Evaluation of the Initiation Phase of Blood Coagulation Using Ultrasensitive Assays for Serine Proteases
1997; Elsevier BV; Volume: 272; Issue: 34 Linguagem: Inglês
10.1074/jbc.272.34.21527
ISSN1083-351X
AutoresSaulius Butenas, Cornelis van ‘t Veer, Kenneth G. Mann,
Tópico(s)Hemophilia Treatment and Research
ResumoThe 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. 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, 15Lawson J.H. Kalafatis M. Stram S. Mann K.G. J. Biol. Chem. 1994; 269: 23357-23366Abstract Full Text PDF PubMed Google Scholar, 16Beguin S. Lindhout T. Hemker H.C. Thromb. Haemostasis. 1989; 61: 25-29Crossref PubMed Scopus (97) Google Scholar). The high concentrations and amidolytic activity of thrombin allow quantitation with high precision. However, direct evaluation of the activation of the other proteins involved in the coagulation cascade is not a simple task even during the propagation phase, due to their low concentrations (15Lawson J.H. Kalafatis M. Stram S. Mann K.G. J. Biol. Chem. 1994; 269: 23357-23366Abstract Full Text PDF PubMed Google Scholar, 18Jones K.C. Mann K.G. J. Biol. Chem. 1994; 269: 23367-23373Abstract Full Text PDF PubMed Google Scholar) and/or their relatively low (if any) activities toward synthetic substrates (19McRae B.J. Kurachi K. Heimark R.L. Fujikawa K. Davie E.W. Powers J.C. Biochemistry. 1981; 20: 7196-7206Crossref PubMed Scopus (78) Google Scholar, 20Cho K. Tanaka T. Cook R.R. Kisiel W. Fujikawa K. Kurachi K. Powers J.C. Biochemistry. 1984; 23: 644-650Crossref PubMed Scopus (52) Google Scholar, 21Krishnaswamy S. J. Biol. Chem. 1992; 267: 23696-23706Abstract Full Text PDF PubMed Google Scholar, 22Shigematsu Y. Miyata T. Higashi S. Miki T. Sadler J.E. Iwanaga S. J. Biol. Chem. 1992; 267: 21329-21337Abstract Full Text PDF PubMed Google Scholar, 23Butenas S. Ribarik N. Mann K.G. Biochemistry. 1993; 32: 6531-6538Crossref PubMed Scopus (56) Google Scholar). The "initiation phase" is defined in these studies as the period in which little thrombin amidolytic activity is observed or a "lag" time. There is no doubt, however, that enzymatic reactions start at the inception of reagent mixing, but the sensitivities of chromogenic assays do not allow the quantitation of the enzymes present. The most common methods used for the evaluation of low concentrations of serine proteases are multistage, coupled assays that employ additional protein(s) (24Astrup T. Mullerty S. Arch. Biochem. Biophys. 1952; 40: 346-351Crossref PubMed Scopus (1171) Google Scholar, 25Kessner A. Troll W. Arch. Biochem. Biophys. 1976; 176: 411-416Crossref PubMed Scopus (15) Google Scholar, 26Seligsohn U. Osterud B. Rapaport S.I. Blood. 1978; 52: 978-988Crossref PubMed Google Scholar, 27Ruf W. Edgington T.S. Thromb. Haemostasis. 1991; 66: 529-533Crossref PubMed Scopus (85) Google Scholar). These assays are complicated and have inherent difficulties in interpretation due to almost ubiquitous feedback activation reactions (28Lawson J.H. Mann K.G. J. Biol. Chem. 1991; 266: 11317-11327Abstract Full Text PDF PubMed Google Scholar, 29Radcliffe R. Nemerson Y. J. Biol. Chem. 1976; 251: 4797-4802Abstract Full Text PDF Google Scholar, 30Barlow G.H. Francis C.W. Marder V.J. Thromb. Res. 1981; 23: 54-61Abstract Full Text PDF Scopus (35) Google Scholar, 31Gyzander E. Eriksson E. Teger-Nilsson A.C. Thromb. Res. 1984; 35: 547-558Abstract Full Text PDF PubMed Scopus (28) Google Scholar, 32Verheijen J.H. de Jong Y.F. Chang G.T.G. Thromb. Res. 1985; 39: 281-288Abstract Full Text PDF PubMed Scopus (19) Google Scholar, 33Rao L.V.M. Rapaport S.I. Blood. 1988; 72: 396-401Crossref PubMed Google Scholar). One strategy to overcome these problems is the development of synthetic substrates and assays that allow direct and selective quantitation of enzymes at picomolar and lower concentrations. In a previous report (34Butenas S. Orfeo T. Lawson J.H. Mann K.G. Biochemistry. 1992; 31: 5399-5411Crossref PubMed Scopus (37) Google Scholar), we presented substrates containing the fluorescent aminonaphthalenesulfonamide leaving groups, which are easily modifiable in the sulfonamide moiety to gain selectivity for a given serine protease. Using these substrates, structure-efficiency correlations for a number of the serine proteases involved in blood coagulation and fibrinolysis have been established (23Butenas S. Ribarik N. Mann K.G. Biochemistry. 