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Regulation of Tissue Factor Initiated Thrombin Generation by the Stoichiometric Inhibitors Tissue Factor Pathway Inhibitor, Antithrombin-III, and Heparin Cofactor-II

1997; Elsevier BV; Volume: 272; Issue: 7 Linguagem: Inglês

10.1074/jbc.272.7.4367

1083-351X

Cornelis van’t Veer, Kenneth G. Mann,

Coagulation, Bradykinin, Polyphosphates, and Angioedema

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. Pestka C.A. Blood. 1985; 66: 769-774Crossref PubMed Google Scholar). Thrombin is the only procoagulant reported to be inhibited by HC-II. The inhibition of thrombin by HC-II is potentiated by heparin and by dermatan sulfate (for a review on HC-II see Ref. 16Tollefsen D.M. Thromb. Haemostasis. 1995; 74: 1209-1214Crossref PubMed Scopus (99) Google Scholar). A heterozygous deficiency of HC-II (activity levels < 60%) is found in approximately 1% of the healthy population and does not appear to be a risk factor for thrombosis. A homozygous deficient individual has not yet been identified (16Tollefsen D.M. Thromb. Haemostasis. 1995; 74: 1209-1214Crossref PubMed Scopus (99) Google Scholar).We have described reconstituted empirical (17Lawson J.H. Kalafatis M. Stram S. Mann K.G. J. Biol. Chem. 1994; 269: 23357-23366Abstract Full Text PDF PubMed Google Scholar) and mathematical (18Jones K.C. Mann K.G. J. Biol. Chem. 1994; 269: 23367-23373Abstract Full Text PDF PubMed Google Scholar) models for the tissue factor pathway to thrombin using purified coagulation factors and computer simulations based upon the known kinetic constants for the reactions thought to be essential in the procoagulant scheme. The reaction could be divided into two phases, an "initiation phase," in which factor V and factor VIII were quantitatively cleaved and trivial amounts of factor Xa and factor IXa were produced, and a "propagation phase" in which prothrombin was quantitatively activated (17Lawson J.H. Kalafatis M. Stram S. Mann K.G. J. Biol. Chem. 1994; 269: 23357-23366Abstract Full Text PDF PubMed Google Scholar). As the concentration of initiator (factor VIIa·TF) was reduced, the initiation phase was prolonged while the rate of thrombin generation in the propagation phase varied by only 5-fold over a 1000-fold range of factor VIIa·TF concentration. The initiation phase was shortened when the reaction was initiated in the presence of factor Va while the propagation phase was dependent upon factor VIII and factor IX at factor VIIa·TF concentrations below 100 pM. The data obtained with the reconstituted empirical model using purified coagulation factors were reasonably approximated by the mathematical model. The data presented here extend the empirical tissue factor pathway to thrombin studies to include TFPI, AT-III, and HC-II.DISCUSSIONTFPI is a major inhibitor of thrombin generation, extending the "lag" time, or initiation phase, of thrombin generation and reducing the rate of thrombin generation during the propagation phase. The delay in the propagation phase of thrombin generation shows that TFPI exerts a relatively rapid inhibitory effect on the reaction, which is associated with delayed factor V activation. In the presence of TFPI as the only inhibitor, the factor IXa·factor VIIIa complex ultimately generates sufficient factor Xa, resulting in a bolus of prothrombinase activity independent of the initiator concentration. In the absence of the factor IXa·factor VIIIa pathway, TFPI reduces the maximally formed prothrombinase activity to 1% of that observed in the absence of the inhibitor at low (≤5 pM) factor VIIa·TF concentrations. Thus, the propagation phase of thrombin generation in the presence of TFPI is totally dependent upon factor IXa·factor VIIIa activity at low concentrations of factor VIIa·TF. These results provide quantitative support for the hypothesis that the failure of the hemostatic response upon injury in patients with hemophilia A or B is, in part, caused by the inactivation of low concentrations of factor VIIa·TF by TFPI (34Broze Jr., G.J. Warren L.A. Novotny W.F. Higuchi D.A. Girard J.J. Miletich J.P. Blood. 