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• W2053186743 abstract "The activation of factor X by the extrinsic coagulation system results from the action of an enzyme complex composed of factor VIIa bound to tissue factor on phospholipid membranes in the presence of calcium ions (extrinsic Xase complex). Proteolysis at the Arg52-Ile53 peptide bond in the heavy chain of factor X leads to the formation of the serine protease, factor Xa, and the generation of a heavily glycosylated activation peptide comprising residues 1–52 of the heavy chain. The role of the activation peptide region in mediating substrate recognition and cleavage by the extrinsic Xase complex is unclear. The protease Agkistrodon rhodostoma hydrolase γ (ARHγ), from the venom of the Malayan pit viper, was used to selectively cleave human factor X in the activation peptide region. Three cleavage sites were found within this region and gave products designated Xdes1-34, Xdes1-43, and Xdes1-49. The products were purified to yield Xdes 1–49 and a mixture of Xdes 1–34 and Xdes 1–43. Reversed phase high pressure liquid chromatography analysis indicated that the cleaved portion of the activation peptide was likely removed during purification. All cleaved species were inactive and could be completely activated to factor Xa by the extrinsic Xase complex or by a purified activator from Russell's viper venom. Steady state kinetic studies using tissue factor reconstituted into membranes yielded essentially equivalent kinetic constants for the activation of intact factor X and the cleaved derivatives under a wide range of conditions. Since Xdes 1–49 lacks all but three residues of the activation peptide and is devoid of the carbohydrate present in this region, the data suggest that the specific recognition of human factor X by the extrinsic Xase complex is not achieved through specific interactions with residues 1–49 of the activation peptide or with carbohydrate structures attached to these residues. The activation of factor X by the extrinsic coagulation system results from the action of an enzyme complex composed of factor VIIa bound to tissue factor on phospholipid membranes in the presence of calcium ions (extrinsic Xase complex). Proteolysis at the Arg52-Ile53 peptide bond in the heavy chain of factor X leads to the formation of the serine protease, factor Xa, and the generation of a heavily glycosylated activation peptide comprising residues 1–52 of the heavy chain. The role of the activation peptide region in mediating substrate recognition and cleavage by the extrinsic Xase complex is unclear. The protease Agkistrodon rhodostoma hydrolase γ (ARHγ), from the venom of the Malayan pit viper, was used to selectively cleave human factor X in the activation peptide region. Three cleavage sites were found within this region and gave products designated Xdes1-34, Xdes1-43, and Xdes1-49. The products were purified to yield Xdes 1–49 and a mixture of Xdes 1–34 and Xdes 1–43. Reversed phase high pressure liquid chromatography analysis indicated that the cleaved portion of the activation peptide was likely removed during purification. All cleaved species were inactive and could be completely activated to factor Xa by the extrinsic Xase complex or by a purified activator from Russell's viper venom. Steady state kinetic studies using tissue factor reconstituted into membranes yielded essentially equivalent kinetic constants for the activation of intact factor X and the cleaved derivatives under a wide range of conditions. Since Xdes 1–49 lacks all but three residues of the activation peptide and is devoid of the carbohydrate present in this region, the data suggest that the specific recognition of human factor X by the extrinsic Xase complex is not achieved through specific interactions with residues 1–49 of the activation peptide or with carbohydrate structures attached to these residues. INTRODUCTIONHuman coagulation factor X is a serine protease zymogen, which circulates in blood as a two-chain molecule. It is composed of a 303-residue heavy chain, which is covalently linked by a disulfide bond to a 139-residue light chain. The light chain contains a γ-carboxyglutamic acid-containing domain that is critical for the binding of calcium ions and phospholipid (3Jackson C.M. Prog. Hemost. Thromb. 1984; 7: 55-109PubMed Google Scholar). This subunit also contains two domains homologous to the epidermal growth factor precursor that are considered important for interdomain or protein-protein interactions (4Valcarce C. Holmgren A. Stenflo J. J. Biol. Chem. 1994; 269: 26011-26016Abstract Full Text PDF PubMed Google Scholar, 5Hertzberg M.S. Ben Tal O. Furie B. Furie B.C. J. Biol. Chem. 1992; 267: 14759-14766Abstract Full Text PDF PubMed Google Scholar, 6Rezaie A.R. Neuenschwander P.F. Morrissey J.H. Esmon C.T. J. Biol. Chem. 1993; 268: 8176-8180Abstract Full Text PDF PubMed Google Scholar). The heavy chain contains the serine protease module, and glycosylation occurs exclusively in this chain (3Jackson C.M. Prog. Hemost. Thromb. 