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The effects of the stoichiometric inhibitors tissue factor pathway inhibitor (TFPI), antithrombin-III (AT-III) and heparin cofactor-II (HC-II) on thrombin generation were evaluated in a reaction system composed of coagulation factors VIIa, X, IX, VIII, and V and prothrombin initiated by tissue factor (TF) and phospholipids. Initiation of the reaction in the absence of inhibitors resulted in explosive thrombin generation for factor VIIa·TF concentrations varying from 100 to 0.25 pM with the lag time or initiation phase of thrombin generation increasing from 0 to 180 s with decreasing factor VIIa·TF concentrations. During the propagation phase, prothrombin is quantitatively activated to 1.4 μMα-thrombin. At normal plasma concentration (2.5 nM) full-length recombinant TFPI prolonged the initiation phase of thrombin generation 2-fold, and the rate of thrombin generation in the propagation phase of the reaction was 25-50% that of the uninhibited reaction when the reaction was initiated with 1.25-20 pM factor VIIa·TF. Inhibition of the reaction by TFPI is associated with a delay in factor V activation. In the presence of TFPI no explosive thrombin generation was observed when factor VIII was omitted from reactions initiated by factor VIIa·TF concentrations ≤20 pM. This indicates that in the presence of TFPI the factor IXa·factor VIIIa pathway becomes essential at low factor VIIa·TF concentrations. In the reconstituted system, AT-III (3.4 μM) did not prolong the initiation phase of thrombin generation when the reaction was initiated with 1.25 pM factor VIIa·TF, nor did AT-III delay factor V activation. The rate of thrombin formation in the presence of AT-III was reduced to 30% that of the uninhibited reaction, and the α-thrombin formed was rapidly inhibited subsequent to its generation. The addition of HC-II alone at its physiological concentration (1.38 μM) to the procoagulant mixture did not alter the rate or extent of thrombin generation. Subsequently, the thrombin formed was slowly inhibited by HC-II. The slow inactivation of thrombin by HC-II does not contribute to thrombin inhibition in the presence of AT-III. In contrast, the combination of physiological levels of AT-III and TFPI inhibited explosive thrombin generation initiated by 1.25 pM factor VIIa·TF completely. The absence of prothrombin consumption indicated that the combination of TFPI and AT-III is able to prevent the formation of prothrombinase activity at low factor VIIa·TF concentrations. The data indicate that TFPI potentiates the action of AT-III by decreasing the rate of formation and thus the amount of catalyst formed in the reaction, enabling AT-III to effectively scavenge the limited traces of factor IXa and factor Xa formed in the presence of TFPI. The initiation of thrombin generation by increasing factor VIIa·TF concentrations in the presence of physiological concentrations of TFPI and AT-III showed dramatic changes in the maximal rates of thrombin generation over small changes in initiator concentration. These data demonstrate that significant thrombin generation becomes a “threshold-limited” event with regard to the initiating factor VIIa·TF concentration in the presence of TFPI and AT-III. The effects of the stoichiometric inhibitors tissue factor pathway inhibitor (TFPI), antithrombin-III (AT-III) and heparin cofactor-II (HC-II) on thrombin generation were evaluated in a reaction system composed of coagulation factors VIIa, X, IX, VIII, and V and prothrombin initiated by tissue factor (TF) and phospholipids. Initiation of the reaction in the absence of inhibitors resulted in explosive thrombin generation for factor VIIa·TF concentrations varying from 100 to 0.25 pM with the lag time or initiation phase of thrombin generation increasing from 0 to 180 s with decreasing factor VIIa·TF concentrations. During the propagation phase, prothrombin is quantitatively activated to 1.4 μMα-thrombin. At normal plasma concentration (2.5 nM) full-length recombinant TFPI prolonged