The Significance of Circulating Factor IXa in Blood
2004; Elsevier BV; Volume: 279; Issue: 22 Linguagem: Inglês
10.1074/jbc.m400531200
ISSN1083-351X
AutoresSaulius Butenas, Thomas Orfeo, Matthew Gissel, Kathleen E. Brummel, Kenneth G. Mann,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoThe presence of activation peptides (AP) of the vitamin K-dependent proteins in the phlebotomy blood of human subjects suggests that active serine proteases may circulate in blood as well. The goal of the current study was to evaluate the influence of trace amounts of key coagulation proteases on tissue factor-independent thrombin generation using three models of coagulation. With procoagulants and select coagulation inhibitors at mean physiological concentrations, concentrations of factor IXa, factor Xa, and thrombin were set either equal to those of their AP or to values that would result based upon the rates of AP/enzyme generation and steady state enzyme inhibition. In the latter case, numerical simulation predicts that sufficient thrombin to produce a solid clot would be generated in ∼2 min. Empirical data from the synthetic plasma suggest clotting times of 3–5 min, which are similar to that observed in contact pathway-inhibited whole blood (4.3 min) initiated with the same concentrations of factors IXa and Xa and thrombin. Numerical simulations performed with the concentrations of two of the enzymes held constant and one varied suggest that the presence of any pair of enzymes is sufficient to yield rapid clot formation. Modeling of states (numerical simulation and whole blood) where only one circulating protease is present at steady state concentration shows significant thrombin generation only for factor IXa. The addition of factor Xa and thrombin has little effect (if any) on thrombin generation induced by factor IXa alone. These data indicate that 1) concentrations of active coagulation enzymes circulating in vivo are significantly lower than can be predicted from the concentrations of their AP, and 2) expected trace amounts of factor IXa can trigger thrombin generation in the absence of tissue factor. The presence of activation peptides (AP) of the vitamin K-dependent proteins in the phlebotomy blood of human subjects suggests that active serine proteases may circulate in blood as well. The goal of the current study was to evaluate the influence of trace amounts of key coagulation proteases on tissue factor-independent thrombin generation using three models of coagulation. With procoagulants and select coagulation inhibitors at mean physiological concentrations, concentrations of factor IXa, factor Xa, and thrombin were set either equal to those of their AP or to values that would result based upon the rates of AP/enzyme generation and steady state enzyme inhibition. In the latter case, numerical simulation predicts that sufficient thrombin to produce a solid clot would be generated in ∼2 min. Empirical data from the synthetic plasma suggest clotting times of 3–5 min, which are similar to that observed in contact pathway-inhibited whole blood (4.3 min) initiated with the same concentrations of factors IXa and Xa and thrombin. Numerical simulations performed with the concentrations of two of the enzymes held constant and one varied suggest that the presence of any pair of enzymes is sufficient to yield rapid clot formation. Modeling of states (numerical simulation and whole blood) where only one circulating protease is present at steady state concentration shows significant thrombin generation only for factor IXa. The addition of factor Xa and thrombin has little effect (if any) on thrombin generation induced by factor IXa alone. These data indicate that 1) concentrations of active coagulation enzymes circulating in vivo are significantly lower than can be predicted from the concentrations of their AP, and 2) expected trace amounts of factor IXa can trigger thrombin generation in the absence of tissue factor. Five vitamin K-dependent proteins serve as precursors for serine proteases, which are essential for normal hemostasis (factors VII, IX, and X, prothrombin, and protein C) (1Jenny N.S. Mann K.G. Thrombosis and Hemorrhage. 