Role of the α-Helix 163-170 in Factor Xa Catalytic Activity

Abstract

Factor Xa (FXa) is a key protease of the coagulation pathway whose activity is known to be in part modulated by binding to factor Va (FVa) and sodium ions. Previous investigations have established that solvent-exposed, charged residues of the FXa α-helix 163-170 (h163-170), Arg165 and Lys169, participate in its binding to FVa. In the present study we aimed to investigate the role of the other residues of h163-170 in the catalytic functions of the enzyme. FX derivatives were constructed in which point mutations were made or parts of h163-170 were substituted with the corresponding region of either FVIIa or FIXa. Purified FXa derivatives were compared with wild-type FXa. Kinetic studies in the absence of FVa revealed that, compared with wild-type FXa, key functional parameters (catalytic activity toward prothrombin and tripeptidyl substrates and non-enzymatic interaction of a probe with the S1 site) were diminished by mutations in the NH2-terminal portion of h163-170. The defective amidolytic activity of these FXa derivatives appears to result from their impaired interaction with Na+ because using a higher Na+ concentration partially restored normal catalytic parameters. Furthermore, kinetic measurements with tripeptidyl substrates or prothrombin indicated that assembly of these FXa derivatives with an excess of FVa in the prothrombinase complex improves their low catalytic efficiency. These data indicate that residues in the NH2-terminal portion of the FVa-binding h163-170 are energetically linked to the S1 site and Na+-binding site of the protease and that residues Val163 and Ser167 play a key role in this interaction. Factor Xa (FXa) is a key protease of the coagulation pathway whose activity is known to be in part modulated by binding to factor Va (FVa) and sodium ions. Previous investigations have established that solvent-exposed, charged residues of the FXa α-helix 163-170 (h163-170), Arg165 and Lys169, participate in its binding to FVa. In the present study we aimed to investigate the role of the other residues of h163-170 in the catalytic functions of the enzyme. FX derivatives were constructed in which point mutations were made or parts of h163-170 were substituted with the corresponding region of either FVIIa or FIXa. Purified FXa derivatives were compared with wild-type FXa. Kinetic studies in the absence of FVa revealed that, compared with wild-type FXa, key functional parameters (catalytic activity toward prothrombin and tripeptidyl substrates and non-enzymatic interaction of a probe with the S1 site) were diminished by mutations in the NH2-terminal portion of h163-170. The defective amidolytic activity of these FXa derivatives appears to result from their impaired interaction with Na+ because using a higher Na+ concentration partially restored normal catalytic parameters. Furthermore, kinetic measurements with tripeptidyl substrates or prothrombin indicated that assembly of these FXa derivatives with an excess of FVa in the prothrombinase complex improves their low catalytic efficiency. These data indicate that residues in the NH2-terminal portion of the FVa-binding h163-170 are energetically linked to the S1 site and Na+-binding site of the protease and that residues Val163 and Ser167 play a key role in this interaction. Factor X (FX) 4The abbreviations used are: FX, factor X; FV, factor V; FVII, factor VII; FVIII, factor VIII; FIX, factor IX; h163-170, α-helix 163-170; RVV-X, purified FX-activating enzyme from Russell's viper venom; pd-FX, human plasma-derived factor X; wt-FX, wild-type factor X; PS, phosphatidylserine; PC, phosphatidylcholine; BSA, bovine serum albumin; S-2238, H-d-phenylalanyl-l-pipecolyl-l-arginine-para-nitroanilide dihydrochloride (H-d-Phe-Pip-Arg-pNA·2HCl); S-2765, benzyloxycarbonyl-d-arginyl-l-glycyl-l-arginine-para-nitroanilide dihydrochloride (Z-d-Arg-Gly-Arg-pNA); SpeFXa, methoxycarbonyl-cyclohexylglycyl-glycyl-arginine-para-nitroanilide dihydrochloride (CH3CO2-d-CHGly-Gly-Arg-pNA), product name Spectrozyme FXa; PAB, para-aminobenzamidine; d-FFR-CK, methanesulfonyl-d-phenyl-phenyl-arginylchloromethyl ketone; ATIII, antithrombin III. is a vitamin K-dependent, two-chain glycoprotein that plays a central role in blood coagulation. During this process, FX is activated to FXa and forms a high affinity macromolecular complex with other components of the prothrombinase complex, factor Va (FVa), negatively charged phospholipid surfaces, and calcium to activate prothrombin to thrombin (1Jackson C.M. Kim Y. Annu. Rev. Biochem. 