The Use of Prothrombin(S525C) Labeled with Fluorescein to Directly Study the Inhibition of Prothrombinase by Antithrombin during Prothrombin Activation

Abstract

Serine 525 of human prothrombin was mutated to cysteine and covalently labeled with fluorescein to make II(S525C)-fluorescein. Kinetics of cleavage of this derivative by prothrombinase are identical to those of wild-type prothrombin. Cleavage is coincident with a 50% increase in fluorescence intensity and the product is catalytically inactive. Thus, it allows convenient monitoring of prothrombin activation without generating active thrombin. The kinetics of inhibition of factor Xa (FXa) by antithrombin (AT) and AT-heparin were measured by monitoring activation of II(S525C)-fluorescein and the hydrolysis of the chromogenic substrate S2222 in the presence of AT. With S2222 as the substrate the rate constant for inhibition of FXa, Ca2+, and unilamellar vesicles of phosphatidylcholine and phosphatidylserine (75:25) (PCPS) vesicles by AT was 3.51 × 103m−1 s−1; when factor Va (FVa) was included the rate constant was 1.55 × 103m−1 s−1. In the absence of FVa, II(S525C)-fluorescein had no effect on inhibition. When II(S525C)-fluorescein was the substrate, however, FVa at saturating concentrations profoundly protected FXa from inhibition by AT, increasing the half-life from 3 min with FXa, Ca2+, PCPS, and II(S525C)-fluorescein, to greater than 69 min when FVa was included. Thus, both FVa and prothrombin are necessary for this level of protection. In the absence of prothrombin, FVa decreased the second order rate constant for inhibition by the AT-heparin complex from 1.58 × 107m−1s−1, for FXa, Ca2+, and PCPS, to 7.72 × 106m−1 s−1. II(S525C)-fluorescein and factor Va together reduced the rate constant to less than 1% of that for FXa, Ca2+, and PCPS. At a heparin concentration of 0.2 unit/ml, this corresponds to a half-life increase from 1 s to 136 s. Serine 525 of human prothrombin was mutated to cysteine and covalently labeled with fluorescein to make II(S525C)-fluorescein. Kinetics of cleavage of this derivative by prothrombinase are identical to those of wild-type prothrombin. Cleavage is coincident with a 50% increase in fluorescence intensity and the product is catalytically inactive. Thus, it allows convenient monitoring of prothrombin activation without generating active thrombin. The kinetics of inhibition of factor Xa (FXa) by antithrombin (AT) and AT-heparin were measured by monitoring activation of II(S525C)-fluorescein and the hydrolysis of the chromogenic substrate S2222 in the presence of AT. With S2222 as the substrate the rate constant for inhibition of FXa, Ca2+, and unilamellar vesicles of phosphatidylcholine and phosphatidylserine (75:25) (PCPS) vesicles by AT was 3.51 × 103m−1 s−1; when factor Va (FVa) was included the rate constant was 1.55 × 103m−1 s−1. In the absence of FVa, II(S525C)-fluorescein had no effect on inhibition. When II(S525C)-fluorescein was the substrate, however, FVa at saturating concentrations profoundly protected FXa from inhibition by AT, increasing the half-life from 3 min with FXa, Ca2+, PCPS, and II(S525C)-fluorescein, to greater than 69 min when FVa was included. Thus, both FVa and prothrombin are necessary for this level of protection. In the absence of prothrombin, FVa decreased the second order rate constant for inhibition by the AT-heparin complex from 1.58 × 107m−1s−1, for FXa, Ca2+, and PCPS, to 7.72 × 106m−1 s−1. II(S525C)-fluorescein and factor Va together reduced the rate constant to less than 1% of that for FXa, Ca2+, and PCPS. At a heparin concentration of 0.2 unit/ml, this corresponds to a half-life increase from 1 s to 136 s. 5-iodoacetamidofluorescein base pair(s) polymerase chain reaction Factor Va Factor Xa polyacrylamide gel electrophoresis antithrombin unilamellar vesicles of phosphatidylcholine and phosphatidylserine (75:25) N-benzoyl-l-isoleucyl-l-glutamyl-glycyl-l-arginine-p-nitroaniline hydrochloride H-d-phenylalanyl-l-pipecolyl-l-arginine-p-nitroaniline dihydrochloride One of the key steps of coagulation involves activation of prothrombin to its active form thrombin by the multicomponent complex, prothrombinase. Prothrombinase comprises the serine protease factor Xa, the activated protein cofactor factor Va, calcium ions, and an appropriate cell membrane or phospholipid surface (1Tracy P.B. Eide L.L. Mann K.G. J. Biol. Chem. 1985; 260: 2119-2124Abstract Full Text PDF PubMed Google Scholar, 2Tracy P.B. Nesheim M.E. Mann K.G. J. Biol. Chem. 1981; 256: 743-751Abstract Full Text PDF PubMed Google Scholar, 3Mann K.G. Jenny R.J. Krishnaswamy S. Ann. Rev. Biochem. 