2nd Ed. Williams & Wilkins, Baltimore, MD1998: 3-27Google Scholar). The zymogens are converted to fully functional enzymes by the proteolytic removal of corresponding activation peptides. The only exception is factor VII, which is converted to an active enzyme by a single cleavage not followed by the release of an activation peptide (2Hagen F.S. Gray C.L. O'Hara P. Grant F.J. Saari G.C. Woodbury R.G. Hart C.E. Insley M. Kisiel W. Kurachi K. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2412-2416Crossref PubMed Scopus (318) Google Scholar). For the release of activation peptides from factor X, protein C, and prothrombin, single cleavages are required, whereas the activation peptide of factor IX is generated by two proteolytic cleavages (3Ichinose A. Davie E.W. Hemostasis and Thrombosis. 3rd Ed. J. B. Lippincott Co., Philadelphia, PA1994: 19-54Google Scholar). The length of activation peptides varies from 12 amino acids for protein C to 271 amino acids for prothrombin (Fragment 1.2). There are no structural domains observed for activation peptides of factor IX, factor X, and protein C. However, fragment 1.2 of prothrombin contains a Gla domain and two kringle domains. The former one is rich in γ-carboxyglutamic acid residues and facilitates a calcium-dependent binding of prothrombin to the membranes containing acidic phospholipids (1Jenny N.S. Mann K.G. Thrombosis and Hemorrhage. 2nd Ed. Williams & Wilkins, Baltimore, MD1998: 3-27Google Scholar). The kringle domain 1 contains two of three prothrombin glycosylation sites (4Mizuochi T. Fujii J. Kisiel W. Kobata A. J. Biochem. (Tokyo). 1981; 90: 1023-1031Crossref PubMed Scopus (24) Google Scholar).The detection of activation peptides of the vitamin K-dependent proteins in plasma of human subjects suggests that active serine proteases may circulate in blood either as a result of continuous in situ production ("idling motor") or as a consequence of production during localized coagulation ("historical record"). The relationship between the concentrations of enzymes and their corresponding activation peptides is not clear in part because the source(s) of these peptides is not identified. A possible trigger for the generation of these peptides and corresponding enzymes can be active tissue factor hypothetically circulating in blood (5Giesen P.L. Rauch U. Bohrmann B. Kling D. Roque M. Fallon J.T. Badimon J.J. Himber J. Riederer M.A. Nemerson Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2311-2315Crossref PubMed Scopus (892) Google Scholar). Alternatively, they can be generated due to the initiation of the coagulation cascade during phlebotomy and processing of blood. The key problem for the evaluation of active enzymes is that direct assays of these serine proteases are difficult (if possible at all) due to their rapid neutralization by the natural inhibitors present in blood.To circumvent this obstacle, assays for the activation peptides released during the generation of serine proteases involved in blood coagulation as well as for the reaction and inhibition products of these enzymes have been developed (6Lau H.K. Rosenberg J.S. Beeler D.L. Rosenberg R.D. J. Biol. Chem. 1979; 254: 8751-8761Abstract Full Text PDF PubMed Google Scholar, 7Hoek J.A. Nurmohamed M.T. ten Cate J.W. Buller H.R. Knipscheer H.C. Hamelynck K.J. Marti R.K. Sturk A. Thromb. Haemost. 