Removal of B-domain Sequences from Factor V Rather than Specific Proteolysis Underlies the Mechanism by Which Cofactor Function Is Realized

Factor V, the precursor of factor Va, circulates in plasma with little or no procoagulant activity. Activity is generated following limited proteolysis indicating that the conversion of factor V to factor Va results in appropriate structural changes, which impart cofactor function. We have produced recombinant partial B-domain-truncated derivatives of factor V (FVdes811-1491 and FVdes811-1491 with Arg709 and Arg1545 mutated to Gln) to investigate whether discrete proteolysis within the B-domain followed by a conformational transition is responsible for activation. Direct binding fluorescence measurements as well as steady-state kinetic assays were employed to assess the ability of these factor V derivatives to assemble and function in prothrombinase. In contrast to human factor V, single-chain B-domain-truncated factor V bound to FXa membranes with an affinity that was identical to factor Va. Additionally, it was found that, once this modified derivative was assembled in prothrombinase, it functioned in an equivalent manner to factor Va. Taken together these data support the hypothesis that proteolysis within the B-domain of factor V, although necessary, is incidental to the mechanism by which cofactor function is realized. Instead, our results are more consistent with the interpretation that proteolytic activation of factor V simply eliminates steric and/or conformational constraints contributed by the B-domain that otherwise interfere with discrete binding interactions that govern the eventual function of factor Va. Factor V, the precursor of factor Va, circulates in plasma with little or no procoagulant activity. Activity is generated following limited proteolysis indicating that the conversion of factor V to factor Va results in appropriate structural changes, which impart cofactor function. We have produced recombinant partial B-domain-truncated derivatives of factor V (FVdes811-1491 and FVdes811-1491 with Arg709 and Arg1545 mutated to Gln) to investigate whether discrete proteolysis within the B-domain followed by a conformational transition is responsible for activation. Direct binding fluorescence measurements as well as steady-state kinetic assays were employed to assess the ability of these factor V derivatives to assemble and function in prothrombinase. In contrast to human factor V, single-chain B-domain-truncated factor V bound to FXa membranes with an affinity that was identical to factor Va. Additionally, it was found that, once this modified derivative was assembled in prothrombinase, it functioned in an equivalent manner to factor Va. Taken together these data support the hypothesis that proteolysis within the B-domain of factor V, although necessary, is incidental to the mechanism by which cofactor function is realized. Instead, our results are more consistent with the interpretation that proteolytic activation of factor V simply eliminates steric and/or conformational constraints contributed by the B-domain that otherwise interfere with discrete binding interactions that govern the eventual function of factor Va. The generation of thrombin at the appropriate time and place is crucial for balancing normal hemostasis and thrombosis. Thrombin is formed subsequent to proteolysis of two peptide bonds in prothrombin by the macromolecular enzyme complex prothrombinase, which assembles through reversible interactions between the serine protease factor Xa (FXa), 1The abbreviations used are: FXa, activated FX; FX, factor X; PD-FV, plasma-derived factor V; FVa, activated FV; rFVa, recombinant factor Va; rFV-DT, recombinant factor V with amino acids 811-1491 deleted from the B-domain; rFV-DTQQ, rFV-DT with Arg709 and Arg1545 mutated to Gln; PAR, protease-activated receptor; OG488-FXa, human FXa modified with Oregon Green-488; BHK, baby hamster kidney; PCPS, small unilamellar vesicles composed of 75% (w/w) phosphatidylcholine and 25% (w/w) phosphatidylserine; DAPA, dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide; S2238, H-d-phenylalanyl-l-pipecolyl-l-arginyl-p-nitroanilide; APMSF, 4-amidinophenylmethanesulfonyl fluoride hydrochloride; ITS, insulin-transferrin-sodium selenite; FPR-CH2Cl, d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone. and the cofactor protein factor Va (FVa), on a suitable membrane surface in the presence of Ca2+ ions (1Mann K.G. Nesheim M.E. Church W.R. Haley P.E. Krishnaswamy S. Blood. 