1988; 71: 335-343Crossref PubMed Google Scholar). In flow studies using purified proteins Repke et al. (35Repke D. Gemmel C.H. Guha A. Turrito V.T. Broze G.J. Nemerson Y. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7623-7627Crossref PubMed Scopus (60) Google Scholar) showed that in the presence of TFPI, ongoing factor Xa generation by factor VIIa·TF was only obtained in the presence of the factor IXa·factor VIIIa pathway.Several groups have reported the rate constants for the inhibition of factor Xa by full-length TFPI (36Huang Z.-F. Wun T.-C. Broze Jr., G.J. J. Biol. Chem. 1993; 268: 26950-26955Abstract Full Text PDF PubMed Google Scholar, 37Nordvang O. Bjorn S.E. Valintin S. Nielsen L.S. Wildgoose P. Beck T.C. Hedner U. Biochemistry. 1991; 30: 10371-10376Crossref PubMed Scopus (96) Google Scholar, 38Lindhout T. Willems G. Blezer R. Hemker H.C. Biochem. J. 1994; 297: 131-136Crossref PubMed Scopus (45) Google Scholar), and a recent study by Jesty et al. (39Jesty J. Wun T.C. Lorenz A. Biochemistry. 1994; 33: 12686-12694Crossref PubMed Scopus (67) Google Scholar) reports the rate constants for the inhibition of the factor VIIa·TF complex by the preformed factor Xa·TFPI complex. The complexity of the procoagulant reactions (no steady state conditions established and enzyme occupation by multiple substrates) combined with the complexity of the inhibition reactions by TFPI makes it difficult to determine whether the observed inhibition of the reaction by TFPI can be explained by the reported kinetic constants. Significant (∼90%) inhibition of factor VIIa·TF activity by TFPI·factor Xa was observed by Jesty et al. (39Jesty J. Wun T.C. Lorenz A. Biochemistry. 1994; 33: 12686-12694Crossref PubMed Scopus (67) Google Scholar) after 60 s. This rapid effect of TFPI on factor VIIa·TF is in agreement with the rapid effect of TFPI on the initiation of the reaction in the reconstituted model.AT-III inhibits serine proteases by trapping the enzyme at an intermediate stage of proteolysis of the Arg393-Ser394 peptide bond (40Jornvall H. Fish W.W. Björk I. FEBS Lett. 1979; 106: 358-362Crossref PubMed Scopus (49) Google Scholar, 41Björk I. Danielsson A. Fenton II, J.W. Jornvall H. FEBS Lett. 1981; 126: 257-260Crossref PubMed Scopus (47) Google Scholar, 42Björk I. Jackson C.M. Jornvall H. Lavine K.K. Nordling K. Salsgiver W.J. J. Biol. Chem. 1982; 257: 2406-2411Abstract Full Text PDF PubMed Google Scholar). Speculation based upon the relatively high (μM) physiological concentrations of AT-III compared with the trace amounts (2.5 nM) of TFPI in the circulation have suggested a potential role for AT-III as an inhibitor of factor VIIa·TF. However, no empirical comparisons on the relative potencies of TFPI and AT-III as inhibitors of factor VIIa·TF-dependent thrombin generation have been reported. The inhibitor AT-III displays a completely different profile of inhibition compared with TFPI. At 1.25 pM factor VIIa·TF, no increase in lag time is observed, while the rate of prothrombin consumption during the propagation phase was reduced by 50% in the presence of physiological concentrations of AT-III. These data demonstrate that full-length TFPI is the major inhibitor of factor VIIa·TF-initiated thrombin formation, while AT-III has no significant influence upon the initiation phase of the reaction, which is almost totally a function 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 Scholar) is too slow to significantly inhibit the reaction during its early phase.In experiments initiated with 5 pM factor VIIa·TF, AT-III inhibited factor Xa generation by 50% during the initiation phase. This is consistent with a slight decrease in prothrombin consumption observed in the presence of AT-III. However, the reduced amounts of factor Xa and factor IXa generated by factor VIIa·TF when TFPI is present seem to become efficiently scavenged by AT-III, thus preventing explosive thrombin generation (Fig. 7, open diamonds).Factor Va has been reported to protect 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 cleaved by 2 min in the