood Clotting Proteins The structure and organization of the genes coding for the and Their Encoding Genes blood coagulation proteins emphasize that the evolution The plasma proteins involved in hemostasis can be sepa- of new protein function occurs via gene duplication, gene rated into three functional groups with interrelated but dis- modification, and exon shuffling (Gilbert, 1978; Patthy, tinct physiologic roles (Figure 1B and Table 1). One group 1985). Each of the exons may be considered a module consists of the blood coagulation proteins, which promote coding for a homologous domain in each protein (Figures clot formation; this group includes cofactors (e.g., factor 2 and 3). The three-dimensional structures of the polypep- VIII and factor V), structural proteins (e.g., fibrinogen), and tide backbones of these homologous domains are likely proenzymes (e.g., factor Xl, prothrombin). A second group to be nearly identical, but substitution of side consists of the regulatory proteins that modulate and chains on the protein surfaces gives definition to unique localize clot formation to regions of tissue injury; this cate- properties of substrate recognition, cofactor binding, or gory includes the plasma protease inhibitors (e.g., an- membrane interaction (Furie et al., 1982). The blood tithrombin Ill) and specific enzyines that inactivate pro- coagulation proteins remain a prime example of the devel- coagulant components (e.g., activated protein C). A third opment of a family of proteins with diverse functional prop- group comprises the fibrinolytic proteins, which dissolve erties but common, unified structural elements. fibrin clots as a late component of the healing process; Like trypsin, the enzymes involved in blood coagulation these components include enzymes (e.g., tissue plas- are ordinarily maintained in their zymogen, or proenzyme minogen activator) and protease inhibitors (e.g., as-anti- forms. Each is converted to an active enzyme by proteoly- plasmin). sis of one or two peptide bonds. A common feature of Cell 508

Factor VII

Factor IX

Factor X

Protein C

Prothrombin CATALYTIC 0

REPEAT El Factor XI TYPE I ii

FIBRONECTIN TYPE ,I Factor XII II

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 ac- Many of the proteins involved in hemostasis contain one tive site of the “”: a serine, histidine, and or more regions, known as EGF domains, that are homolo- aspartic acid critical to the mechanism of catalysis by gous with a module in the epidermal growth factor (EGF) these enzymes. The plasma blood clotting serine pro- precursor. This module, a 53 amino acid peptide contain- teases are about twice the size of trypsin. The domain in ing three bonds, binds to specific cell-surface the blood clotting enzymes showing marked sequence receptors. The receptor-binding region of EGF modules homology and presumed three-dimensional structural ho- has been localized to residues 19 through 31, and residues mology (Furie et al., 1982) with trypsin is known as the 13-19 and 31-33 show the highest sequence conserva- catalytic domain. The blood clotting serine proteases have tion (Appella et al., 1987). The EGF domains may be an and internal core nearly identical to those of responsible for the high-affinity interaction of proteins with trypsin, but different molecular surfaces surrounding the specific cell surfaces or, alternatively, with receptor do- enzyme active sites are likely responsible for extended mains on other proteins, such as factors VIII and V. substrate binding sites and the unique substrate specifici- The , another structure common to sev- ties of these enzymes. eral proteins involved in hemostasis, is about 100 amino The amino termini of the vitamin K-dependent blood acids long and contains three disulfide bonds. Its three- clotting proteins (Figure 2) contain between ten and twelve dimensional structure resembles an eccentric oblate ellip- v-carboxyglutamic acid (Gla) residues within the first forty- soid, and its folding is defined by close contacts between two amino acids. Two, or possibly three, of these amino the sulfur atoms of two of the disulfide bridges, which form acids bind a single calcium ion (Furie et al., 1979), thus a sulfur cluster in the center of the “kringle” (Park and forming a noncovalent intramolecular bridge between Tulinsky, 1986). Internal amino acids are well conserved regions of the polypeptide backbone. This bond stabilizes among various proteins containing kringles; again, differ- the tertiary structure of this region of the protein (Tai et al., ences in the molecular surfaces probably distinguish the 1984). Upon binding by calcium, the proteins undergo two functional properties of these proteins. The function of conformational changes that expose a membrane-binding these domains remain uncertain, but, as a structural unit, site (Nelsestuen, 1976; Prendergast and Mann, 1977; the kringle likely contains recognition elements important Borowski et al., 1986; Liebman et al., 1987). Although the for macromolecular assembly. G/a domain is clearly required for these conformational A short linking segment observed in numerous blood transitions, it remains uncertain whether some of the clotting proteins, termed the aromatic amino acid stack, y-carboxyglutamic acids directly bind to membrane sur- contains the sequence Phe-Trp-X-X-Tyr. Although hydro- faces through calcium ion bridges (Mann et al., 1982) or phobic, this region is oriented toward the surface in pro- whether metal binding to y-carboxyglutamic acid in the thrombin fragment 1 (Park and Tulinsky, 1986) and may Gla domain alters the structure of adjacent domains, ex- play a role in membrane binding. posing a membrane-binding surface (Borowski et al., Type I fibronectin homology regions, containing two in- 1986). ternal disulfide bonds, are about 50 residues long and are Review: Molecular Basis of Blood Coagulation 509