1988; 57: 915-956Crossref PubMed Scopus (447) Google Scholar, 4Mann K.G. Trends Biochem. Sci. 1987; 12: 229-233Abstract Full Text PDF Scopus (82) Google Scholar, 5Nesheim M.E. Taswell J.B. Mann K.G. J. Biol. Chem. 1979; 254: 10952-10962Abstract Full Text PDF PubMed Google Scholar). Although factor Xa alone can slowly generate thrombin by an extremely inefficient reaction, the rate of thrombin generation is enhanced by several orders of magnitude by incorporation of the cofactor protein factor Va and the procoagulant surface (5Nesheim M.E. Taswell J.B. Mann K.G. J. Biol. Chem. 1979; 254: 10952-10962Abstract Full Text PDF PubMed Google Scholar). The activities of the clotting serine proteases, such as thrombin, are regulated, in part, by plasma protease inhibitors of the serpin superfamily. Of these, antithrombin appears to be the most important. Antithrombin targets both the product of prothrombin activation, thrombin, and the enzyme responsible for the reaction, factor Xa. Inhibition of serine proteases by serpins occurs through the formation of a stable complex between the serpin and the active site of the serine protease. Inhibition by antithrombin is markedly enhanced by the glycosaminoglycan cofactor, heparin. Heparin accelerates rates of inhibition of both thrombin and factor Xa several thousand fold (6Jordon R. Beeler D. Rosenburg R. J. Biol. Chem. 1979; 254: 2902-2913Abstract Full Text PDF PubMed Google Scholar). Upon binding, heparin induces a conformational change in antithrombin (7Olson S.T. Bjork I. Sheffer R. Craig P.A. Shore J.D. Choay J. J. Biol. Chem. 1992; 267: 12528-12538Abstract Full Text PDF PubMed Google Scholar, 8Owen B.A. Owen W.G. Biochemistry. 1990; 29: 9412-9417Crossref PubMed Scopus (16) Google Scholar, 9Craig P.A. Olson S.T. Shore J.D. J. Biol. Chem. 1989; 264: 5452-5461Abstract Full Text PDF PubMed Google Scholar), which is sufficient to accelerate the inhibition of factor Xa through an allosteric mechanism. The acceleration of thrombin inhibition, however, is dependent on the formation of a ternary complex between heparin, antithrombin, and thrombin (10Nesheim M.E. J. Biol. Chem. 1982; 258: 14708-14717Abstract Full Text PDF Google Scholar, 11Griffith M.J. J. Biol. Chem. 1982; 257: 7360-7365Abstract Full Text PDF PubMed Google Scholar, 12Pomerantz M.W. Owen W.G. Biochim. Biophys. Acta. 1978; 535: 66-77Crossref PubMed Scopus (80) Google Scholar). Several studies demonstrated that incorporation of factor Xa into the prothrombinase complex changes the ability of the antithrombin-heparin complex to inhibit it. Calcium ion accelerates the heparin catalyzed inactivation of factor Xa by antithrombin through a template mechanism (13Rezaie A.R. J. Biol. Chem. 1998; 273: 16824-16827Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Phospholipids and factor Va play protective roles and decrease the ability of the heparin-antithrombin complex to inhibit factor Xa both on synthetic surfaces (14Speijer H. Billy D. Willems G. Hemker H.C. Lindhout T. Thromb. Haemost. 1995; 73: 648-653Crossref PubMed Scopus (13) Google Scholar, 15Schoen P. Lindhout T. Blood. 1991; 78: 118-124Crossref PubMed Google Scholar, 16Schoen P. Lindhout T. Willems G. Hemker H.C. J. Biol. Chem. 1989; 264: 10002-10007Abstract Full Text PDF PubMed Google Scholar, 17Lindhout T. Baruch D. Schoen P. Franssen J. Hemker H.C. Biochemistry. 1986; 25: 5962-5969Crossref PubMed Scopus (41) Google Scholar, 18Ellis V. Scully M.F. Kakkar V.V. Biochemistry. 1984; 23: 5882-5887Crossref PubMed Scopus (34) Google Scholar, 19Walker F.J. Esmon C.T. Biochem. Biophys. Res. Commun. 1979; 90: 641-647Crossref PubMed Scopus (32) Google Scholar) and in whole blood clots (20Eisenburg P.R. Siegel J.E. Abendschein D.R. Miletich J.P. J. Clin. Invest. 1993; 91: 1877-1883Crossref PubMed Scopus (169) Google Scholar). Herault et al. (21Herault J.P. Bernat A. Pflieger A.M. Lormeau J.C. Herbert J.M. J. Pharmacol. Exp. Ther. 1997; 283: 16-22PubMed Google Scholar), however, indicated that the antithrombin-heparin complex is an effective inhibitor for phospholipid/factor Va-bound factor Xa in solution and on blood cells. The substrate prothrombin also plays an important role in the protection observed when factor Xa is incorporated into the prothrombinase complex (14Speijer H. Billy D. Willems G. Hemker H.C. Lindhout T. Thromb. Haemost. 