Proexosite-1 on Prothrombin Is a Factor Va-dependent Recognition Site for the Prothrombinase Complex
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m302707200
ISSN1083-351X
AutoresLin Chen, Likui Yang, Alireza R. Rezaie,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoAlthough the contribution of basic residues of exosite-1 to the catalytic function of thrombin has been studied extensively, their role in the specificity of prothrombin recognition by factor Xa in the prothrombinase complex (factor Xa, factor Va, phosphatidylcholine/phosphatidylserine vesicles, and Ca2+) has not been examined. In this study, we prepared several mutants of prethrombin-1 (prothrombin lacking Gla and Kringle-1 domains) in which basic residues of this site (Arg35, Lys36, Arg67, Lys70, Arg73, Arg75, and Arg77 in chymotrypsinogen numbering) were individually substituted with a Glu. Following expression in mammalian cells and purification to homogeneity, these mutants were characterized with respect to their ability to function as zymogens for both factor Xa and the prothrombinase complex. Factor Xa by itself exhibited similar catalytic activity toward both the wild type and mutant substrates; however, its activity in the prothrombinase complex toward most of mutants was severely impaired. Further kinetic studies in the presence of Tyr63-sulfated hirudin-(54–65) peptide suggested that although the peptide inhibits the prothrombinase activation of the wild type zymogen with a K D of 0.5–0.7 μm, it is ineffective in inhibiting the activation of mutant zymogens (K D = 2–30 μm). These results suggest that basic residues of proexosite-1 on prothrombin are factor Va-dependent recognition sites for factor Xa in the prothrombinase complex. Although the contribution of basic residues of exosite-1 to the catalytic function of thrombin has been studied extensively, their role in the specificity of prothrombin recognition by factor Xa in the prothrombinase complex (factor Xa, factor Va, phosphatidylcholine/phosphatidylserine vesicles, and Ca2+) has not been examined. In this study, we prepared several mutants of prethrombin-1 (prothrombin lacking Gla and Kringle-1 domains) in which basic residues of this site (Arg35, Lys36, Arg67, Lys70, Arg73, Arg75, and Arg77 in chymotrypsinogen numbering) were individually substituted with a Glu. Following expression in mammalian cells and purification to homogeneity, these mutants were characterized with respect to their ability to function as zymogens for both factor Xa and the prothrombinase complex. Factor Xa by itself exhibited similar catalytic activity toward both the wild type and mutant substrates; however, its activity in the prothrombinase complex toward most of mutants was severely impaired. Further kinetic studies in the presence of Tyr63-sulfated hirudin-(54–65) peptide suggested that although the peptide inhibits the prothrombinase activation of the wild type zymogen with a K D of 0.5–0.7 μm, it is ineffective in inhibiting the activation of mutant zymogens (K D = 2–30 μm). These results suggest that basic residues of proexosite-1 on prothrombin are factor Va-dependent recognition sites for factor Xa in the prothrombinase complex. Prothrombin is a vitamin K-dependent serine protease zymogen that is proteolytically converted to thrombin by the prothrombinase complex (factor Xa, cofactor Va, negatively charged phospholipid vesicles, and Ca2+) in the final step of the blood clotting cascade (1Mann K.G. Jenny R.J. Krishnaswamy S. Annu. Rev. Biochem. 