known as finger domains (Petersen et al., 1983). They may play a role in fibrin binding. The type II fibronectin homol- ogy regions are approximately 60 residues long and also contain two internal disulfide bonds (Petersen et al., 1983). This region in fibronectin contains the collagen , 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 ho- mology. The human (Friezner Degen and Davie, 1987) and bovine (Irwin et al., 1985) prothrombin genes, for ex- ample, demonstrate partial homology with genes encod- ing other vitamin K-dependent proteins (Figure 3). The prothrombin gene contains fourteen exons: exon I en- codes the ; exon II encodes the propeptide, Figure 4. Model for a Macromolecular Complex Associated with Zymogen Activation including the y-carboxylation recognition site (@ZRS; see A central feature of the proteins involved in blood coagulation is their below) (Jorgensen et al., 1987a; Foster et al., 1987), and assembly on membrane surfaces. The interaction of calcium ions (cir- the Gla domain; exon III codes the aromatic amino acid cles) with the ~carboxyglutamic acids (“v” structures) on the vitamin stack domain. Exons I, II, and Ill are homologous in struc- K-dependent proteins leads to the exposure of membrane-binding ture and organization to corresponding exons in the factor sites (drawn in black) on these proteins. The assembly of these pro- IX gene family, which includes factor IX, factor X, factor teins on membranes in a geometry organized by the protein cofactor facilitates enzyme-substrate interaction. The protein cofactor likely VII, and protein C. Exons IV-VII code for the two kringle plays an important regulatory role in this complex. Factor Xa, enzyme regions; these domains have no parallel with structures in (L, light chain; H, heavy chain); prothrombin, substrate; factor Va, the factor IX family, but are observed in other proteins in- cofactor. volved in hemostasis, including tissue plasminogen acti- vator, plasminogen, , and factor XII (Figure 2). FactorX (Leytus et al., 1986) factor IX (Yoshitake et al., Exons VIII and IX code for the sites of proteolytic cleavage 1986), factor VII (O’Hara et al., 1987) and protein C (Foster by factor Xa, which converts prothrombin to thrombin. The et al., 1985) show marked structural homology in both catalytic thrombin domain, responsible for enzyme cataly- gene structure and gene organization. The genes encod- sis and substrate specificity, is encoded by exons X-XIV. ing these proteins contain eight exons and seven introns The organization of this portion of the gene differs from (Figure 3). Exon I codes for the signal peptide domain. that coding for the analogous domain in the factor IX gene Exon II codes for the propeptide domain and the Gla do- family despite the marked structural homology of the cata- main: the coding of the propeptide, containing the +RS lytic domains in the proteins. In prothrombin, the amino (Jorgensen et al., 1987b; Foster et al., 1987), and the Gla acids that form the catalytic triad of serine proteases are domain on the same exon emphasizes the functional in- each on a separate exon- histidine on exon X, aspartic terplay of these two regions. The small exon III codes for acid on exon XI, and serine on exon XIII. In the factor IX the aromatic amino acid stack. Exon IV and exon V each gene family the entire catalytic domain is encoded by two code for an EGF domain. In each case the activation of exons, not five. This may suggest that the factor IX gene the proenzyme to an enzyme requires proteolytic cleav- and related genes were derived ancestrally from a precur- age, and the cleavage site and the surface features that sor of the prothrombin gene, but that reorganization of define the substrate specificity for this reaction are en- these genes by intron deletion led to consolidation of the coded in exon VI. The catalytic domain is encoded by exon exons encoding the serine protease domain. The introns VII and exon VIII. The components of the catalytic triad are in the prothrombin gene range in size from 84 to 9447 dispersed between these exons: histidine is encoded by base pairs (Friezner Degan and Davie, 1987), and 40% of exon VII, aspartic acid and serine by exon VIII. Intervening the prothrombin gene is composed of repetitive DNA of sequences occur in similar or identical positions in all of the Alu and Kpn family of repeats. these genes. However, these introns demonstrate no ho- Prothrombin (M, 72,000) assembles with factor Xa and mology in length or in sequence. In the case of factor VII, factor Va on the membrane surface to form a catalytic unit two alternative genetic organizations have been identi- known as the prothrombinase complex (Mann et al., fied. One form of preprofactor VII arises from a gene anal- 1982). In the presence of calcium ions, prothrombin is ogous in organization to the factor X, factor IX, and protein converted to thrombin by the cleavage of two peptide C genes. The second form arises from a gene containing bonds and removal of the Gladomain, the aromatic amino a ninth exon, exon IA. This exon codes for a polypeptide acid stack domain, and the kringle domains. Thrombin is extension of the amino terminus that elongates the released from the membrane phase into solution. The for- prepropeptide from 38 to 60 residues. The significance of mation of the activating complex on membrane surfaces these two preprofactor VII forms is unknown. is typical of many of the blood clotting reactions, and a Factor IX is a single-chain plasma zymogen (M, 56,000) possible model for this complex is shown in Figure 4. that is converted to its enzyme form, factor IXa, by the Cell 510

