Evaluation of the Initiation Phase of Blood Coagulation Using Ultrasensitive Assays for Serine Proteases

The initiation phase of enzyme generation in a reconstituted model of the tissue factor (TF) pathway to thrombin was evaluated. At 1.25 pm added TF, no thrombin generation was observed in the absence of factor V. The substitution of factor Va for factor V increased the rate of thrombin generation. Factor X activation during the initiation phase was not influenced by the absence of factor VIII or thrombin, leading to the conclusion that initially factor Xa is generated exclusively by the factor VIIa-TF complex. When thrombin was eliminated from the system, no contribution of the factor IXa-factor VIIIa complex to factor X activation was observed during the propagation phase. Similarly, factor V activation was also not observed in the absence of thrombin, indicating that thrombin is the only enzyme responsible for factor V and factor VIII activation. Only subnanomolar amounts of factor VII were activated when prothrombin activation was almost complete. In the absence of coagulation inhibitors, factor XI did not influence thrombin generation initiated by 1.25 pm factor VIIa-TF complex. The termination of factor XIa generation by added hirudin in the factor XI experiment indicates that factor XI activation occurs exclusively by thrombin. The initiation phase of enzyme generation in a reconstituted model of the tissue factor (TF) pathway to thrombin was evaluated. At 1.25 pm added TF, no thrombin generation was observed in the absence of factor V. The substitution of factor Va for factor V increased the rate of thrombin generation. Factor X activation during the initiation phase was not influenced by the absence of factor VIII or thrombin, leading to the conclusion that initially factor Xa is generated exclusively by the factor VIIa-TF complex. When thrombin was eliminated from the system, no contribution of the factor IXa-factor VIIIa complex to factor X activation was observed during the propagation phase. Similarly, factor V activation was also not observed in the absence of thrombin, indicating that thrombin is the only enzyme responsible for factor V and factor VIII activation. Only subnanomolar amounts of factor VII were activated when prothrombin activation was almost complete. In the absence of coagulation inhibitors, factor XI did not influence thrombin generation initiated by 1.25 pm factor VIIa-TF complex. The termination of factor XIa generation by added hirudin in the factor XI experiment indicates that factor XI activation occurs exclusively by thrombin. The blood coagulation cascade is thought to be triggered when subendothelial tissue factor (TF) 1The abbreviations used are: TF, tissue factor; PNS, 6-peptidylamino-1-naphthalenesulfonamide; PCPS, phospholipid vesicles composed of 75% phosphatidylcholine and 25% phosphatidylserine; TAP, tick anticoagulant protein; HBS, Hepes-buffered saline; mLGRnds, 6-(methanesulfonyl-d-Leu-Gly-Arg)-amino-1-naphthalenediethylsulfonamide; FPRnbs, 6-(d-Phe-Pro-Arg)-amino-1-naphthalenebutylsulfonamide; VPRnbs, 6-(d-Val-Pro-Arg)-amino-1-naphthalenebutylsulfonamide; LPRnps, 6-(d-Leu-Pro-Arg)-amino-1-naphthalenepropylsulfonamide. is exposed as a consequence of vascular damage (1Ploplis V.A. Edgington T.S. Fair D.S. J. Biol. Chem. 1987; 262: 9503-9508Abstract Full Text PDF PubMed Google Scholar, 2Lee D.T. Rapaport S.I. Rao L.V.M. J. Biol. Chem. 1992; 267: 15447-15454Abstract Full Text PDF PubMed Google Scholar). TF forms an enzymatic complex with preexistent plasma factor VIIa, and the resulting factor VIIa-TF complex activates factors X and IX (3Jesty J. Silverberg S.A. J. Biol. Chem. 1979; 254: 12337-12345Abstract Full Text PDF PubMed Google Scholar, 4Komiyama Y. Pedersen A.H. Kisiel W. Biochemistry. 1990; 29: 9418-9425Crossref PubMed Scopus (153) Google Scholar, 5Lawson J.H. Mann K.G. J. Biol. Chem. 1991; 266: 11317-11327Abstract Full Text PDF PubMed Google Scholar). Factor IXa in complex with its cofactor, factor VIIIa, activates factor X at an ∼50-fold higher rate than the factor VIIa-TF complex (6Mann K.G. Krishnaswamy S. Lawson J.H. Semin. Hematol. 1992; 29: 213-226PubMed Google Scholar). In turn, factor Xa with phospholipids may activate factor VII (7Radcliffe R. Nemerson Y. J. Biol. Chem. 1975; 250: 388-395Abstract Full Text PDF PubMed Google Scholar) and further enhance factor IX and factor X activation. The major function of factor Xa is to form the prothrombinase complex with factor Va and a phospholipid membrane surface, leading to prothrombin activation (8Mann K.G. Jenny R.J. Krishnaswamy S. Annu. Rev. Biochem. 