1984; 7: 55-109PubMed Google Scholar).The proteolytic activation of factor X via either the intrinsic or the extrinsic pathways is a requisite step of the blood coagulation cascade (7Nemerson Y. Blood. 1988; 71: 1-8Crossref PubMed Google Scholar, 8Ruf W. Edgington T.S. FASEB J. 1994; 8: 385-390Crossref PubMed Scopus (216) Google Scholar, 9Mann K.G. Jenny R.J. Krishnaswamy S. Annu. Rev. Biochem. 1988; 57: 915-956Crossref PubMed Scopus (448) Google Scholar). The enzyme complex responsible for factor X activation by the extrinsic pathway is composed of factor VIIa, tissue factor (TF), 1The abbreviations used are: TFtissue factorARHγ and ARHβA. rhodostoma hydrolase γ and β, respectivelyCAPS3-(cyclohexylamino)propanesulfonic acidEBLelderberry bark lectinMES2-(N-morpholino)ethanesulfonic acidPEG-8000polyethylene glycol 8000PCPSvesicles composed of 75% (w/w) L-α-phosphatidylcholine and 25% (w/w) L-α-phosphatidylserineRVVX-CPfactor X activator from Russell's viper venomS2238D-phenylalanyl-pipecoyl-arginyl p-nitroanilideSpXacyclohexylglycyl-glycyl-arginyl p-nitroanilideTween 20polyoxyethylenesorbitan monolaurateXdes 1–49human factor X lacking residues 1–49 of the heavy chainXdes 1–34, Xdes 1–43a mixture of proteolyzed forms of factor X lacking residues 1–34 and 1–43 of the heavy chainPAGEpolyacrylamide gel electrophoresisHPLChigh pressure liquid chromatographyMAL IIMaackia amurensis lectin II. phospholipid, and calcium and is referred to as the extrinsic Xase complex. The formation of the protease, factor Xa, results from specific cleavage of the Arg52-Ile53 peptide bond 2Since factor X circulates as a two-chain molecule, we prefer to use a residue numbering system that begins at the NH2 terminus of each polypeptide chain. As proteolysis is confined to the heavy chain of factor X, the sites of cleavage are designated relative to the NH2 terminus of the heavy chain. Therefore, the species Xdes 1–49 is equivalent to the derivative of porcine factor X designated Xdes 143–191 by Duffy and Lollar (1Duffy E.J. Lollar P. J. Biol. Chem. 1992; 267: 7821-7827Abstract Full Text PDF PubMed Google Scholar). in the heavy chain of the zymogen with the generation of a 52-residue glycosylated activation peptide (10Di Scipio R.G. Hermodson M.A. Davie E.W. Biochemistry. 1977; 16: 5253-5260Crossref PubMed Scopus (115) Google Scholar). The activation peptide of human factor X is heavily glycosylated with carbohydrate linked to Asn39 and Asn49 in human factor X (3Jackson C.M. Prog. Hemost. Thromb. 1984; 7: 55-109PubMed Google Scholar, 10Di Scipio R.G. Hermodson M.A. Davie E.W. Biochemistry. 1977; 16: 5253-5260Crossref PubMed Scopus (115) Google Scholar). The presence of additional O-linked carbohydrate sites at Thr17 and Thr29 has also been recently reported (11Inoue K. Morita T. Eur. J. Biochem. 1993; 218: 153-163Crossref PubMed Scopus (39) Google Scholar). Further proteolysis at Arg237-Gly238 of the heavy chain is responsible for the removal of a 65-residue glycopeptide from the carboxyl terminus of the heavy chain (12Mertens K. Bertina R.M. Biochem. J. 1980; 185: 647-658Crossref PubMed Scopus (51) Google Scholar). This reaction evidently results from autoproteolysis by Xa and converts the α-form of either factor X or Xa to the β form. No difference in function has yet been observed for these two forms of the zymogen or protease (13Pryzdial E.L.G. Kessler G.E. Blood. 1994; 84 (abstr.): 194Crossref Google Scholar).The high specificity of the cleavage reaction that leads to factor X activation by the VIIa-TF complex raises the possibility that the Arg52-Ile53 peptide bond in the substrate is specifically recognized as a result of extended site interactions between the substrate and the enzyme complex involving residues additional to the P1 3Nomenclature of Schechter and Berger (2Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4730) Google Scholar). arginine. An alternative possibility is that these types of extended interactions are limited but that the scissile bond in the substrate is highly accessible, leading to selective cleavage at this site. The first possibility is consistent with the developing structural information regarding other highly specific serine proteases of coagulation (14Stubbs M.T. Bode W. Trends Biochem. Sci. 1995; 20: 23-28Abstract Full Text PDF PubMed Scopus (176) Google Scholar, 15Padmanabhan K. Padmanabhan K.P. Tulinsky A. Park C.H. Bode W. Huber R. Blankenship D.T. Cardin A.D. Kisiel W. J. Mol. Biol. 1993; 232: 947-966Crossref PubMed Scopus (401) Google Scholar) as well as with the demonstration of residues in TF that are important for factor X binding and cleavage by the extrinsic Xase complex (16Ruf W. Miles D.J. Rehemtulla A. Edgington T.S. J. Biol. Chem. 1992; 267: 6375-6381Abstract Full Text PDF PubMed Google Scholar, 17Ruf W. Miles D.J. Rehemtulla A. Edgington T.S. J. Biol. Chem. 1992; 267: 22206-22210Abstract Full Text PDF PubMed Google Scholar). The second possibility is consistent with the ability of several different proteases to specifically catalyze the same cleavage reaction, including the intrinsic Xase complex of coagulation (18Fujikawa K. Coan M.H. Legaz M.E. Davie E.W. Biochemistry. 