1988; 57: 915-956Crossref PubMed Scopus (448) Google Scholar, 2Furie B. Furie B.C. Cell. 1988; 53: 505-518Abstract Full Text PDF PubMed Scopus (980) Google Scholar, 3Rosing J. Tans G. Govers-Riemslag J.W.P. Zwaal R.F.A.Z. Hemker H.C. J. Biol. Chem. 1980; 255: 274-283Abstract Full Text PDF PubMed Google Scholar, 4Nesheim M.E. Kettner C. Shaw E. Mann K.G. J. Biol. Chem. 1981; 256: 6537-6540Abstract Full Text PDF PubMed Google Scholar, 5Pei G. Powers D.D. Lentz B.R. J. Biol. Chem. 1993; 268: 3226-3233Abstract Full Text PDF PubMed Google Scholar, 6Tracy P.B. Rohrbach M.S. Mann K.G. J. Biol. Chem. 1983; 258: 7264-7267Abstract Full Text PDF PubMed Google Scholar). Factor Xa specifically catalyzes the cleavage of two peptide bonds after the two basic residues, Arg273 and Arg322, to convert prothrombin to thrombin (1Mann K.G. Jenny R.J. Krishnaswamy S. Annu. Rev. Biochem. 1988; 57: 915-956Crossref PubMed Scopus (448) Google Scholar). Although factor Xa by itself can catalyze the cleavage of both peptide bonds on the substrate, its catalytic efficiency is improved by greater than 5 orders of magnitude when it is assembled into the prothrombinase complex (1Mann K.G. Jenny R.J. Krishnaswamy S. Annu. Rev. Biochem. 1988; 57: 915-956Crossref PubMed Scopus (448) Google Scholar, 3Rosing J. Tans G. Govers-Riemslag J.W.P. Zwaal R.F.A.Z. Hemker H.C. J. Biol. Chem. 1980; 255: 274-283Abstract Full Text PDF PubMed Google Scholar). Results of several kinetic studies have indicated that such a dramatic improvement in the rate of prothrombin activation by the prothrombinase complex is derived from an ∼100-fold decrease in the apparent K m and a greater than 1000-fold enhancement in the k cat of the activation reaction (3Rosing J. Tans G. Govers-Riemslag J.W.P. Zwaal R.F.A.Z. Hemker H.C. J. Biol. Chem. 1980; 255: 274-283Abstract Full Text PDF PubMed Google Scholar, 7Nesheim M.E. Taswell J.B. Mann K.G. J. Biol. Chem. 1979; 254: 10952-10962Abstract Full Text PDF PubMed Google Scholar). The improvement in the apparent K m of the activation reaction is mediated through the Ca2+-dependent assembly of both prothrombin and factor Xa on negatively charged membrane surfaces via their N-terminal Gla domains (3Rosing J. Tans G. Govers-Riemslag J.W.P. Zwaal R.F.A.Z. Hemker H.C. J. Biol. Chem. 1980; 255: 274-283Abstract Full Text PDF PubMed Google Scholar, 7Nesheim M.E. Taswell J.B. Mann K.G. J. Biol. Chem. 1979; 254: 10952-10962Abstract Full Text PDF PubMed Google Scholar). However, the improvement in the k cat of the activation reaction is believed to arise from the factor Va-mediated protein-protein interaction between factor Xa and prothrombin in the prothrombinase complex (7Nesheim M.E. Taswell J.B. Mann K.G. J. Biol. Chem. 1979; 254: 10952-10962Abstract Full Text PDF PubMed Google Scholar, 8Boskovic D.S. Giles A.R. Nesheim M.E. J. Biol. Chem. 1990; 265: 10497-10505Abstract Full Text PDF PubMed Google Scholar).The mechanism of the factor Va-mediated protein-protein interaction that improves the catalytic efficiency of factor Xa in the prothrombinase complex is under intensive investigation. Based on recent kinetic data, it has been hypothesized that factor Va binding to factor Xa allosterically exposes a secondary binding site”exosite“remote from the catalytic pocket on the protease that is a specific recognition site for interaction with the prothrombin lacking the Gla and both Kringle-1 and -2 domain (prethrombin-2) 1The abbreviations used are: prethrombin-2, prothrombin lacking the Gla and both Kringle-1 and -2 domains; prethrombin-1, prothrombin mutant in which the Gla and Kringle-1 domains have been deleted by recombinant DNA methods; TM, thrombomodulin; TM4–6, TM fragment containing the epidermal growth factor-like domains 4, 5, and 6; GPR-pNA, N-p-tosyl-Gly-Pro-Arg-p-nitroanilide; TBS, Tris-buffered saline; PC, phosphatidylcholine; PS, phosphatidylserine; Hir, hirudin.1The abbreviations