1995; 73: 648-653Crossref PubMed Scopus (13) Google Scholar). Prothrombinase activity is usually inferred through measurements of thrombin activity over time. Therefore, studying heparin-dependent antithrombin inhibition of prothrombinase is challenging because the inhibitor recognizes both the product of the reaction, thrombin, and the enzyme component of prothrombinase, factor Xa. To resolve this problem in this study, a mutant form of prothrombin (II(S525C)), in which serine 525 (the active site serine of thrombin) is replaced with cysteine, was expressed in a mammalian expression system, isolated, and covalently labeled at Cys525with fluorescein. II(S525C)-fluorescein exhibits an increase in fluorescence intensity upon activation to thrombin but is catalytically inactive to both small and macromolecular substrates. Thus, it allows convenient monitoring of prothrombin activation in the presence of antithrombin without generating active thrombin. In this study inhibition of free factor Xa or factor Xa bound to vesicles and factor Va, by antithrombin or the antithrombin heparin complex was determined by monitoring over time the hydrolysis of the small factor Xa substrate S2222 or the activation of II(S525C)-fluorescein. DNA restriction and modification enzymes were obtained from New England BioLabs (Mississauga, Ontario) or Life Technologies, Inc. (Burlington, Ontario) and Pyrococcus furiosus(Pfu) DNA polymerase was obtained from Stratagene (La Jolla, CA). Baby hamster kidney cells and the mammalian expression vector pNUT were graciously provided by Dr. Ross MacGillivray (University of British Columbia). Newborn calf serum, Dulbecco's modified Eagle's medium/F-12 nutrient mixture (1:1), Opti-MEM, penicillin/streptomycin/Fungizone mixture, and reduced glutathione were obtained from Life Technologies, Inc. Methotrexate and heparin were at the prothrombin was from was from and was from S2222 and were from (Mississauga, was from vesicles were as by et al. Nesheim M.E. Mann K.G. Biochemistry. 1979; PubMed Scopus Google Scholar). prothrombin J.C. Nesheim M.E. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar), thrombin J.B. Nesheim M.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), Factor Xa J.C. Nesheim M.E. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar), Factor Va M.E. P.B. Mann K.G. 1981; PubMed Scopus Google Scholar), and antithrombin (10Nesheim M.E. J. Biol. Chem. 1982; 258: 14708-14717Abstract Full Text PDF Google Scholar) were as human prothrombin Nesheim M.E. MacGillivray J. Biol. Chem. Full Text PDF PubMed Google Scholar) was with and restriction The was into 1 and were to of in a serine to cysteine at in the expressed protein DNA with 1 and the a product that with and the a product product was into the site of and in were and and The were at and into with and The presence of and were by DNA the and The mutated was into prothrombin to make expression in mammalian the was from with by with DNA and into the pNUT vector MacGillivray Esmon C.T. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar) at site is of the and of the human of the was determined restriction and DNA 3 and The pNUT vector a modified which allows for in this and are are in a cells were in Dulbecco's modified Eagle's medium/F-12 nutrient mixture with calf and of was the calcium H. Biol. 1987; PubMed Scopus Google Scholar). the was with to for were for by with prothrombin were into for At the was replaced by with and penicillin/streptomycin/Fungizone The was at with glutathione and at was at and × and × in at or The was with of by of with were a and were protein was from the was on this by a of from a solution of and the for at in the The II(S525C)-fluorescein was from the by of to a concentration of by of a solution to a concentration of The solution was at for 1 and The was with the from a in The protein was by the in 0.2 of the The was at and to on a at was to resolve from of II(S525C)-fluorescein Nesheim M.E. MacGillivray J. Biol. Chem. Full Text PDF PubMed Google P. J.D. 1990; PubMed Scopus Google Scholar). The protein was with a in rate The the II(S525C)-fluorescein at the second between and from the were and protein concentrations and were determined by at and II(S525C)-fluorescein