1989; 62: 1050-1052Crossref PubMed Scopus (41) Google Scholar, 8Kockum C. Frebelius S. Thromb. Res. 1980; 19: 589-598Abstract Full Text PDF PubMed Scopus (117) Google Scholar, 9Owen J. Thromb. Haemost. 1989; 62: 807-810Crossref PubMed Scopus (9) Google Scholar, 10Bauer K.A. Kass B.L. ten Cate H. Hawiger J.J. Rosenberg R.D. Blood. 1990; 76: 731-736Crossref PubMed Google Scholar, 11Bauer K.A. Kass B.L. ten Cate H. Bednarek M.A. Hawiger J.J. Rosenberg R.D. Blood. 1989; 74: 2007-2015Crossref PubMed Google Scholar). Products, including activation peptides of prothrombin, factor X, and factor IX, related to the processes leading to thrombin generation and clot formation have been used as markers for the assessment of the coagulation state of human beings. Elevated levels of activation peptides are observed in patients suffering from disseminated intravascular coagulation (11Bauer K.A. Kass B.L. ten Cate H. Bednarek M.A. Hawiger J.J. Rosenberg R.D. Blood. 1989; 74: 2007-2015Crossref PubMed Google Scholar, 12Bauer K.A. Rosenberg R.D. Blood. 1984; 64: 791-796Crossref PubMed Google Scholar), deep vein thrombosis (13Bucek R.A. Reiter M. Quehenberger P. Weltermann A. Kyrle P.A. Minar E. Br. J. Haematol. 2003; 120: 123-128Crossref PubMed Scopus (12) Google Scholar, 14Boneu B. Bes G. Pelzer H. Sie P. Boccalon H. Thromb. Haemost. 1991; 65: 28-31Crossref PubMed Scopus (169) Google Scholar), coronary heart disease (15Miller G.J. Bauer K.A. Barzegar S. Cooper J.A. Rosenberg R.D. Thromb. Haemost. 1996; 75: 767-771Crossref PubMed Scopus (177) Google Scholar, 16Ardissino D. Merlini P.A. Bauer K.A. Bramucci E. Ferrario M. Coppola R. Fetiveau R. Lucreziotti S. Rosenberg R.D. Mannucci P.M. Blood. 2001; 98: 2726-2729Crossref PubMed Scopus (68) Google Scholar), unstable angina, myocardial infarction (17Merlini P.A. Bauer K.A. Oltrona L. Ardissino D. Cattaneo M. Belli C. Mannucci P.M. Rosenberg R.D. Circulation. 1994; 90: 61-68Crossref PubMed Scopus (565) Google Scholar, 18Minnema M.C. Peters R.J.G. de Winter R. Lubbers Y.P.T. Barzegar S. Bauer K.A. Rosenberg R.D. Hack C.E. ten Cate H. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2489-2493Crossref PubMed Scopus (71) Google Scholar), and other coagulation-related disorders (19Bauer K.A. Bailliere's Clin. Haematol. 1999; 12: 387-406Crossref PubMed Scopus (26) Google Scholar). The concentrations of activation peptides are also affected by deficiencies or mutations in procoagulants and coagulation inhibitors (10Bauer K.A. Kass B.L. ten Cate H. Hawiger J.J. Rosenberg R.D. Blood. 1990; 76: 731-736Crossref PubMed Google Scholar, 20Mannucci P.M. Bauer K.A. Santagostino E. Faioni E. Barzegar S. Coppola R. Rosenberg R.D. Blood. 1994; 84: 1314-1319Crossref PubMed Google Scholar, 21Bauer K.A. Humphries S. Smillie B. Li L. Cooper J.A. Barzegar S. Rosenberg R.D. Miller G.J. Thromb. Haemost. 2000; 84: 396-400Crossref PubMed Scopus (22) Google Scholar, 22Bauer K.A. Broekmans A.W. Bertina R.M. Conard J. Horellou M.H. Samama M.M. Rosenberg R.D. Blood. 1988; 71: 1418-1426Crossref PubMed Google Scholar, 23Mannucci P.M. Tripodi A. Bottasso B. Baudo F. Finazzi G. De Stefano V. Palareti G. Manotti C. Mazzucconi M.G. Castaman G. Thromb. Haemost. 1992; 67: 200-202Crossref PubMed Scopus (74) Google Scholar, 24Santagostino E. Mannucci P.M. Gringeri A. Tagariello G. Baudo F. Bauer K.A. Rosenberg R.D. Thromb. Haemost. 1994; 71: 737-740Crossref PubMed Scopus (25) Google Scholar), by smoking (25Miller G.J. Bauer K.A. Cooper J.A. Rosenberg R.D. Thromb. Haemost. 1998; 79: 549-553Crossref PubMed Scopus (90) Google Scholar), and by age (26Mari D. Mannucci P.M. Coppola R. Bottasso B. Bauer K.A. Rosenberg R.D. Blood. 