1980; 49: 765-811Crossref PubMed Scopus (430) Google Scholar, 2Rosing J. Kim G. Govers-Riemslag J.W. Zwaal R.F. Hemker H.C. J. Biol. Chem. 1980; 255: 274-283Abstract Full Text PDF PubMed Google Scholar, 3Nesheim M.E. Kim C. Shaw E. Mann K.G. J. Biol. Chem. 1981; 256: 6537-6540Abstract Full Text PDF PubMed Google Scholar, 4Furie B. Kim B.C. Cell. 1988; 53: 505-518Abstract Full Text PDF PubMed Scopus (995) Google Scholar, 5Mann K.G. Kim M.E. Church W.R. Haley P. Krishnaswamy S. Blood. 1990; 76: 1-16Crossref PubMed Google Scholar, 6Davie E.W. Kim K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1636) Google Scholar). These macromolecular interactions lead to an increase of 5 orders of magnitude in the catalytic efficiency of FXa toward prothrombin (2Rosing J. Kim G. Govers-Riemslag J.W. Zwaal R.F. Hemker H.C. J. Biol. Chem. 1980; 255: 274-283Abstract Full Text PDF PubMed Google Scholar, 7Nesheim M.E. Kim J.B. Mann K.G. J. Biol. Chem. 1979; 254: 10952-10962Abstract Full Text PDF PubMed Google Scholar). Enhancement of the kcat of the reaction is mainly due to the cofactor function of FVa. Two basic residues of h163-170 5Amino acid sequence numbering of FXa and of all the proteases mentioned in this study is based on the three-dimensional topological equivalences with chymotrypsin. of the protease domain of FXa, namely Arg165 and Lys169, directly interact with FVa (8Rezaie A.R. J. Biol. Chem. 2000; 275: 3320-3327Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 9Rudolph A.E. Kim R. Miletich J.P. Biochemistry. 2000; 39: 2861-2867Crossref PubMed Scopus (30) Google Scholar). All known sequences from different species in this surface-exposed helix of FXa are similar. Interestingly despite being stimulated by different cofactors, the catalytic domains of other blood coagulation proteins, such as factor IXa (FIXa) and factor VIIa (FVIIa), share the same cofactor-dependent activity binding site based on the structural equivalences with chymotrypsin (10Dickinson C.D. Kim C.R. Ruf W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14379-14384Crossref PubMed Scopus (179) Google Scholar, 11Kolkman J.A. Kim P.J. Mertens K. Biochem. J. 1999; 339: 217-221Crossref PubMed Scopus (52) Google Scholar, 12Mathur A. Kim S.P. J. Biol. Chem. 1999; 274: 18477-18486Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Like other serine proteases of blood coagulation, small ligands such as calcium and sodium can allosterically modulate the activity and the specificity of FXa (13Furie B.C. Kim K.G. Furie B. J. Biol. Chem. 1976; 251: 3235-3241Abstract Full Text PDF PubMed Google Scholar, 14Orthner C.L. Kim D.P. Arch. Biochem. Biophys. 1978; 185: 400-406Crossref PubMed Scopus (47) Google Scholar, 15Sugo T. Kim I. Holmgren A. Stenflo J. J. Biol. Chem. 1984; 259: 5705-5710Abstract Full Text PDF PubMed Google Scholar, 16Persson E. Kim P.J. Stenflo J. J. Biol. Chem. 1993; 268: 22531-22539Abstract Full Text PDF PubMed Google Scholar, 17Rezaie A.R. Kim C.T. J. Biol. Chem. 1994; 269: 21495-21499Abstract Full Text PDF PubMed Google Scholar, 18Sabharwal A.K. Kim K. Tulinsky A. Mathur A. Gorka J. Bajaj S.P. J. Biol. Chem. 1997; 272: 22037-22045Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 19Underwood M.C. Kim D. Mathur A. Heyduk T. Bajaj S.P. J. Biol. Chem. 2000; 275: 36876-36884Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 20Camire R.M. J. Biol. Chem. 2002; 277: 37863-37870Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) by binding to several exposed surface loops near or remote from the catalytic pocket of the enzyme (21Brandstetter H. Kim A. Bode W. Huber R. von der Saal W. Wirthensohn K. Engh R.A. J. Biol. Chem. 1996; 271: 29988-29992Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). According to the three-dimensional of FXa K. Kim Tulinsky A. Bode W. Huber R. Kisiel W. J. Biol. 1993; PubMed Scopus Google the FXa Na+-binding site is to the catalytic pocket of the enzyme and to the FVa-binding h163-170 is that the and Na+-binding of FXa are energetically linked