6 Molecular biology of factor X

6 Molecular biology of factor X ROSS T. A. M A C G I L L I V R A Y M A R I O N R. FUNG

The intrinsic and extrinsic clotting pathways converge at the activation of factor X (see Figure 1). The mechanisms of blood coagulation have been reviewed recently (Davie et al, 1979; Jackson and Nemerson, 1980). In the intrinsic pathway, factor IXa in the presence of factor Villa, calcium ions and phospholipid cleaves a single peptide bond in factor X (Fujikawa et al, 1975). This limited proteolysis converts factor X from an inactive zymogen to the active serine protease called factor Xa (Figure 2). In the intrinsic pathway, the same peptide bond in factor X is cleaved by factor VIIa in the presence of tissue factor, calcium ions and phospholipid (Fujikawa et al, 1975). Factor X is also activated by various proteases including factor IXa, trypsin and a protease from Russell's viper venom (Fujikawa et al, 1972b; Jesty and Esnouf, 1973; Jesty and Nemerson, 1974). Since the development of recombinant DNA techniques in the mid-1970s, it has become possible to characterize eukaryotic genes at the nucleotide level. This has led to a wealth of information on promoter and transcription factor function, gene organization, chromosomal localizations of genes, tissue-specific expression of genes, and regulation of gene expression. Much of this information has resulted from the ability to isolate cloned DNA coding for a particular structural gene. In this chapter, we briefly review the factor X polypeptide chain, and describe recent progress in understanding factor IXa Ca + + phospholipid Factor X

factor VIIa tissue factor

Factor X

Factor Xa factor Va

Ca+ + phospholipid

prothrombin

thrombin

Figure 1. Role of factor X in blood coagulation. Baillibre's Clinical Haernatotogy--Vol. 2, No. 4, October1989

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Figure 2. Schematic diagram of the structure of factor X. From Leytus et al (1986a), with permission. See text for details.

the factor X gene, its chromosomal localization, and tissue-specific expression. Recent reviews of the molecular biology of blood coagulation (Furie and Furie, 1988; MacGillivray et al, 1988) and the vitamin K-dependent clotting factor genes (Fung and MacGillivray, 1988) have been published. FACTOR X POLYPEPTIDE CHAIN Isolation of factor X Factor X is present in plasma at a concentration of approximately 8 Ixg/ml (Bajaj and Mann, 1973). Factor X was first identified by its deficiency in individuals with bleeding disorders; the deficient protein was called the Stuart (Hougie et al, 1957) or Prower (Teller et al, 1956) factor. Subsequently, factor X has been highly purified and characterized from bovine (Jackson and Hanahan, 1968; Fujikawa et al, 1972a; Jackson, 1972; Esnouf et al, 1973), pig (Dupe and Howell, 1973), rat (Graves et al, 1982;

M O L E C U L A R BIOLOGY OF FACTOR X

899

Willingham and Matschiner, 1984), and human (Aronson et al, 1969; Rosenburg et al, 1975; Kosow, 1976; Vician and Tishkoff, 1976; DiScipio et al, 1977) plasma. Fractionation of bovine factor X by barium sulphate adsorption and ion exchange chromatography yields two forms of the protein designated X1 and X2 (Jackson and Hanahan, 1968; Fujikawa et al, 1972a). Both species possess similar chemical and biological properties. The only distinguishing feature is the sulphated tyrosine residue near the aminoterminal end of the higher molecular weight chain of the factor Xa variant (Morita and Jackson, 1979). As discussed above, factor X is activated to the serine protease factor Xa by cleavage of a single peptide bond. In a second, slower reaction, factor Xa (also designated as factor Xao0 is converted to factor Xal3 by hydrolysis of an Arg-Gly peptide bond in the carboxyterminal region of the protein (Fujikawa et al, 1975) (Figure 2). This autocatalytic reaction requires calcium ions and phospholipids to proceed, but is unrelated to the activation process (Jesty et al, 1974). Enzymatically, the degradation product exhibits no loss of coagulant activity (Fujikawa et al, 1975); thus the carboxy-terminal 17 amino acids of bovine factor X are not essential for physiological function. Functions of factor X The primary enzymatic function of factor Xa is the conversion of prothrombin to thrombin in the presence of the cofactors calcium, phospholipids, and factor Va via two specific proteolytic events (Jackson, 1981). The initial cleavage releases an Mr 33 500 glycopeptidyl intermediate possessing no coagulant activity (Esmon et al, 1974). The second proteolysis generates two-chained thrombin with full activity but no associated peptide loss (Jesty and Esnouf, 1973). The proteolytic action of factor Xa is not limited to prothrombin but is significant in the activation of factor VII by a positive feedback loop mechanism (Radcliffe and Nemerson, 1975; 1976). Initial hydrolysis of factor VII by factor Xa generates factor VIIa which activates the extrinsic pathway in vitro; however, prolonged exposure to factor Xa results in the formation of a three-chained molecule with esterase, but no proteolytic activity (Radcliffe and Nemerson, 1975; 1976). Therefore, the activation of factor X via the extrinsic pathway may be viewed as self-terminating. In vitro, factor V, factor VIII, as well as factor Xa, act as substrates for factor Xa (Davie et al, 1979; Jackson, 1984). Regulation of enzymatic activity Regulation of the coagulation reactions in which factor X participates is complex. Three mechanisms control the proteolytic activity of factor Xa: (1) the interrelationship between the reactants in activation complex formation including cofactor and surface membrane binding (see Jackson, 1984); (2) factor Va and factor VIIIa inactivation by activated protein C both at the stage of factor X activation and factor Xa activation of prothrombin (see Esmon, 1987); and (3) regulation by association with an irreversible

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R. T. A . M A C G I L L I V R A Y A N D M. R. F U N G

protease inhibitor (see Jackson and Nemerson, 1980). Regulation is further complicated by the positive feedback of factor Xa on factor VII. The third mechanism may act more as a sink for dissociated proteases than as a regulator. Antithrombin III is a serine protease inhibitor of several of the blood coagulation enzymes, including factor Xa (Kurachi et al, 1976; Jesty, 1978). Although antithrombin III appears to neutralize free protease effectively, the accessibility of factor Xa to the inhibitor is severely restricted when bound either to factor Va and phospholipids (Marciniak, 1973) or to platelets (Miletich et al, 1978). Thus, antithrombin III may only inhibit plasma free enzymes, and therefore remove discarded proteins, but is not a regulator of their action in complex form (see Jackson and Nemerson, 1980). In vitro, soybean trypsin inhibitor and trypsin are commonly used inhibitory agents of factor Xa (Jackson and Hanahan, 1968; Fujikawa et al, 1972b). Structure of factor X