1974; 13: 5290-5299Crossref PubMed Scopus (109) Google Scholar), the factor X-activating enzyme from Russell's viper venom (RVVX-CP) (10Di Scipio R.G. Hermodson M.A. Davie E.W. Biochemistry. 1977; 16: 5253-5260Crossref PubMed Scopus (115) Google Scholar), and trypsin (18Fujikawa K. Coan M.H. Legaz M.E. Davie E.W. Biochemistry. 1974; 13: 5290-5299Crossref PubMed Scopus (109) Google Scholar). The VIIa-TF complex also catalyzes the activation of factor IX to IXa with kinetic constants comparable with the activation of factor X (19Komiyama Y. Pedersen A.H. Kisiel W. Biochemistry. 1990; 29: 9418-9425Crossref PubMed Scopus (149) Google Scholar, 20Zur M. Nemerson Y. J. Biol. Chem. 1980; 255: 5703-5707Abstract Full Text PDF PubMed Google Scholar). There are differences between the sequences beyond P1-P3 surrounding the two scissile bonds in factor IX and the bond cleaved in factor X (10Di Scipio R.G. Hermodson M.A. Davie E.W. Biochemistry. 1977; 16: 5253-5260Crossref PubMed Scopus (115) Google Scholar, 21Di Scipio R.G. Kurachi K. Davie E.W. J. Clin. Invest. 1978; 61: 1528-1538Crossref PubMed Scopus (173) Google Scholar). The data therefore imply that macromolecular substrate specificity of the extrinsic Xase complex is not completely explained by the recognition of extended sequences surrounding the scissile bond by the VIIa-TF complex.Recent studies have suggested an important role for carbohydrate structures present in the activation peptide in the recognition and cleavage of factor X by the extrinsic Xase complex (11Inoue K. Morita T. Eur. J. Biochem. 1993; 218: 153-163Crossref PubMed Scopus (39) Google Scholar, 22Sinha U. Wolf D.L. J. Biol. Chem. 1993; 268: 3048-3051Abstract Full Text PDF PubMed Google Scholar). Sinha and Wolf (22Sinha U. Wolf D.L. J. Biol. Chem. 1993; 268: 3048-3051Abstract Full Text PDF PubMed Google Scholar), observed large effects on the rate and extent of human factor X activation by both the extrinsic and intrinsic Xase complexes following enzymatic removal of the sialic acids. Removal of both sialic acids and O-linked carbohydrates in bovine factor X led to a 5-fold decrease in the catalytic efficiency for activation by the VIIa-TF complex, which could be partially restored by subsequent removal of N-linked sugars (11Inoue K. Morita T. Eur. J. Biochem. 1993; 218: 153-163Crossref PubMed Scopus (39) Google Scholar). In contrast, more recent studies have shown that removal of sialic acids has no detectable effect on factor X activation (23Bharadwaj D. Harris R.J. Kisiel W. Smith K.J. J. Biol. Chem. 1995; 270: 6537-6542Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar).Evidence for a functional role for the activation peptide domain of factor X has also been developed in studies of Xa formation by the intrinsic Xase complex (IXa-VIIIa-membranes) (1Duffy E.J. Lollar P. J. Biol. Chem. 1992; 267: 7821-7827Abstract Full Text PDF PubMed Google Scholar, 24Iino M. Takeya H. Nishioka J. Nakagaki T. Tamura K. Suzuki K. J. Biochem. (Tokyo). 1994; 116: 335-340Crossref PubMed Scopus (13) Google Scholar). Duffy and Lollar (1Duffy E.J. Lollar P. J. Biol. Chem. 1992; 267: 7821-7827Abstract Full Text PDF PubMed Google Scholar), prepared a proteolytic derivative of factor X by the action of a protease (ARHγ) isolated from the venom of the Malayan pit viper, Agkistrodon rhodostoma (25Lollar P. Parker C.G. Kajenski P.J. Litwiller R.D. Fass D.N. Biochemistry. 1987; 26: 7627-7636Crossref PubMed Scopus (26) Google Scholar). This protease cleaves porcine factor X in the activation peptide region three residues NH2-terminal to the activation site, yielding a zymogen that can be completely activated to factor Xa upon subsequent cleavage by RVVX-CP or the intrinsic Xase complex (1Duffy E.J. Lollar P. J. Biol. Chem. 1992; 267: 7821-7827Abstract Full Text PDF PubMed Google Scholar). Initial velocity studies by Duffy and Lollar (1Duffy E.J. Lollar P. J. Biol. Chem. 1992; 267: 7821-7827Abstract Full Text PDF PubMed Google Scholar) indicated that prior cleavage at this site in the activation peptide reduced the rate of activation of porcine factor X by the VIIIa-IXa complex. The reduced catalytic efficiency observed with the porcine factor X derivative lacking most of the activation peptide could be attributed to a decreased kcat compared with native factor X observed in the presence of the cofactor, factor VIIIa. More recent studies have also indicated an important role for the activation peptide in substrate recognition by the human VIIIa-IXa complex (24Iino M. Takeya H. Nishioka J. Nakagaki T. Tamura K. Suzuki K. J. Biochem. (Tokyo). 