reaction and the picomolar amounts of factor Xa generated contemporaneously are saturated by this excess of factor Va and thus relatively protected against inactivation by AT-III. However, factor Xa inactivation can occur prior to factor V activation and/or prothrombinase complex formation. In this respect, the observed delay of factor V activation in the presence of TFPI may play an important role in the potentiation of the action of AT-III by TFPI (Fig. 7).In the absence of TFPI, protection of factor IXa by factor VIIIa against inactivation by AT-III is probably of less importance because of the relatively low concentration and the transient presence of active factor VIIIa due to its dissociation (26Lollar P. Parker C.G. J. Biol. Chem. 1990; 265: 1688-1692Abstract Full Text PDF PubMed Google Scholar, 27Fay P.J. Smudzin T.M. J. Biol. Chem. 1992; 267: 13246-13250Abstract Full Text PDF PubMed Google Scholar, 28Lollar P. Parker E.T. Fay P.J. J. Biol. Chem. 1992; 267: 23652-23657Abstract Full Text PDF PubMed Google Scholar) and/or proteolytic inactivation (29Vehar G.A. Davie E.W. Biochemistry. 1980; 19: 401-410Crossref PubMed Scopus (286) Google Scholar, 30Lollar P. Knutson G.J. Fass D.N. Biochemistry. 1985; 24: 8056-8064Crossref PubMed Scopus (77) Google Scholar, 31Fay P.J. Smudzin T.M. Walker F.J. J. Biol. Chem. 1991; 266: 20139-20145Abstract Full Text PDF PubMed Google Scholar, 32Lamphear B.J. Fay P.J. Blood. 1992; 80: 3120-3126Crossref PubMed Google Scholar, 33O'Brien D.P. Johnson D. Byfield P. Tuddenham E.G.D. Biochemistry. 1992; 31: 2805-2812Crossref PubMed Scopus (41) Google Scholar). In contrast to prothrombinase, the amount of factor VIIIa cofactor is probably the limiting factor for the formation of the factor IXa·factor VIIIa complex. In the presence of TFPI, however, the observed inhibitory effect of AT-III is probably also due to scavenging of the trace amounts of factor IXa generated. In this model the threshold for explosive thrombin generation in the presence of 2.5 nM TFPI and 3.4 μM AT-III is ∼20 pM factor VIIa·TF. The threshold level coincides with the minimal factor VIIa·TF concentration (20 pM) needed to generate considerable thrombin in the presence of TFPI and the absence of the factor IXa·VIIIa pathway (Fig. 4). This is an indication that AT-III prevents explosive thrombin generation by inhibiting the factor IXa·VIIIa pathway. This coincidence may, however, also be the result of a change in the reaction in the range of 10-20 pM from a reaction with no lag time to a reaction with a considerable lag time, which allows the effects of the additional factor Xa generation by factor IXa·factor VIIIa and the effects of the inhibitors to become more prominent. Overall, the effects of TFPI and AT-III acting in concert seem to result in a synergistic inhibition of thrombin generation at low concentrations of initiator, leading to a 70-fold higher inhibition than expected if their combined action would have been multiplicative. Although it seems that TFPI and AT-III act in synergy under these conditions, such claims of synergism must be made with caution without full knowledge of the mechanisms involved (43Berenbaum M.C. Pharmacol. Rev. 1989; 41: 93-141PubMed Google Scholar). At present the complexity of the reaction does not allow a proof of true synergism between TFPI and AT-III.The major inhibitory effect of TFPI on factor VIIa·TF-initiated thrombin generation in the reconstituted model predicts that a TFPI deficiency would be a major risk factor for thrombosis. Titration of the effect of TFPI on thrombin generation revealed that TFPI exerts a significant inhibitory effect at 1 nM (Fig. 6). This TFPI concentration is approximately 50% of the normal plasma concentration, suggesting that an individual with a 50% TFPI level would derive a significant inhibitory benefit. Our data suggest, however, that a homozygous TFPI deficiency would result in massive thrombin formation, which may not be compatible with life. However this hypothesis does not take into account that the dependence on TFPI of inhibition of factor