1995; 85: 3144-3149Crossref PubMed Google Scholar).Although there is a strong correlation between the prothrombotic state of individuals and estimated levels of activation peptides, the question of whether these levels reflect concentrations of active enzymes circulating in vivo remains open. The validity of this question can be illustrated by the observation that estimated activation peptide levels are dependent upon the conditions of sample collection (27Tripodi A. Chantarangkul V. Bottasso B. Mannucci P.M. Thromb. Haemost. 1994; 73: 548-550Google Scholar, 28Leroy-Mathurin C. Gouault-Heilmann M. Thromb. Res. 1994; 74: 399-407Abstract Full Text PDF PubMed Scopus (16) Google Scholar).In the current study, we evaluated the capability of thrombin, factor Xa, and factor IXa to trigger thrombin generation and clot formation, when these enzymes are present at concentrations relevant to those reported for their activation peptides in healthy individuals. Three in vitro models of coagulation developed in our laboratory were used to achieve this goal (29Jones K.C. Mann K.G. J. Biol. Chem. 1994; 269: 23367-23373Abstract Full Text PDF PubMed Google Scholar, 30Hockin M.F. Jones K.C. Everse S.J. Mann K.G. J. Biol. Chem. 2002; 277: 18322-18333Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 31Lawson J.H. Kalafatis M. Stram S. Mann K.G. J. Biol. Chem. 1994; 269: 23357-23366Abstract Full Text PDF PubMed Google Scholar, 32Butenas S. van't Veer C. Mann K.G. J. Biol. Chem. 1997; 272: 21527-21533Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 33Rand M.D. Lock J.B. van't Veer C. Gaffney D.P. Mann K.G. Blood. 1996; 88: 3432-3445Crossref PubMed Google Scholar, 34Cawthern K.M. van't Veer C. Lock J.B. DiLorenzo M.E. Branda R.F. Mann K.G. Blood. 1998; 91: 4581-4592Crossref PubMed Google Scholar).EXPERIMENTAL PROCEDURESMaterialsHuman coagulation factors VII, X, and IX and prothrombin were isolated from fresh frozen plasma using the general methods of Bajaj et al. (35Bajaj S.P. Rapaport S.I. Prodanos C. Prep. Biochem. 1981; 11: 397-412Crossref PubMed Scopus (155) Google Scholar) and were purged of trace contaminants and traces of active enzymes as described (36van't Veer C. Mann K.G. J. Biol. Chem. 1997; 272: 4367-4377Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Human factor V and antithrombin III (AT-III) were isolated from freshly frozen plasma (37Katzmann J.A. Nesheim M.E. Hibbard L.S. Mann K.G. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 162-166Crossref PubMed Scopus (143) Google Scholar, 38Griffith M.J. Noyes C.M. Church F.C. J. Biol. Chem. 1985; 260: 2218-2225Abstract Full Text PDF PubMed Google Scholar). Recombinant factor VIII and recombinant tissue factor (TF) (residues 1–242) were provided as gifts by Drs. Shu Len Liu and Roger Lundblad (Hyland Division, Baxter Healthcare Corp., Duarte, CA). Recombinant human factor VIIa was provided as a gift by Dr. Ula Hedner (Novo Nordisk, Denmark). Recombinant full-length tissue factor pathway inhibitor (TFPI) produced in Escherichia coli was provided as a gift by Dr. K. Johnson (Chiron Corp., Emeryville, CA). Factor IXa, factor Xa, and α-thrombin were provided as a gift by Dr. R. Jenny (Hematologic Technologies, Essex Junction, VT). Corn trypsin inhibitor (CTI) 1The abbreviations used are: AT-III, antithrombin III; CTI, corn trypsin inhibitor; PS, 1,2-dioleolyl-sn-glycero-3-phospho-l-serine; PC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; PCPS, phospholipid vesicle composed of 25% PS and 75% PC; FPRck, d-Phe-Pro-ArgCH2Cl; VPRnbs, 6-(d-Val-Pro-Arg)amino-1-naphthalene(n-butyl)sulfonamide; PPP, platelet-poor plasma; AP, activation peptide(s); TAT, thrombin-AT-III; TF, tissue factor; TFPI, tissue factor pathway inhibitor. was isolated from popcorn as described elsewhere (34Cawthern K.M. van't Veer C. Lock J.B. DiLorenzo M.E. Branda R.F. Mann K.G. Blood. 