1994; 116: 335-340Crossref PubMed Scopus (13) Google Scholar). Product inhibition studies of the VIIIa-IXa complex by factor Xa in the presence and absence of the isolated activation peptide have indicated that the activation peptide may mediate interactions between factor X and factor IXa in a carbohydrate-dependent manner within the intrinsic Xase complex (24Iino M. Takeya H. Nishioka J. Nakagaki T. Tamura K. Suzuki K. J. Biochem. (Tokyo). 1994; 116: 335-340Crossref PubMed Scopus (13) Google Scholar).In this study, proteolytic derivatives of human factor X were prepared by cleavage with ARHγ. These derivatives contain an intact activation site but lack various portions of the activation peptide NH2-terminal to this site (Scheme 1). These species were zymogens that could be fully activated to factor Xa by either RVVX-CP or the extrinsic Xase complex. One of these truncated derivatives contained no carbohydrate attachment site, whereas the other two contained one and two sites of N-linked carbohydrate, respectively. Kinetic studies with these purified substrate analogs were used to assess the contributions of the polypeptide sequences and carbohydrate structures in the activation peptide domain to factor X activation by the extrinsic Xase complex.DISCUSSIONThe venom protease from Malayan pit viper, ARHγ, was found to cleave human factor X at multiple sites to yield derivatives of the zymogen that were differentially truncated in the activation peptide domain. These proteolytic derivatives could be completely activated to factor Xa by the action of either RVVX-CP or the VIIa-TF extrinsic Xase complex. Three cleaved species, Xdes 1–34, Xdes 1–43, and Xdes 1–49 (Scheme 1), identified by NH2-terminal sequence analysis, were further purified and used in kinetic studies to assess the possible role of structures present in the activation peptide toward the recognition and cleavage of factor X by the extrinsic Xase complex.Steady state kinetic studies comparing the activation of human factor X with the proteolytic derivatives by RVVX-CP or by the extrinsic Xase complex yielded equivalent kinetic constants (Table II). Since minor effects on Km and kcat were observed following cleavage of the activation peptide at residues 34, 43, or 49, the data suggest that the amino acid side chains or carbohydrate structures present in this region do not significantly contribute to the binding of factor X to the extrinsic Xase complex or to the rate-limiting step in catalysis that yields factor Xa. These observations imply a limited role for the NH2-terminal 49 residues in determining the cleavage of factor X by the extrinsic Xase complex.The highly specific cleavage of factor X by the extrinsic Xase complex implies that the recognition of the scissile bond may be achieved by structures in the substrate that lead to binding interactions at extended sites in factor VIIa or possibly through additional interactions with TF. Mutagenesis experiments with TF have been able to resolve residues that affect the ability of the extrinsic Xase complex to cleave protein substrates such as factor X without affecting the ability of TF to interact with factor VIIa or enhancing its activity toward peptidyl substrates (16Ruf W. Miles D.J. Rehemtulla A. Edgington T.S. J. Biol. Chem. 1992; 267: 6375-6381Abstract Full Text PDF PubMed Google Scholar, 17Ruf W. Miles D.J. Rehemtulla A. Edgington T.S. J. Biol. Chem. 1992; 267: 22206-22210Abstract Full Text PDF PubMed Google Scholar). One interpretation of these findings is that TF contains sites for substrate binding. Studies examining the ability of synthetic peptides derived from the heavy chain of factor X to inhibit factor X activation by the VIIa-TF complex have provided evidence for sites on the substrate that are removed from the scissile bond but contribute to substrate recognition by the extrinsic Xase complex (47Chattopadhyay A. Fair D.S. J. Biol. Chem. 1989; 264: 11035-11043Abstract Full Text PDF PubMed Google Scholar). More recent work has also implicated oligosaccharide structures present at the four potential glycosylation sites in the activation peptide domain of factor X (Scheme 1) as being important for factor X activation by the extrinsic Xase complex (11Inoue K. Morita T. Eur. J. Biochem. 1993; 218: 153-163Crossref PubMed Scopus (39) Google Scholar, 22Sinha U. Wolf D.L. J. Biol. Chem. 1993; 268: 3048-3051Abstract Full Text PDF PubMed Google Scholar).The present kinetic results argue against a significant contribution of the first 49 residues of the activation peptide toward factor X recognition by the extrinsic Xase complex. Thus, specific cleavage at Arg52-Ile53 in the heavy chain, which results in the conversion of factor X to factor Xa by the extrinsic Xase complex, does not apparently result from substrate-protease or substrate-cofactor interactions that involve heavy chain residues that are NH2-terminal to the P3 position. Therefore, the substrate specificity of this enzyme complex may be dictated entirely by the P1-P3 residues and/or residues COOH-terminal to the scissile bond in the heavy chain of the molecule or possibly even by structural elements in the light chain of factor X. Studies with synthetic peptidyl substrates have previously implied a role for P′ residues for efficient cleavage by the VIIa-TF complex (48Butenas S. Ribarik N. Mann K.G. Biochemistry. 