Plasma factor X has been fully characterized, in both the bovine (Jackson and Hanahan, 1968; Fujikawa et al, 1972a; Jackson, 1972; Esnouf et al, 1973) and human (Aronson et al, 1969; Rosenberg et al, 1975; Kosow, 1976; Vician and Tishkoff, 1976; DiScipio et al, 1977) species. Factor X is present in plasma as an inactive zymogen comprised of two polypeptide chains (Figure 2). This feature is unique among the blood coagulation proteases, but is present in the regulatory protein called protein C (see Esmon, 1987). Bovine factor X is a glycoprotein, Mr 55 000 consisting of a light chain (Mr 16 500) and a heavy chain (Mr 39 300) linked by a disulphide bond (Fujikawa et al, 1972a; Jackson, 1972). The human counterpart has a somewhat larger molecular weight (Mr 59 500) which is largely attributable to a higher carbohydrate content and an amino-terminal extension associated with the heavy chain (DiScipio et al, 1977). In both species, activation occurs at a Arg-Ile bond, releasing an Mr 10 000-14000 fragment from the amino terminus of the heavy chain (Fujikawa et al, 1972a; 1974; Jesty et al, 1974; DiScipio et al, 1977) (Figure 2). The complete amino acid sequence of bovine factor X has been reported (Titani et al, 1975; Enfield et al, 1980) as well as the organization of the disulphide bonds (Hojrup and Magnusson, 1987). The amino acid sequence of the light chain of human factor X has also been reported (McMullen et al, 1983a). Comparison of the light chains shows an overall sequence identity of 70% between the two species (McMullen et al, 1983a). Post-translational modifications

After synthesis, but before secretion from liver cells, many of the blood coagulation factors are highly modified to facilitate their specialized functions (Burgess and Esnouf, 1985). In the case of factor X, three modifications are required. Glycosylation is associated exclusively with the heavy chain (Jackson and Hanahan, 1968; DiScipio et al, 1977) and occurs at Asn-36 (N-linked) and Thr-300 (O-linked) in the bovine molecule (Titani et al, 1975).

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Both carbohydrate moieties are removed following activation and autocatalytic cleavage (Fujikawa et al, 1975). In the amino-terminal region, several glutamic acid residues of factor X are converted to ~/-carboxyglutamic acid residues (Gla) (Jackson and Nemerson, 1980; Burgess and Esnouf, 1985). Historically, Gla residues were first identified in prothrombin (Stenflo et al, 1974) and subsequently in the other vitamin K-dependent plasma proteins factors VII, IX, and X and proteins C, S, and Z (Suttie, 1985). Gla modification follows glycosylation (Swanson and Suttie, 1985) and is catalysed by a vitamin K-dependent carboxylase found in the hepatic cellular endoplasmic reticulum (Suttie, 1985). Gla residues are necessary for the biological activity of these clotting factors. An impaired state may be brought about either by the absence of the essential vitamin or by inhibition of the carboxylation process by coumarol drugs (e.g. warfarin). Under such conditions, Gla-less factor X is activated at a minimal rate (Burgess and Esnouf, 1985). In each of the vitamin K-dependent proteins, with the exception of prothrombin, an aspartic acid residue is converted to [3-hydroxyaspartic acid in a post-translational reaction (Asp-63 in the case of factor X) (Fernlund and Stenflo, 1983; McMullen et al, 1983b). The catalytic mechanism involved is unknown (McMullen et al, 1983a; 1983b). Conserved arginine positions and a single tyrosine/phenylalanine residue (Stenflo et al, 1987a,b) may reflect a unique recognition site for post-translational hydroxylation. The functional significance of the modification is not clear, but recent speculation suggests that the 13-hydroxyaspartic acid residue may have calcium binding capacity independent of the Gla domain (Esmon et al, 1983; McMullen et al, 1983b; Cook et al, 1987). Studies involving Gla-less factor X correlate the presence of a single 13-hydroxyaspartic acid with the binding of a single calcium ion (Sugo et al, 1984). Adjacent aspartic residues may combine with the modified residue to form a calcium binding site (Cook et al, 1987).

Functional domains

Factor X is a complex protein that is composed of multiple structural domains, each of which is essential for the physiological action of the protein. The amino,terminal 40 amino acids of the light chain encode the Gla domain of factor X (Enfield et al, 1980; McMullen et al, 1983a). Gla residues are required for the calcium-dependent interaction between the protease and negatively charged phospholipid surface which is essential for the activation of the zymogen (Suttie, 1985). The mechanism of calcium action remains controversial. Some postulate that the Gla residues function in binding calcium ions, thus forming an ionic bridge between factor X and the phospholipid membranes at the site of injury (Stenflo and Suttie, 1977). Others theorize that the binding of calcium ions induces one or possibly two conformational changes in the protein, favouring dimerization or protein association to acidic lipid vesicles; however, the primary calcium binding site is located elsewhere on the molecule (Borowski et al, 1986). Still others

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R. T. A. MACGILL1VRAY A N D M. R. F U N G

propose the presence of multiple classes of binding sites exhibiting both positive and negative co-operativity (Jackson and Brenkle, 1980). The Gla domain is followed by a twofold repeat of approximately 40 amino acids, each demonstrating homology to epidermal growth factor (EGF) (Blomquist et al, 1984; Doolittle et al, 1984). The EGF-like domains consist of three conserved glycine residues as well as six cysteines which form three structurally distinctive disulphide bridges. Similar regions exist in the dotting proteins factors VII, IX, XI1, protein C, protein S, plasminogen activator, and urokinase (Doolittle et al, 1984; Cool et al, 1985; Stenflo et al, 1987a,b). The post-translationally modified [3-hydroxyaspartate residue has been identified in the first EGF element in factors VII IX, X and proteins C and S (aspartate residues 63, 64, 63, 71, and 95, respectively) (Fernlund and Stenflo, 1983; McMullen et al, 1983b; Stenflo et al, 1987a,b). In addition, EGF homologies were recently found in such diverse proteins as the Mr 19 000 protein from vaccinia virus (Blomquist et al, 1984; Reisner, 1985), the low density lipoprotein receptor (Sudhoff et al, 1985), thrombomodulin (Wen et al, 1987), complement protein Clr (Leytus et al, 1986b), the Notch gene product in Drosophila melanogaster (Wharton et al, 1985) and the lin-12 gene product in Caenorhabditis elegans (Greenwald, 1985). Whether this structural unit has any binding affinity similar to that of EGF is unclear (Doolittle et al, 1984). However, immuno-isolation of the EGF region of protein C identified a Gla-independent high affinity calcium binding site possibly associated with the [3-hydroxyaspartic acid contained in the first EGF homology (Ohlin and Stenflo, 1987; Stenfio et al, 1987a,b). The heavy chain of factor X contains the serine protease region essential for proteolytic activity. The histidine, aspartic acid, and serine residues of the charge relay network found in trypsinogen, chymotrypsin, and proelastase are found in similar positions in factor X and the other blood coagulation proteases, indicating a common evolutionary ancestor (Kraut, 1977; Jackson and Nemerson, 1980). In factor X, the catalytic triad is represented by His-93, Asp-138, and Ser-235 (Titani et al, 1975). In each case, except for factor VII, enzymatic activity is triggered by cleavage at an Arg-X bond and loss of a peptide fragment from the aminoterminus of the zymogen (Davie et al, 1979). No peptide toss is associated with the proteolysis of factor VII. The resulting conformational change induces activity. The highest degree of sequence similarity is observed in the activation recognition region and around the serine active site (Davie et al, 1979). The active site region, Gly-Asp-Ser-Gly-Gly-Pro is found in each protease (see Jackson and Nemerson, 1980). However, in contrast to the digestive serine proteases, factor Xa and the other blood clotting proteases display a more limited substrate specificity.