1990; 76: 1-16Crossref PubMed Google Scholar). Factor Va is essential for the rapid generation of thrombin, and its removal from this enzyme complex profoundly reduces the rate of prothrombin activation (2Mann K.G. Kalafatis M. Blood. 2002; 101: 20-30Crossref PubMed Scopus (181) Google Scholar). The precursor of FVa is factor V (FV), a large (Mr = 330,000) heavily glycosylated, single-chain, multidomain (A1-A2-B-A3-C1-C2) protein, which is synthesized in the liver and is homologous to factor VIII (2Mann K.G. Kalafatis M. Blood. 2002; 101: 20-30Crossref PubMed Scopus (181) Google Scholar, 3Ortel T.L. Kane W.H. Keller F.G. High K.A. Roberts H.R. Molecular Basis of Thrombosis and Hemostasis. Marcel Dekker, New York1995: 119-146Google Scholar). Factor V is considered a procofactor and is not known to function within the prothrombinase complex (4Nesheim M.E. Taswell J.B. Mann K.G. J. Biol. Chem. 1979; 254: 10952-10962Abstract Full Text PDF PubMed Google Scholar, 5Foster W.B. Nesheim M.E. Mann K.G. J. Biol. Chem. 1983; 258: 13970-13977Abstract Full Text PDF PubMed Google Scholar). This is consistent with the observation that FV binds very weakly, if at all, to FXa and prothrombin (6Suzuki K. Dahlbäck B. Stenflo J. J. Biol. Chem. 1982; 257: 6556-6564Abstract Full Text PDF PubMed Google Scholar, 7Guinto E.R. Esmon C.T. J. Biol. Chem. 1984; 259: 13986-13992Abstract Full Text PDF PubMed Google Scholar, 8Esmon C.T. Owen W.G. Duiguid D. Jackson C.M. Biochim. Biophys. Acta. 1973; 310: 289-294Crossref PubMed Scopus (53) Google Scholar) and indicates that proteolytic conversion of FV to FVa leads to appropriate structural changes that impart cofactor function. Thrombin is established as the most robust activator of FV (2Mann K.G. Kalafatis M. Blood. 2002; 101: 20-30Crossref PubMed Scopus (181) Google Scholar). Proteolysis occurs at Arg709, Arg1018, and Arg1545 generating FVaIIa, a heterodimer composed of an N-terminal 105-kDa heavy chain associated via Ca2+ ions to the C-terminal 74/71-kDa light chain (6Suzuki K. Dahlbäck B. Stenflo J. J. Biol. Chem. 1982; 257: 6556-6564Abstract Full Text PDF PubMed Google Scholar, 9Esmon C.T. J. Biol. Chem. 1979; 254: 964-973Abstract Full Text PDF PubMed Google Scholar, 10Nesheim M.E. Mann K.G. J. Biol. Chem. 1979; 254: 1326-1334Abstract Full Text PDF PubMed Google Scholar, 11Jenny R.J. Pittman D.D. Toole J.J. Kriz R.W. Aldape R.A. Hewick R.M. Kaufman R.J. Mann K.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4846-4850Crossref PubMed Scopus (346) Google Scholar). The large, heavily glycosylated B-domain, spanning amino acids 710-1545, is not necessary for cofactor activity and is released during activation (9Esmon C.T. J. Biol. Chem. 1979; 254: 964-973Abstract Full Text PDF PubMed Google Scholar). The mechanism by which FV is maintained in an inactive state is not well understood. It is also not clear how macromolecular binding sites on the heavy and/or light chains are expressed following thrombin-mediated proteolysis. At least two mechanisms could underlie these processes. One possibility, drawn from the known mechanism of serine protease zymogen, protease-activated receptor (PAR), and fibrinogen activation, implies that discrete proteolysis followed by the release of a new N terminus and a conformational change accounts for activation (12Khan A.M. James M.N.G. Protein Sci. 1998; 7: 815-836Crossref PubMed Scopus (387) Google Scholar, 13Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2173) Google Scholar, 14Doolittle R.F. J. Thromb. Haemostasis. 2003; 1: 1559-1565Crossref PubMed Scopus (44) Google Scholar). Support for this mechanism comes from studies using either recombinant FV or proteolyzed derivatives of FV, which demonstrate that cleavage at Arg1545 is sufficient for activation and that release of the B-domain from the heavy chain is not necessary for expression of cofactor activity (6Suzuki K. Dahlbäck B. Stenflo J. J. Biol. Chem. 1982; 257: 6556-6564Abstract Full Text PDF PubMed Google Scholar, 15Keller F.G. Ortel T.L. Quinn-Allen M.A. Kane W.H. Biochemistry. 1995; 34: 4118-4124Crossref PubMed Scopus (56) Google Scholar, 16Thorelli E. Kaufman R.J. Dahlbäck B. Eur. J. Biochem. 