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• W2053168161 abstract "Formation of thrombin is triggered when membrane-localized tissue factor (TF) is exposed to blood. In closed models of this process, thrombin formation displays an initiation phase (low rates of thrombin production cause platelet activation and fibrinogen clotting), a propagation phase (>95% of thrombin production occurs), and a termination phase (prothrombin activation ceases and free thrombin is inactivated). A current controversy centers on whether the TF stimulus requires supplementation from a circulating pool of blood TF to sustain an adequate procoagulant response. We have evaluated the requirement for TF during the progress of the blood coagulation reaction and have extended these analyses to assess the requirement for TF during resupply (“flow replacement”). Elimination of TF activity at various times during the initiation phase indicated: a period of absolute dependence (<10 s); a transitional period in which the dependence on TF is partial and decreases as the reaction proceeds (10–240 s); and a period in which the progress of the reaction is TF independent (>240 s). Resupply of reactions late during the termination phase with fresh reactants, but no TF, yielded immediate bursts of thrombin formation similar in magnitude to the original propagation phases. Our data show that independence from the initial TF stimulus is achieved by the onset of the propagation phase and that the ensemble of coagulation products and intermediates that yield this TF independence maintain their prothrombin activating potential for considerable time. These observations support the hypothesis that the transient, localized expression of TF is sufficient to sustain a TF-independent procoagulant response as long as flow persists. Formation of thrombin is triggered when membrane-localized tissue factor (TF) is exposed to blood. In closed models of this process, thrombin formation displays an initiation phase (low rates of thrombin production cause platelet activation and fibrinogen clotting), a propagation phase (>95% of thrombin production occurs), and a termination phase (prothrombin activation ceases and free thrombin is inactivated). A current controversy centers on whether the TF stimulus requires supplementation from a circulating pool of blood TF to sustain an adequate procoagulant response. We have evaluated the requirement for TF during the progress of the blood coagulation reaction and have extended these analyses to assess the requirement for TF during resupply (“flow replacement”). Elimination of TF activity at various times during the initiation phase indicated: a period of absolute dependence (<10 s); a transitional period in which the dependence on TF is partial and decreases as the reaction proceeds (10–240 s); and a period in which the progress of the reaction is TF independent (>240 s). Resupply of reactions late during the termination phase with fresh reactants, but no TF, yielded immediate bursts of thrombin formation similar in magnitude to the original propagation phases. Our data show that independence from the initial TF stimulus is achieved by the onset of the propagation phase and that the ensemble of coagulation products and intermediates that yield this TF independence maintain their prothrombin activating potential for considerable time. These observations support the hypothesis that the transient, localized expression of TF is sufficient to sustain a TF-independent procoagulant response as long as flow persists. Tissue factor (TF) 2The abbreviations used are: TFtissue factorAT-IIIantithrombin IIIFPRckd-Phe-Pro-ArgCH2ClHSPGheparan sulfate proteoglycansPC1,2-dioleoyl-sn-glycero-3-phosphocholinePS1,2-dioleoyl-sn-3-glycero-3-phospho-l-serinePCPS vesiclessingle bilayer phospholipid vesicles composed of 75% phosphatidylcholine and 25% phosphoserineTFPItissue factor pathway inhibitorTATthrombin-antithrombin III complexAPCactivated protein C. is a 263-amino acid glycoprotein with three major domains: 1) an extracellular domain (residues 1–219) that binds with high affinity to factor VIIa; 2) a transmembrane domain (residues 220–242) that anchors TF to the membrane surface; and 3) a cytoplasmic domain (residues 243–263) (1Spicer E.K. Horton R. Bloem L. Bach R. Williams K.R. Guha A. Kraus J. Lin T.C. Nemerson Y. Konigsberg W.H. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5148-5152Crossref PubMed Scopus (196) Google Scholar, 2Ruf W. Rehemtulla A. Morrissey J.H. Edgington T.S. J. Biol. Chem. 1991; 266: 16256Abstract Full Text PDF PubMed Google Scholar, 3Komiyama Y. Pedersen A.H. Kisiel W. Biochemistry. 1990; 29: 9418-9425Crossref PubMed Scopus (153) Google Scholar). Binding of plasma factor VIIa to membrane-bound TF results in an ∼2 × 107-fold increase in the enzymatic activity of factor VIIa toward its natural substrates factor IX and factor X (4Bjoern S. Foster D.C. Thim L. Wiberg F.C. Christensen M. Komiyama Y. Pedersen A.H. Kisiel W. J. Biol. Chem. 1991; 266: 11051-11057Abstract Full Text PDF PubMed Google Scholar). Most studies have concluded that membrane-bound TF, expressed by inflammatory cells and cells outside the vasculature, are the key initiators of the blood coagulation process (5Nemerson Y. Blood. 1988; 71: 1-8Crossref PubMed Google Scholar, 6Morrissey J.H. Thromb. Haemostasis. 2001; 86: 66-74Crossref PubMed Scopus (180) Google Scholar, 7Edgington T.S. Mackman N. Brand K. Ruf W. Thromb. Haemostasis. 1991; 66: 67-79Crossref PubMed Scopus (513) Google Scholar). tissue factor antithrombin III d-Phe-Pro-ArgCH2Cl heparan sulfate proteoglycans 1,2-dioleoyl-sn-glycero-3-phosphocholine 1,2-dioleoyl-sn-3-glycero-3-phospho-l-serine single bilayer phospholipid vesicles composed of 75% phosphatidylcholine and 25% phosphoserine tissue factor pathway inhibitor thrombin-antithrombin III complex activated protein C. The generation of thrombin, the enzyme responsible for clot formation as well as other procoagulant and anticoagulant functions during the blood coagulation process occurs in a nonlinear fashion (8Mann K.G. Butenas S. Brummel K.E. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 17-25Crossref PubMed Scopus (435) Google Scholar). During an initiation phase, tiny amounts of thrombin are generated, platelets, zymogens, and procofactors are activated, and complex enzymes assembled (9Butenas S. van't Veer C. Mann K.G. J. Biol. Chem. 1997; 272: 21527-21533Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 10Brummel K.E. Paradis S.G. Butenas S. Mann K.G. Blood. 2002; 100: 148-152Crossref PubMed Scopus (320) Google Scholar). Subsequently, a propagation phase of thrombin generation occurs, characterized by dramatic increases in both the rate of thrombin generation and levels of thrombin. The duration of the initiation phase, which roughly corresponds to the clotting time of blood and plasma, is predominantly dependent upon the concentration of the factor VIIa-TF enzyme complex and tissue factor pathway inhibitor (TFPI) (11Lawson J.H. Kalafatis M. Stram S. Mann K.G. J. Biol. Chem. 1994; 269: 23357-23366Abstract Full Text PDF PubMed Google Scholar, 12van't Veer C. Mann K.G. J. Biol. Chem. 1997; 272: 4367-4377Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 13Rand M.D. Lock J.B. van't Veer C. Gaffney D.P. Mann K.G. Blood. 1996; 88: 3432-3445Crossref PubMed Google Scholar). Thrombin generation during the propagation phase, however, is almost independent of this complex and TFPI (12van't Veer C. Mann K.G. J. Biol. Chem. 1997; 272: 4367-4377Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). TFPI is a multivalent Kunitz-type plasma proteinase inhibitor (15Wun T.C. Kretzmer K.K. Girard T.J. Miletich J.P. Broze Jr., G.J. J. Biol. Chem. 1988; 263: 6001-6004Abstract Full Text PDF PubMed Google Scholar, 16Novotny W.F. Palmier M. Wun T.C. Broze Jr., G.J. Miletich J.P. Blood. 