1998; 91: 4581-4592Crossref PubMed Google Scholar). Washed platelets were prepared by the procedure of Mustard et al. (39Mustard J.F. Perry D.W. Ardlie N.G. Packham M.A. Br. J. Haematol. 1972; 22: 193-204Crossref PubMed Scopus (650) Google Scholar). Preparation of the TF/lipid reagent was done as described elsewhere (34Cawthern K.M. van't Veer C. Lock J.B. DiLorenzo M.E. Branda R.F. Mann K.G. Blood. 1998; 91: 4581-4592Crossref PubMed Google Scholar). 1,2-Dioleolyl-sn-glycero-3-phospho-l-serine (PS) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (PC) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL), and EDTA (Ca2+ quencher) was purchased from Sigma. Phospholipid vesicles (PCPS) composed of 25% PS and 75% PC were prepared as described (40Lawson J.H. Krishnaswamy S. Butenas S. Mann K.G. Methods Enzymol. 1993; 222: 177-195Crossref PubMed Scopus (42) Google Scholar). Spectrozyme TH was purchased from American Diagnostica, Inc. (Greenwich, CT). d-Phe-Pro-ArgCH2Cl (FPRck) and the fluorogenic substrate 6-(d-Val-Pro-Arg)amino-1-naphthalene(n-butyl)sulfonamide (VPRnbs) (41Butenas S. DiLorenzo M.E. Mann K.G. Thromb. Haemost. 1997; 78: 1193-1201Crossref PubMed Scopus (29) Google Scholar) were synthesized in house. Recombinant hirudin was a gift from Genentech. An enzyme-linked immunosorbent assay thrombin-AT-III (TAT) kit (Enzygnost TAT) was purchased from Behring (Marburg, Germany).Numerical SimulationThe present effort is based upon prior publications by Jones et al. (29Jones K.C. Mann K.G. J. Biol. Chem. 1994; 269: 23367-23373Abstract Full Text PDF PubMed Google Scholar) and Hockin et al. (30Hockin M.F. Jones K.C. Everse S.J. Mann K.G. J. Biol. Chem. 2002; 277: 18322-18333Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). An additional step of factor X activation by factor IXa was incorporated using kinetic constants published by Walsh and co-workers (42Rawala-Sheikh R. Ahmad S.S. Ashby B. Walsh P.N. Biochemistry. 1990; 29: 2606-2611Crossref PubMed Scopus (90) Google Scholar) (Km = 1.4 × 10–7 m; kcat = 8.0 × 10–4 s–1).Synthetic Coagulation ModelThe procedure used was a modification of Lawson et al. (31Lawson J.H. Kalafatis M. Stram S. Mann K.G. J. Biol. Chem. 1994; 269: 23357-23366Abstract Full Text PDF PubMed Google Scholar) and van't Veer et al. (36van't Veer C. Mann K.G. J. Biol. Chem. 1997; 272: 4367-4377Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar).Procofactor Solution—4 × 108/ml platelets or 4 μm PCPS were incubated in HBS (20 mm HEPES and 150 mm NaCl, pH 7.4), 2 mm CaCl2 for 10 min at 37 °C. When desired, 10 pm relipidated TF was added (molar ratio of PCPS/TF = 5000). Factor V (40 nm) and factor VIII (1.4 nm) were added prior to the initiation of the reaction.Zymogen-Inhibitor Solution—Prothrombin (2.8 μm), factors VII (20 nm), VIIa (0.2 nm), X (340 nm), IX (180 nm), and XI (60 nm), TFPI (5 nm), and AT-III (6.8 μm) were preheated in HBS, 2 mm CaCl2 at 37 °C for 3 min. Thrombin, factor IXa, and factor Xa were added at 2× desired concentrations prior to the initiation of the reaction.The reaction was started by mixing equal volumes of both solutions, resulting in physiological concentrations of the zymogens, pro-cofactors, inhibitors, and platelets (or 2 μm PCPS) and desired concentrations of enzymes. Following initiation of the reaction, at selected time points, 10-μl aliquots were withdrawn from the reaction mixture and quenched in 20 mm EDTA in HBS (pH 7.4) containing 0.2 mm Spectrozyme TH and assayed immediately for thrombin activity. The hydrolysis of the substrate was monitored by the change in absorbance at 405 nm using a Vmax spectrophotometer (Molecular Devices Corp., Menlo Park, CA). Thrombin generation was calculated from a standard curve prepared by serial dilutions of α-thrombin.Whole Blood ModelThe protocol used was a modification of Rand et al. (33Rand M.D. Lock J.B. van't Veer C. Gaffney D.P. Mann K.G. Blood. 