1997; 247: 12-20Crossref PubMed Scopus (65) Google Scholar, 17Steen M. Dahlbäck B. J. Biol. Chem. 2002; 277: 38424-38430Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 18Camire R.M. Kalafatis M. Tracy P.B. Biochemistry. 1998; 37: 11896-11906Crossref PubMed Scopus (41) Google Scholar, 19Kalafatis M. Beck D.O. Mann K.G. J. Biol. Chem. 2004; 278: 33550-33561Abstract Full Text Full Text PDF Scopus (44) Google Scholar). A second possibility, employed by proteins such as aspartic and cysteine proteases, implies that the prosegment or activation peptide sterically blocks functional binding sites and activation results from the removal of these (12Khan A.M. James M.N.G. Protein Sci. 1998; 7: 815-836Crossref PubMed Scopus (387) Google Scholar). of these steric by changes in or interactions with B. Nature. PubMed Scopus Google Scholar). Kane F.G. Ortel T.L. Quinn-Allen M.A. Kane W.H. Biochemistry. 1995; 34: 4118-4124Crossref PubMed Scopus (56) Google Scholar, W.H. D. Ortel T.L. Biochemistry. 1990; PubMed Scopus Google Scholar) have a recombinant partial of FV that at least support for this This FV derivative is as a single-chain protein and to have partial activity of with the with this derivative also an for discrete activity was following cleavage by thrombin or V activator from have to cleavage in FV with the generation of cofactor activity (9Esmon C.T. J. Biol. Chem. 1979; 254: 964-973Abstract Full Text PDF PubMed Google Scholar, 10Nesheim M.E. Mann K.G. J. Biol. Chem. 1979; 254: 1326-1334Abstract Full Text PDF PubMed Google Scholar, 15Keller F.G. Ortel T.L. Quinn-Allen M.A. Kane W.H. Biochemistry. 1995; 34: 4118-4124Crossref PubMed Scopus (56) Google Scholar, 16Thorelli E. Kaufman R.J. Dahlbäck B. Eur. J. Biochem. 1997; 247: 12-20Crossref PubMed Scopus (65) Google Scholar, 17Steen M. Dahlbäck B. J. Biol. Chem. 2002; 277: 38424-38430Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 18Camire R.M. Kalafatis M. Tracy P.B. Biochemistry. 1998; 37: 11896-11906Crossref PubMed Scopus (41) Google Scholar, 19Kalafatis M. Beck D.O. Mann K.G. J. Biol. Chem. 2004; 278: 33550-33561Abstract Full Text Full Text PDF Scopus (44) Google Scholar, M.E. W.B. Hewick Mann K.G. J. Biol. Chem. 1984; 259: Full Text PDF PubMed Google Scholar, K.A. Pittman D.D. Kaufman R.J. Blood. 1995; PubMed Google Scholar). of these studies have on functional measurements with thrombin generated in This the for cofactor activation, it is to proteolysis of Additionally, it that the as to which of are considered M. Mann K.G. Thromb. Biol. 1997; PubMed Scopus (44) Google Scholar). to the of the between proteolysis in FV and the of cofactor activity. In the have expressed recombinant B-domain-truncated derivatives of FV and have used a of and kinetic measurements to assess the of B-domain in to the mechanism by which FV is maintained as a and 4-amidinophenylmethanesulfonyl fluoride were from was from and its was using = Jackson C.M. Biochim. Biophys. Acta. 1983; PubMed Scopus Google Scholar). The affinity used to human FV and were from were from insulin-transferrin-sodium which was from and were from unilamellar vesicles composed of 75% (w/w) phosphatidylcholine and 25% (w/w) were as Mann K.G. J. Biol. Chem. 1983; 258: Full Text PDF PubMed Google Scholar). The of the vesicles was by J. Scholar). Oregon and were from chloromethyl was from was from and plasma was from FX, and FV were from plasma as Krishnaswamy S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (53) Google Scholar, Krishnaswamy S. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, Nesheim M.E. Mann K.G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). plasma-derived FVa was by proteolytic activation of by thrombin and as M. Krishnaswamy S. Mann K.G. PubMed Scopus Google Scholar). was by proteolysis of human prothrombin and by established K.G. J. R.J. M. Nesheim M.E. PubMed Scopus Google Scholar). of thrombin were from by on Oregon FXa was as Krishnaswamy S. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). thrombin and a the light chain of FVa were from The used for FV was from Molecular and of the proteins used were as and K.G. PubMed Scopus Google and K.G. PubMed Scopus Google thrombin, and Mann K.G. PubMed Scopus Google FXa, and M.A. Biochemistry. PubMed Scopus Google and on measurements in the and rFVa, and and rFV-DT, and of FV in the expression was from The was with and the expression which was a from R.J. D. PubMed Scopus Google Scholar). A partial derivative of FV, was using the of by R.M. PubMed Scopus Google Scholar). This derivative FV to amino acids 811-1491 from the B-domain used were as to the for amino in which the to for and the to for in which the to for and the to for and to the FV for The was with and and with the the of the modified FV was This derivative by Kane W.H. D. Ortel T.L. Biochemistry. 1990; PubMed Scopus Google and the protein to and was to rFV-DT on the used in the A. J. E. U. S. M. Thromb. Haemostasis. PubMed Scopus Google Scholar). A rFV-DT with Arg709 and Arg1545 by was generated with the using two the the for the and for the and of V expression were baby hamster using and as the from were for in an for FV were in and for protein by and activity. Protein expression of the from to were and in modified with ITS, and was for and at in the presence of protease of was at followed by the of protease APMSF, FPR-CH2Cl, The was an in The was with and with FV activity were and and by The was of of and the protein was at Protein was by using followed by with of rFV-DT was with at for followed by the of activation of rFV-DT, was by as M. Krishnaswamy S. Mann K.G. PubMed Scopus Google Scholar). were in a using was at with a protein of in at with an and were by Biochem. PubMed Scopus Google Scholar). were by by the by and J. E. Biochem. PubMed Scopus Google Scholar). derivatives were in in which with thrombin was were at for with thrombin, followed by the of were to in with and activity using plasma was as R.M. Kalafatis M. Tracy P.B. Biochemistry. 1998; 37: 11896-11906Crossref PubMed Scopus (41) Google Scholar). in were maintained at in and steady-state fluorescence was using = and = with a in the were as Krishnaswamy S. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, A. Krishnaswamy S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of of or prothrombin cleavage by prothrombinase were at as S. Biochemistry. 1997; PubMed Scopus Google Scholar, R.M. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). of activation were using the following and were with the in and the was with FXa At time of the were thrombin generation was by using the S2238, and were also for for FXa binding to cofactor were using the following and were with of cofactor in and the was with FXa The kinetic of prothrombin or activation and were as using prothrombin as a and the were with of prothrombin and the was with FXa using as a cofactor and of and the was with FXa and was to and to at V for The membrane was with a the FVa light chain as R.M. Kalafatis M. Tracy P.B. Biochemistry. 1998; 37: 11896-11906Crossref PubMed Scopus (41) Google Scholar). The used was a to of was by of using The was with light using a and the were with the of The and for the were to a were to the by least using the and for the New Scholar). The of the were by the M. PubMed Scopus Google Scholar). are and for the between FXa and FVa were from the of the fluorescence or rate of activation on the of cofactor S. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). measurements of macromolecular or cleavage by prothrombinase were by the data to the and of and New to for and and of rFV-DT and and were expressed in and to by with of that rFV-DT and with an of was to following with thrombin, was not FV and used in this as are on the FVa used as an was following of rFV-DT with thrombin and on the in a manner to of studies were to assess as well as The data for and well with for FVa K.G. Kalafatis M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Mann K.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Esmon C.T. Biochemistry. 1984; PubMed Scopus Google Scholar). The of rFV-DT was consistent with and with the results of (Mr = using a N-terminal that rFV-DT was by as the N terminus was found to in a new of Activity by on the activity of rFV-DT and to and following with thrombin of with thrombin in a to in cofactor with to with and In rFV-DT and to FVa and of these FV derivatives with thrombin in a change in results that rFV-DT and as and not an is that these proteins are more activated by thrombin or produced during the with of FVa and rFV-DT to ability of the FV derivatives to assemble the membrane complex was by binding fluorescence a of OG488-FXa, subsequent with of or a in fluorescence with and to using Krishnaswamy S. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). In no in fluorescence was using single-chain human if the was to also established that the procofactor not for binding to not The data a for the between FV and FXa on a membrane the conversion of FV to FVa leads to a large change in its affinity for FXa on the membrane binding for prothrombinase in a new In contrast to PD-FV, single-chain rFV-DT and bound to FXa on the membrane surface with an affinity identical to FVa and of of the FV derivatives at the and of the from that were not during the data that rFV-DT and with affinity to FXa membranes and that proteolysis of these proteins is not a for affinity the data are consistent with the that the of a FXa binding on FVa results from removal of steric and/or conformational constraints contributed by of the B-domain in FV not of with functional measurements is rapid proteolysis of rFV-DT or by FXa and thrombin during of this were to and a reversible of thrombin was in the of the conversion of to thrombin were generated using of cofactor and FXa these very little and the was by an indicating an in the rate of thrombin with time This observation to the proteolytic activation of the procofactor by thrombin or FXa during the (4Nesheim M.E. Taswell J.B. Mann K.G. J. Biol. Chem. 1979; 254: 10952-10962Abstract Full Text PDF PubMed Google Scholar, 5Foster W.B. Nesheim M.E. Mann K.G. J. Biol. Chem. 1983; 258: 13970-13977Abstract Full Text PDF PubMed Google Scholar). In thrombin generation with no with rFV-DT and The of thrombin generation for rFVa, and were the rate with rFV-DT was A more of functional activity was using of cofactor at a of FXa and of the data was to the for membrane bound cofactor to FXa data with the and from binding measurements in the of and that rFV-DT and to with an affinity identical to function of of the derivatives was by studies using as a of membranes and cofactor the steady-state kinetic that once assembled in prothrombinase rFV-DT and function to and results were prothrombin was used as the macromolecular for macromolecular cleavage by in a new of Proteolysis during proteolysis during the to FXa or cleavage by thrombin or FXa during the of the could for the with rFV-DT and that rFV-DT and were proteolyzed during the rate The of proteolysis was time and the of cofactor and rFV-DT was proteolyzed to a proteolysis could not the between cleavage and expression of cofactor activity was was by and the of was of the of to the of FXa, following the that the protein was not proteolyzed results were using an the FVa heavy chain not of FXa, a of proteolysis was the time The was a that is from the C-terminal of the on the of the the sites of cleavage within the B-domain or a cleavage with at Ortel T.L. Quinn-Allen M.A. Kane W.H. Biochemistry. PubMed Scopus Google Scholar). was to assess the of and whether this with cofactor activity. A of the rate of thrombin generation using either or derivative of data from and the of the time is in the rate of thrombin generation at time during the for either or rFVa, indicating that these proteins have a of activity the In the of the indicating that cleavage of not with the expression of cofactor of the activity is to the of this a of the of the time that the of this peptide not with the expression of activity not Taken together these data that rFV-DT and have cofactor activity that is to FVa in the of proteolysis. The of proteins in the are synthesized as inactive binding sites to function following proteolysis and of or activation The for this of activation mechanism is the to protease transition in the serine protease cleavage at a a new N which as an a change that leads to activation (12Khan A.M. James M.N.G. Protein Sci. 1998; 7: 815-836Crossref PubMed Scopus (387) Google Scholar). This of mechanism is also employed in the activation of and in the of fibrinogen S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2173) Google Scholar, 14Doolittle R.F. J. Thromb. Haemostasis. 