cleavage of two peptide bonds and the release of an acti- al., 1987). The four”repeat”domains in the amino-terminal vation peptide. Twelve y-carboxyglutamic acid residues region of the protein are likely responsible for macro- are located near the amino terminus of the mature zymo- molecular assembly. The protein shows marked se- gen. A single f3-hydroxyaspartic acid at position 64 in the quence homology with prekallikrein, and both factor Xl EGF domain has uncertain function. Factor X (M, 56,000) and prekallikrein circulate in plasma as complexes with is synthesized as a single chain (Fung et al., 1985; Leytus HMWK. Accordingly, the tandem repeats in both proteins et al., 1984) but is converted to a two-chain zymogen that may be responsible for HMWK interaction. circulates in the plasma. The light chain of human factor X The factor XII gene contains fourteen exons and thir- includes eleven y-carboxyglutamic acid residues and the teen introns (Figure 3) (Cool and MacGillivray, 1987). Exon EGF domain, containing a single f3-hydroxyaspartic acid. I encodes the signal peptide. Exon II encodes a region The activation peptide is cleaved from the heavy chain with no known structural homologies. Exons Ill and IV en- during activation of factor X by the factor IXa-factor Villa code regions homologous to the type II fibronectin struc- complex or the factor Vlla-tissue factor complex to yield ture. Exon V and exon VII each encode EGF domains, the enzyme factor Xa. The remaining portion of the heavy exon VI encodes a region homologous to the fibrin finger chain contains the catalytic region homologous to trypsin. domain of fibronectin, and exons VIII and IX code for the Factor VII, a single-chain zymogen (M, 56,000) that is a two kringle domains. The catalytic domain is encoded by component of the extrinsic pathway of blood coagulation, exons XI-XIV. The organization of these exons, like those differs from other blood coagulation proenzymes in that encoding the catalytic domain of factor XI, parallels the the zymogen expresses significant enzyme activity. The structure of the exons encoding the urokinase and tissue generation of factor Xa can lead to activation of factor VII plasminogen activator catalytic domains. The factor XII to factor Vlla, a two-chain enzyme that, in complex with gene lacks the consensus TATA and CAAT sequences im- tissue factor, activates factor X. Factor VII contains ten mediately upstream of the transcription initiation site, sug- y-carboxyglutamic acid residues, and two EGF domains gesting alternative sites or alternative structures for the containing a single 5-hydroxyaspartic acid. promoters of this gene. Protein C is a vitamin K-dependent regulatory protein Factor XII, the first component of the intrinsic pathway, (M, 56,000) containing nine y-carboxyglutamic acid resi- is converted from a single-chain zymogen (M, 80,000) dues and a single 6-hydroxyaspartic acid residue. The ac- into the enzyme factor Xlla, a two-chain disulfide-linked tivation of the zymogen to the enzyme form is catalyzed enzyme. This reaction can be accomplished by kallikrein, by the thrombin-thrombomodulin complex on endothelial but, as noted in the Overview, the relevant physiologic ac- cells (Esmon et al., 1982). Thrombomodulin is an integral tivator has not been identified with certainty. Factor Xlla membrane protein whose primary structure has recently is capable of activating the fibrinolytic system, generating been determined (Jackman et al., 1986; Wen et al., 1987). and initiating blood coagulation through the activa- Activated protein C destroys both factor Villa and factor tion of factor Xl. A region homologous to the type II region Va, in each case by cleavage of the polypeptide chain. In of fibronectin may be responsible for factor XII binding to this manner, factor Villa and factor Va activities are turned anionic surfaces. off, and the activity of the coagulation pathway is dampened. It is interesting to note that structurally homologous pro- Protein Cofactors teins have been adapted to serve both coagulant (e.g., fac- The majority of the serine proteases of blood coagulation tor IX, factor X) and anticoagulant (e.g., protein C) func- require protein cofactors for efficient proteolytic activity. tions. Tissue factor is a transmembrane protein (M, 37,000) that The factor XI gene contains fifteen exons and fourteen activates blood coagulation through the extrinsic pathway intervening sequences (Asakai et al., 1987). Of these by forming a complex with factor Vlla that activates factor exons, fourteen code for translated regions of the mRNA. X. This protein is not present in plasma, but rather is a Exon II encodes an 18 residue signal sequence. There is component of many cell surfaces with the exception of no evidence for a propeptide (Fujikawa et al., 1986). Four resting endothelial cells and monocytes. The amino acid “repeat” sequences are each encoded by two exons: re- sequence, predicted from isolated cDNA (Spicer et al., peat 1 by exons III and IV, repeat 2 by exons V and VI, re- 1987; Morrissey et al., 1987) includes a 32 residue signal peat 3 by exons VII and VIII, and repeat 4 by exons IX and peptide, an extracellular domain of 219 amino acids, a X. The catalytic domain is encoded in exons XII-XV. The short membrane-spanning domain, and a cytoplasmic do- organization of the exons coding for the catalytic domain main. No propeptide is encoded. A key event in the initia- is similar to that of corresponding regions of the genes for tion of blood coagulation involves the expression of tissue urokinase and tissue plasminogen activator. factor activity on the cell surface to form a complex with The factor Xl (M, 160,000) zymogen contains two identi- plasma factor VII. cal chains that are disulfide-linked. Upon activation by fac- Protein S is a vitamin K-dependent protein (M, 80,000) tor Xlla in a reaction stimulated by HMWK, two peptide that functions as a plasma anticoagulant. This protein bonds are cleaved to generate two- heavy chain-light facilitates the action of activated protein C on factor Villa chain homodimers linked through the heavy chain by a di- and factor Va. Protein S is a membrane-binding protein, sulfide bond. The catalytic domain is located on the light interacting with cell membranes through a mechanism re- chain, and the heavy chain contains the HMWK binding quiring y-carboxyglutamic acid. Protein S does not con- site and the recognition site for interaction of factor Xla tain a