used are: prethrombin-2, prothrombin lacking the Gla and both Kringle-1 and -2 domains; prethrombin-1, prothrombin mutant in which the Gla and Kringle-1 domains have been deleted by recombinant DNA methods; TM, thrombomodulin; TM4–6, TM fragment containing the epidermal growth factor-like domains 4, 5, and 6; GPR-pNA, N-p-tosyl-Gly-Pro-Arg-p-nitroanilide; TBS, Tris-buffered saline; PC, phosphatidylcholine; PS, phosphatidylserine; Hir, hirudin. portions of the substrate (9Betz A. Krishnaswamy S. J. Biol. Chem. 1998; 273: 10709-10718Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In support of this hypothesis, it has been demonstrated that factor Va enhances the k cat of both prothrombin and prethrombin-2 activation by factor Xa to a similar extent (9Betz A. Krishnaswamy S. J. Biol. Chem. 1998; 273: 10709-10718Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 10Rezaie A.R. Yang L. Biochim. Biophys. Acta. 2001; 1528: 167-176Crossref PubMed Scopus (33) Google Scholar). The putative cofactor-mediated interaction sites, on either the protease or the substrate, have not been identified. However, an active site inhibited thrombin and a C-terminal fragment derived from the cleavage of thrombin by chymotrypsin at Trp148 have been shown to inhibit competitively the activation of prethrombin-2 by factor Xa in the prothrombinase complex (9Betz A. Krishnaswamy S. J. Biol. Chem. 1998; 273: 10709-10718Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Based on such results, it has been hypothesized that the factor Va-mediated factor Xa interactive site on prothrombin is located on the protease domain that does not include either the fibrinogen recognition (exosite-1) or the heparin-binding site (exosite-2) (9Betz A. Krishnaswamy S. J. Biol. Chem. 1998; 273: 10709-10718Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar).Other studies (11Dharmawardana K.R. Bock P.E. Biochemistry. 1998; 37: 13143-13152Crossref PubMed Scopus (49) Google Scholar, 12Dharmawardana K.R. Olson S.T. Bock P.E. J. Biol. Chem. 1999; 274: 18635-18643Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) have demonstrated recently that the heavy chain of factor Va contains a binding site for exosite-1 of thrombin and that this site is also present in a low affinity precursor state “proexosite-1” on prothrombin. Thus, an alternative hypothesis for the mechanism of the cofactor function of factor Va is that the cofactor in the prothrombinase complex may provide a binding site for direct interaction with the proexosite-1 of prothrombin (13Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16428-16434Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 14Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16435-16442Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In support of this hypothesis, it has been demonstrated that the factor Va-mediated acceleration of prothrombin activation by the prothrombinase complex can be specifically inhibited by an exosite-1-specific peptide ligand derived from the C-terminal domain of the leech inhibitor, hirudin (14Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16435-16442Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar).The exosite-1 of thrombin plays a pivotal role in the catalytic function of thrombin. This site has several basic residues that can interact with nearly all natural substrates, inhibitors, and cofactors of thrombin including fibrinogen, factors V and VIII, PAR-1, heparin cofactor II, and thrombomodulin (TM) (15Fenton II, J.W. Thromb. Haemostasis. 1995; 74: 493-498Crossref PubMed Scopus (46) Google Scholar, 16Rydel T.J. Ravichandran K.G. Tulinsky A. Bode W. Huber R. Roitsch C. Fenton II, J.W. Science. 