1988; 57: 915-927Crossref PubMed Scopus (451) Google Scholar). Thrombin cleaves soluble fibrinogen, forming fibrin, which polymerizes to form an insoluble clot (9Blomback B. Blomback M. Ann. N. Y. Acad. Sci. 1972; 202: 77-97Crossref PubMed Scopus (118) Google Scholar). The blood coagulation cascade is down-regulated by the synergistic action of the natural inhibitors of blood coagulation: tissue factor pathway inhibitor, antithrombin III, and the activated protein C system (10van ‘t Veer C. Mann K.G. J. Biol. Chem. 1997; 272: 4367-4377Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 11van ‘t Veer C. Golden N.J. Kalafatis M. Mann K.G. J. Biol. Chem. 1997; 272: 7983-7994Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The procoagulant processes described above may be divided into two phases (see Fig. 1): (a) an initiation phase, which is characterized by the appearance of small amounts of thrombin (<1 nm) and other enzymes (<10 pm) and by low rates of their generation; and (b) a propagation phase, which may be characterized by rapid, quantitative prothrombin activation. Coincidentally, when the inhibitors are present, thrombin generation is terminated, and extant thrombin is inhibited by the anticoagulation system. The propagation and termination processes have been investigated and described in studies in various reconstituted systems (10van ‘t Veer C. Mann K.G. J. Biol. Chem. 1997; 272: 4367-4377Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 11van ‘t Veer C. Golden N.J. Kalafatis M. Mann K.G. J. Biol. Chem. 1997; 272: 7983-7994Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 12van ‘t Veer C. Kalafatis M. Bertina R. Simioni P. Mann K.G. J. Biol. Chem. 1997; 272: 20721-20729Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 13Hoffman M. Monroe D.M. Oliver J.A. Roberts H.R. Blood. 1995; 86: 1794-1801Crossref PubMed Google Scholar, 14Rao L.V.M. Nordfang O. Hoang A.D. Pendurthi U.R. Blood. 1995; 85: 121-129Crossref PubMed Google Scholar, 15Lawson J.H. Kalafatis M. Stram S. Mann K.G. J. Biol. Chem. 1994; 269: 23357-23366Abstract Full Text PDF PubMed Google Scholar), in blood plasma (16Beguin S. Lindhout T. Hemker H.C. Thromb. Haemostasis. 1989; 61: 25-29Crossref PubMed Scopus (97) Google Scholar), and in minimally altered whole blood (17Rand M.D. Lock J.B. van ‘t Veer C. Gaffney D.P. Mann K.G. Blood. 1996; 88: 3432-3445Crossref PubMed Google Scholar). The major regulatory steps that occur during the initiation phase are major factor V and factor VIII and limited factor IX and factor X activation (15Lawson J.H. Kalafatis M. Stram S. Mann K.G. J. Biol. Chem. 1994; 269: 23357-23366Abstract Full Text PDF PubMed Google Scholar). The studies presently available, however, do not identify the sources of the initial factors Va, VIIIa, and Xa required to generate the thrombin that is essential in the catalytic feedback activation leading to explosive thrombin generation. Knowledge of these early reactions is critical to the understanding of blood coagulation, and the answers lie in understanding the dynamics of the initiation phase of the process. The most common methods used for the evaluation of the different phases of the coagulation process have employed peptidyl-p-nitroanilide substrates (10van ‘t Veer C. Mann K.G. J. Biol. Chem. 1997; 272: 4367-4377Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 11van ‘t Veer C. Golden N.J. Kalafatis M. Mann K.G. J. Biol. Chem. 1997; 272: 7983-7994Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 12van ‘t Veer C. Kalafatis M. Bertina R. Simioni P. Mann K.G. J. 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Biochem. 1995; PubMed Scopus Google Scholar). substrates were initially in to a of and phosphatidylcholine were from vesicles composed of 75% phosphatidylcholine and 25% were as described Mann K.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The thrombin hirudin J. Biochemistry. PubMed Scopus Google Scholar) was a from and the factor Xa S. Biochemistry. 1994; Scopus Google Scholar) was as a from Krishnaswamy of and factor VIII of were as from and thrombin and factors and XI were as from Factor XI was with and to of factor XIa in the factor XI and and were from the of factor was from factors and X were from plasma as described Rapaport S.I. C. Biochem. 1981; PubMed Scopus Google Scholar) and were and of enzymes as described (10van ‘t Veer C. Mann K.G. J. Biol. Chem. 1997; 272: 4367-4377Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). factor V was by the of J.A. Mann K.G. Acad. Sci. S. 1981; PubMed Scopus Google Scholar) and activated to factor Va as described J. Biol. Chem. 1981; Full Text PDF PubMed Google K. B. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). TF vesicles and the factor VIIa-TF complex were as described J.H. Krishnaswamy S. S. Mann K.G. PubMed Scopus Google Scholar, S. Mann K.G. Biochemistry. 