1996; 88: 3432-3445Crossref PubMed Google Scholar). Two healthy donors were recruited and advised according to a protocol approved by the University of Vermont Human Studies Committee (33Rand M.D. Lock J.B. van't Veer C. Gaffney D.P. Mann K.G. Blood. 1996; 88: 3432-3445Crossref PubMed Google Scholar, 34Cawthern K.M. van't Veer C. Lock J.B. DiLorenzo M.E. Branda R.F. Mann K.G. Blood. 1998; 91: 4581-4592Crossref PubMed Google Scholar), and their consent was obtained. Individuals selected exhibited normal values for the parameters of blood coagulation, protein levels, and platelet counts. Experiments were performed in tubes placed on a rocking table enclosed in a 37 °C temperature-controlled glove box using fresh CTI-inhibited (100 μg/ml CTI) blood. Blood was drawn by venipuncture and immediately delivered into the reagent-loaded tubes.Time Course—All tubes (four series per experiment) were loaded with CTI. No additional reagents are added to the phlebotomy control series (four tubes). Six tubes were loaded with relipidated TF (TF/PCPS, 5 pm/25 nm) in HBS, 2 mm CaCl2; eight tubes were loaded with 97 pm factor IXa; and another eight tubes were loaded with 97 pm factor IXa, 5.5 pm factor Xa, and 5.3 pm thrombin. The zero-time tube of each series was pretreated with 1 ml of 50 mm EDTA and 10 μl of 10 mm FPRck (diluted in 10 mm HCl). After blood was delivered, the tubes were periodically (2–20 min) quenched with EDTA and FPRck.Final Levels of the TAT—In these experiments, all tubes were loaded in duplicates with CTI and the desired concentrations of thrombin, factor IXa, and factor Xa (individually or all three together). The TF control tubes are loaded with 5 pm relipidated TF. Tubes were quenched with EDTA and FPRck 20 min after fresh blood was added.In all experiments, no more than 35 μl of reagents were loaded in each tube. The clotting time was observed visually by two observers and was recorded when "clumps" were observed on the side of the tube. After the experiment, tubes were centrifuged, and the supernatants were aliquoted and analyzed for the TAT concentration.Plasma Preparation for the Fluorogenic AssayBlood (10 ml) from a healthy volunteer was drawn into a syringe loaded with 10 ml of HBS containing 50 mm EDTA, pH 7.4. The platelet-poor plasma (PPP) was made by centrifugation at 3000 rpm for 20 min followed by the centrifugation at 11,000 rpm (20,000 × g) for 10 min. When kept on ice, plasma did not clot for at least 10 h. One fraction of the PPP was used for the fluorogenic assay immediately and another one was spiked with 1 pm α-thrombin and kept for 1 h before the assay.Fluorogenic Thrombin Activity Assay in the PPPFluorogenic substrate VPRnbs at a final 50 μm concentration was added to a cuvette containing PPP. This substrate allows the quantitation of α-thrombin at concentrations as low as 20 fm (41Butenas S. DiLorenzo M.E. Mann K.G. Thromb. Haemost. 1997; 78: 1193-1201Crossref PubMed Scopus (29) Google Scholar). Change in fluorescence over time representing substrate hydrolysis was monitored in a spectrofluorometer FluoroMax-2 (Jobin Yvon-Spex Instruments S.A., Inc.) at 25 °C for 15 min using λex = 350 nm and λem = 470 nm. Light-scattering artifacts were minimized with a 450-nm cut-off filter in the emission light beam. Hirudin at 1 μm was added to the reaction mixture, and a change in fluorescence was monitored again. Rates of substrate hydrolysis were established from a calibration line constructed using the detecting group (6-amino-1-naphthalene-(n-butyl)sulfonamide).Estimation of the