1993; 32: 6531-6538Crossref PubMed Scopus (55) Google Scholar).The venom protease ARHγ has previously been shown to cleave porcine factor X and yield a product equivalent to the Xdes 1–49 derivative identified in the present work (1Duffy E.J. Lollar P. J. Biol. Chem. 1992; 267: 7821-7827Abstract Full Text PDF PubMed Google Scholar). Kinetic studies of the activation of this derivative with intact factor X by the porcine intrinsic Xase complex (IXa-VIIIa-PCPS-Ca2+) yielded the same Km but a 100-fold lower kcat, suggesting a significant contribution of the activation peptide region to substrate hydrolysis, possibly through interactions with the protease and/or the cofactor (1Duffy E.J. Lollar P. J. Biol. Chem. 1992; 267: 7821-7827Abstract Full Text PDF PubMed Google Scholar). In contrast, experimentally indistinguishable kinetic constants were obtained for the cleavage of human factor X or Xdes 1–49 by the extrinsic Xase complex. Since the activation of factor X by the extrinsic and the intrinsic Xase complexes results from the cleavage of the same peptide bond, one explanation for the results is that the determinants for the recognition and cleavage of the same substrate by the two enzyme complexes are different. Other explanations for these data include the possibility that cleavage(s) in the activation peptide region has a deleterious effect on substrate structure at distant sites that is significant in the case of porcine factor X but kinetically undetectable with human factor X. This possibility is supported by the significant decrease in kcat and in Km for activation by RVVX-CP following cleavage of porcine factor X by ARHγ. In contrast, the kinetic constants for human factor X activation by the same enzyme were essentially unaltered following cleavage by ARHγ. This interpretation would suggest that the kinetic consequences of cleavage or removal of the first 49 residues of the activation peptide region are actually related to the indirect effects of this region on structures about the scissile bond or on other domains of factor X rather than the deletion of sites directly involved in binding the protease and/or cofactor in the cognate enzyme complex.At least two other studies have provided indirect evidence suggesting the importance of the carbohydrates present in the activation peptide domain to factor X activation by the extrinsic Xase complex (11Inoue K. Morita T. Eur. J. Biochem. 1993; 218: 153-163Crossref PubMed Scopus (39) Google Scholar, 22Sinha U. Wolf D.L. J. Biol. Chem. 1993; 268: 3048-3051Abstract Full Text PDF PubMed Google Scholar). In these cases, contradictory information regarding the importance of O-linked versus N-linked carbohydrate for factor X activation by the extrinsic Xase complex has been developed following partial deglycosylation of factor X by glycosidases or by the use of specific lectins to inhibit factor X activation (11Inoue K. Morita T. Eur. J. Biochem. 1993; 218: 153-163Crossref PubMed Scopus (39) Google Scholar, 22Sinha U. Wolf D.L. J. Biol. Chem. 1993; 268: 3048-3051Abstract Full Text PDF PubMed Google Scholar). More recently, evidence has been presented to show that removal of sialic acids has no effect on factor X cleavage by the extrinsic Xase complex (23Bharadwaj D. Harris R.J. Kisiel W. Smith K.J. J. Biol. Chem. 1995; 270: 6537-6542Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar).The proteolytic derivatives of human factor X prepared in the present work (Scheme 1) have provided reagents to assess this possibility more directly. Since the kinetic constants for the activation of these derivatives by the extrinsic Xase complex were equivalent to those obtained for intact factor X, our data are not consistent with these previous suggestions of a specific role for carbohydrates linked to the activation peptide in substrate recognition by this enzyme complex (22Sinha U. Wolf D.L. J. Biol. Chem. 1993; 268: 3048-3051Abstract Full Text PDF PubMed Google Scholar). This conclusion is also supported by the observations of heterogeneity in carbohydrate structures present in the zymogen as detected by lectin binding. Although it is possible that this heterogeneity arises from subtle differences in sugars distinct from those involved in substrate recognition, it is difficult to envision how heterogeneity in specific macromolecular recognition elements could yield a substrate preparation that could be adequately characterized by a single set of kinetic constants by initial velocity measurements as well as by analysis of complete progress curves.Reversible, high affinity interactions have been documented between the fragment 1.2 activation peptide domain of prothrombin and prethrombin 2 (49Myrmel K.H. Lundblad R.L. Mann K.G. Biochemistry. 