trypsin-like catalytic domain, but rather has a with its substrate, factor IX (Sinha et al., 1987; Liebman et 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 (Kane and Davie, 1986; Kane et al., 1987; Jenny et al., S is synthesized as a single chain containing a signal pep- 1987). tide, a propeptide, a Gla domain, four EGF domains con- taining 8-hydroxyaspartic acid and 8-hydroxyasparagine, Fibrinogen and von Willebrand Factor and an additional large domain likely responsible for rec- The structural protein fibrinogen is unique among the ognition of activated protein C (Lundwall et al., 1986; blood clotting proteins in that each of its three subunits is Hoskins et al., 1987). Protein S exists in two forms in the encoded by a separate gene. These three genes are blood: as a species bound to C4b-binding binding protein clustered on a 50 kb region of (Kant et al., and as a free form. 1985): the a chain gene is 10 kb upstream of the y chain The gene encoding facror V//l is one of the largest gene, which in turn is 13 kb upstream of the 8 chain gene. known, spanning 186 kb on the chromosome. The coding The a chain, y chain, and 8 chain genes contain nine, five, DNA is divided into twenty-six exons, including several of and eight exons, respectively. The significant homology almost 3 kb, and twenty-five introns, some of almost 32 kb. among these genes suggests that they arose by triplica- The open reading frame of the factor VIII cDNA codes for tion of a common ancestral gene (Crabtee and Kant, 1981; a 19 amino acid signal peptide and a 2332 amino acid ma- Chung et al., 1983a, 1983b). The regulation of these ture factor VIII molecule synthesized as a single chain genes is coordinated at the transcriptional level, and three (Gitschier et al., 1984; Toole et al., 1984). Including carbo- separate mRNA species can be identified (Crabtree and hydrate, a molecular weight of 330,000 has been esti- Kant, 1982); however, the synchronous regulation of syn- mated. thesis of these mRNAs is largely unexplained. Factor VIII is a pro-cofactor that is critical to the full ex- Fibrinogen represents about 2%-3% of the plasma pro- pression of the enzymatic activity of factor IXa for activa- tein. It is composed of two pairs of each chain: the Aa tion of factor X. Its large size, its very low concentration in chain, the B8 chain, and the y chain. Removal of fibrino- plasma, and its instability during purification have com- peptide A from the Aa chain and removal of fibrinopeptide promised study of its biochemistry, but isolation of factor B from the B8 chain leads to the generation of fibrin mono- VIII cDNA has allowed prediction of its primary structure. mer. Fibrin is a prototypic example of protein self-assembly: Two internal repeat sequences characterize factor VIII. it rapidly polymerizes to form long structural strands that The A domain is repeated three times, the C domain represent the fibrin clot. The clot is stabilized by the cross- twice: A A B A C C. Factor VIII shows marked sequence linking action of factor Xllla, a transglutaminase. homology with factor V and , the copper- The gene for van Wilebrand factor, an adhesive protein binding plasma protein (Church et al., 1984). The homol- located in plasma and the blood vessel wall, is quite large, ogy with factor V extends to the functions of these proteins in excess of 175 kb (Collins et al., 1987). This gene in- in blood coagulation: each protein acts as a cofactor in the cludes more than forty-two exons, but final definition of the assembly of vitamin K-dependent proteins on membrane exon and intron structures is incomplete (Mancuso et al., surfaces. Removal of much of the large B domain, repre- 1987). The genomic clone includes a 25 kb sequence up- senting close to 40% of the factor VIII sequence, does not stream of the vWF initiator and a 5 kb sequence alter biologic activity (Toole et al., 1986). Unlike most of the downstream of the vWF termination codon (Collins et al., blood clotting proteins, which are synthesized in the liver, 1987). A typical TATA box, required for transcription initia- factor VIII is synthesized in the liver, spleen, lymph nodes, tion of many genes, is approximately 30 bp upstream of , , and muscle, as shown by the expres- the transcription start site. sion of factor VIII mRNA in these tissues (Wion et al., VWF plays a critical role in the adhesion of platelets to 1985). the subendothelium during vascular injury. Synthesized Proteolytic conversion of factor VIII to the active cofac- in endothelial cells, vWF is packaged in the Weibel-Palade tor, factor Villa, occurs by thrombin cleavage of the pep- body prior to secretion (Wagner et al., 1982). vWF (M, tide bonds defined by residues 372-373 and 1686-1689 225,000) is synthesized as a single chain but dimerizes in (Pittman and Kaufman, 1988). Factor Villa, bound to mem- the . The amino acid sequence of brane surfaces, allows the assembly of the enzyme factor pro-vWF, derived from the cDNA sequence (Verweij et al., IXa onto the complex in the presence of calcium ions. Fac- 1986; Ginsberg et al., 1985; Bonthron et al., 1986; Shel- tor Villa is inactivated by the action of activated protein C. ton-lnloes et al., 1986) predicts a signal peptide, a large, Factor V is a plasma cofactor (M, 330,000) that partici- 741 amino acid propeptide, and a mature vWF protein of pates in complex with factor Xa to activate prothrombin to 2050 residues. Within the Pans Golgi and the Weibel- thrombin (Figure 4). This protein is synthesized and circu- Palade body, vWF dimers multimerize into high molecular lates in the blood as an inactive single-chain species. weight species that exhibit the highest biological activity. Thrombin activates factor V proteolytically to the active vWF circulates in the blood in a noncovalent complex with cofactor, factor Va, which contains a heavy and light chain factor VIII. derived from the amino terminus and carboxyl terminus, respectively. The heavy chain-light chain interaction is Synthesis and Posttranslational Processing noncovalent and is Ca2+-dependent. A central carbohy- drate-rich domain is released as an activation peptide. Biosynthesis of the Vitamin K-Dependent Factor Va is inactivated by activated protein C. The full- Blood Clotting Proteins length cDNA coding sequence indicates a 28 amino acid Six of the plasma proteins involved in blood coagulation signal peptide and a mature protein of 2196 amino acids require vitamin K for their complete synthesis. These Cell 512