was activated with PCPS, FVa, FXa, and in intensity was at with an of and an of of and and a in were from the reaction and to an of 0.2 were and reduced and to on a which was over II(S525C)-fluorescein or plasma prothrombin was activated in the presence FVa with PCPS, FXa, and in The were in the absence of FVa, with were from the and to on which were with was to the of prothrombin at time were AT with FXa FXa PCPS, FXa FVa, or FXa 1 II(S525C)-fluorescein in were by the of FXa, and were and in a which had with 1% The with and in sufficient to concentrations of and at was over time in a at rates were The of were time and the was to the second order rate of inhibition. S2222 at concentrations and in were at to in a S2222 was and the were at rates were determined and and for the inhibition of FXa by the product of S2222 hydrolysis was determined to the is the concentration of S2222 In is the rate of hydrolysis of is is the FXa is the concentration of S2222 is the constant for hydrolysis of is the constant for the S2222 and is the concentration of the S2222 The for hydrolysis of S2222 was determined by measurements of reaction rates in the absence of S2222 at FXa and in were at at rates were determined and to were for FXa FXa PCPS, and The were for In were to determined for for the was to be identical to the S2222 with FXa FXa FXa PCPS, FXa FVa, or FXa 1 in the presence and absence of AT, were in a which had with 1% were by of FXa and at to were to the order rate The was into that the product of S2222 hydrolysis as as AT inhibit the In this is the is the substrate, the is the inhibitor and is the order rate constant for inhibition of FXa by AT. is to the second order rate constant by the inhibitor product and of When the reaction with AT and Therefore, can be and and can be determined from with no AT. With the that of over time is as were to to to of between and from which the of was The second order rate constant was II(S525C)-fluorescein, and FVa at concentrations were in a which had with and were in a fluorescence at and of and with a in were by the of for no AT, were to the to and for order rate were and the second order rate To stable cell were in in of were as the for as to wild-type prothrombin in of the was to be in at a concentration of of however, was II(S525C)-fluorescein was with a of II(S525C)-fluorescein with plasma prothrombin when to Upon activation of II(S525C)-fluorescein, an increase in fluorescence intensity of was which was to with cleavage The fluorescence intensity a in which is of the Nesheim M.E. MacGillivray J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. Mann K.G. Nesheim M.E. J. Biol. Chem. 1986; Full Text PDF PubMed Google Scholar). upon the that the corresponds to the of thrombin, which the active site that the fluorescein to When with plasma prothrombin, II(S525C)-fluorescein activated at rates in both the presence and absence of FVa The thrombin from activation of II(S525C)-fluorescein was catalytically inactive small as determined by in which II(S525C)-fluorescein was observed to at less activity II(S525C)-fluorescein was also catalytically inactive macromolecular such as Factor and of II(S525C)-fluorescein with plasma prothrombin in the presence and absence of II(S525C)-fluorescein or plasma prothrombin were activated with PCPS, FVa, FXa, and in were from the and were to on polyacrylamide was to the of prothrombin at time II(S525C)-fluorescein or plasma prothrombin were activated with FXa, and in were and as The second order rate for inhibition of FXa by antithrombin in the presence and absence of Ca2+, and FVa were determined from the time of hydrolysis of the FXa substrate were One at and FXa activity with the The monitoring the the substrate to and the of the inhibition from the level of substrate at the of the reaction in the presence of In the the time of of activity is the are In the the of the inferred on the of the to the The second was with this substrate in of the with II(S525C)-fluorescein, in which the be because of the of FXa Thus, the of measurements with S2222 were to the of the The are in time of the with without AT, with the substrate included are in In the of the of the activity were and order rate were measured from the In the AT to because the enzyme was which of the order rate constant for inhibition from the substrate concentration and for the to by of the were because the