R.M. J. Biol. Chem. 2002; 277: 37863-37870Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, A.R. Kim J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). these that is an the Na+-binding site and FVa-binding h163-170. In the the h163-170, which is a FVa-binding and the Na+-binding site were this FXa derivatives were in which h163-170 either substituted by the corresponding region of FVIIa and or residues based on the three-dimensional of FXa, to be or to be to the Na+-binding site and The FXa derivatives were in and the purified activated forms were were from and and were from and from product name S-2238, and benzyloxycarbonyl-d-arginyl-l-glycyl-l-arginine-para-nitroanilide dihydrochloride product name S-2765, were from The methoxycarbonyl-cyclohexylglycyl-glycyl-arginine-para-nitroanilide dihydrochloride (CH3CO2-d-CHGly-Gly-Arg-pNA), product name Spectrozyme from bovine serum sodium vitamin and were from bovine and were from serum from and and were from and were from and were from and were from were from from from and and were from FX or with were from Purified human plasma-derived FX antithrombin human FX-activating enzyme from Russell's viper bovine and human thrombin were from The FVa from using used to and FX FX in is the present in of normal human were by the of Biochem. 1976; PubMed Scopus Google Scholar) using as a of FXa or and were by site FX human FX and wild-type FX Kim P.J. G. R. M.E. A. J. S. R. D. Mertens K. Blood. PubMed Scopus Google Scholar) used as a to FX residues of or and The the were by sequence using the on an to the All were using calcium and were in with and FX derivatives and were as Kim P.J. G. R. M.E. A. J. S. R. D. Mertens K. Blood. PubMed Scopus Google Scholar) and in in with 5 vitamin and and were to a concentration of and and the to and to a concentration of 5 The to the concentration to The with to a concentration of were with and FX in the and were with of of The in and The on a with of and with of FX from the with a a of FX were on and and a a with and used to of FXa that have or of the to as a FX derivatives were with to of FXa. indicated that in and with other using of the FX derivatives and by with NH2-terminal sequence the purified FX derivatives were and a The were to an and with S. The were and using an in the of the indicated that all FX derivatives the sequence and the were and In sequence that the the acid residues the of the of the the of these data indicate that the residues were of FX by FX derivatives and were activated by W. Kim E.W. Biochemistry. 1976; PubMed Scopus Google of to of to the FX derivatives were with in and of the by indicated that all FX were to their forms these the reaction by of FX derivatives were a and with and 5 The and and and by of to The were in and and the the same and FXa by and from to with to its and kcat the of the substrates and as as the of prothrombin and were compared in all in this study and to be of FXa with the interaction and or FXa derivatives as with used to the concentration of in in kinetic 5 BSA, and with of The reaction in kinetic S-2238, and the enzyme concentration from the of The of as a function of the of in a The of were from the to the of a of the activity the of a of activated enzyme from the of FXa of Kim E.W. Biochemistry. PubMed Scopus Google Scholar) to a amidolytic activity of in a of to 5 in the of a concentration of in kinetic of were used that to and the concentration to that the reaction the of or and the amidolytic activity in a the of as a function of the of and its a using the of the enzyme can be The parameters and the activity the activity and the activity the is to the concentration of by its the enzyme. of site of FXa and activated FX were by with known of FXa and enzyme from the of FXa Kim E.W. Biochemistry. PubMed Scopus Google with to in kinetic The the reaction a of were The of the reaction is to the of by the product of the of FXa derivatives and the concentration of the the of this enzyme with as The amidolytic activity in a the of as a function of a which the the to the concentration of the enzyme. of by of prothrombin by FXa and activated FX in the absence and of FVa as Kim G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in the absence of the the concentration of prothrombin by FXa with of the in the of a concentration of phospholipid in and with 5 and of were by the of Kim D. Biophys. 