Factor IX Prothrombin Ser Leu Vat His Ser Ctn Factor x Leu Leu Leu Leu Gty Ctu Protein C Thr Pro Ata Pro Leu Asp Factor VII Trp Lys Pro Cty Pro His Protein S Vat Leu Pro Vat Leu Glu Bone Cta protein 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 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, FLEEL (K, 2200 PM) (Suttie and Hageman, 1976), a 20 and protein S-contain between ten and twelve residues residue peptide corresponding to residues -10 to +lO in of y-carboxyglutamic acid (Stenflo et al., 1974; Nelsestuen prothrombin, and a 10 amino acid peptide analogous to et al., 19i4) near their amino termini. They represent a residues +l to +lO in prothrombin are all poor substrates unique class of calcium-binding proteins that assemble for the carboxylase. In vitro, recombinant protein C ex- on membrane surfaces in the presence of calcium ions. pressed in E. coli incorporated 14COz into y-carboxyglu- These proteins are each synthesized in a precursor form; tamic acid only minimally better if the protein substrate after translocation of the precursor to the rough endoplas- also included a segment (residues -10 to -1) of the pro- mic reticulum, the glutamic acid residues in the prozy- peptide (Suttie et al., 1987). Given that the propeptide of mogen are selectively y-carboxylated. This reaction, cata- protein C extends from -24 to -1 (Foster et al., 1987), lyzed by a membrane-bound y-carboxylase, requires these results and those of Ulrich et al. (1988) indicate the reduced vitamin K, molecular oxygen, Con, and the pro- importance of the intact y-CRS for efficient carboxylation tein precursor substrate (Suttie, 1980). both in vivo and in vitro. Determination of the length of the propeptide of factor Recently, Knobloch and Suttie (1987) have shown that IX, the observation of incomplete carboxylation associ- an 18 residue peptide based on the predicted structure of ated with a propeptide point , and the marked se- the propeptide of factor X stimulates 8-fold the carboxyla- quence homology of this domain in y-carboxyglutamic tion of the pentapeptide FLEEL by the partially purified acid-containing proteins (Diuguid et al., 1986; Pan and carboxylase. These results raise the possibility that the Price, 1985) (Figure 5) led to the proposal of a role for the propeptide plays a role as an allosteric regulator of the car- propeptide in designating protein precursors for subse- boxylase in addition to its role as a recognition element. quent y-carboxylation. Using site-specific mutagenesis, An unanswered question is whether the propeptide is suf- Jorgensen et al. (1987b) demonstrated that factor IX lack- ficient to designate proteins for y-carboxylation. ing the 18 amino acid propeptide was not carboxylated. Similarly, point at position -16 (Phe-Ala) or P-Hydroxylation -10 (Ala-Glu) eliminate y-carboxylation. These results An unusual amino acid, erythro$-hydroxyaspartic acid, indicate that the propeptide contains a recognition ele- has been found in the amino-terminal EGF domains of ment, termed the y-CRS, which designates factor IX and protein C (Drakenberg et al., 1983) and factors IX, X, and the other vitamin K-dependent proteins for y-carboxyl- VII. Recently, erythro$-hydroxyasparagine has been iden- ation. Foster et al. (1987) have demonstrated that the tified in protein S (Stenflo et al., 1987). These amino acids expression of deletion mutants of proprotein C lacking are formed by posttranslational hydroxylation of aspartic residues in the -17 to -12 portion of the propeptide is as- acid and asparagine. Their function remains unknown, al- sociated with impaired y-carboxylation. In a single known though proposals have been made suggesting that they instance, the y-CRS is found not in a propeptide sequence are involved in defining the metal-binding properties of but in the sequence of y-carboxylated bone matrix Gla these proteins. protein (Price et al., 1987). Unlike posttranslational y-carboxylation, f%hydroxylation Through use of synthetic as substrates for in of factor IX is not directed by the propeptide, nor does this vitro carboxylation with a partially purified enzyme prepa- process require vitamin K or concomitant y-carboxylation. ration (Soute et al., 1987) Ulrich et al. (1988) have shown Synthesisof factor IX in the presence of vitamin K antago- that the vitamin K-dependent carboxylase itself, or a nists such as sodium impairs y-carboxylation but closely associated protein, has the ability to recognize the not f3-hydroxylation (Rabiet et al., 1987). Moreover, factor y-CRS. A 28 residue synthetic peptide based on residues IX mutants lacking the propeptide or containing point mu- -18 to +lO in prothrombin is efficiently carboxylated, with tations that eliminate y-carboxylation are still substrates a K, of 3 PM. In contrast, peptides lacking the y-CRS, the for 6-hydroxylation (Rabiet et al., 1987). commonly used carboxylase pentapeptide substrate f3-Hydroxylation occurs in domains homologous to the Review: Molecular Basis of Blood Coagulation 513