the initiation phase of thrombin generation 2-fold, and the rate of thrombin generation in the propagation phase of the reaction was 25-50% that of the uninhibited reaction when the reaction was initiated with 1.25-20 pM factor VIIa·TF. Inhibition of the reaction by TFPI is associated with a delay in factor V activation. In the presence of TFPI no explosive thrombin generation was observed when factor VIII was omitted from reactions initiated by factor VIIa·TF concentrations ≤20 pM. This indicates that in the presence of TFPI the factor IXa·factor VIIIa pathway becomes essential at low factor VIIa·TF concentrations. In the reconstituted system, AT-III (3.4 μM) did not prolong the initiation phase of thrombin generation when the reaction was initiated with 1.25 pM factor VIIa·TF, nor did AT-III delay factor V activation. The rate of thrombin formation in the presence of AT-III was reduced to 30% that of the uninhibited reaction, and the α-thrombin formed was rapidly inhibited subsequent to its generation. The addition of HC-II alone at its physiological concentration (1.38 μM) to the procoagulant mixture did not alter the rate or extent of thrombin generation. Subsequently, the thrombin formed was slowly inhibited by HC-II. The slow inactivation of thrombin by HC-II does not contribute to thrombin inhibition in the presence of AT-III. In contrast, the combination of physiological levels of AT-III and TFPI inhibited explosive thrombin generation initiated by 1.25 pM factor VIIa·TF completely. The absence of prothrombin consumption indicated that the combination of TFPI and AT-III is able to prevent the formation of prothrombinase activity at low factor VIIa·TF concentrations. The data indicate that TFPI potentiates the action of AT-III by decreasing the rate of formation and thus the amount of catalyst formed in the reaction, enabling AT-III to effectively scavenge the limited traces of factor IXa and factor Xa formed in the presence of TFPI. The initiation of thrombin generation by increasing factor VIIa·TF concentrations in the presence of physiological concentrations of TFPI and AT-III showed dramatic changes in the maximal rates of thrombin generation over small changes in initiator concentration. These data demonstrate that significant thrombin generation becomes a “threshold-limited” event with regard to the initiating factor VIIa·TF concentration in the presence of TFPI and AT-III. INTRODUCTIONThe extrinsic pathway of blood coagulation involves the activation of multiple coagulation factors leading to thrombin generation. The procoagulant reaction starts with the binding of activated factor VII (factor VIIa) to its cofactor, tissue factor (TF). 1The abbreviations used are:TFtissue factorTFPItissue factor pathway inhibitorAT-IIIantithrombin-IIIHC-IIheparin cofactor-IIFPR-ckD-phenylalanyl-L-arginine chloromethyl ketoneTBSTris-buffered salinePAGEpolyacrylamide gel electrophoresisF1fragment 1F2fragment 2IIaα-thrombin. TF is an integral membrane protein that is exposed as a result of vessel wall injury or cytokine activation of endothelial cells or peripheral blood monocytes. The membrane-bound factor VIIa·TF enzyme complex activates the zymogens factor X and factor IX by limited proteolysis (1Osterud B. Rapaport S.I. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5260-5264Crossref PubMed Scopus (588) Google Scholar). Factor IXa combines with factor VIIIa on the membrane surface to form a second complex that activates factor X. Once activated, factor Xa associates with factor Va on a membrane surface to form prothrombinase, which converts prothrombin into thrombin. (For reviews on blood coagulation and membrane-dependent reactions in blood coagulation see, respectively, Refs. 2Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1616) Google Scholar and 3Mann K.G. Krishnaswamy S. Lawson J.H. Semin. Hematol. 1992; 29: 213-227PubMed Google Scholar). The thrombin initially formed accelerates further thrombin generation by feedback activation of the procofactors factor V and factor VIII. Thrombin may also activate factor XI (4Gailani D. Broze Jr., G.J. Science. 