1993; 32: 6531-6538Crossref PubMed Scopus (56) Google Scholar, 34Butenas S. Orfeo T. Lawson J.H. Mann K.G. Biochemistry. 1992; 31: 5399-5411Crossref PubMed Scopus (37) Google Scholar, 35Butenas S. Drungilaite V. Mann K.G. Anal. Biochem. 1995; 225: 231-241Crossref PubMed Scopus (11) Google Scholar). In this study, we describe the use of 6-peptidylamino-1-naphthalenesulfonamide substrates with selective inhibitors in single enzyme microassays of the blood coagulation serine proteases. The assays were employed to reveal the sequence of events occurring during the initiation phase of zymogen activation in a tissue factor-initiated model of blood coagulation. The fluorogenic 6-peptidylamino-1-naphthalenesulfonamide substrates (PNS substrates) were synthesized and characterized as described previously (23Butenas S. Ribarik N. Mann K.G. Biochemistry. 1993; 32: 6531-6538Crossref PubMed Scopus (56) Google Scholar, 34Butenas S. Orfeo T. Lawson J.H. Mann K.G. Biochemistry. 1992; 31: 5399-5411Crossref PubMed Scopus (37) Google Scholar, 35Butenas S. Drungilaite V. Mann K.G. Anal. Biochem. 1995; 225: 231-241Crossref PubMed Scopus (11) Google Scholar). For all assays, substrates were initially dissolved in dimethyl sulfoxide to a stock concentration of 10 mm. Phosphatidylserine and phosphatidylcholine were purchased from Sigma. Phospholipid vesicles (PCPS) composed of 75% phosphatidylcholine and 25% phosphatidylserine were prepared as described (36Higgins D.L. Mann K.G. J. Biol. Chem. 1983; 258: 6503-6508Abstract Full Text PDF PubMed Google Scholar). The thrombin inhibitor hirudin (37Stone R.S. Hofsteenge J. Biochemistry. 1986; 25: 4622-4628Crossref PubMed Scopus (514) Google Scholar) was a gift from Genentech, and the factor Xa inhibitor TAP (38Krishnaswamy S. Vlasuk G.P. Bergum P.W. Biochemistry. 1994; 33: 7879-7907Crossref Scopus (56) Google Scholar) was provided as a gift from Sriram Krishnaswamy (Hematology/Oncology Division, Department of Medicine, Emory University). Recombinant human TF-(1–242) and recombinant human factor VIII (free of albumin) were provided as gifts from Shu-Len Liu and Rodger Lundblad (Hyland Division, Baxter Healthcare Corp.). Human thrombin and factors Xa, XIa, and XI were provided as gifts from Hematologic Technologies Inc. Factor XI was treated with 20 μmd-Phe-Pro-Arg chloromethyl ketone and 1 mm diisopropyl fluorophosphate to inhibit traces of factor XIa in the factor XI preparation and subsequently dialyzed. Monoclonal antibodies αHFV-9 and αHFV-17 were from the Biochemistry Antibody Core Laboratory (University of Vermont). Recombinant human factor VIIa was purchased from NOVO Pharmaceuticals. Human factors II, IX, and X were isolated from fresh frozen plasma as described previously (39Bajaj S.P. Rapaport S.I. Prodanos C. Prep. Biochem. 1981; 11: 397-412Crossref PubMed Scopus (156) Google Scholar) and were additionally purified and depleted of trace 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). Human factor V was isolated by the method of Katzmann et al. (40Katzmann J.A. Nesheim M.E. Hibbard L.S. Mann K.G. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 162-166Crossref PubMed Scopus (147) Google Scholar) and activated to factor Va as described previously (41Kane W.H. Majerus P.W. J. Biol. Chem. 1981; 256: 1002-1007Abstract Full Text PDF PubMed Google Scholar,42Suzuki K. Dahlback B. Stenflo J. J. Biol. Chem. 1982; 257: 6556-6564Abstract Full Text PDF PubMed Google Scholar). TF relipidation on PCPS vesicles and the factor VIIa-TF complex formation were accomplished as described previously (43Lawson J.H. Krishnaswamy S. Butenas S. Mann K.G. Methods Enzymol. 1993; 222: 177-194Crossref PubMed Scopus (42) Google Scholar, 44Butenas S. Mann K.G. Biochemistry. 1996; 35: 1904-1910Crossref PubMed Scopus (94) Google Scholar). Experiments were designed to evaluate each enzyme in mixtures similar to those encountered during mixed coagulation factor activation experiments. For validation/recovery studies of factors Xa and VIIa, 10 nm thrombin, 30 pm factor VIIa, 10 pm factor IXa, and 10 pm factor Xa were incubated at room temperature in HBS/CaCl2 (HBS and 2 mm CaCl2, pH 7.4) containing 200 μm PCPS for 5 min (total volume of 800 μl) (mixture A). A 200-μl aliquot of mixture A was added to 988 μl of HBS/ETDA (HBS and 20 mm EDTA, pH 7.4) containing 6 μm hirudin (standard concentration). 