EGF precursor in certain vitamin K-dependent proteins the carboxy-terminal four amino acids of the propeptide (Stenflo et al., 1987) as well as in proteins outside of this (Foster et al., 1987). The propeptidase responsible for family, including the complement proteins Clr and Cls, cleavage of the propeptide in COS cells secreting vWF is thrombomodulin, uromodulin, and the low density lipopro- highly specific for certain substrates. An Arg+Lys substi- tein receptor (Stenflo et al., 1987, 1988; Przysiecki et al., tution at residue -1 in the propeptide of vWF precludes 1987). A consensus sequence encompassing the P-hy- its cleavage, although this mutation does not inhibit mul- droxylated Asp and Asn residues within a number of EGF timerization (Wise et al., 1988). domains has been noted by Stenflo et al. (1987): Molecular Genetics of Hemophilia Cys-X-AspIAsn-X-X-X-X-PhelTyr-X-Cys-X-Cys. Although a wide diversity of bleeding disorders as- EGF domains that lack the consensus sequence do not sociated with almost all phases of clot formation and its contain this posttranslational modification. 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 Multimerization of von Willebrand Factor with a bleeding disorder known as hemophilia. Defects in vWF is initially synthesized as a large precursor consist- factor VIII (hemophilia A) account for about 85% of this ing of a signal peptide, a large propeptide (M, 80,000), heredita.ry disorder, while defects in factor IX (hemophilia and the mature vWF subunit (M, 225,000). The major 9) are responsible for 100/o-12%. Other defects in coagu- functional form of vWF is a large, multimeric, disulfide- lation proteins are rare, but deficiency states have been bonded complex, and molecular weights of some species recognized for most of the blood clotting proteins. These approach 107. vWF is synthesized and secreted by en- “experiments of nature” allow appreciation of the physio- dothelial cells via two separate pathways: the constitutive logic import of each of these proteins. Furthermore, corre- pathway produces primarily dimeric vWF, which ha’s lit- lation of the structural defect in the gene and the protein tle platelet adherence activity; the regulated pathway contributes to our understanding of structure-function releases vWF multimers from Weibel-Palade bodies. In a relationships in these proteins and to our knowledge of the process that requires the propeptide, dimers of vWF mul- basis of hereditary disease. timerize in the tram Golgi or secretory granules. The pro- Hemophilia A is due to diminished or defective factor peptide is cleaved, and the vWF and propeptide (known VIII molecules in the blood. Because of their considerable as VW Agll) circulate in the plasma (Wagner et al., 1987). size and low plasma concentration, mutant factor VIII mol- If the propolypeptide is deleted by mutagenesis, the se- ecules have not been purified and have not been amena- creted vWF is unable to form multimers (Verweij et al., ble to direct protein analysis. Rather, defective factor VIII 1987). Interestingly, the propeptide can facilitate multimer- has been studied indirectly by analysis of factor VIII ization in trans; that is, multimerization activity can be res- genomic DNA isolated from hemophilia A patients. By the cued if cDNAs coding for the propeptide and vWF are nature of the analytical methods used, factor VIII genes cotransfected into heterologous cells on separate plas- characterized by gross structural changes, such as partial mids (Wise et al., 1988). These results support the original gene deletions, are more likely to be detected by the re- hypothesis of Wagner and Marder (1984) that the propep- striction analyses employed. tide is required for dimer multimerization. However, the As shown in Table 2, gene deletions from 2.5 to 80 kb mechanism by which the propeptide participates in this have been described in eight mutant factor VIII genes posttranslational process is unknown. leading to factor VIII deficiency and hemophilia A (Gits- chier et al., 1985; Antonarakis et al., 1985; Youssoufian et Cleavage of Prosequences al., 1987). Exons encoding either the heavy chain (exons The propeptides in the vitamin K-dependent proteins and 6 and 7) or the light chain (exons 23-26) of the protein ap- vWF are critical to posttranslational processing. After pro- pear essential to the expression of normal factor VIII activ- cessing, these peptides are cleaved and the mature pro- ity. In other families hemophilia has arisen as a result of teins circulate in the plasma. Two contiguous basic amino nonsense mutations. The recognition sequence of the re- acids appear to the amino-terminal side of the site of striction enzyme Taql includes the CpG dimer, a major site propeptidase cleavage in most of the vitamin K-depen- of methylation in mammalian DNA. CpG dimers are muta- dent proteins (Figure 5). Although the propeptidases have tion “hotspots” in the factor VIII gene (Youssoufian et al., not been identified, their specificity for arginine suggests 1986). As noted by Gitschier et al. (1985), five of the seven a serine protease in the trypsin family. An Arg+Ser muta- Taql sites in exons of the factor VIII gene can be converted tion at -1 in a naturally occurring mutant, factor IX Cam- to nonsense mutations via C-to-T transitions and the intro- bridge, inhibits propeptide cleavage (Diuguid et al., 1986). duction of in-frame stop codons. Mutations in Taql sites in The properties of a naturally occurring factor IX mutant, exons 18, 24, and 26 have been associated with hemo- factor IX Oxford 3, suggest that the propeptidase sub- philia (Table 2) (Gitschier et al., 1985; Antonarakis et al., strate specificity is not limited to the basic residues at -1 1985). A point mutation leading to the substitution of a and -2 but also includes residue -4 in the propeptide glutamine for arginine 2307, encoded by exon 26, is re- (Bentley et al., 1986), since an Arg-Gln substitution at sponsible for a mild form of hemophilia A (Gitschier et al., this position precludes propeptide cleavage. Deletion mu- 1986). The factor VIII molecule synthesized appears to be tations in protein C further emphasize the importance of fully active, but its plasma level is significantly decreased. Cell 514

