Role of Thrombin Compared With Factor Xa in the Procoagulant Activity of Whole Blood Clots
Background Thrombi are known to induce activation of the coagulation system, which may be a mechanism for progression of thrombosis and its recurrence after thrombolysis. This study was designed to characterize the relative role of thrombin and activated factor X (factor Xa) as mediators of procoagulant activity of whole blood clots in blood and plasma.
Methods and Results Clots formed from human blood were incubated in recalcified (25 mmol/L CaCl2) citrated plasma or nonanticoagulated blood with increasing concentrations of recombinant desulfatohirudin (hirudin) to inhibit thrombin activity, recombinant tick anticoagulant peptide (TAP) or recombinant tissue factor pathway inhibitor (TFPI) to inhibit factor Xa, or heparin. Fibrinopeptide A (FPA) was assayed serially as an index of procoagulant (thrombin) activity. FPA generation was greatly accelerated by addition of clots to recalcified plasma (from 1251±211 ng/mL after 15 minutes without clot to 5916±1412 ng/mL with clot, n=7, P<.01) or whole blood (4803±761 ng/mL with clot compared with 546±233 without clot, n=5, P<.05) and was attenuated by inhibitors of thrombin (>90% inhibition of FPA with 0.05 μmol/L hirudin and 1.0 U/mL heparin) and factor Xa (>90% inhibition of FPA with 1.0 μmol/L TAP and 0.15 μmol/L TFPI) in a concentration-dependent manner. Preincubation of clots with tissue-type plasminogen activator sufficient to induce partial clot lysis increased the rate of thrombin-induced FPA generation by increasing the surface area of clot exposed to plasma. However, procoagulant activity induced by partially lysed clots was attenuated by lower concentrations of both thrombin and Xa inhibitors, presumably because access of the inhibitors to bound procoagulant molecules was facilitated. Comparable results were obtained with incubations in nonanticoagulated blood.
Conclusions These results indicate that factor Xa is primarily responsible for the procoagulant activity of clots in vitro and suggest a potential molecular mechanism for the observed efficacy of inhibitors of factor Xa in preventing recurrent thrombosis after coronary thrombolysis.
Thrombi are known to induce activation of the coagulation system,1 2 which may play a role in the recurrence of thrombosis after thrombolysis and the progression of thrombotic complications of atherosclerotic coronary vascular disease. Because a platelet-rich thrombus is the principal procoagulant surface exposed to blood after rupture of an atherosclerotic plaque or after coronary thrombolysis, the procoagulant activity of specific components of the thrombus will determine the progression of coronary thrombosis and the response to anticoagulant therapy.
Specific binding of thrombin to fibrin has generally been thought to be the primary mediator of thrombus-associated procoagulant activity, and it is known that fibrin-bound thrombin is resistant to antithrombin III–mediated inhibition.3 Thrombus-associated thrombin has been shown to induce fibrin formation3 and may activate coagulation cofactors V and VIII as well as platelets. The efficacy of the specific thrombin inhibitor hirudin in attenuating recurrent coronary reocclusion after thrombolysis in experimental and clinical studies is consistent with the critical role of thrombin in recurrent platelet-rich coronary thrombosis.4 5 6 However, we have recently shown that whole blood clots formed in vitro and thrombi recovered after arterial injury in vivo express sufficient factor Xa activity to induce marked activation of prothrombin.7 In addition, specific inhibitors of factor Xa have been shown to have efficacy at least equal to inhibition of thrombin in preventing recurrent thrombosis after thrombolysis in experimental animals.8 These results are consistent with the clinical observation that factor Xa–induced activation of prothrombin increases in patients treated with tissue-type plasminogen activator (TPA) for acute myocardial infarction.9 However, previous studies have not addressed the extent to which the procoagulant activity of arterial platelet-rich thrombi is attributable to the activity of bound thrombin compared with factor Xa. Furthermore, the relative efficacy of inhibitors of thrombin and factor Xa in attenuating thrombus-associated procoagulant activity has not been defined.
Because the extent of local procoagulant activity is likely to be attributable, at least in part, to thrombus-associated procoagulants, the current study was designed to characterize the relative efficacy of inhibition of thrombin compared with factor Xa in attenuating thrombus-induced activation of the coagulation system. To accomplish this objective, we used whole blood clots prepared in vitro; these have been shown previously to exhibit procoagulant activity similar to that of thrombi recovered after arterial injury in vivo.7 The extent of attenuation of procoagulant activity then was characterized when the clots were incubated in recalcified citrated plasma or nonanticoagulated whole blood containing concentrations of antithrombin-dependent (heparin) and antithrombin-independent (hirudin) inhibitors of thrombin or inhibitors of factor Xa (recombinant tick anticoagulant peptide [TAP] or tissue factor pathway inhibitor [TFPI]) in the range of those documented as effective in vivo. The potential impact of fibrinolysis on the intensity of clot-associated procoagulant activity and its inhibition by the specific anticoagulants was assessed by preincubation of the clots with concentrations of TPA similar to those achieved during pharmacological fibrinolysis in vivo. We sought to determine the relative efficacy of specific inhibition of thrombin compared with that of factor Xa in attenuating the procoagulant activity of whole blood clots and the impact of fibrinolysis on the efficacy of specific inhibitors.