1990; 249: 277-280Crossref PubMed Scopus (635) Google Scholar, 17Esmon C.T. Thromb. Haemostasis. 1993; 70: 1-5Crossref Scopus (51) Google Scholar, 18Liu L. Vu T.H. Esmon C.T. Coughlin S.R. J. Biol. Chem. 1991; 266: 16977-16980Abstract Full Text PDF PubMed Google Scholar, 19Ye J. Liu L. Esmon C.T. Johnson A.E. J. Biol. Chem. 1992; 267: 11023-11028Abstract Full Text PDF PubMed Google Scholar). Previous mutagenesis of basic residues of exosite-1 has been demonstrated to dramatically impair the reactivity of thrombin with all of these macromolecules (20Wu Q. Sheehan J.P. Tsiang M. Lentz S.R. Birktoft J.J. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6775-6779Crossref PubMed Scopus (107) Google Scholar, 21Tsiang M. Jain A.K. Dunn K.E. Rojas M.E. Leung L.L.K. Gibbs C.S. J. Biol. Chem. 1995; 270: 16854-16863Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 22Myles T. Yun T.H. Hall S.W. Leung L.L.K. J. Biol. Chem. 2001; 276: 25143-25149Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 23Myles T. Yun T.H. Leung L.L.K. Blood. 2002; 100: 2820-2826Crossref PubMed Scopus (58) Google Scholar). Despite an extensive characterization of basic residues of exosite-1 in thrombin, the contribution of these residues to the specificity of prothrombin recognition by factor Xa in the prothrombinase complex has not been studied. In this study, we substituted all basic residues of this site including Arg35, Lys36, Arg67, Lys70, Arg73, Arg75, and Arg77 (chymotrypsinogen numbering (24Bode W. Mayr I. Baumann U. Huber R. Stone S.R. Hofsteenge J. EMBO J. 1989; 8: 3467-3475Crossref PubMed Scopus (817) Google Scholar)) with Glu in individual constructs in prethrombin-1 (prothrombin lacking both the Gla and Kringle-1 domains) and expressed the mutant proteins in mammalian cells as described (25Rezaie A.R. Protein Sci. 1998; 7: 349-357Crossref PubMed Scopus (29) Google Scholar). Following purification to homogeneity, the properties of mutant proteins were analyzed with respect to their ability to function as zymogens for factor Xa in both the absence and presence of factor Va on phospholipid vesicles. It was discovered that whereas factor Xa activates the wild type and prethrombin-1 mutant zymogens with a comparable rate in the absence of factor Va, the protease exhibits a dramatic catalytic defect toward mutant substrates in the presence of the cofactor. Further kinetic studies in the presence of the proexosite-specific peptide, Tyr63-sulfated hirudin-(54–65) (Hir-(54–65)(SO3- ), suggested that the hirudin peptide inhibits the prothrombinase activation of prethrombin-1 with a K D of 0.7 μm. However, the competitive inhibitory effect of the hirudin peptide in the prothrombinase activation of mutants was impaired at varying degrees, which correlated well with the extent of impairments observed in the activation of mutant zymogens. These results suggest that basic residues of proexosite-1 are specific recognition sites for factor Xa in the prothrombinase complex. Interestingly, we also discovered that the epidermal growth factor-like domains 4–6 of TM (TM4–6) can inhibit the prothrombinase activation of prethrombin-1 with a K D of 0.5 μm. The possible physiological significance of this finding is discussed.EXPERIMENTAL PROCEDURESConstruction and Expression of Mutant Proteins—The expression of wild type prethrombin-1 (prothrombin lacking both Gla and Kringle-1 domains) and prethrombin-2 (prothrombin lacking Gla, Kringle-1, and Kringle-2 domains) by the pNUT-PL2 expression/purification vector system in baby hamster kidney cells has been described previously (10Rezaie A.R. Yang L. Biochim. Biophys. Acta. 2001; 1528: 167-176Crossref PubMed Scopus (33) Google Scholar, 26Rezaie A.R. Biochemistry. 