1991; 253: 909-912Crossref PubMed Scopus (633) Google Scholar, 5Naito K. Fujikawa K. J. Biol. Chem. 1991; 266: 7353-7358Abstract Full Text PDF PubMed Google Scholar), which, in turn, activates more factor IX. Deficiencies in factors VII, X, IX, V, or VIII or prothrombin are associated with abnormal bleeding. Factor XI-deficient individuals rarely suffer from spontaneous bleeding; however, homozygotes may require replacement therapy during significant surgical challenge. Thrombin also activates platelets, which secrete their granule contents and aggregate upon activation. In addition, thrombin cleaves fibrinogen to generate the fibrin network and activates the protransglutaminase factor XIII. The fibrin-platelet aggregate, stabilized by factor XIIIa-catalyzed cross-links, forms the hemostatic plug, which maintains the integrity of the circulatory system following vessel wall perforation.In normal hemostasis, the procoagulant system is in balance with anticoagulant systems involved in the termination of the hemostatic reaction and the fibrinolytic system, which dissolves clots once they are formed. The anticoagulant systems consist of several stoichiometric protease inhibitors, the tissue factor pathway inhibitor (TFPI), antithrombin-III (AT-III), and heparin cofactor-II (HC-II), and the dynamic protein C pathway, which involves thrombin, activated protein C, protein S, and thrombomodulin.TFPI is a reversible, active site-directed inhibitor of factor Xa, which regulates coagulation by inhibiting factor VIIa·TF in a factor Xa-dependent manner (for a review on TFPI see Ref. 6Rapaport S.I. Thromb. Haemostasis. 1991; 66: 6-15Crossref PubMed Scopus (171) Google Scholar). The TFPI·factor Xa complex binds to the factor VIIa·TF complex, resulting in the formation of a TF·factor VIIa·TFPI·factor Xa quaternary complex (7Girard T.J. Warren L.A. Novotny W.F. Likert K.M. Brown S.G. Miletich J.P. Broze Jr., G.J. Nature. 1989; 338: 518-520Crossref PubMed Scopus (414) Google Scholar). Although no human deficiencies have been reported, the in vivo relevance of TFPI is supported by experiments that showed the sensitization of rabbits to TF-triggered disseminated intravascular coagulation after immunodepletion of TFPI (8Sandset P.M. Warner-Cramer B.J. Rao L.V.M. Maki S.L. Rapaport S.I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 708-712Crossref PubMed Scopus (185) Google Scholar).AT-III is a serine protease inhibitor whose importance in hemostasis is confirmed by the association of thrombosis with heterozygous AT-III deficiency (for reviews on AT-III see Refs. 9Blajchman M.N. Austin R.C. Fernandez-Rachubinski F. Sheffield W.P. Blood. 1992; 80: 2159-2171Crossref PubMed Google Scholar and 10Olson S.T. Bjork I. Shore J.D. Methods Enzymol. 1993; 222: 525-559Crossref PubMed Scopus (261) Google Scholar). AT-III inhibits thrombin, factor Xa, factor IXa, factor VIIa, factor XIa, and factor XIIa in vitro by forming covalent complexes in which the active site of the protease is trapped. The action of AT-III is potentiated by heparinoids. The rate of inhibition by AT-III and the potentiation of the inhibition reaction by heparinoids varies for each target protease (10Olson S.T. Bjork I. Shore J.D. Methods Enzymol. 1993; 222: 525-559Crossref PubMed Scopus (261) Google Scholar). Factor Xa is protected from inactivation by AT-III when in a membrane-associated complex with factor Va (11Marciniack E. Br. J. Hematol. 1973; 24: 391-400Crossref PubMed Scopus (78) Google Scholar, 12Rosenburg R.D. Rosenburg J.S. J. Clin. Invest. 1984; 74: 1-6Crossref PubMed Scopus (185) Google Scholar). In contrast, factor VIIa inactivation by AT-III is only significant when the protease is bound to TF (13Lawson J.H. Butenas S. Ribarik N. Mann K.G. J. Biol. Chem. 1993; 268: 767-770Abstract Full Text PDF PubMed Google Scholar). The inactivation of TF-bound factor VIIa by AT-III probably involves the opening of the active site of factor VIIa upon TF binding, allowing factor VIIa inactivation by the active site-directed AT-III. The low enzymatic activity of free factor VIIa provides a mechanism by which traces of factor VIIa may circulate in blood (14Morrissey J. Macik B.G. Neuenschwander P.F. Comp P.C. Blood. 