1991; 78: 394-400Crossref PubMed Google Scholar). It is the principal stoichiometric inhibitor of the factor VIIa-TF complex and thus is a key regulator of the initiation phase of thrombin generation (12van't Veer C. Mann K.G. J. Biol. Chem. 1997; 272: 4367-4377Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). TFPI inhibits the factor VIIa-TF complex in a factor Xa-dependent manner (17Broze 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, 18Jesty J. Wun T.C. Lorenz A. Biochemistry. 1994; 33: 12686-12694Crossref PubMed Scopus (68) Google Scholar, 19Baugh R.J. Broze Jr., G.J. Krishnaswamy S. J. Biol. Chem. 1998; 273: 4378-4386Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). The mechanistic dependence of factor VIIa-TF inhibition by TFPI on one of the products of factor VIIa-TF catalysis works against premature neutralization of the TF stimulus. The centrality of the TFPI regulatory mechanism to normal hemostasis is apparent by the lack of reports of individuals with a TFPI–/– genotype and the lethality in utero of the TFPI–/– genotype in transgenic mice (20Huang Z.F. Higuchi D. Lasky N. Broze Jr., G.J. Blood. 1997; 90: 944-951Crossref PubMed Google Scholar). Recently, controversy has emerged concerning the presence and functionality of TF species circulating in blood. Reports of circulating TF can be divided into those showing TF localized on the surface of blood cells and microparticles and those describing a TF species that circulates as a soluble protein (21Diamant M. Nieuwland R. Pablo R.F. Sturk A. Smit J.W. Radder J.K. Circulation. 2002; 106: 2442-2447Crossref PubMed Scopus (337) Google Scholar, 22Siddiqui F.A. Desai H. Amirkhosravi A. Amaya M. Francis J.L. Platelets. 2002; 13: 247-253Crossref PubMed Scopus (133) Google Scholar, 23Giesen 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 (912) Google Scholar, 24Takahashi H. Satoh N. Wada K. Takakuwa E. Seki Y. Shibata A. Am. J. Hematol. 1994; 46: 333-337Crossref PubMed Scopus (40) Google Scholar, 25So A.K. Varisco P.A. Kemkes-Matthes B. Herkenne-Morard C. Chobaz-Peclat V. Gerster J.C. Busso N. J. Thromb. Haemostasis. 2003; 1: 2510-2515Crossref PubMed Scopus (154) Google Scholar, 26Bogdanov V.Y. Balasubramanian V. Hathcock J. Vele O. Lieb M. Nemerson Y. Nat. Med. 2003; 9: 458-462Crossref PubMed Scopus (392) Google Scholar, 27Falati S. Liu Q. Gross P. Merrill-Skoloff G. Chou J. Vandendries E. Celi A. Croce K. Furie B.C. Furie B. J. Exp. Med. 2003; 197: 1585-1598Crossref PubMed Scopus (667) Google Scholar). These blood TF species are invoked in support of a new hypothesis that TF-dependent thrombin generation requires a continuous infusion of this cofactor. It has, for example, been hypothesized that circulating TF plays an important role in clot growth (26Bogdanov V.Y. Balasubramanian V. Hathcock J. Vele O. Lieb M. Nemerson Y. Nat. Med. 2003; 9: 458-462Crossref PubMed Scopus (392) Google Scholar). However, data from our laboratory and others have indicated that there is little or no TF-related activity in the blood of healthy individuals (28Santucci R.A. Erlich J. Labriola J. Wilson M. Kao K.J. Kickler T.S. Spillert C. Mackman N. Thromb. Haemostasis. 2000; 83: 445-454Crossref PubMed Scopus (56) Google Scholar, 29Berckmans R.J. Neiuwland R. Boing A.N. Romijn F.P. Hack C.E. Sturk A. Thromb. Haemostasis. 2001; 85: 639-646Crossref PubMed Scopus (579) Google Scholar, 30Butenas S. Mann K.G. Nat. Med. 2004; 10: 1155-1156Crossref PubMed Scopus (63) Google Scholar, 31Butenas S. Bouchard B.A. Brummel-Ziedins K.E. Parhami-Seren B. Mann K.G. Blood. 2005; 105: 2764-2770Crossref PubMed Scopus (237) Google Scholar) or in the blood of mice (32Day S.M. Reeve J.L. Pedersen B. Farris D.M. Myers D.D. Im M. Wakefield T.W. Mackman N. Fay W.P. Blood. 2005; 105: 192-198Crossref PubMed Scopus (249) Google Scholar). The goals of this study were to define the duration of the TF stimulus necessary to yield normal thrombin generation and to assess the requirement for additional TF during the progress of the blood coagulation reaction. We used three models of blood coagulation developed by our laboratory: numerical simulation (33Jones K.C. Mann K.G. J. Biol. Chem. 1994; 269: 23367-23373Abstract Full Text PDF PubMed Google Scholar, 34Hockin 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 (328) Google Scholar), synthetic plasma (11Lawson J.H. Kalafatis M. Stram S. Mann K.G. J. Biol. Chem. 1994; 269: 23357-23366Abstract Full Text PDF PubMed Google Scholar, 12van't Veer C. Mann K.G. J. Biol. Chem. 1997; 272: 4367-4377Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), and whole blood (13Rand M.D. Lock J.B. van't Veer C. Gaffney D.P. Mann K.G. Blood. 