VIIa·TF-initiated thrombin generation might be less in the presence of the dynamic protein C pathway.Experiments with physiological concentrations of HC-II (1.38 μM) predict an insignificant role for this inhibitor as compared with AT-III. Thrombin generation proceeds in a similar fashion whether or not HC-II is present. Although a marginal effect of HC-II was observed on the activity of thrombin after the quantitative activation of prothrombin, no additional thrombin-inhibitory potential was observed when HC-II was combined with physiological concentrations of AT-III. This result supports the hypothesis that HC-II is not important as a coagulation inhibitor and is in agreement with the lack of a thrombotic tendency in individuals with reduced HC-II levels (16Tollefsen D.M. Thromb. Haemostasis. 1995; 74: 1209-1214Crossref PubMed Scopus (99) Google Scholar).The combined effects of TFPI and AT-III prevent explosive thrombin generation by traces of factor VIIa·TF in the fully reconstituted system. The dramatic change in the rate of thrombin generation over a small change in the initiating factor VIIa·TF concentration (Fig. 13, Fig. 14) clearly demonstrates 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 presence of traces of fibrinopeptide A, prothrombin F1·2, and thrombin·AT-III complexes in the plasma of normal individuals indicates that very low, but constant triggering of the coagulation cascade occurs in the unperturbed circulation. The present data suggest that the low level of basal activation of the coagulation system is largely controlled by the combined action of inhibitors like TFPI and AT-III, which prevent this apparent basal activity from turning into massive thrombin formation. 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. Pestka C.A. Blood. 1985; 66: 769-774Crossref PubMed Google Scholar). Thrombin is the only procoagulant reported to be inhibited by HC-II. The inhibition of thrombin by HC-II is potentiated by heparin and by dermatan sulfate (for a review on HC-II see Ref. 16Tollefsen D.M. Thromb. Haemostasis. 1995; 74: 1209-1214Crossref PubMed Scopus (99) Google Scholar). A heterozygous deficiency of HC-II (activity levels < 60%) is found in approximately 1% of the healthy population and does not appear to be a risk factor for thrombosis. A homozygous deficient individual has not yet been identified (16Tollefsen D.M. Thromb. Haemostasis. 1995; 74: 1209-1214Crossref PubMed Scopus (99) Google Scholar).We have described reconstituted empirical (17Lawson J.H. Kalafatis M. Stram S. Mann K.G. J. Biol. Chem. 1994; 269: 23357-23366Abstract Full Text PDF PubMed Google Scholar) and mathematical (18Jones K.C. Mann K.G. J. Biol. Chem. 1994; 269: 23367-23373Abstract Full Text PDF PubMed Google Scholar) models for the tissue factor pathway to thrombin using purified coagulation factors and computer simulations based upon the known kinetic constants for the reactions thought to be essential in the procoagulant scheme. The reaction could be divided into two phases, an "initiation phase," in which factor V and factor VIII were quantitatively cleaved and trivial amounts of factor Xa and factor IXa were produced, and a "propagation phase" in which prothrombin was quantitatively activated (17Lawson J.H. Kalafatis M. Stram S. Mann K.G. J. Biol. Chem. 1994; 269: 23357-23366Abstract Full Text PDF PubMed Google Scholar). As the concentration of initiator (factor VIIa·TF) was reduced, the initiation phase was prolonged while the rate of thrombin generation in the propagation phase varied by only 5-fold over a 1000-fold range of factor VIIa·TF concentration. The initiation phase was shortened when the reaction was initiated in the presence of factor Va while the propagation phase was dependent upon factor VIII and factor IX at factor VIIa·TF concentrations below 100 pM. The data obtained with the reconstituted empirical model using purified coagulation factors were reasonably approximated by the mathematical model. The data presented here extend the empirical tissue factor pathway to thrombin studies to include TFPI, AT-III, and HC-II.