time of product formation were of the substrate which is of of both the substrate and product to the Thus, was by rate measurements and the for inhibition by product was by rates of substrate hydrolysis in the presence of the product at concentrations was determined to be the of the presence or absence of Ca2+, PCPS, and The inferred by of the from to the was The for product inhibition obtained for the in was hydrolysis over time. S2222 and FXa or FXa FVa, were in a in both the absence and presence of AT. were by the of FXa and at were to the second order rate of with S2222 of FXa, and in were at rates were determined and to of FXa, and in were at in a S2222 was and the reaction was at rates were determined and and a was determined as The for the inhibition of FXa by antithrombin were determined for FXa FXa Ca2+, FXa PCPS, FXa FVa, and FXa II(S525C)-fluorescein. determined both The determined were in with Thus, was determined to be a for and was in with II(S525C)-fluorescein. Upon of Ca2+, FXa was to inhibition by AT, which was (13Rezaie A.R. J. Biol. Chem. 1998; 273: 16824-16827Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). of or II(S525C)-fluorescein to the had to no effect when with FXa of FVa decreased the second order rate constant by when with that for FXa PCPS. In the absence of FVa, II(S525C)-fluorescein had no effect on inhibition of FXa by as determined by and × in a II(S525C)-fluorescein is a substrate for monitoring inhibition of prothrombinase, because activation of the mutant can be without the generation of active thrombin. a no AT, was and the were to the to and S2222 prothrombin activation to product inhibition. and for FXa, Ca2+, PCPS, FVa at and II(S525C)-fluorescein. The for the AT increase with increasing FVa, increasing protection. at saturating of FVa, prothrombinase protected and the for inhibition by AT to The half-life at a FVa concentration of is 69 which can be with the half-life in the absence of FVa of this is to the half-life from the FXa II(S525C)-fluorescein prothrombin alone protection of FXa from inhibition by AT. When a of FVa was S2222 as the substrate, the were observed to by at saturating of FVa Therefore, in the presence of and PCPS, FXa was observed to a half-life of which to min at a FVa concentration of that FVa alone protection of FXa from inhibition by Thus, FVa and prothrombin both be to of FVa FVa at FXa, PCPS, 1 II(S525C)-fluorescein, in the absence and presence of AT were at and of and with a in was as were of FVa, FXa, PCPS, and S2222 in the absence and presence of AT were was as were In the FVa in which II(S525C)-fluorescein was the substrate, a effect was obtained at a FVa concentration of of the effect was also observed when S2222 was the substrate, but the concentration of FVa at effect was Heparin accelerates inhibition of FXa by AT by several orders of the order rate for a of heparin concentrations determined by monitoring II(S525C)-fluorescein activation by prothrombinase. The was determined from the of the to be × s−1. The in the order rate obtained for of the heparin-dependent AT inhibition of FXa FVa determined by S2222 The was determined to be 7.72 × The second order rate constant for FXa was also determined in a and to be 1.58 × the determined in both the absence and presence of heparin. Heparin the inhibition of FXa in the presence of and by increase was also observed for FXa FVa as determined by S2222 was to a for prothrombinase inhibition in the presence of II(S525C)-fluorescein, because prothrombinase appears to be protected at saturating of FVa in the absence of heparin but is with a second order rate constant of × upon heparin also the in the presence of heparin to that for FXa PCPS. The for prothrombinase in the presence of II(S525C)-fluorescein was to be of that for FXa PCPS, protection of FXa, when it is incorporated into the prothrombinase complex and in the presence of prothrombin, in the presence of heparin. for prothrombinase as determined by S2222 was that obtained with FXa PCPS. was greater than that obtained with prothrombinase II(S525C)-fluorescein. The profoundly observed when II(S525C)-fluorescein is the substrate that the presence of both FVa and prothrombin protection of FXa from AT, in the presence of determined in the absence and presence of are to for FXa in the presence of × × × × FVa × × are to for FXa in the presence of heparin. in a FXa FVa and