100 μm6-(methanesulfonyl-d-Leu-Gly-Arg)-amino-1-naphthalenediethylsulfonamide (mLGRnds) was added, and the rate of substrate hydrolysis was evaluated. A second 200-μl aliquot of mixture A was treated the same way, except that 600 nm TAP (standard concentration) was added. The rate observed for the second aliquot was subtracted from the rate observed for the first aliquot, and the difference was assigned to the factor Xa amidolytic activity. Factor Xa concentrations were estimated from a standard curve prepared using serial dilutions of purified factor Xa. The third 200-μl aliquot of mixture A was mixed with 988 μl of HBS/EDTA containing hirudin (6 μm) and TAP (600 nm). 6-(d-Phe-Pro-Arg)-amino-1-naphthalenebutylsulfonamide (FPRnbs) (100 μm final concentration) was added, and the rate of substrate hydrolysis was evaluated. A fourth 200-μl aliquot of mixture A was treated the same way, except that 20 nm TF was added, and the mixture was incubated for 20 min at room temperature to allow complete factor VIIa-tissue factor complexation. 100 μm FPRnbs was added, and the rate of substrate hydrolysis was evaluated. The difference in substrate hydrolysis rates in the presence and absence of TF is specific for factor VIIa (45Butenas S. Lawson J.H. Kalafatis M. Mann K.G. Biochemistry. 1994; 33: 3449-3456Crossref PubMed Scopus (37) Google Scholar). Factor VIIa concentrations were estimated from a standard curve prepared using serial dilutions of purified factor VIIa. Similar factor Xa and factor VIIa assays were carried out for an enzyme mixture that contained 1.4 μm thrombin, 0.3 nm factor VIIa, 1 nm factor Xa, and 1 nm factor IXa (mixture B). 1 pm thrombin was added to HBS/CaCl2 containing 200 μm PCPS (total volume of 400 μl) and incubated for 5 min at room temperature. A 200-μl aliquot of this mixture was added to 994 μl of HBS/EDTA containing 600 nm TAP; 50 μm6-(d-Val-Pro-Arg)-amino-1-naphthalenebutylsulfonamide (VPRnbs) was added; and the rate of substrate hydrolysis was evaluated. A second 200-μl aliquot was treated the same way, except that hirudin (6 μm) was added to the mixture. The difference in rates is specific for thrombin amidolytic activity. Thrombin concentrations were evaluated from a standard curve prepared using serial dilutions of purified thrombin. 400 nm thrombin, 30 pm factor VIIa, 30 pm factor IXa, and 30 pm factor Xa were incubated at room temperature in HBS/CaCl2 containing 200 μm PCPS for 5 min (total volume of 200 μl). This mixture was added to 994 μl of HBS/EDTA containing hirudin (6 μm) and TAP (600 nm), followed by the addition of 50 μm6-(d-Leu-Pro-Arg)-amino-1-naphthalenepropylsulfonamide (LPRnps). The rate of substrate hydrolysis was evaluated. In the second part of this assay, 1 pm factor XIa was added to the enzyme mixture described above, and this mixture was treated the same way as the previous one. The factor XIa concentration was evaluated from the difference in substrate hydrolysis rates and from a standard curve prepared using serial dilutions of purified factor XIa. A similar factor XIa assay was carried out with a mixture containing 1.4 μm thrombin, 1 nm factor VIIa, 1 nm factor IXa, 1 nm factor Xa, and 15 pm factor XIa. These experiments were carried out using the procedures described in previous publications (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). 1) For experiments designed to evaluate the earliest events occurring during TF-induced thrombin generation, prothrombin (1.4 μm), factor IX (90 nm), factor X (170 nm), factor VII (10 nm), and factor VIIa (100 pm) were preincubated at 37 °C for 3 min and added to 20 nm factor V or factor Va, 0.7 nm factor VIII, and 1.25 pm TF relipidated on 200 μm PCPS (final concentrations in the reaction mixture). When indicated, factor V, factor VIII, or prothrombin was omitted, or 3 μm hirudin was added. At selected time points, three aliquots were removed for the following assays: (a) a 20-μl aliquot for the evaluation of factor V activation; (b) a 5-μl aliquot for the thrombin chromogenic assay using 200 μm substrate Spectrozyme TH (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 (c) a 1200-μl aliquot for factor VIIa, factor Xa, and thrombin fluorogenic assays. Prothrombinase concentrations were calculated using a kcat for prothrombin activation of 83.6 s−1 (46Tans G. Janssen-Claessen T. Hemker H.C. Zwaal R.F.A. Rosing J. J. Biol. Chem. 1991; 266: 21864-21873Abstract Full Text PDF PubMed Google Scholar). 