in Vivo Concentrations of Free EnzymesThe concentration of a given free enzyme in a dynamic system containing stoichiometric inhibitors is determined by the balance between the rate of enzyme generation and the rate(s) of inhibition. Assuming that the concentrations of inhibitors are relatively constant in blood under normal flow, the concentration of free enzyme can be described by the following equation: v = k1[I1]·[Ef] + k2[I2]·[Ef] +... + kn[In]·[Ef], where v is the rate of enzyme generation; k1, k2,... kn are the second order rate constants defining the rates of reactions of inhibitors I1, I2,... In with a particular enzyme (30Hockin M.F. Jones K.C. Everse S.J. Mann K.G. J. Biol. Chem. 2002; 277: 18322-18333Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar); and Ef is free enzyme.The rate of generation of each enzyme was assumed to be equal to the rate of removal of its activation peptide from blood (19Bauer K.A. Bailliere's Clin. Haematol. 1999; 12: 387-406Crossref PubMed Scopus (26) Google Scholar). The latter was assumed to be a first order process, and the rate constant (kAP) could be calculated using the reported half-life for each activation peptide (AP) (see Table I). Ongoing rates of activation peptide removal and equal rates of enzyme formation are then defined as the product kAP[AP] using reported steady state concentrations of activation peptides (13Bucek R.A. Reiter M. Quehenberger P. Weltermann A. Kyrle P.A. Minar E. Br. J. Haematol. 2003; 120: 123-128Crossref PubMed Scopus (12) Google Scholar, 14Boneu B. Bes G. Pelzer H. Sie P. Boccalon H. Thromb. Haemost. 1991; 65: 28-31Crossref PubMed Scopus (169) Google Scholar, 15Miller G.J. Bauer K.A. Barzegar S. Cooper J.A. Rosenberg R.D. Thromb. Haemost. 1996; 75: 767-771Crossref PubMed Scopus (177) Google Scholar, 16Ardissino D. Merlini P.A. Bauer K.A. Bramucci E. Ferrario M. Coppola R. Fetiveau R. Lucreziotti S. Rosenberg R.D. Mannucci P.M. Blood. 2001; 98: 2726-2729Crossref PubMed Scopus (68) Google Scholar, 17Merlini P.A. Bauer K.A. Oltrona L. Ardissino D. Cattaneo M. Belli C. Mannucci P.M. Rosenberg R.D. Circulation. 1994; 90: 61-68Crossref PubMed Scopus (565) Google Scholar, 20Mannucci P.M. Bauer K.A. Santagostino E. Faioni E. Barzegar S. Coppola R. Rosenberg R.D. Blood. 1994; 84: 1314-1319Crossref PubMed Google Scholar, 21Bauer K.A. Humphries S. Smillie B. Li L. Cooper J.A. Barzegar S. Rosenberg R.D. Miller G.J. Thromb. Haemost. 2000; 84: 396-400Crossref PubMed Scopus (22) Google Scholar, 22Bauer K.A. Broekmans A.W. Bertina R.M. Conard J. Horellou M.H. Samama M.M. Rosenberg R.D. Blood. 1988; 71: 1418-1426Crossref PubMed Google Scholar, 23Mannucci P.M. Tripodi A. Bottasso B. Baudo F. Finazzi G. De Stefano V. Palareti G. Manotti C. Mazzucconi M.G. Castaman G. Thromb. Haemost. 1992; 67: 200-202Crossref PubMed Scopus (74) Google Scholar, 24Santagostino E. Mannucci P.M. Gringeri A. Tagariello G. Baudo F. Bauer K.A. Rosenberg R.D. Thromb. Haemost. 1994; 71: 737-740Crossref PubMed Scopus (25) Google Scholar, 25Miller G.J. Bauer K.A. Cooper J.A. Rosenberg R.D. Thromb. Haemost. 1998; 79: 549-553Crossref PubMed Scopus (90) Google Scholar, 26Mari D. Mannucci P.M. Coppola R. Bottasso B. Bauer K.A. Rosenberg R.D. Blood. 1995; 85: 3144-3149Crossref PubMed Google Scholar). These rate estimates are used to calculate [Ef] for each enzyme using the above equation and reported concentrations of inhibitors and their second order rate constants of inhibition for the particular enzyme.Table IEstimated in vivo concentrations of active enzymesZymogenActivation peptides[I]kcSecond order rate constants of inhibition. Data from Refs. 50-52.EfdFree enzyme.ConcentrationHalf-lifeaData from Ref. 19.Removal ratebEqual to the enzyme generation rate.msm/smm-1 s-1pmProthrombin1.0 × 10-954001.3 × 10-133.4 × 10-6eAT-III.7.1 × 1035.3Factor IX2.1 × 10-109001.6 × 10-133.4 × 10-6eAT-III.4.9 × 10297Factor X9.4 × 10-1118003.6 × 10-123.4 × 10-6eAT-III.1.5 × 1032.5 × 10-9fTFPI.9.0 × 1055.5a Data from Ref. 19Bauer K.A. Bailliere's Clin. Haematol. 