Three-dimensional structure

To date, factor X has not been crystallized in forms suitable for X-ray diffraction studies. The structure of part of fragment 1 of prothrombin has been elucidated (Park and Tulinsky, 1986). It is postulated that several

M O L E C U L A R B I O L O G Y OF FACTOR X

903

stacked aromatic residues contained in the carboxy terminus of the Gla domain may form a receptor recognition site for the carboxylase. By inference, corresponding amino acids in factor X may have a similar function. Attempts to examine the crystal structure of EGF have been unsuccessful. However, solution structures of human EGF have been analysed by NMR with extrapolation to the first, more homologous, EGF-like domain in factors IX and X (Cook et al, 1987). The model indicates two distinct domains within the homologous region; the amino-terminal domain contains the first two disulphide bridge structures and the carboxy-terminal domain contains the third. These units adopt a triple-stranded t3-sheet conformation allowing the aspartic acid and [3-hydroxyaspartic acid residues to lie in close proximity on the one face of the sheet where they can easily form a calcium binding site (Cook et al, 1987). As discussed, the catalytic heavy chain of factor X (and the other clotting proteases) share sequence homology with trypsin (Davie et al, 1979; Jackson and Nemerson, 1980). The homology to the pancreatic enzymes has allowed the development of computer models of the three-dimensional structures of factor IXa, factor Xa, thrombin, and factor XIIa (Furie et al, 1982; Cool et al, 1985; Geddes et al, 1989) based upon the known tertiary structures of the pancreatic proteases. The models project each serine protease as enzymes possessing highly individualistic, charged surfaces enveloping a strictly conserved active site core. A conserved catalytic mechanism between the pancreatic and coagulation proteases is implied by the invariant three-dimensional structure of the His-Asp-Set catalytic triad, whereas the unique substrate specificity of the clotting factors is thought to be defined by the structural differences found in the substrate binding pockets as well as on the surfaces of the proteins (Furie et al, 1982).

Single-chain factor X

Factor X and protein C circulate in plasma as two-chained molecules (Jackson and Hanahan, 1968; Fujikawa et al, 1972a). However, prothrombin and other homologous serine proteases are found in plasma as a single polypeptide chain. This has led several investigators to propose that factor X is synthesized as a single-chain precursor comprised of both the light and heavy chains (see Jackson and Nemerson, 1980). Studies from several laboratories have shown that factor X is synthesized by rat (Graves et al, 1982; Willingham and Matschiner, 1984) and human (Rosenberg et al, 1975; Fair and Bahnak, 1984) hepatoma cells as a precursor consisting of a single polypeptide chain of Mr 63 000 (rat). After secretion into the tissue culture medium, the single-chain form is converted to the two-chain form found in plasma, but the nature of this conversion was not established in these studies. Recent evidence indicates that approximately 10% of the total factor X antigen present in normal rat plasma is found in the single-chain form (Willingham and Matschiner, 1984), raising the possibility of a physiological role for the factor X precursor.

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ISOLATION OF FACTOR X cDNA CLONES Although the complete amino acid sequence of bovine factor X had been determined by using classical protein chemistry techniques, the characterization of factor X complementary D N A (cDNA) clones offered the advantage that the nature of the primary translation product could be predicted from the cDNA sequence. In the case of factor X, this offered information on the presence of a leader peptide sequence (as had been found with many other secreted proteins (Blobel et al, 1979)) and on the nature of the single-chain form of factor X. Bovine factor X cDNAs

Fung et al (1984) screened a bovine liver cDNA library by using a mixture of synthetic oligodeoxyribonucleotides as a hybridization probe. The oligonucleotides coded for part of bovine factor X as predicted by the amino acid sequence (Titani et al, 1975). Of 30000 colonies screened, five positives were obtained. Subsequent D N A sequence analysis showed that each of these clones contained D N A complementary to part of factor X mRNA. One of the bovine factor X cDNA clones (pBX1) contained D N A coding for 75 base pairs of putative 5' untranslated sequence, a putative leader peptide of 40 amino acids, and a single polypeptide chain consisting of 447 amino acids. Comparison with the amino acid sequence of bovine factor X (Titani et al, 1975; Enfield et al, 1980) suggested that pBX1 was lacking 14 base pairs of coding sequence, a probable stop codon, and the 3' untranslated sequence. Analysis of the other four positives showed that they too lacked this region. The factor X cDNA sequence predicted that factor X mRNA encodes a single polypeptide chain in which the light and heavy chains of plasma factor X are linked by the dipeptide Arg-Arg (Figure 3). Analysis of the 5' end of factor X cDNA revealed the presence of a leader peptide. A single ATG codon occurred in the same reading frame as the factor X amino acid sequence. Comparison with the leader sequences of other vitamin Kdependent clotting factors suggests that this ATG encodes the initiator methionine residue. The amino-terminal region of the leader peptide consisted of a highly hydrophobic region (residues - 3 7 to - 2 2 , Figure 4) Bovine Human Bovine Human

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Figure 3. Peptide sequences that join the light and heavy chains of single-chain factor X and protein C. The sequences of bovine factor X (Fung et al, 1984), human factor X (Leytus et al, 1984; Fung et al, 1985; Kaul et al, 1986), bovine protein C (Long et al, 1984), and human protein C (Beckman et al, 1985; Foster et al, 1985) were predicted from the corresponding cDNA sequences.