1996; 88: 3432-3445Crossref PubMed Google Scholar). Human coagulation factors VII, X, IX, and prothrombin, were isolated from fresh frozen plasma using the methods of Bajaj et al. (35Bajaj S.P. Rapaport S.I. Prodanos C. Prep. Biochem. 1981; 11: 397-412Crossref PubMed Scopus (156) Google Scholar), and were purged of trace contaminants and traces of active enzymes as described (12van'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 and antithrombin III (AT-III) were isolated from freshly frozen plasma (36Katzmann 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, 37Griffith 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 TF (residues 1–242) were provided as gifts from Drs. Shu Len Liu and Roger Lundblad (Hyland division, Baxter Healthcare Corp., Duarte, CA). Recombinant human factor VIIa was provided as a gift from Dr. Ula Hedner (Novo Nordisk, Denmark). Recombinant full-length TFPI was provided as a gift from Dr. K. Johnson (Chiron Corp., Emeryville, CA). Corn trypsin inhibitor was isolated from popcorn and the preparation of the TF/lipid reagent was performed as described elsewhere (38Cawthern 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 (39Higgins D.L. Mann K.G. J. Biol. Chem. 1983; 258: 6503-6508Abstract Full Text PDF PubMed Google Scholar). Spectrozyme TH was purchased from American Diagnostica, Inc. (Greenwich, CT). d-Phe-Pro-ArgCH2Cl (FPRck) was prepared in house, and monoclonal anti-TF (αTF-5) and anti-factor VII/VIIa (αFVII-1) antibodies were produced by the Biochemistry Antibody Core Laboratory (University of Vermont). Enzyme-linked immunosorbent assay thrombin-AT-III (TAT) kit (Enzygnost TAT) was purchased from Behring (Marburg, Germany). The current numerical model is based upon prior publications by Jones et al. (33Jones K.C. Mann K.G. J. Biol. Chem. 1994; 269: 23367-23373Abstract Full Text PDF PubMed Google Scholar), Hockin et al. (34Hockin 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 (328) Google Scholar), and Butenas et al. (40Butenas S. Orfeo T. Gissel M.T. Brummel K.E. Mann K.G. J. Biol. Chem. 2004; 279: 22875-22882Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) and yields concentration versus time profiles for selected species when electronic mixtures of the procoagulant factors II, IX, X, VII, VIIa, V, and VIII and the anticoagulants TFPI and AT-III are exposed to picomolar concentrations of TF. The standard simulation sets zymogen, cofactor, and inhibitor concentrations at their mean physiological values and the concentration of the TF stimulus at 5 pm. Simulations designed to define the minimum time interval of TF function were conducted by arresting the standard simulation at selected times, setting the concentrations of free TF and all TF containing species to zero, and then allowing the electronic simulation to proceed. Numerical simulations of resupply were constructed to mimic the equal volume addition of fresh “plasma” material used in the empirical models. Electronic reactions were allowed to proceed for a given time and then stopped. The starting concentrations of all species for the resupplied reaction were then calculated as follows: the concentration of each product or intermediate species at any time (t = x) in the initial reaction was divided in half for the start of the resupplied reaction; for each zymogen, cofactor, and inhibitor, the concentration present at the start of the resupplied reaction was calculated as follows: ((unreacted component concentration at t = x)/2 + (resupply component concentration)/2). TF was not included in the resupplying material. With the initial state of the resupplied reaction defined, the simulation was then restarted. The procedure used is a modification of Lawson et al. (11Lawson 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. (12van't Veer C. Mann K.G. J. Biol. Chem. 1997; 272: 4367-4377Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Platelets were prepared by the method of Mustard et al. (41Mustard J.F. Perry D.W. Ardlie N.G. Packham M.A. Br. J. Haematol. 