1999; 12: 387-406Crossref PubMed Scopus (26) Google Scholar.b Equal to the enzyme generation rate.c Second order rate constants of inhibition. Data from Refs. 50Baugh R.J. Broze Jr., G.J. Krishnaswamy S. J. Biol. Chem. 1998; 273: 4378-4386Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 51Chuang Y.J. Swanson R. Raja S.M. Olson S.T. J. Biol. Chem. 2001; 276: 14961-14971Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 52Schoen P. Lindhout T. J. Biol. Chem. 1987; 262: 11268-11274Abstract Full Text PDF PubMed Google Scholar.d Free enzyme.e AT-III.f TFPI. Open table in a new tab Operational DefinitionsThe initiation phase of thrombin generation was defined as a time interval from the start of the reaction (represented by 0 on the x axis in the figures) to the point of intersection of the x axis and a tangent to the maximum slope of thrombin generation. It is characterized by relatively slow prothrombin activation and the proteolytic processing steps required for the assembly of the vitamin K-dependent protein activating complexes. The propagation phase of thrombin generation is defined as a time interval from the end of the initiation phase to the maximum thrombin concentration. It is characterized by robust prothrombin activation principally by factor Xa·factor Va·membrane (the prothrombinase complex) with factor Xa provided predominantly by the factor IXa·factor VIIIa·membrane complex (the intrinsic factor Xase).RESULTSNumerical Simulations Predicting the Influence of Simultaneous Variations of Thrombin, Factor Xa, and Factor IXa on Thrombin Generation in the Absence of TFFig. 1 represents thrombin generation over time initiated with the mixture of all three enzymes. Reported concentrations of activation peptides of serine protease zymogens involved in blood coagulation circulating in vivo for healthy individuals varied from 94 pm for factor X to 210 pm for factor IX and to 1.0 nm for prothrombin (F1.2). In the presence of serine proteases at these concentrations, thrombin generation entered the propagation phase after a very short initiation phase (i.e. blood containing these concentrations of enzymes would clot in a few seconds (Fig. 1, solid line). The maximum concentration of active thrombin produced reached almost 1.0 μm, with the maximum rate of generation as high as 28 nm/s. A decrease in the concentration of all three enzymes by 1 order of magnitude (9.4 pm for factor Xa, 21 pm for factor IXa, and 100 pm for thrombin) prolonged the initiation phase to 100 s and decreased both maximum concentration and maximum rate of thrombin generation to 630 nm and 8.5 nm/s, respectively (long dashed line). A further decrease of the enzyme concentrations to 1% of those reported for activation peptides (0.94 pm factor Xa, 2.1 pm factor IXa, and 10 pm thrombin) prolonged the initiation phase to 600 s, decreased maximum concentration of active thrombin to 260 nm, and provided a maximum rate of thrombin generation of 1.1 nm/s (*). No active thrombin was observed in 20 min of the reaction when concentrations of all three enzymes were decreased to 0.1% (94 fm for factor Xa, 0.21 pm for factor IXa, and 1 pm for thrombin) of those detected for their activation peptides (short dashed line).The enzyme concentrations selected for the initial studies of thrombin generation were either equal to those of activation peptides or decreased in sequential studies by orders of magnitude. However, the free enzymes continuously genera
Referência(s)