1996; PubMed Scopus Google Scholar). were to enzyme in to during coagulation factor activation studies of factors Xa and VIIa, thrombin, pm factor VIIa, pm factor and pm factor Xa were at in and for of of was added to of and hirudin was and the rate of was evaluated. of was the that was The rate observed for the was from the rate observed for the and the was to the factor Xa amidolytic Factor Xa concentrations were from a of factor The of was with of hirudin and was and the rate of was evaluated. of was the that TF was and the was for at to allow factor factor was and the rate of was evaluated. The in rates in the and absence of TF is for factor S. Lawson J.H. Kalafatis M. Mann K.G. Biochemistry. 1994; PubMed Scopus Google Scholar). Factor concentrations were from a of factor factor Xa and factor were for an enzyme that thrombin, factor VIIa, factor and factor IXa pm thrombin was added to of and for at of was added to of was and the rate of was evaluated. was the that hirudin was added to the The in rates is for thrombin amidolytic Thrombin concentrations were from a of thrombin. thrombin, pm factor VIIa, pm factor and pm factor Xa were at in for of was added to of hirudin and by the of The rate of was evaluated. In the of pm factor XIa was added to the enzyme described and was the as the The factor XIa was from the in rates and from a of factor factor XIa was with a thrombin, factor VIIa, factor factor and pm factor were the described in (10van ‘t Veer C. Mann K.G. J. Biol. Chem. 1997; 272: 4367-4377Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 15Lawson J.H. Kalafatis M. Stram S. Mann K.G. J. Biol. Chem. 1994; 269: 23357-23366Abstract Full Text PDF PubMed Google Scholar). to the during thrombin prothrombin factor IX factor X factor VII and factor pm) were at for and added to factor V or factor Va, factor and 1.25 pm TF concentrations in the When factor factor or prothrombin was or hirudin was At were for the (a) a for the evaluation of factor V (b) a for the thrombin (10van ‘t Veer C. Mann K.G. J. Biol. Chem. 1997; 272: 4367-4377Abstract Full Text Full Text PDF PubMed Scopus (174) Google and a for factor VIIa, factor and thrombin concentrations were a for prothrombin activation of T. Hemker H.C. J. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). In an experiment to the influence of factor XI the activation 1.25 pm factor was in for at with TF factors V and VIII at the concentrations in the experiment as as factor XI were and the activation was by the of factor and factor X at the above At were for the thrombin (10van ‘t Veer C. Mann K.G. J. Biol. Chem. 1997; 272: 4367-4377Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), and were for a factor XIa The factor XIa was the that factor XI not the thrombin generation rate in system. In were in of concentrations of hirudin and The were into was and the rate of was evaluated. The rate of the from the experiment in the absence of factor XI was also and the of factor XIa was from the in of the were in of and and for at were to as described by PubMed Scopus Google Scholar). the proteins were to for the of T. J. Acad. Sci. S. 1979; PubMed Scopus Google Scholar). were for with in of factor Va was by for with the or of the The of factor V by these were and the were in a and for a The quantitation of relatively low concentrations of various enzymes generated during the activation process is the the high of the for and the of inhibitors as hirudin and The factor is the in amidolytic activity of enzyme by TF that activation of the in the system S. Lawson J.H. Kalafatis M. Mann K.G. Biochemistry. 1994; PubMed Scopus Google Scholar). The of with enzyme in the of PCPS, TAP, and/or hirudin at the concentrations in the enzyme in coagulation factor activation that the amidolytic activities of factor VIIa, factor thrombin, and factor XIa are almost is for low concentrations of factors and Xa in the of thrombin for pm factor XIa in the of thrombin, and for factor and factor Xa or pm factor XIa in the of thrombin. The amidolytic activities of thrombin and factor XIa were at and The amidolytic activities of factor Xa and factor were at and thrombin and factor XIa be at factor Xa at and factor at pm concentrations and K. Thromb. in and in the of relatively high concentrations of other of factors Xa and by the evaluation of their amidolytic activities in of enzymes and inhibitors the of In the of in a In an experiment initiated with 1.25 when factors VII and were added to the the initiation or phase of thrombin generation the for of phase a thrombin generation pm) the the were However, the quantitation of the