1972; 22: 193-204Crossref PubMed Scopus (650) Google Scholar). Procofactor Solution—Relipidated TF (10 pm; molar ratio PCPS:TF = 5000) was incubated with 4 μm PCPS or 4 × 108 platelets/ml in HBS (20 mm HEPES and 150 mm NaCl, pH 7.4), 2 mm CaCl2 for 10 min at 37 °C. 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), factor VII (20 nm), factor VIIa (0.2 nm), factor X (340 nm), factor IX (180 nm), factor 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. The reaction was started by mixing equal volumes of both Ca+2 preequilibrated solutions resulting in physiological concentrations of the zymogens, pro-cofactors, and inhibitors, 5 pm TF, 2 mm CaCl2, and 2 μm PCPS or 2 × 108 platelets/ml. Following the start 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. In the antibody quenching experiments αTF-5 and αFVII-1 (0.25 mg of each; diluted in HBS, 2 mm CaCl2, pH 7.4) were added at selected time points to the reaction mixture. Resupply was conducted at selected times by the addition of an equal volume of freshly constituted, TF-free, procofactor/zymogen-inhibitor mixture to an ongoing reaction. The same protocol of sampling, quenching, and assay was then followed. The protocol used is a modification of Rand et al. (13Rand M.D. Lock J.B. van't Veer C. Gaffney D.P. Mann K.G. Blood. 1996; 88: 3432-3445Crossref PubMed Google Scholar). A healthy donor was recruited, advised according to a protocol approved by the University of Vermont Human Studies Committee, and his consent obtained. The individual 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 blood. Blood was drawn by venipuncture and immediately delivered into the reagent-loaded tubes. All tubes (two series per experiment) were loaded with corn trypsin inhibitor (0.1 mg/ml). No additional reagents were added to the phlebotomy control series (2 tubes). Thirty-two tubes (15 tubes in series A and 17 tubes in series B) were loaded with relipidated TF (TF/PCPS, 5 pm/25 nm) in HBS, 2 mm CaCl2. The zero time tube of each series is 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 (60–1,200 s) quenched with EDTA and FPRck. In TF quenching experiments, antibodies αTF-5 and αFVII-1 (0.25 mg of each; diluted in HBS, 2 mm CaCl2, pH 7.4) were added to two series B tubes at 0 s and to 12 tubes (quenching time 180–1,200 s) at 120 s. An equal volume of HBS, 2 mm CaCl2 (pH 7.4) was added to the A series tubes. In all experiments, no more than 35 μl of reagents (3.5% of blood volume) were loaded in each tube. The clotting time was observed visually by two observers and was called when “clumps” were observed on the side of the tube. After the experiment, tubes were centrifuged and the supernatants were aliquoted and analyzed for TAT levels. In resupply experiments, the setup was similar but the B series tubes were not quenched during the first 20 min. At 20 min after initiation with TF, these tubes all received an equal volume of new blood, drawn from the same individual 1 min prior to its use. In some experiments, inhibitory antibodies (αTF-5 and αFVII-1: 0.25 mg of each; diluted in HBS, 2 mm CaCl2, pH 7.4) were added to the ongoing reactions 1 min prior to resupply. These tubes were then periodically quenched (1260–2400 s) with EDTA and FPRck. Within this experimental format several additional tubes were quenched at 20 min, prior to resupply, and at 40 min with EDTA only. Sera derived from TF-initiated whole blood quenched only with EDTA was assessed for the presence of active thrombin and for its residual capacity to inhibit thrombin. Active thrombin was measured in a clotting based assay using purified fibrinogen (42Lundblad R.L. Kingdon H.S. Mann K.G. Methods Enzymol. 