2) In an experiment designed to evaluate the influence of factor XI on the activation process, 1.25 pm factor VIIa was incubated in HBS/CaCl2 for 20 min at 37 °C with 0.25 nm TF relipidated on 200 μm PCPS. Following incubation, factors V and VIII at the concentrations indicated in the previous experiment as well as 30 nm factor XI were added, and the activation reaction was started by the addition of prothrombin, factor IX, and factor X at the above concentrations. At selected time points, 5-μl aliquots were removed for the chromogenic thrombin assay (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 400-μl aliquots were taken for a factor XIa assay. The factor XIa assay was based upon the observation that factor XI does not alter the thrombin generation rate in this experimental system. In this assay, 400-μl aliquots were quenched in 794 μl of HBS/EDTA containing standard concentrations of hirudin and TAP. The aliquots were transferred into cuvettes; 50 μm substrate LPRnps was added; and the rate of substrate hydrolysis was evaluated. The rate of the corresponding assay from the control experiment performed in the absence of factor XI was also evaluated, and the concentration of factor XIa was estimated from the difference in rates. 20-μl samples of the reaction mixture were quenched in 20 μl of 2% SDS, 0.06 m Tris, 10% glycerol, and 0.1% bromphenol blue, pH 6.8, and heated for 5 min at 95 °C. 17-μl subsamples were subjected to SDS-polyacrylamide gel electrophoresis under nonreducing conditions on 4–12% polyacrylamide gel as described by Laemmli (47Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207227) Google Scholar). Following SDS-polyacrylamide gel electrophoresis, the proteins were transferred to nitrocellulose membranes for immunoblot analyses using the general techniques of Towbin et al. (48Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44923) Google Scholar). Membranes were blocked for nonspecific binding with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween. Generation of factor Va was followed by incubating blocked membranes for 1.5 h with either monoclonal antibody αHFV-9 (directed against the light chain) or αHFV-17 (directed against fragment 307–506 of the heavy chain). The products of factor V recognized by these antibodies were visualized using peroxidase-conjugated horse anti-mouse IgG and the chemiluminescence reagent Luminol (DuPont). Films were developed in a Kodak X-Omat and scanned for densitometric analysis using a ScanJet 4c/T scanner (Hewlett-Packard Co.). The specific quantitation of relatively low concentrations of various enzymes generated during the activation process is based upon the substrate's selectivity, the high sensitivity of the substrate leaving group for detection, and the selectivity of specific serine protease inhibitors such as hirudin (thrombin) and TAP (factor Xa). The factor VIIa assay is based upon the increase in amidolytic activity of this enzyme caused by TF under conditions that prevent activation of the zymogens present in the system (10 mm EDTA) (45Butenas S. Lawson J.H. Kalafatis M. Mann K.G. Biochemistry. 1994; 33: 3449-3456Crossref PubMed Scopus (37) Google Scholar). The results of control experiments performed with enzyme mixtures in the presence of PCPS, TAP, and/or hirudin at the concentrations present in the enzyme assays in coagulation factor activation experiments showed that the amidolytic activities of factor VIIa, factor Xa, thrombin, and factor XIa are recovered almost quantitatively. This observation is valid for low picomolar concentrations of factors VIIa and Xa measured in the presence of 10 nm thrombin (Table I), for 1 pm factor XIa measured in the presence of 400 nm thrombin, and for 0.3 nm factor VIIa and 1 nm factor Xa or 15 pm factor XIa measured in the presence of 1.4 μm thrombin. The amidolytic activities of thrombin and factor XIa were