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MOLECULAR BIOLOGY OF FACTOR X -46

Bovine prothrombin Human prothrombln Bovine factor X Human factor X Human factor IX

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Figure 4. Comparison of the leader peptide sequences of the vitamin K-dependent clotting factors. The amino acid sequences were predicted from c D N A sequences. Amino acid residues that are common to at least five of the sequences are boxed. References are given in the text.

followed by a region containing glycine, alanine and proline. This type of sequence is typical of signal peptides found in many secreted proteins (Blobel et al, 1979). However, the conversion of the pre-factor X to the form found in plasma involves the cleavage of an Arg-Arg-Ala bond, where the Ala represents the amino-terminal residue of plasma factor X. This type of cleavage is not consistent with the substrate specificity of signal peptidase, which has an elastase-like activity (von Heijne, 1983; 1985). Several other proteins are processed by proteolysis that occurs carboxy-terminal to two basic residues, such as albumin (Strauss et al, 1978), apolipoprotein A-II (Gordon et al, 1983), and several peptide hormones (Steiner et al, 1980). In the cases of albumin and apolipoprotein A-II, the site of signal peptidase cleavage has been identified (Strauss et al, 1978; Gordon et al, 1983). After the signal peptide has been removed, a short peptide called the propeptide remains. It is this propeptide that is removed by the trypsin-like proteolysis that occurs carboxy-terminal to the double basic residues. The cDNA sequences of the other vitamin K-dependent clotting factors predict that these proteins are also synthesized as pre-proproteins. Recent experiments on factor IX and protein C have shown that the propeptide consists, at least in part, of the recognition site for the vitamin K-dependent carboxylase (see next section). By analogy, the factor X propeptide probably also functions in this way.

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g . T. A. MACGILLIVRAY AND M. R. F U N G

Human factor X cDNAs

Several groups have isolated cDNA clones coding for human factor X (Leytus et al, 1984; Fung et al, 1985; Kaul et al, 1986; Leytus et al, 1986a; Bahnak et al, 1987). Leytus et al (1984) screened a human liver cDNA library in Xgt 11 with an antibody against human factor X. Of 2 x 10~ phage screened, ten positives were isolated. The largest insert (from phage hX-1137) was 1137 base pairs in size and coded for amino acid residues 75--445, a stop codon, a 3' untranslated region of 10 base pairs, and a poly(A) tail. These investigators then reported the nucleotide sequence of a full-length cDNA clone that included 25 base pairs of 5' untranslated sequence, and D N A coding for the pre-propeptide (Leytus et al, 1986a). Fung et al (1985) screened a human liver cDNA library in pKT218 (Prochownik et al, 1983) by using the bovine factor X cDNA (Fung et al, 1984) as a hybridization probe under conditions of reduced stringency. Of 240 000 colonies screened, nine positives were isolated. The largest insert (designated pcHX14) was 1441 base pairs in size, and coded for residues - 2 8 to 448 of factor X in addition to the stop codon, 3' untranslated region and poly(A) tail. Several differences were noted between the nucleotide sequence of pcHX14 and the partial clone isolated by Leytus et al (1984). Two of these differences were point mutations that probably represent polymorphisms between the two sequences. The other difference was an apparent deletion of nine nucleotides in hX-1137. Examination of other factor X cDNA clones and the factor X genornic D N A have shown that this deletion was the result of a cloning artefact (Leytus et al, 1986a). Kaul et al (1986) also screened a human liver cDNA library constructed in pKT218 by using two overlapping synthetic oligonucleotides as a hybridization probe. Four positive clones were isolated; the longest one was similar in size to that isolated by Fung et al (1985), and contained DNA coding for amino acids - 2 2 to 448, the stop codon, 3' untranslated region and poIy(A) tail. Bahnak et al (1987) also screened a human liver cDNA library in hgt 11 using a polyclonal antibody against factor X. Of 300 000 recombinant phage screened, four positives were obtained. One clone was picked for further analysis, and reacted with a mixture of five mouse monoclonal antibodies against factor X. Partial D N A sequence analysis of the 1100 base-pair insert confirmed that it coded for factor X. This clone was then used for measuring factor X m R N A levels in HepG2 cells (see later section). Northern blot analysis showed that human factor X mRNA in both liver and HepG2 cells is approximately 1700 nucleotides in size. D N A sequence analysis of these cDNA clones showed that human factor X has a similar structure to bovine factor X. The protein is synthesized as a single polypeptide chain containing a leader peptide of 40 amino acids. This leader peptide appears to consist of a signal peptide and a propeptide. Figure 4 shows a comparison of the leader peptides of the vitamin Kdependent proteins, including bovine prothrombin (MacGillivray and Davie, 1984), human prothrombin (Degen et al, 1983; Degen and Davie,

MOLECULAR BIOLOGY OF FACTOR X

907

1987), bovine factor X (Fung et al, 1984), human factor X (Fung et al, 1985; Kaul et al, 1986; Leytus et al, 1986a), human factor IX (Kurachi and Davie, 1982; Jaye et al, 1983; McGraw et al, 1985), bovine protein C (Long et al, 1984), human protein C (Beckman et al, 1985), bovine protein S (Dahlback et al, 1986), and human protein S (Lundwall et al, 1986; Hoskins et al, 1987). Little sequence homology is found in the signal peptide regions; however, they retain the general hydrophobic structure that is found in these peptides (von Heijne, 1983; 1985). In contrast, the putative propeptides retain a high level of sequence identity, suggesting that there are functional restraints on the structure of _this region. There is now considerable evidence that the propeptides represent (at least in part) the substrate recognition site for the vitamin K-dependent carboxylase. This evidence includes homologies with a region of the bone ~/-carboxyglutamic acid-containing proteins (Pan and Price, 1985), an analysis of mutations in the propeptide (Diuguid et al, 1986; Foster et al, 1987; Jorgensen et al, 1987), and a direct assay of the effect of the propeptide on the efficiency of carboxylation (Knobloch and Suttie, 1987; Suttie et al, 1987; Ulrich et al, 1988). Although these latter studies were performed with factor IX and protein C, it is very probable that the propeptide of factor X functions in a similar way. The heavy and light chains of human factor X are joined by the tripeptide Arg-Lys-Arg (Leytus et al, 1984; Fung et al, 1985; Kaul et al, 1986). Factor X and protein C are unique amongst the clotting factors in being present in plasma as two-chain molecules. The linkages between the bovine and human proteins are shown in Figure 3. In each case, the chains are linked by basic amino acid residues. Human factor X is secreted by HepG2 cells as the single-chain form, but is converted to the two-chain form on exposure to the tissue culture medium (Fair and Bahnak, 1984). Although the nature of the protease(s) responsible is unknown, it has been postulated that a trypsin-like protease found in plasma may cleave the single-chain form, with the basic residues being removed subsequently by a carboxypeptidase B-like activity (Fung et al, 1984). Any biological significance of the two-chain nature of plasma factor X and plasma protein C is not understood at present. The 3'-untranslated region of human factor X is only ten nucleotides in length (Leytus et al, 1984; Fung et al, 1985; Kaul et al, 1986). As a result, the putative polyadenylation signal A T T A A A is located in the coding region of factor X mRNA. The mRNAs coding for the [3 subunit of human chorionic gonadotropin (Fiddes and Goodman, 1980) and the abnormal oL-globin Constant Spring (Proudfoot and Longley, 1976) also have short 3' untranslated regions (16 nucleotides). In these two proteins, the polyadenylation signal contains the U A A codon that is used as a stop codon. Comparison of the amino acid sequences of bovine and h u m a n factor X