thrombin generated from to the of thrombin the thrombin increased at a relatively rate prothrombin activation was observed an when factor V was from the The substitution of factor Va for factor V the in which thrombin generation was from to The to the explosive propagation phase of thrombin generation in the experiment with factor Va than observed in the experiment with factor V and In the absence of factor VIII the thrombin generation rate during the initiation phase was to that observed in the of factor of the thrombin generation rates during the initial the of prothrombinase from in the factor Va experiment increased from to In the of factor factor VIII was or the in prothrombinase at When factor Va was for factor the in prothrombinase at a In with and factor the amounts of factor Va were observed at and at the when the amounts of thrombin pm) were However, the amounts of factor Va nm) were observed a to the appearance of the at The of factor V in the or absence of factor VIII was at the were activation was thrombin in the of no activation of factor V was observed a In the factor system, the amounts pm) of factor Xa were observed the initial rate of factor Xa generation was not altered by the of hirudin the absence of prothrombin or the absence of factor VIII prothrombinase activity to form at the at which pm factor Xa was in the rate of factor Xa generation was observed a only in the system In when factor VIII was or when factor VIII activation by thrombin was factor Xa generation almost a Factor X activation rates in the absence of prothrombin or factor VIII or in the of hirudin were to the conclusion that thrombin is the only enzyme responsible for factor VIII activation these The in Fig. during the initiation phase, the of prothrombinase is than that of factor Va or factor Xa When factor Va subnanomolar and factor Xa was at the of prothrombinase increased to the of the propagation phase, the of prothrombinase to that of factor The process of factor VII activation is the of thrombin in the system. In the absence of factor the thrombin rate of the propagation phase of prothrombin activation is from to in with the system, and thrombin generation during phase at a rate (15Lawson J.H. Kalafatis M. Stram S. Mann K.G. J. Biol. Chem. 1994; 269: 23357-23366Abstract Full Text PDF PubMed Google Scholar). in factor and were observed at in the system and at in the absence of factor VIII At that however, the thrombin concentrations were and In the activation of factor VII at In thrombin was or by hirudin the initial rates of factor generation were than when thrombin was allow the conclusion that the of factor VII is thrombin. In to the influence of factor the was initiated by 1.25 pm factor VIIa-TF and the generation of thrombin and factor XIa was during the The substitution of 1.25 pm factor VIIa-TF complex for 1.25 pm TF added to the protein factor the initiation phase from to The initiation phase of thrombin generation was by the propagation phase to for the complex and from to for the TF The thrombin generation in the factor VIIa-TF experiment were in the and absence of factor The was employed for the factor XI activation to factor XIa at The in Fig. that is no pm) factor XIa observed the of the thrombin is at relatively low The pm) of factor XIa were observed at In at that thrombin generation was in an explosive at The rate of factor XI activation was observed at the of the and was for at of the rate of factor XIa generation was At the only nm) of factor XI was activated to factor When hirudin was added to the at the initiation of further generation of factor XIa was to the conclusion that thrombin is the only of factor XI in the absence of inhibitors in the system. The substrates with inhibitors and the evaluation of thrombin and factors VIIa, and XIa generated during the initiation and propagation phases of the tissue procoagulant The been to the of The into the during phase, which to explosive thrombin generation during the propagation phase of the system. The that be with the of are the the initiation phase, the for prothrombinase complex is factor Va the initiation phase of the factor Xa is generated by the factor VIIa-TF complex. factor generation during the initiation phase of the not occur by factor VIIa-TF or by factor In the absence of factor the of factor Xa is of thrombin pm at The initial that thrombin is factor V or factor Va, with the from factor Va of factor V Thrombin is the only of factors and The of the is the of thrombin as an essential in thrombin generation. of the initiation phase of the tissue factor pathway to thrombin the conclusion during the initiation phase, the of the prothrombinase complex in the absence of