1997; 36: 7437-7446Crossref PubMed Scopus (20) Google Scholar). Prethrombin-1 mutants in the chymotrypsinogen numbering system (24Bode W. Mayr I. Baumann U. Huber R. Stone S.R. Hofsteenge J. EMBO J. 1989; 8: 3467-3475Crossref PubMed Scopus (817) Google Scholar): Arg35 → Glu and Ala (R35E and R35A), Lys36 → Glu (K36E), Arg67 → Glu (R67E), Lys70 → Glu (K70E), Arg73 → Glu (R73E), Arg75 → Glu (R75E), and Arg77 → Glu (R77E) were prepared by PCR mutagenesis methods as described (26Rezaie A.R. Biochemistry. 1997; 36: 7437-7446Crossref PubMed Scopus (20) Google Scholar). Following confirmation of accuracy of mutations by DNA sequencing, the mutant constructs were expressed in baby hamster kidney cells using the same expression/purification vector system described above. All derivatives were purified to homogeneity by immunoaffinity chromatography using the Ca2+-dependent monoclonal antibody, HPC4, as described (26Rezaie A.R. Biochemistry. 1997; 36: 7437-7446Crossref PubMed Scopus (20) Google Scholar). TM4–6 was expressed in HEK293 cells and purified to homogeneity as described (27Rezaie A.R. Esmon C.T. J. Biol. Chem. 1992; 267: 26104-26109Abstract Full Text PDF PubMed Google Scholar).Human plasma proteins, antithrombin, and factors Va and Xa were purchased from Hematologic Technologies Inc. (Essex Junction, VT). Phospholipid vesicles containing 80% phosphatidylcholine and 20% phosphatidylserine (PC/PS) were prepared as described (28Smirnov M.D. Esmon C.T. J. Biol. Chem. 1994; 269: 816-819Abstract Full Text PDF PubMed Google Scholar). The chromogenic substrate S2238 was purchased from Kabi Pharmacia/Chromogenix (Franklin, OH). The chromogenic substrate N-p-tosyl-Gly-Pro-Arg-p-nitroanilide (GPR-pNA) and Tyr63-sulfated hirudin-(54–65) (Hir-(54–65)(SO3- ) were purchased from Sigma.Prethrombin-1 Activation—The initial rate of prethrombin-1 activation by factor Xa was studied in both the absence and presence of factor Va on PC/PS vesicles. In the absence factor Va, the time course of activation of each prethrombin-1 derivative (2 μm) by factor Xa (5 nm) was measured at room temperature in 0.1 m NaCl, 0.02 m Tris-HCl (pH 7.5) containing 0.1 mg/ml bovine serum albumin, 0.1% polyethylene glycol 8000, and 2.5 mm CaCl2 (TBS/Ca2+). At different time intervals, small aliquots of activation reactions were transferred to wells of a 96-well assay plate containing 20 mm EDTA, and the rate of thrombin generation was determined from the cleavage of S2238 (100 μm) at 405 nm by a V max Kinetic Microplate Reader (Molecular Devices, Menlo Park, CA). The concentration of thrombin generated was determined from standard curves prepared from the cleavage rate of S2238 (100 μm) by known concentrations of wild type and mutant thrombins. In the presence of factor Va, the concentration dependence of prethrombin-1 (0.3–20 μm) activation by human factor Xa (0.05–1 nm) in complex with a saturating concentration of human factor Va (30 nm) was measured on PC/PS vesicles (35 μm) in TBS/Ca2+. After 1–30 min of incubation at room temperature, the reactions were terminated by addition of EDTA, and the initial rate of thrombin generation was measured from the cleavage rate of S2238 as described above. The apparent K m and k cat values for prethrombin-1 activation were calculated from the Michaelis-Menten equation. It was ensured that the factor Va concentration (30 nm) was in excess in all activation reactions. Thus, the factor Va (0.1–10 nm) concentration dependence of activation reactions by factor Xa (0.2 nm) were carried out on PC/PS vesicles (35 μm) in TBS/Ca2+ using 1 μm wild type or mutant prethrombin-1. The K d(app) values were calculated from hyperbolic dependence of activation rates on the concentrations of factor Va as described (29Rezaie A.R. He X. Biochemistry. 