When the amino acid sequences of bovine and human factor X are compared (Fung et al, 1985), extensive sequence identities are found throughout the polypeptide chains. Overall, the two sequences display 65% sequence identity when a single gap is inserted into the bovine activation peptide to maximize the homology. The leader peptides exhibit 39% sequence identity

908

R. T. A . M A C G I L L I V R A Y A N D M. R. F U N G

at the amino acid level, but 63% identity at the nucleotide level. The light chains (residues 1-139) and heavy chains (residues 194-429) exhibit 70% and 84% identity at the amino acid level, respectively. Presumably, this reflects the constraints on the sequence due to the functional regions of the polypeptides. In contrast, the activation peptide and carboxy-terminal region show only 14% and 5% sequence identity, respectively. Presumably, this reflects the lack of functional importance of these regions of the factor X molecule. Indeed, a carboxy-terminal peptide can be removed from factor Xa without altering its enzymatic activity (Fujikawa et al, 1975). C H A R A C T E R I Z A T I O N OF THE H U M A N F A C T O R X GENE

By using the human factor X cDNA as a hybridization probe, Leytus et al (1986a) and Fung (1988) isolated recombinant phage clones spanning most of the human factor X gene. Leytus et al (1986a) screened two human genomic libraries with h X-1137 and a full-length cDNA as hybridization probes. Of 3 × 106 phage screened, seven positives were obtained. Restriction endonuclease mapping and partial D N A sequence analysis showed that the phage contained D N A coding for seven introns and seven exons; however, the phage did not contain D N A coding for the 5' untranslated region of the m R N A and residues - 4 0 to - 1 8 of the leader peptide. If these regions are found on a single exon, then the human factor X gene would consist of eight exons. The seven exons contained in the phage spanned 22 000 base pairs of human DNA. Fun g (1988) also screened several human genomic D N A libraries. Of 1 × 10" clones screened, 32 positives were isolated. Six of these clones were analysed further, and were found to contain overlapping DNA covering 32 000 base pairs of human genomic DNA. DNA sequence analysis showed that these phage covered most of the human factor X gene, but were again lacking the exon encoding the 5' untranslated region of the m R N A and residues - 40 to - 18 of the leader peptide. Analysis of the other 32 positives showed that the missing exon was not present in these phage either. Despite screening two other genomic phage libraries, Fung (1988) was unable to isolate the genomic DNA encoding the putative exon I. The reason(s) why both Leytus et al and Fung were unable to isolate this region of D N A is unknown. The overall organization of the human factor X gene is shown in Figure 5. The intron/exon boundaries conform to the consensus sequences reported by Mount (1982). The introns vary in size from about 950 base pairs (intron C) to about 7400 base pairs (intron B). The exons vary in size from 25 base pairs (exon 3) to 612 base pairs (exon 8); however, the average size (176 base pairs) is consistent with data previously collected on eukaryotic exon lengths (Naora and Deacon, 1982). As has been found with other genes, the introns in the factor X gene do not occur randomly (Figure 5). Instead, the introns appear to separate exons coding for discrete regions of the polypeptide chain. Thus, the first exon

909

M O L E C U L A R BIOLOGY OF FACTOR X

prothrombin [] ~1 I] I-I [] [ ] ~

[~ ~ r-~ ~

[---1 r~

D

S

[]

I-~

I-~ I'

[]

[]

~

I~ I'

'

factor X :::] ~'1 0 []

[]

~"

~ I?

s I]

factor IX 13 ~

I1 []

' I~'~A D

protein CIIl-1 ~ 1 1

D

factorW,' a

S

S

[]

Figure 5. Schematic representation of the genes coding for the vitamin K-dependent clotting factors. Only the exons are shown. The 5' untranslated regions are denoted by the solid bars, protein-encoding regions by the open bars, and 3' untranslated regions by the slashed bars. The -y-carboxyglutamic acid-containing region is denoted by % the kringles by K and K2, the epidermal growth factor-like regions by E, the codons for the active site residues by D (aspartic acid), H (histidine) and S (serine), the propeptide protease cleavage sites by ?, the activation sites by ~ , and the sites giving rise to the two-chain forms of factor X and protein C by ~. The scale at the bottom represents 200 base pairs. Modified from Fung and MacGillivray (1988). References are given in the text.

probably encodes the 5' untranslated region and the signal peptide, and exon 2 encodes the presumed propeptide and the ~-carboxyglutamic acid region. Exon 3 includes a stretch of eight amino acids that link this calcium binding region to the EGF-like regions, which are encoded by exons 4 and 5. Exon 6 encodes a connecting region including the activation peptide, while exons 7 and 8 encode the serine protease domain of factor Xa. Figure 5 also shows the organization of the genes for several vitamin K-dependent proteins, including factor X (Leytus et al, 1986a; Fung, 1988), prothrombin (Degen and Davie, 1987; Irwin et al, 1988), factor IX (Anson et al, 1984; Yoshitake et al, 1985), factor VII (O'Hara et al, 1987), and protein C (Foster et al, 1985; Plutzky et al, 1986). Because of the similarity in structure of the factor X, factor VII, factor IX, and protein C polypeptides, it is not surprising that the gene organizations of these proteins are very similar. Indeed, introns occur in corresponding positions in these genes, with the exception of introns I and II of the protein C gene. Intron I occurs in the 5' untranslated region and appears to be unique. Intron II has been displaced by 6 base pairs, and appears to be an example of intron sliding (see Irwin et al (1988) for a full discussion). These data suggest that the genes for factors IX, X and VII, and protein C have evolved relatively recently by means of gene duplication events. Interestingly, exons 1-3 of the prothrombin gene have a similar organization to the other vitamin K-dependent clotting factors (Figure 5), although the remainder of the prothrombin gene (including the homologous serine protease region) differs in organization from these other genes. This has been taken to suggest that ancient gene duplications gave rise to multiple serine protease genes that have subsequently diverged to give the

910

R.T.A.