inhibitors is factor Va conclusion is the that substitution of factor V by factor Va the initiation phase and that amounts of prothrombinase to of factor are generated only factor Va at relatively high during the of the propagation phase, factor Xa the of the prothrombinase complex. The factor Xa not the of factor Va the of the prothrombin been The of factor V and/or factor Va activity in the initiation phase of the an The activation of prothrombin a factor V or factor Va with factor Xa in the the factor Xa activation of factor V be by the in is with the conclusion that factor V is in forming prothrombinase or that amounts of factor Va are in the factor V to the initial complex at factor V the activity of factor Va as a to prothrombinase complex and activity J.B. Mann K.G. J. Biol. Chem. 1979; 254: Full Text PDF PubMed Google Scholar). M. that activity is to the initial amounts of thrombin in system. the other these also that the of of factor Va in a factor V The of factor Xa generation during the initiation phase of activation that factor Xa is initially generated only by the of the factor VIIa-TF complex in the amounts of the factor IXa-factor VIIIa complex the factor Xa generation and the rate of factor X activation The in the of hirudin as as in the absence of factor VIII or the initial rate of factor Xa generation is to that in a system the conclusion during the initiation phase, factor X is activated only by the factor VIIa-TF complex of the factor IXa-factor VIIIa complex. rate of factor Xa generation is observed in the absence of factor VIII and in the absence of thrombin. to the conclusion that thrombin is the only enzyme responsible for factor VIII activation. the that is no factor V activation in the absence of prothrombin or thrombin indicates that is also activated only by thrombin these The of pm factor Xa not in factor V pm thrombin of factor V. The relatively appearance of generated factor at the TF the activation of factor VII not in the generation of thrombin. factor VIIa, in the of limited concentrations of TF, is to thrombin generation in an explosive generation of factor occurs during the to the initiation phase at the TF have in a S. Mann K.G. Biochemistry. 1996; PubMed Scopus Google Scholar) that the most of factor VII of blood is factor Xa a phospholipid Thrombin also activity factor however, is than factor The in in the reconstituted system, factor VII is activated by thrombin. may be by the at the of the initial phase of factor VII thrombin is at the of factor Xa the low the relatively low of thrombin in factor VII activation is for by the high of the amounts of thrombin generated are involved in the of proteins at low and subnanomolar concentrations and the amounts of factor Xa are involved in the of at factor and factor of which only prothrombin is at a S. Mann K.G. J. Biol. Chem. 1987; 262: Full Text PDF PubMed Google Scholar). the factor Xa generated during the experiment is involved in the prothrombinase and in is inhibited from factor VII S. Mann K.G. Biochemistry. 1996; PubMed Scopus Google Scholar). been that factor XI activation may be by thrombin K. Fujikawa K. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar, 1991; PubMed Scopus Google Scholar). The of process for in is Acad. Sci. S. 1992; PubMed Scopus Google Scholar, T. C. Silverberg M. Blood. PubMed Google Scholar, Blood. PubMed Google Scholar). Lawson J.H. Kalafatis M. Stram S. Mann K.G. J. Biol. Chem. 1994; 269: 23357-23366Abstract Full Text PDF PubMed Google Scholar) that factor XI influenced the explosive propagation phase of thrombin generation in procoagulant system. Factor XI activation however, were not evaluated. The in that thrombin generation initiated by 1.25 pm factor VIIa-TF complex in the absence of coagulation inhibitors is not influenced at plasma concentrations of factor is due to a relatively low rate of factor XI activation by thrombin and the to factor IXa generation in the initial phase of the coagulation process. In the prothrombin activation in system not of influence of the factor XIa generated process. The only enzyme in the system to generate factor XIa is thrombin. In the of experiment that concentrations of factor XIa may be in an a The of is than that of the by Thromb. Haemostasis. 1994; PubMed Scopus Google Scholar), which an In the enzyme the of substrates allow quantitation of the initiation phase of blood coagulation in a reconstituted system. The to the of enzymes or enzymatic responsible for the generation of the initial amounts of of blood coagulation and their are to for in enzymatic evaluation of substrates and to and for Krishnaswamy for and for tissue factor and factor for to factor and Jenny for coagulation