2000; 39: 1817-1825Crossref PubMed Scopus (63) Google Scholar). In all reactions, it was ensured that less than 10% of prethrombin-1 was activated at all concentrations of the substrates.Prethrombin-1 Activation in the Presence of Hir-(54–65)-(SO3- )—The inhibitory effect of the hirudin peptide on the kinetics of prethrombin-1 and prethrombin-2 activation by both factor Xa and prothrombinase was studied. Thus, the rate of activation of each prethrombin-1 derivative (1 μm) by factor Xa alone (5 nm) or factor Xa (0.05–1 nm) in complex with factor Va (30 nm) on PC/PS vesicles was monitored in the presence of increasing concentrations of the hirudin peptide in the same TBS/Ca2+ buffer system. The concentration of thrombin generated in each reaction was calculated from standard curves as described above except that GPR-pNA was used as the chromogenic substrate because the cleavage rate of this substrate has been reported not to be affected by the occupancy of exosite-1 by the hirudin peptide (18Liu L. Vu T.H. Esmon C.T. Coughlin S.R. J. Biol. Chem. 1991; 266: 16977-16980Abstract Full Text PDF PubMed Google Scholar). To simplify comparisons of the hirudin peptide dependence of the activation reactions, the data for all activation reactions were normalized to maximal thrombin generation in the absence of the peptide. The dissociation constants (K D) for the interaction of the hirudin peptide with prethrombin-1 was calculated from Equations 1 and 2 as described (14Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16435-16442Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Vobs=(Vlim-Vo)[Pre-1·Hir][Pre-1]o+Vo(Eq. 1) [Pre-1·Hir]=(KD+[Pre-1]o+[Hir]o)-((KD+[Pre-1]o+[Hir]o)2-4[Pre-1]o[Hir]o)2(Eq. 2) V obs is the observed initial rate of prethrombin-1 (Pre-1) activation; V lim is the limiting rate at a saturating hirudin peptide (Hir) concentration; V o is the initial rate of activation in the absence of the hirudin peptide; K D is the dissociation constant for the hirudin peptide binding to prethrombin-1; and [Pre-1·Hir] represents the prethrombin-1-hirudin peptide complex concentration. The quadratic binding equation assumes that the concentration of prethrombin-1 in complex with either factor Xa or factor Va is small enough to be neglected under experimental conditions where [Pre-1]o is in excess of the initial concentrations of both the enzyme and the cofactor (14Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16435-16442Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar).Cleavage of Chromogenic Substrates—Approximately 1 mg of each prethrombin-1 derivative was activated to completion, and thrombin mutants were purified by cation exchange chromatography on a Mono S column using a linear gradient of 0.1–0.6 m NaCl as described (25Rezaie A.R. Protein Sci. 1998; 7: 349-357Crossref PubMed Scopus (29) Google Scholar). The concentrations of thrombin mutants were determined by their absorbance at 280 nm, assuming a molecular mass of 36.6 kDa and an extinction coefficient (E1cm1&x0025; ) of 17.1 and by stoichiometric titrations with a known concentration of antithrombin as described (25Rezaie A.R. Protein Sci. 1998; 7: 349-357Crossref PubMed Scopus (29) Google Scholar). The steady-state kinetics of hydrolysis of S2238 (1.5–100 μm) by thrombin derivatives (0.5 nm) were studied in TBS/Ca2+ as described (25Rezaie A.R. Protein Sci. 1998; 7: 349-357Crossref PubMed Scopus (29) Google Scholar). The rate of hydrolysis was measured at 405 nm at room temperature in a V max Kinetic Microplate Reader as described above. The K m and k cat values for the chromogenic substrate hydrolysis were calculated from the Michaelis-Menten equation, and the catalytic efficiencies were expressed as the ratios of k cat/K m.RESULTSExpression and Purification of Recombinant Proteins—Wild type and mutant prethrombin-1 derivatives were expressed in baby hamster kidney cells using the pNUT-PL2 expression/purification vector system as described previously (26Rezaie A.R. Biochemistry. 