1979; PubMed Scopus Google Scholar). concentration by of the reaction were and the reaction in in by the amidolytic activity of the toward the in the of to activity of FXa. During the that of prothrombin to and thrombin of of thrombin in the were from a from the of by known of thrombin the same In using of the which activated FX and can activate prothrombin to thrombin in the of a concentration of phospholipid were compared as with Kim G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). phospholipid and FXa were 5 with of FVa The reaction by the of and the in 5 and of the reaction were and in the In using of prothrombin and a concentration of FVa the same as During all that of prothrombin to and thrombin concentration as measurements of prothrombin by FXa derivatives or in the prothrombinase complex were by the data to the to and used to the interaction FXa derivatives and phospholipid FVa using the of thrombin in the of different of FVa and a site by of of and by FXa derivatives were in kinetic with parameters of were using enzyme of or and from to The of in a the interaction FXa derivatives and Na+ were from the of the of on the concentration of Na+ in the of of that the amidolytic activity of FXa is on the of the reaction in the of used M.C. Kim D. Mathur A. Heyduk T. Bajaj S.P. J. Biol. Chem. 2000; 275: 36876-36884Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, A.R. Kim J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A.R. Kim Biochemistry. 2000; 39: PubMed Scopus Google Scholar). to the The data were to a site with a amidolytic activity in the of Na+ using is the of Na+ an FXa in the kinetic is the kcat of the a Na+ concentration by is the kcat in the absence of and kcat is the kcat concentration of In the of a concentration of FVa the concentration of by FXa and with of the on phospholipid a concentration measurements of by FXa derivatives or in the prothrombinase complex were by the data to the to and of FXa or in the by of FXa derivatives or in the prothrombinase complex by measurements of by enzyme using of different of as S. Kim A. Biochemistry. 1997; PubMed Scopus Google Scholar). were in kinetic using enzyme of or with or FVa and phospholipid The were to the prothrombinase with the FXa derivatives based on the The of on the concentration of FXa of FVa and established in measurements of by FXa derivatives or in the prothrombinase complex were to the to the and of FXa or in the by of of FXa derivatives or in the prothrombinase complex by by a the of FXa with FXa or in kinetic to the of the to enzyme the of FXa derivatives in the prothrombinase complex, with FXa FVa and in kinetic to enzyme activity as the of as a function of the of and its a using the of the enzyme In made in the structural of the and mutations near the with and were with D. Kim E. B. G. A.E. J. Chem. PubMed Scopus Google Scholar) based on the of K. Kim Tulinsky A. Bode W. Huber R. Kisiel W. J. Biol. 1993; PubMed Scopus Google The wild-type used as a and in an to the using to the of the to that in the of FXa in the of and and and were to the and The and wild-type were in an with from the using the with and to of using a and parameters. used to a and to a on the interactions with a and the Kim A.E. J. A.E. Huber T. P. J. Chem. A. 1999; Scopus Google Scholar). a these were to residues and and residues with a to a of or the from the and to as The of the and the were using The 2002; Scholar). FX were to by to of All FX derivatives be their and the activated were as by site h163-170 of the protease domain are in with FVa studies have indicated that of basic residues exposed the surface of h163-170 with the of the complex (8Rezaie A.R. J. Biol. Chem. 2000; 275: 3320-3327Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 9Rudolph A.E. Kim R. Miletich J.P. Biochemistry. 2000; 39: 2861-2867Crossref PubMed Scopus (30) Google Scholar, A.E. Kim R. Miletich J.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to the of other residues h163-170 on prothrombin by the this the of thrombin as a function of FVa concentration in the of an excess of phospholipid and prothrombin these prothrombin a concentration of all and the concentration in all the FXa the of thrombin and on the concentration of FVa The affinity of FVa to be and a the In of h163-170 by the corresponding residues of a on the affinity of FVa FXa. The same as (8Rezaie A.R. J. Biol. Chem. 2000; 275: 3320-3327Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 9Rudolph A.E. Kim R. Miletich J.P. Biochemistry. 