II(S525C)-fluorescein at in the presence and absence of AT and heparin, were was and the were that II(S525C)-fluorescein at concentrations from to in that the in the absence of prothrombin be 7.72 × 106m−1 which is than the in Thus, prothrombin to play a protective to FVa, the protective of prothrombin at it is to activation of II(S525C)-fluorescein at concentrations because the change in fluorescence is expressed a of prothrombin in which the serine of the thrombin replaced with a cysteine that was labeled with fluorescein. of the change in fluorescence upon the rate of activation in the presence and absence of FVa and the absence of activity upon activation of this it to be a substrate to its activation by the enzyme FXa in the presence of the inhibitor allows monitoring of the of prothrombin to an inactive thrombin and be a for monitoring prothrombin activation in the absence of thrombin such as the activation of and cleavage of that FXa is less to inhibition by antithrombin and the antithrombin-heparin complex when it is incorporated into the prothrombinase complex (14Speijer H. Billy D. Willems G. Hemker H.C. Lindhout T. Thromb. Haemost. 1995; 73: 648-653Crossref PubMed Scopus (13) Google Scholar, 15Schoen P. Lindhout T. Blood. 1991; 78: 118-124Crossref PubMed Google Scholar, 16Schoen P. Lindhout T. Willems G. Hemker H.C. J. Biol. Chem. 1989; 264: 10002-10007Abstract Full Text PDF PubMed Google Scholar, 17Lindhout T. Baruch D. Schoen P. Franssen J. Hemker H.C. Biochemistry. 1986; 25: 5962-5969Crossref PubMed Scopus (41) Google Scholar, 18Ellis V. Scully M.F. Kakkar V.V. Biochemistry. 1984; 23: 5882-5887Crossref PubMed Scopus (34) Google Scholar, 19Walker F.J. Esmon C.T. Biochem. Biophys. Res. Commun. 1979; 90: 641-647Crossref PubMed Scopus (32) Google Scholar, P.R. Siegel J.E. Abendschein D.R. Miletich J.P. J. Clin. Invest. 1993; 91: 1877-1883Crossref PubMed Scopus (169) Google Scholar). The of this protection and the mechanism however, Inhibition of FXa by antithrombin and the antithrombin-heparin complex was determined in this In the absence of FVa, the of phospholipid vesicles and prothrombin had no effect on the second order rate constant of inhibition of FXa FVa in the absence of prothrombin a the rate constant Upon of both FVa and prothrombin, however, the second order rate constant decreased to an when both FVa and prothrombin are prothrombinase is extremely protected from antithrombin, with its half-life increasing from 3 min in the absence of FVa to greater than 69 min with prothrombin and Upon of heparin, FVa in the absence of prothrombin was observed to decrease the second order rate constant of inhibition by In the presence of both FVa and prothrombin, the second order rate constant decreased to of that for FXa PCPS, protection of prothrombinase in the presence of heparin. In the presence of heparin at 0.2 the half-life from 1 s without prothrombin to 136 s with it. the protection of prothrombinase. studies that the thrombin at the surface antithrombin and its concentration in the of the prothrombinase complex P. Lindhout T. Blood. 1991; 78: 118-124Crossref PubMed Google Scholar). In the study however, active thrombin was and no change in the fluorescence of was observed upon antithrombin that the in this study with and the to which FXa is protected from inhibition by both antithrombin and the antithrombin-heparin complex also be on between prothrombin and antithrombin to a Thus, of the of prothrombin to thrombin by prothrombinase is responsible for the protection. One is that the of FXa with both prothrombin and antithrombin an on FXa from the of the such an on FXa is by the of Krishnaswamy and S. A. Biochemistry. 1997; PubMed Scopus Google Scholar). this the the is by prothrombin in the absence of FVa, because prothrombin alone the protection is in the of the for most of the FXa is in a or in which it this is FXa be for 1% of the because the rate constant for inhibition by a factor of for inhibition of FXa by the antithrombin-heparin of the for the the that it that prothrombin activation be for by substrate and the The serine proteases of multicomponent of the coagulation and factor are also by are protected from inhibition in the presence of the substrate and the for prothrombinase to of the of the this is the the of the at the site of be protected from antithrombin, enzymes that be to inhibition, both thrombin formation or it