2003; 1: 1559-1565Crossref PubMed Scopus (44) Google Scholar). One to this activation is that structural to protein function are sterically by or activation is following removal of these via proteolysis or by (12Khan A.M. James M.N.G. Protein Sci. 1998; 7: 815-836Crossref PubMed Scopus (387) Google Scholar). The that FV activation simply results from the of steric and/or conformational constraints contributed by the B-domain that otherwise interfere with binding interactions with FXa and the mechanism of FV activation from the established for serine protease One of that mechanism is the to proteins or activation are Krishnaswamy S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (53) Google Scholar, J. M. K. 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PubMed Scopus Google Scholar, 1998; PubMed Scopus Google Scholar). that the of FV and to function at least with to the procofactor to support our from that that, in the of rFV-DT and assemble and function in prothrombinase in an equivalent manner to Direct binding fluorescence measurements that single-chain rFV-DT and to FXa membranes with an affinity that is identical to could not binding of FV to FXa using measurements (6Suzuki K. Dahlbäck B. Stenflo J. J. Biol. Chem. 1982; 257: 6556-6564Abstract Full Text PDF PubMed Google Scholar). results contrast the by and Dahlbäck M. Dahlbäck B. J. Biol. Chem. 2002; 277: 38424-38430Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) that bound to FXa membranes with an affinity of our data that removal of of the B-domain in the of proteolysis results in the of the FXa binding on the heavy and/or light of this could simply from removal of the large heavily glycosylated B-domain that is the B-domain could function by with heavy and/or light chain changes that the FXa binding A is that the generation of rFV-DT or and/or such that modified a identical to At our data between these to our that human FV not FXa, it that FXa membranes are known to the indicating the two proteins W.B. Nesheim M.E. Mann K.G. J. Biol. Chem. 1983; 258: 13970-13977Abstract Full Text PDF PubMed Google Scholar, D.D. Tracy P.B. Biochemistry. 1990; PubMed Scopus Google Scholar). that FV and FVa to FXa membranes with interactions a in FV not FVa the from the binding measurements were whether or not these FV derivatives have functional activity a measurements with and were to that and by rFV-DT, have cofactor activity. proteolysis of these FV derivatives was during the rate it not with the expression of functional cofactor activity. We also found that rFV-DT in our assays to have a to in cofactor activity with FVa and rFV-DTQQ, a that was not found in the The for this observation is not clear to rapid and proteolysis of this FV derivative during the of our functional measurements the of of activity to FV derivatives using in which are was by Kane W.H. D. Ortel T.L. Biochemistry. 1990; PubMed Scopus Google Scholar) and was to have partial cofactor activity of to FVa expressed in The for this partial was not Full activity was following proteolysis at Arg1545 by or thrombin, indicating that discrete proteolysis in of the light chain is for activity F.G. Ortel T.L. Quinn-Allen M.A. Kane W.H. Biochemistry. 1995; 34: 4118-4124Crossref PubMed Scopus (56) Google Scholar). of rFV-DT results that to the new and that discrete proteolysis not underlie the mechanism by which cofactor function is realized. A for the is that in the protein are the of which are within the It is that the and of the between and rFV-DT could of activity. A. M. Eur. J. Biochem. 1987; PubMed Scopus Google Scholar) have that removal of from FV in a in activity. these and to changes in functional activity to it is to that in to within the B-domain a in FV as a The generation of FV derivatives with of B-domain whether the of this or its or its to the ability of this to FV in a procofactor The of for recombinant FV derivatives in in this these studies and the of cofactor function by large of In the results of the that the a in the procofactor it is well established that proteolysis within the B-domain of FV with activation, our that it is incidental and that this for the mechanism by which cofactor function is realized. The removal of B-domain either through proteolysis or through recombinant results in the a cofactor that assembles and within prothrombinase. the mechanism of FV activation from that of serine protease activation and the of proteolysis to the mechanism of FV activation. We are to Krishnaswamy of The of and The of for the and We also and for in of FV and for N-terminal