1976; 45: 156-176Crossref PubMed Scopus (233) Google Scholar). Briefly, 50 μl of a5 mg/ml fibrinogen stock in HBS + 5 mm CaCl2 + 0.1% PEG 8000 was brought to 37 °C and then 50 μl of the thrombin containing sample was added. Clotting times were determined using a ST-4 clotting instrument (Diagnostica Stago, Parsippany, NJ). Clotting times obtained in the presence of sera samples were converted to thrombin concentrations by reference to a standard curve constructed with thrombin concentrations ranging from 10 nm to 40 pm. To assess the remaining thrombin inhibitor capacity of these same sera, exogenous thrombin (100 nm or 50 nm final) was added to a 50-μl aliquot of sera at 37 °C. Individual reactions were quenched at different times by a 30-fold dilution with HBS and then assayed immediately as described above. The efficacy of the quenching scheme was established by experiments in which the sera samples were diluted 30-fold prior to the addition of thrombin: assays conducted within 1 min of thrombin addition to a diluted plasma sample showed no decay in thrombin activity. Analysis of the observed decay in thrombin activity as a first-order process yielded a good fit to the data. In empirical models the initiation phase of thrombin generation is defined as a time interval from the start of the reaction (represented by 0 on the horizontal (x) axis in figures) to the point of intersection of the x axis and a tangent to the maximum slope of thrombin generation. This point roughly corresponds to the clotting time in blood and plasma. In numerical simulations the initiation phase is defined as the time to reach an active thrombin level of 2 nm (∼10 nm TAT). The propagation phase of thrombin generation is defined as a time interval from the end of the initiation phase to the maximum thrombin (or TAT) concentration. The termination phase is defined as the period of the reaction in which prothrombin consumption is over and active thrombin levels are falling as a result of inactivation by endogenous protease inhibitors. Data from empirical studies of the regulation of the TF-factor VIIa complex have indicated that TFPI is a rapid and efficient suppressor of the procoagulant consequences of TF expression (12van't Veer C. Mann K.G. J. Biol. Chem. 1997; 272: 4367-4377Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 43Lu G. Broze Jr., G.J. Krishnaswamy S. J. Biol. Chem. 2004; 279: 17241-17249Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Fig. 1 presents simulation data detailing the partitioning of an introduced bolus (5 pm) of TF between catalytically inert (panel A) and active complexes (panel B). Twenty seconds after the introduction of TF to the system, greater than 98% of TF remains inert, either complexed with uncleaved factor VII zymogen (A, •) or unassociated with a protein partner (A, ×). By 100 s into the reaction, ∼80% of TF was bound to factor VII, a condition that persists throughout the propagation phase, whereas ∼9% is free and ∼9% tied up in complexes with TFPI. As the reaction progresses, TF continues to be found primarily in inert complexes, although the predominant species becomes the TF-factor VIIa-factor Xa-TFPI complex (A, ♦). At 1200 s, ∼93% of the TF is bound to TFPI, ∼6% is bound to factor VII, and ∼0.1% (∼5 fm) is unassociated with other proteins. Fig. 1B presents the evolution of functional catalytic TF complexes. This process is characterized by two maxima of functional TF-VIIa complex with each maxima primarily reflecting a different source of factor VIIa. The first occurs 20 s into the reaction, with levels of active TF species (defined as the sum of free TF-factor VIIa complex and TF-factor VIIa complex bound to its substrates and products) of ∼80 fm. This maxima derives primarily from the association of endogenous (circulating) factor VIIa (100 pm) with the introduced TF stimulus (B, ○). This episode of TF-factor VIIa complex formation was short lived because of rapid factor Xa-dependent sequestration by TFPI (B, ♦). The second phase of TF-VIIa accumulation initiates at ∼260 s and