1976; 15: 1767-1773Crossref PubMed Scopus (48) Google Scholar). This interaction is apparently essential for efficient cleavage of prethrombin 2 by prothrombinase as a result of the ability of the fragment 2 domain to bind the cofactor (50Luckow E.A. Lyons D.A. Ridgeway T.M. Esmon C.T. Laue T.M. Biochemistry. 1989; 28: 2348-2354Crossref PubMed Scopus (67) Google Scholar, 51Esmon C.T. Jackson C.M. J. Biol. Chem. 1974; 249: 7791-7797Abstract Full Text PDF PubMed Google Scholar). Similarly, reversible interactions between the activation peptide and factor Xa of high affinity have also been documented following the cleavage of bovine factor X (46Furie B.C. Furie B. Gottlieb A.J. Williams W.J. Biochim. Biophys. Acta. 1974; 365: 121-132Crossref PubMed Scopus (29) Google Scholar). The lack of demonstrable kinetic differences between factor X and the derivatives obtained following cleavage by ARHγ could result from specific binding interactions via the activation peptide domain that are retained through noncovalent interactions between the cleavage products. The HPLC analyses indicate that the complete activation peptide is not present following affinity purification of factor Xa in the presence of EDTA. This is consistent with previous sedimentation velocity studies and the apparent ability of EDTA to disrupt this putative interaction (24Iino M. Takeya H. Nishioka J. Nakagaki T. Tamura K. Suzuki K. J. Biochem. (Tokyo). 1994; 116: 335-340Crossref PubMed Scopus (13) Google Scholar, 52Pryzdial E.L.G. Mann K.G. J. Biol. Chem. 1991; 266: 8969-8977Abstract Full Text PDF PubMed Google Scholar). It is not possible to unequivocally rule out the association between trace amounts of peptidyl fragments and the ARHγ-cleaved derivatives of human factor X. The data are, however, consistent with the reasonable conclusion that the fragments of the activation peptide are likely removed during purification of Xdes 1–49 and are thus unlikely to mediate interactions between the extrinsic Xase complex and the scissile bond in Xdes1-49.In summary, the present data suggest that the activation of human factor X by the extrinsic Xase complex is not significantly influenced by the polypeptide sequence or carbohydrates present in the first 49 residues of the activation peptide domain. Thus, residues NH2-terminal to the P3 position do not play a dominant role in the recognition of factor X by the extrinsic Xase complex. It remains to be established whether the macromolecular substrate specificity of the extrinsic Xase is completely determined by the P1-P3 residues or requires additional interactions with P′ residues in the substrate. INTRODUCTIONHuman coagulation factor X is a serine protease zymogen, which circulates in blood as a two-chain molecule. It is composed of a 303-residue heavy chain, which is covalently linked by a disulfide bond to a 139-residue light chain. The light chain contains a γ-carboxyglutamic acid-containing domain that is critical for the binding of calcium ions and phospholipid (3Jackson C.M. Prog. Hemost. Thromb. 1984; 7: 55-109PubMed Google Scholar). This subunit also contains two domains homologous to the epidermal growth factor precursor that are considered important for interdomain or protein-protein interactions (4Valcarce C. Holmgren A. Stenflo J. J. Biol. Chem. 1994; 269: 26011-26016Abstract Full Text PDF PubMed Google Scholar, 5Hertzberg M.S. Ben Tal O. Furie B. Furie B.C. J. Biol. Chem. 1992; 267: 14759-14766Abstract Full Text PDF PubMed Google Scholar, 6Rezaie A.R. Neuenschwander P.F. Morrissey J.H. Esmon C.T. J. Biol. Chem. 1993; 268: 8176-8180Abstract Full Text PDF PubMed Google Scholar). The heavy chain contains the serine protease module, and glycosylation occurs exclusively in this chain (3Jackson C.M. Prog. Hemost. Thromb. 1984; 7: 55-109PubMed Google Scholar).The proteolytic activation of factor X via either the intrinsic or the extrinsic pathways is a requisite step of the blood coagulation cascade (7Nemerson Y. Blood. 1988; 71: 1-8Crossref PubMed Google Scholar, 8Ruf W. Edgington T.S. FASEB J. 1994; 8: 385-390Crossref PubMed Scopus (216) Google Scholar, 9Mann K.G. Jenny R.J. Krishnaswamy S. Annu. Rev. Biochem. 1988; 57: 915-956Crossref PubMed Scopus (448) Google Scholar). The enzyme complex responsible for factor X activation by the extrinsic pathway is composed of factor VIIa, tissue factor (TF), 1The abbreviations used are: TFtissue factorARHγ and ARHβA. rhodostoma hydrolase γ and β, respectivelyCAPS3-(cyclohexylamino)propanesulfonic acidEBLelderberry bark lectinMES2-(N-morpholino)ethanesulfonic acidPEG-8000polyethylene glycol 8000PCPSvesicles composed of 75% (w/w) L-α-phosphatidylcholine and 25% (w/w) L-α-phosphatidylserineRVVX-CPfactor X activator from Russell's viper venomS2238D-phenylalanyl-pipecoyl-arginyl p-nitroanilideSpXacyclohexylglycyl-glycyl-arginyl p-nitroanilideTween 20polyoxyethylenesorbitan monolaurateXdes 1–49human factor X lacking residues 1–49 of the heavy chainXdes 1–34, Xdes 1–43a mixture of proteolyzed forms of factor X lacking residues 1–34 and 1–43 of the