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

Size Defect (4 Location Phenotype Reference Factor VIII (Hemophika A)

Deletion 21.9 Exon 26 Severe Gitschier et al., 1985 Deletion 7 Exon 6 Severe Youssoufian et al., 1987 Deletion 2.5 Exon 14 Severe Youssoufian et al.. 1987 Deletion 7 Exons 24, 25 Severe Youssoufian et al., 1987 Deletion 16 Exons 23, 24 Severe Youssoufian et al., 1987 Deletion 39 Exons 23, 24, 25 Severe Gitschier et al., 1985 Deletion 80 Exons 7-22 Severe Antonarakis et al., 1985 Deletion 5.5 Exon 22 Moderate Youssoufian et al., 1987 Nonsense mutation Exon 24 Severe Gitschier et al., 1985 Nonsense mutation Exon 26 Severe Gitschier et al., 1985 Nonsense mutation Exon 18 Severe Antonarakis et al., 1985 Nonsense mutation lntron 2 Severe Gitschier et al., 1985 Nonsense mutation lntron 25 Severe Gitschier et al., 1985 Point mutation Glu 2307-Arg Moderate Gitschier et al., 1986 Factor IX (Hemophilia t3)

Deletion 18 Exons I, Ii, III, IV-? Severe Giannelli et al., 1983 Deletion 18 Exons I, II, Ill, IV-? Severe Giannelli et al., 1983 Deletion 9 Exons Ill, IV-? Severe Giannelli et al., 1983 Deletion 10 Exons V, VI Severe Chen et al., 1985 Point mutation Splice 3’ of Exon VI Severe Rees et al., 1985 Deletionlframeshift 0.001 Exon V Severe Schach et al., 1987 Point mutation Arg 145-His Moderate Noyes et al., 1983 Point mutation Arg -I-Ser Severe Diuguid et al., 1986 Point mutation Arg -4-Gln Severe Bentley et al., 1986 Point mutation Arg -4-Gln Severe Ware et al., 1986 Point mutation Asp 47-Gly Moderate Davis et al., 1987