MACG1LLIVRAY A N D M. R. F U N G

classes found today. More recent gene duplications have given rise to the closely related gene families found today, such as the gene family consisting of factors X, IX, VII and protein C. The homology in exons 1-3 suggests that this region has been mobile in the genome, possible by an exon shuffling mechanism (see Irwin et al (1988) for a full discussion).

CHROMOSOMAL LOCALIZATION Several groups have localized the gene for human factor X to chromosome 13. Previous cytogenetic studies had correlated factor X levels with the number of copies of 13q34 in individuals with rearrangements of this region (Pfeiffer et al, 1982; de Grouchy et al, 1984). These studies also showed that the human factor VII gene mapped to this same region. Using a combination of Southern blot analysis of human/rodent cell hybrids, dosage studies on members of a family segregating a chromosome 13 abnormality, and in situ hybridization studies, the human factor X gene was mapped to the 13q34 region (Rocchi et al, 1985; Scambler and Williamson, 1985; Gilgenkrantz et al, 1986; Royle et al, 1986). The linkage of the factor X gene with the factor VII gene within this region has not been reported yet. It is interesting to note that despite the similarity in the organizations of the genes for factors IX, X, VII and protein C, the factor IX gene is located on the X chromosome (Camerino et al, 1984) and the protein C gene is found on chromosome 2 (Kato et al, 1988; Long et al, 1988). Thus, although the duplication events occurred recently enough for the overall intron/exon organization to be still retained, there has been sufficient time for genetic cross-overs to occur such that the factor X and protein C genes have been dispersed in the genome away from the site of the original (presumably tandem) duplication.

RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLPs) IN T H E H U M A N F A C T O R X GENE

RFLPs for several restriction endonucleases have been found in the human factor X gene. Hay et al (1986) found RFLPs for Eco RI and Pst I; the RFLPs, which arein linkage disequilibrium with each other, occur with a frequency of 0.83/0.17 in Caucasians of European descent. A RFLP for TaqI has been reported by Jaye et al (1985); the alleles occur with frequencies of 0.77 and 0.23. Two RFLPs for BclI have been reported by Scambler and Williamson (1986); the alleles occur with frequencies of 0.88/0.12 and 0.87/0.13. Hassan et al (1988) found additional RFLPs for HindlII and PvulI. The HindlII RFLP occurs with a frequency of 0.95/0.05; four polymorphic alleles were identified with Pvu II, occurring with frequencies of 0.72/0.13/0.085/0.057. Hassan et al (1988) also investigated five factor X-deficient families by Southern blot analysis, but could find no evidence of gross gene deletions or rearrangements. Presumably, these factor X deficiencies result from point mutations in the genome.

MOLECULAR BIOLOGY OF FACTOR X

911

None of the RFLPs reported to date reside within the protein coding regions of the factor X gene, as the polymorphic sites were not detected in the cDNA sequences. With the exception of the PstI and E c o R I RFLPs noted above, no linkage disequilibrium has been associated with any of the reported RFLPs; thus, despite their low frequencies, the polymorphisms may be useful in carrier diagnosis in families with factor X deficiency. EXPRESSION OF FACTOR X

Factor X is synthesized in the liver, and is subsequently secreted into the blood (Giddings, 1984). Graves et al (1982) showed that the rat hepatoma cell line H-35 synthesized and secreted a single-chain factor X. Using rapid immunochemical isolation techniques, these authors showed that 40% of rat plasma factor X was in the single-chain form, suggesting that extracellular processing may occur. Graves et al (1982) also showed that the secretion of factor X from H-35 cells was inducible with prothrombin fragments. As it had been shown previously that secretion of prothrombin from H-35 cells was also inducible by its degradation fragments (Graves et al, 1981), Graves et al proposed that there may be a common control mechanism for regulating the levels of the vitamin K-dependent proteins in plasma. However, these results have not been confirmed using other hepatoma cell lines. The human hepatoma cell line HepG2 also secretes single-chain factor X (Fair and Bahnak, 1984). The secretion of factor X by these cells was decreased by warfarin, an inhibitor of the carboxylation reaction. Bahnak et al (1987) used a partial cDNA for human factor X to measure steady-state m R N A levels in HepG2 cells, using an actin cDNA as a control. These authors found no changes in factor X mRNA levels when cells were treated with either vitamin K or warfarin, suggesting that the effects of these two drugs on factor X synthesis must be on post-translational events. REFERENCES Anson DS, Choo KH, Rees D J G et al (1984) The genestructure of human anti-haemophilic factor IX. EMBO Journal 3: 1053-1060. Aronson DL, Mustafa AJ & Mushinski JF (1969) Purification of human factor X and comparison of peptide maps of human factor X and prothrombin. Biochimica et Biophysica Acta 188: 25-30. Bahnak BR, Howk R, Morrissey JH et al (1987) Steady state levels of factor X mRNA in liver and HepG2 cells. Blood 69" 224-230. Bajaj SP & Mann KG (1973) Simultaneous purification of bovine prothrombin and factor X. Journal of Biological Chemistry 248: 7729-7741. Beckmann ILl, Schmidt R J, Santerre RF, Plutzky J, Crabtree G R & Long GL (1985) Structure and evolution of a 461 amino acid human protein C precursor and its messenger RNA based upon the DNA sequence of cloned liver cDNA. Nucleic Acids Research 13: 52335247. Blobel G, Walter P, Chang CN, Goldman BM, Erickson A H & Lingappa R (1979) Translocation of proteins across membranes: the signal hypothesis and beyond. In Hopkin C R & Duncan CJ (eds) Secretory Mechanisms, vol. 33, pp 9-36. London: Cambridge University Press.