1997; 36: 7437-7446Crossref PubMed Scopus (20) Google Scholar). All recombinant proteins were purified to homogeneity by an immunoaffinity chromatography using the Ca2+-dependent monoclonal antibody HPC4 as described (26Rezaie A.R. Biochemistry. 1997; 36: 7437-7446Crossref PubMed Scopus (20) Google Scholar). Except for elevated K m(app) values, factor Xa in the prothrombinase complex activates both prethrombin-1 and prethrombin-2 with similar and normal V max values (10Rezaie A.R. Yang L. Biochim. Biophys. Acta. 2001; 1528: 167-176Crossref PubMed Scopus (33) Google Scholar, 30Krishnaswamy S. Walker R.K. Biochemistry. 1997; 36: 3319-3330Crossref PubMed Scopus (38) Google Scholar). Thus, these truncated substrates are ideal reagents for probing the extent that protein-protein interactions in the prothrombinase complex contribute to the high specificity of the catalytic reaction.Amidolytic Activity—Because the zymogenic properties of prethrombin-1 mutants by the prothrombinase complex are studied from the initial rate of thrombin generation in an amidolytic activity assay, it was essential to determine the kinetic parameters for the cleavage of the chromogenic substrate S2238 by mutant thrombins. Thus, following activation and purification on a Mono S column, the concentration of the active site of mutants was determined by stoichiometric titration with antithrombin as described (25Rezaie A.R. Protein Sci. 1998; 7: 349-357Crossref PubMed Scopus (29) Google Scholar). The concentration of active enzymes correlated well with the concentration of substrates determined based on their absorbance at 280 nm (within 90–100%). Kinetic parameters for the hydrolysis of S2238 are presented in Table I. With the exception of the K70E mutant, which exhibited a dramatically impaired K m value, all other mutants cleaved S2238 with kinetic constants that were similar to those observed for wild type thrombin. These results strongly suggest that with the exception of K70E, the mutations did not adversely affect the folding, charge stabilizing system, or the reactivity of the catalytic pockets of mutant enzymes. The interaction of p-aminobenzamidine with the K70E mutant was also impaired ∼3-fold (K i = 38 and 107 μm for wild type and mutant thrombin, respectively). Thus, the conformation of the P3-P1 binding pocket of the K70E mutant must have been altered.Table IKinetic constants for the cleavage of the chromogenic substrate S2238 by thrombin derivativesKmkcatk cat/KmμMs-1μM s-1WT6.4 ± 0.499.7 ± 7.916 ± 2R35E4.9 ± 0.4108.1 ± 10.222 ± 4R35A3.9 ± 0.897.7 ± 7.925 ± 7K36E4.9 ± 0.293.3 ± 4.819 ± 2R67E4.9 ± 0.4113.0 ± 10.923 ± 4K70E205 ± 1592.5 ± 2.20.45 ± 0.04R73E5.1 ± 1.196.7 ± 4.919 ± 5R75E6.3 ± 0.4123.8 ± 11.620 ± 3R77E5.8 ± 0.5116.4 ± 11.920 ± 4 Open table in a new tab Prethrombin-1 Activation by Factor Xa and the Prothrombinase Complex—The initial rate of activation of prethrombin-1 derivatives by factor Xa was studied both in the absence and presence of factor Va on PC/PS vesicles. As shown in Fig. 1A, factor Xa exhibited similar activity toward both the wild type and mutant zymogens in the absence of factor Va. However, the activation of all mutants by factor Xa in the prothrombinase complex was markedly impaired (Fig. 1B). The concentration dependence of zymogen activation indicated that the prothrombinase complex activated wild type prethrombin-1 