Whether this is due to decreased synthesis or to acceler- peptidase. The presence of this point mutation in the pro- ated clearance of an active but unstable factor VIII species peptide partially inhibits y-carboxylation, thus leading to is unknown. a protein that cannot bind to phospholipid vesicles or ex- Point mutations in the factor VIII introns are also known hibit coagulant activity. Factor IX Oxford 3 (Arg -4-+Gln) to cause hemophilia A. A mutated Taql site located in the (Bentley et al., 1986) and factor IX San Dimas (Arg -4*Gln) intron between exon 2 and exon 3 of the factor VIII gene (Ware et al., 1986) have propeptide mutations that pre- is associated with hemophilia A (Gitschier et al., 1985). clude propeptide cleavage. Factor IX San Dimas contains This defect is not located near the intron-exon splice junc- about 50% of the normal y-carboxyglutamic acids (Ware tions, nor does it include any sequence resembling con- et al., 1986). Factor IX Alabama (Asp 47-Gly) lacks coag- sensus splice donor or acceptor sites, Therefore the ac- ulant activity, probably because it fails to bind to cell mem- tual cause of the defect in mRNA synthesis remains brane surfaces (Davis et al., 1987). Thus, there are now obscure. A new Taql site in an intron between exon 25 and examples of factor IX mutants that interfere with post- exon 26 may be responsible for the factor VIII deficiency translational processing, factor IX-cell interaction, and in another hemophiliac. factor IX zymogen activation. Examples of factor IX forms Hemophilia B is due to diminished or functionally defec- that lack enzyme activity or normal substrate binding tive factor IX. In about one-third of cases, the defective fac- properties are expected to be identified. tor IX circulates in the blood. Factor IX Chapel Hill Alternatively, little or no factor IX antigen may circulate (Arg 145-His) is the prototype for mutant zymogens that in the blood of some hemophiliacs. Hemophilia 6 can re- cannot be activated to their enzyme form because of mu- sult from gross factor IX gene deletions (Table 2) (Gianelli tation of the arginine adjacent to the sessile bond (Noyes et al., 1983; Chen et al., 1985). In the deletion described et al., 1983). In factor IX Chapel Hill, this arginine is adja- by Chen et al. (1985) the 5’ end of the gene is intact, and cent to the cleavage site between the activation peptide a truncated expression product of M, 36,000 is secreted and the light chain of factor IXa. A parallel point mutation but cleared from the blood into the urine (Bray and has been found in prothrombin Barcelona (Arg 273~Cys) Thompson, 1986). In the abnormality described by Rees (Rabiet et al., 1986). Factor IX Cambridge (Diuguid et al., et al. (1985) a point mutation (G-T) is located at base 1986) factor IX Oxford 3 (Bentley et al., 1986) and factor 21,165 within the obligatory GT of a splice junction at the IX San Dimas (Ware et al., 1986) contain point mutations 3’site of exon VI. The change from GT to TT interferes with in the propeptide domain of the factor IX precursor, profac- splicing, causing a fatal defect in transcription. In factor IX tor IX. In factor IX Cambridge (Arg -1 to Ser), the mutation Seattle (Schach et al., 1987) an A at position 17,699 in the prevents proteolytic removal of the propeptide by a pro- 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 thrombin requires two sequential metal-dependent conformational 86. As with hemophilia A, these examples emphasize the transitions to bind phospholipid. J. Biol. Chem. 267, 14969-14975. heterogeneity of hemophilia B. A single-base deletion and Bray, G. L., and Thompson, A. R. (1986). Partial Factor IX protein in a pedigree with hemophilta B due to a partial gene deletion. J. Clin. In- frameshift mutation, a point mutation in the splice junc- vest. 77, 1194-1200. tion, and partial or gross gene deletions are among the Celeste, A. J.. Buecker, J. L., Kriz, R., Wang, E. A., and Wozney, J. M. causes of severe hemophilia B. (1986). Isolation of the human gene for bone gla protein utilizing mouse Von Willebrand’s disease, the most common hereditary and rat cDNA clones. EMBO J. 5, 1885-1890. bleeding disorder, is likely caused by a variety of defects Chen, S.-H., Yoshitake. S., Chance, P F., Bray, G. L., Thompson, A. R., that effect a decrease in the activity of vWF. To date, mo- Scott, C. R., and Kurachi, K. (1985). An intragenic deletion of the factor lecular explanation for this disorder is limited to three IX gene in a family with hemophilia B. J. Clin. Invest. 76, 2181-2164. Choo. K. H., Gould, K. G., Rees, D. J. G., and Brownlee. G. G. (1982). cases of severe von Willebrand’s disease due to the dele- Molecular cloning of the gene for human anti-haemophilic Factor IX. tion of the entire vWF gene (Shelton-lnloes et al., 1987). Nature 299, 178-180. Chung, D. W., Chan, W. Y., and Davie, E. W. (1983a). Characterization Conclusion of a complementary deoxyribonucleic acid coding for the y-chain of hu- man fibrinogen. Biochemistry 22, 3250-3256. Blood coagulation is a complex physiologic process that Chung, D. W., Clue, B. G., Rixon, M. W., Mace, M., Jr., and Davie, E. W. involves the initiation of blood clotting, the localization of (1983b). Characterization of complementary deoxyribonucleic acid and genomic deoxyribonucleic acid for the beta chain of human the process to the area of vascular injury, and the forma- fibrinogen. Biochemistry 22, 3244-3250. tion of the fibrin clot and the platelet plug. The assembly Church, W. R., Jernigan, R. L., Toole, J., Hewick, R. M., Knopf, J., of key components on cell membranes and the interaction Knutson, G. J., Nesheim, M. E., Mann, K. G., and Fass, D. N. (1984). of reactions in the solution phase with reactions on mem- Coagulation factors V and VIII and ceruloplasmin constitute a family brane surfaces are critical to the control of this process. of structurally related proteins, Proc. Natl. Acad. Sci. USA 81, 6934- 6937. Better understanding of the molecular basis of blood Collins, C. J., Underdahl, J. P., Levene, R. B.. Ravera, C. P., Morin. coagulation will continue to give insight into structure- M. J.. Dombalagian, M. J., Ricca, G., Livingston, D. M., and Lynch, function relationships of the components of this system, D. C. (1987). Molecular cloning of the human gene for van Willebrand and the mechanisms by which these component reac- factor and identification of the transcription initiation site. Proc. Natl. tions are regulated. Further study should add to our un- Acad. Sci. USA 84, 4393-4397. derstanding of the molecular and cellular basis of bleed- Cool, D. E., and MacGillivray, R. T. A. (1987). Characterization of the ing and thrombotic disorders. human blood coagulation Factor XII gene. J. Biol. Chem. 262, 13662- 13673. Cool, D. E., Edgell, C. J. S., Louie, G. V., Zoller, M. J., Brayer, G. D., and MacGillivray, R. T. A. (1985). Characterization of human blood coagulation Factor XII cDNA. J. Biol. Chem. 260, 13666-13676. We are especially grateful to members of our laboratory, past and pres- ent, for their contributions to both the scientific effort and the conceptu- Crabtree, G. R., and Kant, J. A. (1981). 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