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Blomquist MC, Hunt LT & Barker NC (1984) Vaccina virus 19-kilodalton protein: relationship to several mammalian proteins, including two growth factors. Proceedings of the National Academy of Sciences USA 81: 7363-7367. Borowski M, Furie BC, Bauminger S & Furie B (1986) Prothrombin requires two sequential metal-dependent conformational transitions to bind phospholipid. Journal of Biological Chemistry 261- 1496%14975. Burgess AI & Esnouf MP (1985) Post-translational modifications in the blood clotting systems. In Freedman RB & Hawkins HC (eds) The Enzymology of Post-Translational Modification of Proteins, vol. 2, pp 299-337. London: Academic Press. Camerino G, Grzeschik KH, Jaye M e t al (1984) Regional localization of the human X chromosome and polymorphism of the coagulation factor IX gene (hemophilia B locus). Proceedings of the National Academy of Sciences USA 81: 498-502. Cook RM, Wilkinson AJ, Baron M e t al (1987) The solution structure of human epidermal growth factor. Nature 327: 339-341. Cool DE, Edgell C-JS, Louie GV, Zoller MJ, Brayer GD & MacGillivray RTA (1985) Characterization of human blood coagulation factor XII cDNA. Journal of Biological Chemistry 260: 12666--13676. Dahlback ]3, Lundwall A & Stenflo J (1986) Primary structure of bovine vitamin K-dependent protein S. Proceedings of the National Academy of Sciences USA 83: 41994203. Davie EW, Fujikawa K, Kurachi K & Kisiel W (1979) The role of serine proteases in the blood coagulation cascade. Advances in Enzymology 48: 277-318. Degen SJF & Davie EW (1987) Nucleotide sequence of the gene for human prothrombin. Biochemistry 26: 6165-6177. Degen SJF, MacGillivray RTA & Davie EW (1983) Characterization of the complementary deoxyribonucleic acid and gene coding for human prothrombin. Biochemistry 22: 20872097. DiScipio RG, Hermodson MA, Yates SG & Davie EW (1977) A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S. Biochemistry 16: 698-706. Diuguid DL, Rabiet MJ, Furie BC, Liebman HA & Furie B (1986) Molecular basis of hemophilia B: a defective enzyme due to an unprocessed propeptide is caused by a point mutation in the factor IX precursor. Proceedings of the National Academy of Sciences USA 83: 5803-5807. Doolittle RF, Feng DF & Johnson MS (1984) Computer-based characterization of epidermal growth factor precursor. Nature 307: 558-560. Dupe R & Howell R (1973) The purification and properties of factor X from pig serum and its role in hypercoagulability in vivo. Biochemical Journal 133: 311-321. Enfield PL, Ericsson LH, Fujikawa D, Walsh KA, Neurath H & Titani K (1980) Amino acid sequence of the light chain of bovine factor X1 (Stuart factor). Biochemistry 19: 659-667. Esmon CT (1987) The regulation of natural anticoagulant pathways. Science 235: 1348-1352. Esmon CT, Owen WG & Jackson CM (1974) A plausible mechanism for prothrombin activation by factor Xa, factor Va, phospholipid and calcium ions. Journal of Biological Chemistry 249: 8045-8947. Esmon NL, DeBault LE & Esmon CT (1983) Proteolytic formation and properties of gammacarboxyglutamic acid-domainless protein C. Journal of Biological Chemistry 258: 55485553. Esnouf MP, Lloyd PH & Jesty J (1973) A method for the simultaneous isolation of factor X and prothrombin. Biochemical Journal 131: 181-789. Fair DS & Bahnak BR (1984) Human hepatoma cells secrete single chain factor X, prothrombin, and antithrombin III. Blood 64: 194-204. Fernlund P & Stenflo J (1983) Beta-hydroxyaspartic acid in vitamin K-dependent proteins.

Journal of Biological Chemistry 258:t2509-12512. Fiddes JC & Goodman HM (1980) The cDNA for the beta-subunit of human chorionic gonadotropin suggests evolution of a gene by readthrough into the 3'-untranslated region. Nature 286" 684-687. Foster DC, Yoshitake S & Davie EW (1985) The nucleotide sequence of the gene for human protein C. Proceedings of the National Academy of Sciences USA 82: 4673-4677. Foster DC, Rudinski MS, Schach BG et al (1987) Propeptide of human protein C is necessary for ~-carboxylation. Biochemistry 26: 7003-7011.

MOLECULAR BIOLOGY OF FACTOR X

913

Fujikawa K, Legaz ME & Davie EW (1972a) Bovine factors X1 and X2 (Stuart factor), isolation and characterization. Biochemistry 11: 4882-4891. Fujikawa K, Legaz ME & Davie EW (1972b) Bovine factor X1 (Stuart factor): mechanisms of activation by a protein from Russell's viper venom. Biochemistry 11: 4892--4899. Fujikawa K, Coan MH, Legaz ME & Davie EW (1974) The mechanism of activation of bovine factor X (Stuart factor) by intrinsic and extrinsic pathways. Biochemistry 13: 5290--5299. Fujikawa K, Titani K & Davie EW (1975) Activation of bovine factor X (Stuart factor): conversion of activation of factor Xaa to factor Xal3. Proceedings of the NationalAcademy of Sciences USA 72: 3359-3363. Fung MR (1988) Molecular genetics of blood coagulationfactor X. PhD dissertation, University of British Columbia, Vancouver, Canada. Fung MR & MacGillivray RTA (1988) Organization of the genes coding for the vitamin K-dependent clotting factors. In Suttie JW (ed.) CurrentAdvances in Vitamin K research, pp 143-151. New York: Elsevier. Fung MR, Campbell RM & MacGillivray RTA (1984) Blood coagulation factor X mRNA encodes a single polypeptide containing a pre-pro leader sequence. NucleicAcids Research 12: 4481-4492. Fung MR, Hay CW & MacGillivray RTA (1985) Characterization of an almost full-length cDNA for human blood coagulation factor X. Proceedings of the National Academy of Sciences USA 82: 3591-3595. Furie B & Furie BC (1988) The molecular basis for blood coagulation. Cell 53: 505-518. Furie B, Bing DH, Feldmann RJ, Robison DJ, Burnier JP & Furie BC (1982) Computergenerated models of blood coagulation factor Xa, factor tXa, and thrombin based upon structural homology with other serine proteases. Journal of Biological Chemistry 257: 3875-3882. Geddes VA, LeBonniec BF, Louie GV, Brayer GD, Thompson AR & MacGillivray RTA (1989) A moderate form of hemophilia B is caused by a novel mutation in the protease domain of factor IXvanco.... . Journal of Biological Chemistry 264: 4689-4697. Giddings JC (1984) Coagulation factors synthesis by the liver with special reference to factor VIII and factor V. In Fondu P & Thijs O (eds) Hemostatic Failure in Liver Disease, pp 5-21. Boston: Martinus Nijhoff Publishers. Gilgenkrantz S, Briquel M-E, Andre E et al (1986) Structural genes of coagulation factor VII and factor X located on 13q34. Annals of Genetics 29: 32-35. Gordon JI, Budelier KA, Sims HF, Edelstein C, Scanu AM & Strauss AW (1983) Biosynthesis of human preproapolipoprotein A-IL. Journal of Biological Chemistry 258: 14054-14059. Graves CB, Munns TW, Carlisle TL, Grant GA & Strauss AW (1981) Induction of prothrombin synthesis by prothrombin fragments. Proceedings of the National Academy of Sciences USA 78: 4772-4776. Graves CB, Munns TW, Willingham AK & Strauss AW (1982) Rat factor X is synthesized as a single chain precursor inducible by prothrombin fragments. Journal of Biological Chemistry 257:13108-13113. Greenwald I (1985) lin-12, a nematode homeotic gene, is homologous to a set of mammalian proteins that includes epidermal growth factor. Cell 43: 583-590. de Grouchy J, Dautzenberg MD, Turleau C, Beguin S & Chavin-Colin F (1984) Regional mapping of clotting factors VII and X to 13q34: expression of factor VII through chromosome 8. Human Genetics 66: 230-233. Hassan HJ, Guerriero R, Chelucci C et al (1988) Multiple polymorphic sites in factor X locus. Blood 71: 1353-1356. Hay CW, Robertson KA, Fung MR & MacGillivray RTA (1986) RFLPs for PstI and EcoRI in the blood dotting factor X gene. Nucleic Acids Research 14: 5118. yon Heijne G (1983) Patterns of amino acids near signal-sequence cleavage sites. European Journal of Biochemistry 133: 17-21. von Heijne G (1985) Signal sequences the limits of variation. Journal of Molecular Biology 184: 99-105. Hojrup P & Magnusson S (1987) Disulfide bridges of bovine factor X. BiochemicalJournal 245: 887-892. Hoskins J, Norman DK, Beckmann ILl & Long GL (1987) Cloning and characterization of human liver cDNA encoding a protein S precursor. Proceedings of the National Academy of Sciences USA 84: 349-353.