heavy chainPAGEpolyacrylamide gel electrophoresisHPLChigh pressure liquid chromatographyMAL IIMaackia amurensis lectin II. phospholipid, and calcium and is referred to as the extrinsic Xase complex. The formation of the protease, factor Xa, results from specific cleavage of the Arg52-Ile53 peptide bond 2Since factor X circulates as a two-chain molecule, we prefer to use a residue numbering system that begins at the NH2 terminus of each polypeptide chain. As proteolysis is confined to the heavy chain of factor X, the sites of cleavage are designated relative to the NH2 terminus of the heavy chain. Therefore, the species Xdes 1–49 is equivalent to the derivative of porcine factor X designated Xdes 143–191 by Duffy and Lollar (1Duffy E.J. Lollar P. J. Biol. Chem. 1992; 267: 7821-7827Abstract Full Text PDF PubMed Google Scholar). in the heavy chain of the zymogen with the generation of a 52-residue glycosylated activation peptide (10Di Scipio R.G. Hermodson M.A. Davie E.W. Biochemistry. 1977; 16: 5253-5260Crossref PubMed Scopus (115) Google Scholar). The activation peptide of human factor X is heavily glycosylated with carbohydrate linked to Asn39 and Asn49 in human factor X (3Jackson C.M. Prog. Hemost. Thromb. 1984; 7: 55-109PubMed Google Scholar, 10Di Scipio R.G. Hermodson M.A. Davie E.W. Biochemistry. 1977; 16: 5253-5260Crossref PubMed Scopus (115) Google Scholar). The presence of additional O-linked carbohydrate sites at Thr17 and Thr29 has also been recently reported (11Inoue K. Morita T. Eur. J. Biochem. 1993; 218: 153-163Crossref PubMed Scopus (39) Google Scholar). Further proteolysis at Arg237-Gly238 of the heavy chain is responsible for the removal of a 65-residue glycopeptide from the carboxyl terminus of the heavy chain (12Mertens K. Bertina R.M. Biochem. J. 1980; 185: 647-658Crossref PubMed Scopus (51) Google Scholar). This reaction evidently results from autoproteolysis by Xa and converts the α-form of either factor X or Xa to the β form. No difference in function has yet been observed for these two forms of the zymogen or protease (13Pryzdial E.L.G. Kessler G.E. Blood. 1994; 84 (abstr.): 194Crossref Google Scholar).The high specificity of the cleavage reaction that leads to factor X activation by the VIIa-TF complex raises the possibility that the Arg52-Ile53 peptide bond in the substrate is specifically recognized as a result of extended site interactions between the substrate and the enzyme complex involving residues additional to the P1 3Nomenclature of Schechter and Berger (2Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4730) Google Scholar). arginine. An alternative possibility is that these types of extended interactions are limited but that the scissile bond in the substrate is highly accessible, leading to selective cleavage at this site. The first possibility is consistent with the developing structural information regarding other highly specific serine proteases of coagulation (14Stubbs M.T. Bode W. Trends Biochem. Sci. 1995; 20: 23-28Abstract Full Text PDF PubMed Scopus (176) Google Scholar, 15Padmanabhan K. Padmanabhan K.P. Tulinsky A. Park C.H. Bode W. Huber R. Blankenship D.T. Cardin A.D. Kisiel W. J. Mol. Biol. 1993; 232: 947-966Crossref PubMed Scopus (401) Google Scholar) as well as with the demonstration of residues in TF that are important for factor X binding and cleavage by the extrinsic Xase complex (16Ruf W. Miles D.J. Rehemtulla A. Edgington T.S. J. Biol. Chem. 1992; 267: 6375-6381Abstract Full Text PDF PubMed Google Scholar, 17Ruf W. Miles D.J. Rehemtulla A. Edgington T.S. J. Biol. Chem. 1992; 267: 22206-22210Abstract Full Text PDF PubMed Google Scholar). The second possibility is consistent with the ability of several different proteases to specifically catalyze the same cleavage reaction, including the intrinsic Xase complex of coagulation (18Fujikawa K. Coan M.H. Legaz M.E. Davie E.W. Biochemistry. 1974; 13: 5290-5299Crossref PubMed Scopus (109) Google Scholar), the factor X-activating enzyme from Russell's viper venom (RVVX-CP) (10Di Scipio R.G. Hermodson M.A. Davie E.W. Biochemistry. 1977; 16: 5253-5260Crossref PubMed Scopus (115) Google Scholar), and trypsin (18Fujikawa K. Coan M.H. Legaz M.E. Davie E.W. Biochemistry. 1974; 13: 5290-5299Crossref PubMed Scopus (109) Google Scholar). The VIIa-TF complex also catalyzes the activation of factor IX to IXa with kinetic constants comparable with the activation of factor X (19Komiyama Y. Pedersen A.H. Kisiel W. Biochemistry. 1990; 29: 9418-9425Crossref PubMed Scopus (149) Google Scholar, 20Zur M. Nemerson Y. J. Biol. Chem. 1980; 255: 5703-5707Abstract Full Text PDF PubMed Google Scholar). There are differences between the sequences beyond P1-P3 surrounding the two scissile bonds in factor IX and the bond cleaved in factor X (10Di Scipio R.G. Hermodson M.A. Davie E.W. Biochemistry. 1977; 16: 5253-5260Crossref PubMed Scopus (115) Google Scholar, 21Di Scipio R.G. Kurachi K. Davie E.W. J. Clin. Invest. 