2000; 39: 2861-2867Crossref PubMed Scopus (30) Google Scholar). to a and the of prothrombin by FXa derivatives in the absence and of FVa and the their interaction with FVa The kinetic kcat and were from the concentration of prothrombin by FXa derivatives on phospholipid in the absence and of concentration of FVa as The the interaction with FVa were from FVa concentration of thrombin by FXa in the of a concentration of prothrombin as are FXa FXa in a with the of thrombin as a function of of prothrombin in the of an excess of and a concentration of FVa the concentration of in these the assembly of the FXa derivatives in the prothrombinase complex partially the catalytic efficiency of the enzyme toward prothrombin to that with and In the absence of FVa a different of prothrombinase activity the FXa The of thrombin in the of of and of prothrombin in the absence of FVa. these the the same catalytic efficiency as toward prothrombin as in the of an excess of FVa and as A.E. Kim R. Miletich J.P. Biochemistry. 2000; 39: 2861-2867Crossref PubMed Scopus (30) Google the same catalytic efficiency as and In of h163-170 by the corresponding residues of and the and mutations a on the catalytic efficiency of FXa toward prothrombin in the absence of FVa and These that h163-170, the residues the part of h163-170 a on the catalytic activity of FXa toward The assembly of the defective FXa derivatives in the prothrombinase complex, to their catalytic efficiency toward of in h163-170 the catalytic of FXa, the amidolytic activity of activated forms of pd-FX, wt-FX, and all FXa derivatives were this of of namely and SpeFXa, as and of catalytic efficiency to of and These indicate that the catalytic of these is to that of and In an increase in and a kcat were and to a compared with of and These data indicate that these mutations the amidolytic activity of FXa, and that the binding site) is in the of substrates by FXa derivatives or in the prothrombinase complex of and by FXa derivatives or in the prothrombinase complex in the of and kcat were using enzyme of or and the in which the prothrombinase complex FXa derivatives were with phospholipid and of of can be are in a of the assembly of the FXa derivatives in the prothrombinase complex restored or partially restored their catalytic efficiency the to that with and with the of and in the kcat were The of the catalytic efficiency and be In the data are with the that prothrombinase complex assembly defective binding of substrates by mutations in h163-170 of FXa. of a with the S1 binding of a S1 PAB, to FXa and affinity to that of or and of an of their S1 specificity pocket were the interaction with and In assembly of the FXa derivatives in the prothrombinase complex, an excess of restored and binding to that with and with the of and In the data that prothrombinase complex assembly defective binding of substrates the S1 pocket by mutations in of by FXa derivatives or in the prothrombinase complex of FXa derivatives or in the prothrombinase complex were from studies with the The of activated forms of the FXa derivatives were The were from the of the toward their with the The are indicated with as a of the the of FXa derivatives were from the of the toward their with the The were by the in the of of of data to a binding the of and of can be are FXa FXa in a The of on R.M. J. Biol. Chem. 2002; 277: 37863-37870Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, A.R. Kim J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) have indicated that is an the FVa-binding and the Na+-binding site of FXa. the that h163-170, which is an FVa-binding is linked to the Na+-binding Na+ of the amidolytic activity toward of and FXa derivatives Na+ the amidolytic activity of and all FXa derivatives low Na+ the kcat and were compared with all the other FXa derivatives kcat to that of which is the concentration in and catalytic efficiency to that of the other FXa derivatives catalytic of the catalytic parameters as a function of Na+ concentration that Na+ diminished the