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Hougie C, Barrow EM & Graham JB (1957) Stuart clotting defect I: segregation of a hereditary hemorrhagic state from the heterogenous group heretofore called 'Stable Factor' (SPCA, proconvertin, factor VII) deficiency. Journal of Clinical Investigation 36: 485-496. Irwin DM, Robertson KA & MacGillivray RTA (1988) Structure and evolution of the bovine prothrombin gene. Journal of Molecular Biology 200: 31-45. Jackson CW (1972) Characterization of two glycoprotein variants of bovine factor X and demonstration that the factor X zymogen contains two polypeptide chains. Biochemistry 11: 4873-4882. Jackson CM (1981) Biochemistry of prothrombin activation. In Bloom AL & Thomas DP (eds) Haemostasis and Thrombosis, pp 140-162. Edinburgh: Churchill Livingstone. Jackson CM (1984) Factor X. In Spaet TH (ed.) Progress in Hemostasis and Thrombosis, vol. 7, pp 55-109. Orlando: Grune & Stratton. Jackson CM & Brenkle GM (1980) Divalent ion binding to bovine prothrombin fragment 1 and its consequences. In Mann KG & Taylor FB (eds) The Regulation of Coagulation, pp 11-18. Amsterdam: Elsevier/North-Holland. Jackson CM & Hanahan DJ (1968) Studies on bovine factor X. II. Observations on some alterations in zone electrophoretic and chromatographic behaviour occurring during purification. Biochemistry 7: 4506--4517. Jackson CM & Nemerson Y (1980) Blood coagulation. Annual Reviews of Biochemistry 49: 765-811. Jaye M, de la Salle H, Schamber F et al (1983) Isolation of anti-haemophilic factor IX cDNA using a unique 52-base synthetic oligonucleotide probe deduced from the amino acid sequence of bovine factor IX. Nucleic Acids Research 11: 2325-2335. Jaye M, Ricca G, Kaplan R et al (1985) Polymorphism associated with the human coagulation factor X (F10) gene. Nucleic Acids Research 13: 8286. Jesty J (1978) The inhibition of activated bovine coagulation factors X and VII by antithrombin. III. Archives of Biochemistry and Biophysics 185: 165-173. Jesty J & Esnouf MP (1973) The preparation of activated factor X and its action on prothrombin. Biochemical Journal 131: 791-799. Jesty J & Nemerson Y (1974) Purification of factor VII from bovine plasma. Reaction with tissue factor and activation of factor X. Journal of Biological Chemistry 249: 509-515. Jesty J, Spencer AK & Nemerson Y (1974) The mechanism of activation of factor X. Journal of Biological Chemistry 249: 5614-5622. Jorgensen MJ, Cantor MA, Furie BC, Brown CL, Shoemaker CB & Furie B (1987) Recognition site directing vitamin K-dependent gamma-earboxylation residues on the propeptide of factor IX. Cell 48: 185-191. Kato A, Miura O, Sumi Y & Aoki N (1988) Assignment of the human protein C gene (PROC) to chromosome region 2q14-q21 by in situ hybridization. Cytogenetics and Cell Genetics 47: 46--47. Kaul RK, Hildebrand B, Roberts S & Jagadeeswaran P (1986) Isolation and characterization of human blood-coagulation factor X cDNA. Gene 41: 311-314. Knobloch JE & Suttie JW (1987) Vitamin K-dependent carboxylase: control of enzyme activity by the 'propeptide' region of factor X. Journal of Biological Chemistry 262: 15334-15337. Kosow DP (1976) Purification and activation of human factor X: cooperative effect of Ca + + on the activation reaction. Thrombosis Research 9: 565-573. Kraut J (1977) Serine proteases: structure and mechanism of catalysis. Annual Reviews of Biochemistry 46: 331-358. Kurachi K & Davie EW (1982) Isolation and characterization of a cDNA coding for human factor IX. Proceedings of the National Academy of Sciences USA 79" 6461-6464. Kurachi K, Fujikawa K, Schmer G & Davie EW (1976) Inhibition of bovine factor IXa and factor Xa~ by antithrombin III. Biochemistry 15: 373-377. Leytus SP, Chung DW, Kisiel W, Kurachi K & Davie EW (1984) Characterization of a cDNA coding for human factor X. Proceedings of the National Academy of Sciences USA 81: 3699-3702. Leytus SP, Foster DC, Kurachi K & Davie EW (1986a) Gene for factor X: a blood coagulation factor whose gene organization is essentially identical with that of factor IX and protein C. Biochemistry 25: 5098-5102. Leytus SP, Kurachi K, Sakariassen KS & Davie EW (1986b) Nucleotide sequence of the cDNA coding for human complement Clr. Biochemistry 25: 4855-4863.

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