1978; 61: 1528-1538Crossref PubMed Scopus (173) Google Scholar). The data therefore imply that macromolecular substrate specificity of the extrinsic Xase complex is not completely explained by the recognition of extended sequences surrounding the scissile bond by the VIIa-TF complex.Recent studies have suggested an important role for carbohydrate structures present in the activation peptide in the recognition and cleavage of factor X by the extrinsic Xase complex (11Inoue K. Morita T. Eur. J. Biochem. 1993; 218: 153-163Crossref PubMed Scopus (39) Google Scholar, 22Sinha U. Wolf D.L. J. Biol. Chem. 1993; 268: 3048-3051Abstract Full Text PDF PubMed Google Scholar). Sinha and Wolf (22Sinha U. Wolf D.L. J. Biol. Chem. 1993; 268: 3048-3051Abstract Full Text PDF PubMed Google Scholar), observed large effects on the rate and extent of human factor X activation by both the extrinsic and intrinsic Xase complexes following enzymatic removal of the sialic acids. Removal of both sialic acids and O-linked carbohydrates in bovine factor X led to a 5-fold decrease in the catalytic efficiency for activation by the VIIa-TF complex, which could be partially restored by subsequent removal of N-linked sugars (11Inoue K. Morita T. Eur. J. Biochem. 1993; 218: 153-163Crossref PubMed Scopus (39) Google Scholar). In contrast, more recent studies have shown that removal of sialic acids has no detectable effect on factor X activation (23Bharadwaj D. Harris R.J. Kisiel W. Smith K.J. J. Biol. Chem. 1995; 270: 6537-6542Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar).Evidence for a functional role for the activation peptide domain of factor X has also been developed in studies of Xa formation by the intrinsic Xase complex (IXa-VIIIa-membranes) (1Duffy E.J. Lollar P. J. Biol. Chem. 1992; 267: 7821-7827Abstract Full Text PDF PubMed Google Scholar, 24Iino M. Takeya H. Nishioka J. Nakagaki T. Tamura K. Suzuki K. J. Biochem. (Tokyo). 1994; 116: 335-340Crossref PubMed Scopus (13) Google Scholar). Duffy and Lollar (1Duffy E.J. Lollar P. J. Biol. Chem. 1992; 267: 7821-7827Abstract Full Text PDF PubMed Google Scholar), prepared a proteolytic derivative of factor X by the action of a protease (ARHγ) isolated from the venom of the Malayan pit viper, Agkistrodon rhodostoma (25Lollar P. Parker C.G. Kajenski P.J. Litwiller R.D. Fass D.N. Biochemistry. 1987; 26: 7627-7636Crossref PubMed Scopus (26) Google Scholar). This protease cleaves porcine factor X in the activation peptide region three residues NH2-terminal to the activation site, yielding a zymogen that can be completely activated to factor Xa upon subsequent cleavage by RVVX-CP or the intrinsic Xase complex (1Duffy E.J. Lollar P. J. Biol. Chem. 1992; 267: 7821-7827Abstract Full Text PDF PubMed Google Scholar). Initial velocity studies by Duffy and Lollar (1Duffy E.J. Lollar P. J. Biol. Chem. 1992; 267: 7821-7827Abstract Full Text PDF PubMed Google Scholar) indicated that prior cleavage at this site in the activation peptide reduced the rate of activation of porcine factor X by the VIIIa-IXa complex. The reduced catalytic efficiency observed with the porcine factor X derivative lacking most of the activation peptide could be attributed to a decreased kcat compared with native factor X observed in the presence of the cofactor, factor VIIIa. More recent studies have also indicated an important role for the activation peptide in substrate recognition by the human VIIIa-IXa complex (24Iino M. Takeya H. Nishioka J. Nakagaki T. Tamura K. Suzuki K. J. Biochem. (Tokyo). 1994; 116: 335-340Crossref PubMed Scopus (13) Google Scholar). Product inhibition studies of the VIIIa-IXa complex by factor Xa in the presence and absence of the isolated activation peptide have indicated that the activation peptide may mediate interactions between factor X and factor IXa in a carbohydrate-dependent manner within the intrinsic Xase complex (24Iino M. Takeya H. Nishioka J. Nakagaki T. Tamura K. Suzuki K. J. Biochem. (Tokyo). 1994; 116: 335-340Crossref PubMed Scopus (13) Google Scholar).In this study, proteolytic derivatives of human factor X were prepared by cleavage with ARHγ. These derivatives contain an intact activation site but lack various portions of the activation peptide NH2-terminal to this site (Scheme 1). These species were zymogens that could be fully activated to factor Xa by either RVVX-CP or the extrinsic Xase complex. One of these truncated derivatives contained no carbohydrate attachment site, whereas the other two contained one and two sites of N-linked carbohydrate, respectively. Kinetic studies with these purified substrate analogs were used to assess the contributions of the polypeptide sequences and carbohydrate structures in the activation peptide domain to factor X activation by the extrinsic Xase complex." @default.
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• W2053186743 title "Role of the Activation Peptide Domain in Human Factor X Activation by the Extrinsic Xase Complex" @default.
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