1993; 81: 734-744Crossref PubMed Google Scholar).HC-II is a serine protease inhibitor circulating in blood plasma at a concentration of ∼1.2 μM (15Tollefsen D.M. Blood. 66: PubMed Google Scholar). Thrombin is the only procoagulant to inhibited by HC-II. The inhibition of thrombin by HC-II is potentiated by heparin and by (for a review on HC-II see Ref. D.M. Thromb. Haemostasis. 74: PubMed Scopus Google Scholar). heterozygous deficiency of HC-II levels is in of the and does not to a factor for not been D.M. Thromb. Haemostasis. 74: PubMed Scopus Google have reconstituted J.H. S. Mann K.G. J. Biol. Chem. Full Text PDF PubMed Google and Mann K.G. J. Biol. Chem. Full Text PDF PubMed Google for the tissue factor pathway to thrombin coagulation factors and upon the for the reactions to essential in the procoagulant The reaction into an in which factor V and factor VIII were quantitatively and of factor Xa and factor IXa were and a in which prothrombin was quantitatively activated J.H. S. Mann K.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the concentration of initiator (factor was the initiation phase was prolonged the rate of thrombin generation in the propagation phase by only over a of factor VIIa·TF concentration. The initiation phase was when the reaction was initiated in the presence of factor Va the propagation phase was upon factor VIII and factor IX at factor VIIa·TF concentrations 100 pM. The data with the reconstituted coagulation factors were by the The data the tissue factor pathway to thrombin to and is a inhibitor of thrombin the or initiation phase, of thrombin generation and the rate of thrombin generation during the propagation The delay in the propagation phase of thrombin generation that TFPI a on the reaction, which is associated with factor V activation. In the presence of TFPI as the only the factor IXa·factor VIIIa complex factor Xa, resulting in a of prothrombinase activity of the initiator concentration. In the absence of the factor IXa·factor VIIIa pathway, TFPI the formed prothrombinase activity to of that observed in the absence of the inhibitor at low factor VIIa·TF concentrations. the propagation phase of thrombin generation in the presence of TFPI is upon factor IXa·factor VIIIa activity at low concentrations of factor VIIa·TF. These for the that the of the hemostatic upon injury in with or in by the inactivation of low concentrations of factor VIIa·TF by TFPI Jr., G.J. Warren L.A. Novotny W.F. Miletich J.P. Blood. PubMed Google Scholar). In D. A. Broze G.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google showed that in the presence of factor Xa generation by factor VIIa·TF was only in the presence of the factor IXa·factor VIIIa have the rate for the inhibition of factor Xa by full-length TFPI Broze Jr., G.J. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, S. U. Biochemistry. 1991; 30: PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar), and a by J. A. Biochemistry. PubMed Scopus Google the rate for the inhibition of the factor VIIa·TF complex by the factor The of the procoagulant reactions and enzyme by multiple with the of the inhibition reactions by TFPI to the observed inhibition of the reaction by TFPI by the inhibition of factor VIIa·TF activity by TFPI·factor Xa was observed by J. A. Biochemistry. PubMed Scopus Google after This of TFPI on factor VIIa·TF is in with the of TFPI on the initiation of the reaction in the reconstituted inhibits serine by the enzyme at an of proteolysis of the I. PubMed Scopus Google Scholar, I. A. PubMed Scopus Google Scholar, I. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar). upon the physiological concentrations of AT-III with the (2.5 nM) of TFPI in the have a for AT-III as an inhibitor of factor VIIa·TF. no on the of TFPI and AT-III as inhibitors of factor thrombin generation have been The inhibitor AT-III a of inhibition with TFPI. At 1.25 pM factor VIIa·TF, no in lag time is the rate of prothrombin consumption during the propagation phase was reduced by in the presence of physiological concentrations of AT-III. These data demonstrate that full-length TFPI is the inhibitor of factor thrombin AT-III no significant upon the initiation phase of the reaction, which is a of the factor VIIa·TF concentration. The inhibition of factor VIIa bound to TF by AT-III (13Lawson J.H. Butenas S. Ribarik N. Mann K.G. J. Biol. Chem. 1993; 268: 767-770Abstract Full Text PDF PubMed Google is slow to the reaction during its experiments initiated with pM factor VIIa·TF, AT-III inhibited factor Xa generation by during the initiation This is with a in prothrombin consumption observed in the presence of AT-III. the reduced of factor Xa and factor IXa by factor VIIa·TF when TFPI is to by thus explosive thrombin generation Va been to factor Xa from inactivation by AT-III (11Marciniack E. Br. J. Hematol. 1973; 24: 391-400Crossref PubMed Scopus (78) Google Scholar, 12Rosenburg R.D. Rosenburg J.S. J. Clin. Invest. 1984; 74: 1-6Crossref PubMed Scopus (185) Google Scholar). Factor V is by in the reaction and the of factor Xa are by of factor Va and thus protected inactivation by AT-III. factor Xa inactivation to factor V activation prothrombinase complex In the observed delay of factor V activation in the presence of TFPI may an in the potentiation of the action of AT-III by TFPI the absence of of factor IXa by factor VIIIa inactivation by AT-III is probably of importance of the low concentration and the presence of active factor VIIIa to its J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1992; Full Text PDF PubMed Google inactivation E.W. Biochemistry. PubMed Scopus Google Scholar, G.J. Biochemistry. 24: PubMed Scopus Google Scholar, J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar, B.J. Blood. 1992; 80: PubMed Google Scholar, D. Biochemistry. 1992; PubMed Scopus Google Scholar). In to prothrombinase, the amount of factor VIIIa is probably the factor for the formation of the factor IXa·factor VIIIa In the presence of however, the observed of AT-III is probably also to of the of factor IXa In the for explosive thrombin generation in the presence of TFPI and μM AT-III is pM factor VIIa·TF. The with the factor VIIa·TF concentration to generate thrombin in the presence of TFPI and the absence of the factor pathway This is an that AT-III explosive thrombin generation by inhibiting the factor This however, also the result of a in the reaction in the of pM from a reaction with no lag time to a reaction with a lag which the effects of the factor Xa generation by factor IXa·factor VIIIa and the effects of the inhibitors to more the effects of TFPI and AT-III in to result in a inhibition of thrombin generation at low concentrations of leading to a inhibition their action have been Although that TFPI and AT-III in of with of the involved 1989; Google Scholar). At the of the reaction does not a of TFPI and of TFPI on factor thrombin generation in the reconstituted that a TFPI deficiency a factor for of the of TFPI on thrombin generation that TFPI a significant at This TFPI concentration is of the normal plasma that an with a TFPI a significant data however, that a TFPI deficiency result in thrombin which may not with does not into that the on TFPI of inhibition of factor thrombin generation in the presence of the dynamic protein C with physiological concentrations of HC-II (1.38 μM) an for inhibitor as with AT-III. Thrombin generation in a or not HC-II is Although a of HC-II was observed on the activity of thrombin after the activation of no was observed when HC-II was with physiological concentrations of AT-III. This result the that HC-II is not as a coagulation inhibitor and is in with the of a in individuals with reduced HC-II levels D.M. Thromb. Haemostasis. 