and to a the kcat that Na+ the the catalytic parameters of the toward the compared with and in kcat and are as a function of Na+ concentration and the of the the Na+ interaction with and FXa derivatives in the amidolytic using a of the affinity of and Na+ compared with these the that in the of Na+ to the concentration in the of the amidolytic activity of FXa is by their impaired interaction with and as a function of Na+ of the amidolytic activity toward of FXa The catalytic parameters of the amidolytic activity toward of FXa or and or or as a function of Na+ are In data were to a site with a kcat in the of Na+ using of the interaction with Na+ are in In data were to a site with a in the of Na+ using kcat by Na+ interaction with FXa derivatives of the interaction of FXa derivatives with Na+ were from the data in the kcat as a function of Na+ of the amidolytic activity of FXa derivatives toward the were using as indicated are in a In the present we the of studies that have the basic residues of h163-170 in the of FXa by (8Rezaie A.R. J. Biol. Chem. 2000; 275: 3320-3327Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 9Rudolph A.E. Kim R. Miletich J.P. Biochemistry. 2000; 39: 2861-2867Crossref PubMed Scopus (30) Google Scholar). data that FXa derivatives with a Arg165 a in thrombin in the of small of FVa. The same to a in the and of h163-170 with the h163-170 on FVa binding is with the of FXa and the data in this and are and from the be to the of h163-170. that basic residues are the interaction of FXa with its cofactor (8Rezaie A.R. J. Biol. Chem. 2000; 275: 3320-3327Abstract Full Text Full Text PDF PubMed Scopus (115) Google and an the and charged have of the three-dimensional of FXa and (21Brandstetter H. Kim A. Bode W. Huber R. von der Saal W. Wirthensohn K. Engh R.A. J. Biol. Chem. 1996; 271: 29988-29992Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, K. Kim Tulinsky A. Bode W. Huber R. Kisiel W. J. Biol. 1993; PubMed Scopus Google Scholar) that the other residues of h163-170 are in the In Ser167 is in by which is to a and the with in this as to the function of h163-170. the absence of on prothrombinase activity in can be by the of a surface-exposed basic or in FXa or the residues and and the Arg165 The present study that FXa other the surface-exposed and residues of h163-170 are the interaction with FVa. Val163 or Ser167 were to FXa a in thrombin in the of small of FVa and The of the mutations because Val163 or Ser167 is directly FVa is by the affinity of and FVa and the same these in interaction A. Kim S.P. J. Biol. Chem. 1999; 274: 18477-18486Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Val163 and Ser167 are on the same of h163-170, with residues the enzyme K. Kim Tulinsky A. Bode W. Huber R. Kisiel W. J. Biol. 1993; PubMed Scopus Google Scholar). is a Ser167 and that appears to the toward h163-170 Val163 der with the of the of the to a in of revealed that in the with a corresponding in with to the wild-type enzyme In the of the same this increase in is of the with and the increase in of this structural FVa binding a in the of of the the to which this is to the Val163 The that a concentration of FVa restored or partially restored the catalytic of FXa derivatives toward prothrombin and and interaction with PAB, d-FFR-CK, and this The of these mutations is in the of the S1 site of the These were FXa derivatives with or mutations of residues the of h163-170 the data that interaction with h163-170, the of the S1 site FXa interaction with its substrates in the have an FVa and Na+ in FVa R.M. J. Biol. Chem. 2002; 277: 37863-37870Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, A.R. Kim J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). study that the of the interaction of and with their substrates or their impaired Na+ binding The same and be that the in and have in the catalytic activity of FXa, the increase in Na+ is with by the the of the study indicate that residues Val163 and Ser167 to a Na+ interaction with FXa 5 and and affinity Na+ of and is the defective of the with substrates that their S1 site and and 5 and These can be in the prothrombinase complex with an excess of FVa and In study the of a functional in FXa h163-170, an FVa-binding and S1 that is modulated by residues Val163 and the of of the and and and