recovered at 100 and 113–120%, respectively. The amidolytic activities of factor Xa and factor VIIa were recovered at 88–112 and 103–109%, respectively. Thus, thrombin and factor XIa can be measured at femtomolar, factor Xa at 0.4 pm, and factor VIIa at 1 pm concentrations individually 2Butenas, S., DiLorenzo, M., and Mann, K. G. (1997) Thromb. Haemostasis, in press . and reliably (±20%) in the presence of relatively high concentrations of other enzymes.Table IActivity recoveries of factors Xa and VIIa established by the evaluation of their amidolytic activities in mixtures of enzymes and inhibitors indicated under "Experimental Procedures"EnzymeSubstrateMixture AMixture BAddedMeasuredRecoveredAddedMeasuredRecoveredpmpm%nmnm%Xa1-aDirect assay.mLGRnds10.08.8881.001.12112VIIa1-bIn the presence of 20 nm TF.FPRnbs30.032.71090.300.31103IIa1 × 104NM1-cNot measured.1400NMIXa10.0NM1.00NM1-a Direct assay.1-b In the presence of 20 nm TF.1-c Not measured. Open table in a new tab In an experiment initiated with 1.25 pmTF when both factors VII and VIIa were added to the zymogen mixture, the initiation or lag phase of thrombin generation evaluated using the chromogenic p-nitroanilide substrate Spectrozyme TH continued for ∼2 min (Fig. 1). Analyses of this phase employing a fluorogenic PNS substrate, VPRnbs (Fig. 2), showed undetectable thrombin generation (<0.2 pm) over the first 20 s after the reactants were mixed. However, the latter substrate allowed quantitation of the thrombin generated from 20 s to 2 min. By 30 s, the concentration of thrombin reached 0.60 pm. Subsequently, the thrombin concentration increased at a relatively constant rate until 60 s (open circles). No prothrombin activation was observed over an 8-min period when factor V was omitted from the reaction mixture (Fig. 2, open triangles). The substitution of 20 nm factor Va for factor V decreased the interval in which thrombin generation was undetectable from 20 to 10 s (closed circles). The transition to the explosive propagation phase of thrombin generation in the experiment started 18–20 s earlier with factor Va than observed in the experiment with factor V (50–55 and 70–73 s, respectively). In the absence of factor VIII (closed triangles), the thrombin generation rate during the initiation phase was similar to that observed in the presence of factor VIII. Analyses of the thrombin generation rates indicated that, during the initial 60 s, the concentration of prothrombinase (deduced from d[IIa]/dt) in the factor Va experiment increased from undetectable levels to 7 fm (Fig. 2, inset,closed circles). In the presence of factor V, whether factor VIII was present (open circles) or absent (closed triangles), the rise in prothrombinase concentration started at ∼50–60 s. When factor Va was substituted for factor V, the rise in prothrombinase concentration occurred at a similar rate, but 20 s earlier. In experiments with and without factor VIII, the first detectable amounts of factor Va heavy chain were observed at 30 s (Fig. 3, A and C, respectively), i.e. approximately at the same time when the first detectable amounts of thrombin (0.6–0.7 pm) (Fig. 2) were observed. However, the first detectable amounts of factor Va light chain (0.36 nm) were observed after a 70-s delay relative to the appearance of the heavy chain, i.e. at 100 s (Fig. 3 B). The cleavage of intact factor V in the presence or absence of factor VIII was complete at 100–120 s after the reactants were mixed (Fig. 3,A–C). This activation was obligately dependent on thrombin since in the presence of 3 μm hirudin, no activation of factor V was observed over a 4-min period (Fig. 3 D). In the complete, factor V-containing system, the first detectable amounts (8.4 pm) of factor Xa were observed 60 s after mixing the reagents (Fig. 4, open circles). This initial rate of factor Xa generation was not altered by the presence of 3 μm hirudin (closed circles), the absence of prothrombin (open triangles), or the absence of factor VIII (closed triangles). Significant prothrombinase activity began to form at 50–60 s (Fig. 2,inset), i.e. the time at which 5–10 pm factor Xa was present. A significant increase in the rate of factor Xa generation was observed a
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