with apparent K m and k cat values of 9.1 ± 0.7 μm and 1529 ± 56 mol/min/mol, respectively. In the case of mutants, these values could only be determined for the R75E mutant (17.1 ± 2.8 μm and 913 ± 87 mol/min/mol) because the rate of thrombin generation was dramatically impaired and remained linear for up to 20 μm substrate (the highest concentration available) for all other mutants (Fig. 1B).Because the saturation of the initial rate of activation was not feasible with mutant substrates for the kinetic analysis, the overall extent of impairment with each proexosite-1 mutant residue was estimated from the initial rate of activation of a limiting concentration of each mutant substrate (at least 10-fold below K m values) in the presence of increasing concentrations of factor Va on PC/PS vesicles. A saturable dependence of thrombin generation on factor Va concentrations was observed yielding both K d(app) values for the factor Xa-factor Va interaction and the maximum rate of thrombin generation with all derivatives. The results presented in Table II suggested that although the K d(app) for the enzyme-cofactor interaction was independent of the substrate, the second-order rate of thrombin generation was impaired at varying degrees with all mutant substrates. The most impairment (75–150-fold) was observed with R67E and K70E mutants. The activation of all other mutants was impaired 4–18-fold (Table II). These results clearly suggest that none of the mutant residues under study interact with factor Xa in the absence of factor Va; however, they are critical recognition sites for the cofactor and/or the protease in the prothrombinase complex.Table IIKinetic constants for interaction of factor Xa with factor Va (Kd(app)), maximum rate of thrombin generation (V), and dissociation constants (KD) for binding of Hir-(54–65)-(SO3-) to prethromnin-1 derivativesK d(app)VKDnMnM/min/nMμMWT1.5 ± 0.2104.2 ± 4.50.7 ± 0.1R35E1.1 ± 0.114.8 ± 0.22.3 ± 0.2K36E0.9 ± 0.17.0 ± 0.319.3 ± 8.4R67E0.7 ± 0.21.4 ± 0.1NDK70E0.8 ± 0.20.7 ± 0.1NDR73E1.5 ± 0.111.0 ± 0.433.3 ± 14.9R75E1.2 ± 0.128.8 ± 1.05.1 ± 0.8R77E0.8 ± 0.15.8 ± 0.211.6 ± 3.8 Open table in a new tab Inhibition of Activation by Hir-(54–65)-(SO3- )—The Tyr63-sulfated hirudin peptide is proven to be a useful probe to analyze the interaction of different ligands with exosite-1 of thrombin (15Fenton II, J.W. Thromb. Haemostasis. 1995; 74: 493-498Crossref PubMed Scopus (46) Google Scholar, 31Liu L. Ye J. Johnson A.E. Esmon C.T. J. Biol. Chem. 1991; 266: 23632-23636Google Scholar). It was demonstrated recently (14Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16435-16442Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) that the hirudin peptide can also interact with the proexosite-1 of the substrate prothrombin. Thus, to determine whether the loss of specific interactions of proexosite-1 residues with prothrombinase accounts for the defective catalytic reactions with different prethrombin-1 derivatives, the initial rates of activation of mutant substrates were measured in the presence of increasing concentrations of the hirudin peptide. The results presented in Fig. 2A and Table II indicated that the hirudin peptide inhibits the initial rate of wild type prethrombin-1 activation with a K D of 0.7 μm. However, the ability of the peptide to inhibit activation of prethrombin-1 mutants was impaired at varying degrees. Interestingly, the degree of impairment in K D values correlated well with the degree of impairment in the observed activation rates of mutant substrates. Thus, the hirudin peptide exhibited no inhibitory effect toward the prothrombinase activation of either R67E or K70E mutants which were also ineffect
Referência(s)