reaches its maximum level, ∼120 fm, at 460 s, a time that corresponds to the active thrombin maxima and the end of the propagation phase (B, □). This factor VIIa derives primarily from activation of factor VII by the rapidly increasing levels of thrombin and factor Xa that characterize the propagation phase (34Hockin 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 (328) Google Scholar). In these simulations, the previously formed pool of TF-factor VII complex supplies the TF via an exchange reaction controlled by off-rate defining the TF factor VII binding equilibrium (t½ = 220 s) (A, •). At 1200 s, functional TF-VIIa constitutes ∼0.5% (∼20 fm) of the total TF pool. Numerical Simulations—Fig. 2A displays simulations in which thrombin generation induced with 5 pm TF was allowed to proceed for selected time periods before the TF stimulus was mathematically removed and the reaction allowed to proceed without further contributions from the factor VIIa-TF complex. In a control experiment (♦) with TF-related activity only regulated by the natural inhibitors of the factor VIIa-TF complex, i.e. TFPI and AT-III, thrombin generation enters the propagation phase after an initiation phase of 240 s. During the propagation phase, thrombin was generated at a maximum rate of 2.0 nm/s and the maximum active thrombin achieved was 275 nm. Electronic nullification of TF activity at 240 s (▪) has little effect on these parameters: the duration of the initiation phase remains 240 s and the maximum rate of thrombin generation and its maximum level were only slightly decreased (1.9 nm/s and 240 nm). Nullification of TF-related activity at 120 s from the start of the reaction (▵) slightly alters the initiation phase with a more pronounced effect on the maximum rate of thrombin generation (1.3 nm/s) and the maximum thrombin level. When TF nullification time occurs earlier (60 s; ▴), all three parameters of thrombin generation undergo more significant changes, especially the initiation phase, which was prolonged to 330 s. No thrombin generation was observed over 1,200 s, when TF-related activity was nullified at the initiation of the reaction (×) or at 10 s post-initiation (•). However, TF nullification at 20 s (not shown) results in significant thrombin generation, indicating that sufficient other procoagulant products have been mobilized to sustain the process between 10 and 20 s. In Fig. 2B, the influence of quenching on the production of factor Xa is shown. Factor Xa generated over the first 300 s of the control simulation was dissected into that produced by the factor VIIa-TF complex (♦) and that by the factor IXa-factor VIIIa complex (⋄). Factor Xa production after the mathematical elimination of the TF containing species has only one significant route in these simulations, the factor IXa-factor VIIIa complex. Thus, time courses of factor Xa production after TF quenching at 10 (•), 20 (○), 60 (▴), and 120 s (▵) are shown emanating from the control simulation of the factor IXa-factor VIIIa derived factor Xa. As can be seen, the post-quenching rate and extent of factor Xa production by the factor IXa-factor VIIIa complex approaches that of the unquenched reaction (⋄) as the interval between initiation and quenching increases. A convenient marker for comparing the performance of the TF-depleted systems with the complete system is the time at which the factor IXa-factor VIIIa complex becomes the predominant source of new factor Xa. In the control simulation this time is ∼210 s (see Fig. 2B, inset). The reaction quenched at 120" @default.
• W2053168161 created "2016-06-24" @default.
• W2053168161 creator A5004717464 @default.
• W2053168161 creator A5015874992 @default.
• W2053168161 creator A5020728232 @default.
• W2053168161 creator A5075389112 @default.
• W2053168161 date "2005-12-01" @default.
• W2053168161 modified "2023-09-29" @default.
• W2053168161 title "The Tissue Factor Requirement in Blood Coagulation" @default.
• W2053168161 cites W110545565 @default.
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