74: PubMed Scopus Google effects of TFPI and AT-III prevent explosive thrombin generation by traces of factor VIIa·TF in the reconstituted The dramatic in the rate of thrombin generation over a small in the initiating factor VIIa·TF concentration that significant thrombin generation becomes a event with regard to the initiating factor VIIa·TF concentration in the presence of TFPI and AT-III. The presence of traces of prothrombin and complexes in the plasma of normal individuals indicates that of the coagulation in the The data that the low of activation of the coagulation system is by the action of inhibitors TFPI and which prevent activity from into thrombin INTRODUCTIONThe extrinsic pathway of blood coagulation involves the activation of multiple coagulation factors leading to thrombin generation. The procoagulant reaction starts with the binding of activated factor VII (factor VIIa) to its cofactor, tissue factor (TF). 1The abbreviations used are:TFtissue factorTFPItissue factor pathway inhibitorAT-IIIantithrombin-IIIHC-IIheparin cofactor-IIFPR-ckD-phenylalanyl-L-arginine chloromethyl ketoneTBSTris-buffered salinePAGEpolyacrylamide gel electrophoresisF1fragment 1F2fragment 2IIaα-thrombin. TF is an integral membrane protein that is exposed as a result of vessel wall injury or cytokine activation of endothelial cells or peripheral blood monocytes. The membrane-bound factor VIIa·TF enzyme complex activates the zymogens factor X and factor IX by limited proteolysis (1Osterud B. Rapaport S.I. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5260-5264Crossref PubMed Scopus (588) Google Scholar). Factor IXa combines with factor VIIIa on the membrane surface to form a second complex that activates factor X. Once activated, factor Xa associates with factor Va on a membrane surface to form prothrombinase, which converts prothrombin into thrombin. (For reviews on blood coagulation and membrane-dependent reactions in blood coagulation see, respectively, Refs. 2Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1616) Google Scholar and 3Mann K.G. Krishnaswamy S. Lawson J.H. Semin. Hematol. 1992; 29: 213-227PubMed Google Scholar). The thrombin initially formed accelerates further thrombin generation by feedback activation of the procofactors factor V and factor VIII. Thrombin may also activate factor XI (4Gailani D. Broze Jr., G.J. Science. 1991; 253: 909-912Crossref PubMed Scopus (633) Google Scholar, 5Naito K. Fujikawa K. J. Biol. Chem. 1991; 266: 7353-7358Abstract Full Text PDF PubMed Google Scholar), which, in turn, activates more factor IX. Deficiencies in factors VII, X, IX, V, or VIII or prothrombin are associated with abnormal bleeding. Factor XI-deficient individuals rarely suffer from spontaneous bleeding; however, homozygotes may require replacement therapy during significant surgical challenge. Thrombin also activates platelets, which secrete their granule contents and aggregate upon activation. In addition, thrombin cleaves fibrinogen to generate the fibrin network and activates the protransglutaminase factor XIII. The fibrin-platelet aggregate, stabilized by factor XIIIa-catalyzed cross-links, forms the hemostatic plug, which maintains the integrity of the circulatory system following vessel wall perforation.In normal hemostasis, the procoagulant system is in balance with anticoagulant systems involved in the termination of the hemostatic reaction and the fibrinolytic system, which dissolves clots once they are formed. The anticoagulant systems consist of several stoichiometric protease inhibitors, the tissue factor pathway inhibitor (TFPI), antithrombin-III (AT-III), and heparin cofactor-II (HC-II), and the dynamic protein C pathway, which involves thrombin, activated protein C, protein S, and thrombomodulin.TFPI is a reversible, active site-directed inhibitor of factor Xa, which regulates coagulation by inhibiting factor VIIa·TF in a factor Xa-dependent manner (for a review on TFPI see Ref. 6Rapaport S.I. Thromb. Haemostasis. 1991; 66: 6-15Crossref PubMed Scopus (171) Google Scholar). The TFPI·factor Xa complex binds to the factor VIIa·TF complex, resulting in the formation of a TF·factor VIIa·TFPI·factor Xa quaternary complex (7Girard T.J. Warren L.A. Novotny W.F. Likert K.M. Brown S.G. Miletich J.P. Broze Jr., G.J. Nature. 1989; 338: 518-520Crossref PubMed Scopus (414) Google Scholar). Although no human deficiencies have been reported, the in vivo relevance of TFPI is supported by experiments that showed the sensitization of rabbits to TF-triggered disseminated intravascular coagulation after immunodepletion of TFPI (8Sandset P.M. Warner-Cramer B.J. Rao L.V.M. Maki S.L. Rapaport S.I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 708-712Crossref PubMed Scopus (185) Google Scholar).AT-III is a serine protease inhibitor whose importance in hemostasis is confirmed by the association of thrombosis with heterozygous AT-III deficiency (for reviews on AT-III see Refs. 9Blajchman M.N. Austin R.C. Fernandez-Rachubinski F. Sheffield W.P. Blood. 1992; 80: 2159-2171Crossref PubMed Google Scholar and 10Olson S.T. Bjork I. Shore J.D. Methods Enzymol. 1993; 222: 525-559Crossref PubMed Scopus (261) Google Scholar). AT-III inhibits thrombin, factor Xa, factor IXa, factor VIIa, factor XIa, and factor XIIa in vitro by forming covalent complexes in which the active site of the protease is trapped. The action of AT-III is potentiated by heparinoids. The rate of inhibition by AT-III and the potentiation of the inhibition reaction by heparinoids varies for each target protease (10Olson S.T. Bjork I. Shore J.D. Methods Enzymol. 1993; 222: 525-559Crossref PubMed Scopus (261) Google Scholar). Factor Xa is protected from inactivation by AT-III when in a membrane-associated complex with factor Va (11Marciniack E. Br. J. Hematol. 1973; 24: 391-400Crossref PubMed Scopus (78) Google Scholar, 12Rosenburg R.D. Rosenburg J.S. J. Clin. Invest. 1984; 74: 1-6Crossref PubMed Scopus (185) Google Scholar). In contrast, factor VIIa inactivation by AT-III is only significant when the protease is bound to TF (13Lawson J.H. Butenas S. Ribarik N. Mann K.G. J. Biol. Chem. 1993; 268: 767-770Abstract Full Text PDF PubMed Google Scholar). The inactivation of TF-bound factor VIIa by AT-III probably involves the opening of the active site of factor VIIa upon TF binding, allowing factor VIIa inactivation by the active site-directed AT-III. The low enzymatic activity of free factor VIIa provides a mechanism by which traces of factor VIIa may circulate in blood (14Morrissey J. Macik B.G. Neuenschwander P.F. Comp P.C. Blood. 1993; 81: 734-744Crossref PubMed Google Scholar).HC-II is a serine protease inhibitor circulating in blood plasma at a concentration of ∼1.2 μM (15Tollefsen D.M. Blood. 66: PubMed Google Scholar). Thrombin is the only procoagulant to inhibited by HC-II. The inhibition of thrombin by HC-II is potentiated by heparin and by (for a review on HC-II see Ref. D.M. Thromb. Haemostasis. 74: PubMed Scopus Google Scholar). heterozygous deficiency of HC-II levels is in of the and does not to a factor for not been D.M. Thromb. Haemostasis. 74: PubMed Scopus Google have reconstituted J.H. S. Mann K.G. J. Biol. Chem. Full Text PDF PubMed Google and Mann K.G. J. Biol. Chem. Full Text PDF PubMed Google for the tissue factor pathway to thrombin coagulation factors and upon the for the reactions to essential in the procoagulant The reaction into an in which factor V and factor VIII were quantitatively and of factor Xa and factor IXa were and a in which prothrombin was quantitatively activated J.H. S. Mann K.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the concentration of initiator (factor was the initiation phase was prolonged the rate of thrombin generation in the propagation phase by only over a of factor VIIa·TF concentration. The initiation phase was when the reaction was initiated in the presence of factor Va the propagation phase was upon factor VIII and factor IX at factor VIIa·TF concentrations 100 pM. The data with the reconstituted coagulation factors were by the The data the tissue factor pathway to thrombin to and HC-II.
Veer et al. (Sat,) studied this question.