Formation of Whole Blood Clots
Clots were formed from human blood as described previously.10 Briefly, venous blood was collected, after discarding the first 3 mL, through a 19-gauge, winged infusion set (Terumo) into a polypropylene syringe without anticoagulants. One-milliliter aliquots were injected slowly into pieces of Tygon tubing (3.2-mm inside diameter, 4.8-mm outside diameter, 27 cm long; Fisher Scientific Products), and the ends were apposed to form a ring held together with latex tubing (4.8-mm inside diameter, 1.6-mm wall, 1 cm long; Fisher). The rings were rotated at 21 rpm and an angle of 80° for 90 minutes at 37°C on a tube rotator (Scientific Equipment Co) to simulate the flow of blood (approximately 30 mL/min). The resulting clots were removed from the tubing rings, rinsed vigorously with 0.9% saline, and placed in aliquots of plasma or whole blood as described below. The washing procedure was shown to remove both nonspecifically bound thrombin and trapped FPA as indicated by the lack of thrombin activity and the absence of measurable FPA in the last wash. We have shown previously that clots formed by these methods have an average diameter of 2 mm, a length of 5 to 10 mm, and weight of 5 to 10 mg (dry weight) and exhibit procoagulant activity identical to that of thrombi formed in vivo after electrical injury to the axillary artery in dogs.7
Incubations of Inhibitors of Thrombin and Factor Xa With Clots in Plasma and Whole Blood
Citrated human plasma (1.5 mL, American Red Cross) was added to polypropylene tubes together with either 0.3 to 1.0 U/mL sodium heparin (LyphoMed, Inc), 0.01 to 1.0 μmol/L recombinant desulfatohirudin (hirudin, CGP39393, CIBA-GEIGY and GEN Therapeutica), 0.01 to 1.0 μmol/L recombinant TAP (Merck Sharp and Dohme), which specifically inhibits factor Xa,11 or 0.015 to 0.15 μmol/L recombinant TFPI (also known as lipoprotein-associated coagulation inhibitor and extrinsic pathway inhibitor; Monsanto), the physiological inhibitor of the complex of tissue factor/VIIa/Xa that also inhibits free factor Xa.12 Whole blood clots were added to one of a pair of tubes with each concentration of inhibitor, and the plasma was recalcified with 25 mmol/L CaCl2 (final concentration). The tubes were placed in a 37°C water bath, and 250-μL aliquots of plasma were removed at 5, 10, and 15 minutes after the addition of calcium. To determine whether procoagulant activity of clots differed in whole blood compared with plasma, a 19-gauge winged infusion set (Terumo) was inserted into an antecubital vein of healthy volunteers and 1 mL of whole blood was allowed to drip into polypropylene test tubes containing the inhibitors. Clots were added, the tubes were incubated at 37°C, and aliquots were removed serially as for plasma. Aliquots of blood and plasma were transferred to tubes containing 25 μL of a solution consisting of EDTA, aprotinin, and d-phe-pro-arg-chloromethylketone (FPA anticoagulant, Byck-Sangtek). Blood samples were centrifuged, and the plasma was frozen at −70°C and assayed subsequently for FPA. Studies involving acquisition of blood from human subjects were approved by the Human Studies Committee at Washington University School of Medicine.
Induction of Fibrinolysis
To determine whether fibrinolysis altered the procoagulant activity of clots, 2.5 μg/mL of TPA (Genentech, Inc) was preincubated in citrated plasma or citrated whole blood with or without added clot and inhibitors of thrombin or factor Xa at 37°C for 30 minutes. The plasma or blood then was recalcified, and serial aliquots were obtained over 15 minutes for assay for FPA, as described above.
Assessment of Clot Lysis
The extent of fibrinolysis induced by incubation of clots with TPA was assessed by addition of fibrinogen labeled with 125I to the whole blood before clots were formed. Fibrinogen was labeled with 125I by the lactoperoxidase method with use of Enzymobeads (Bio-Rad) according to the manufacturer’s instructions. Each clot contained approximately 300 000 cpm (1.6×106 cpm/μg fibrinogen). Several of the clots were washed with saline, and the radioactivity was counted for 1 minute in a gamma counter (NML 5010/5020 Organon Teknika). Other clots were washed with saline and incubated in citrated plasma containing 2.5 μg/mL of TPA for 30 minutes before counting the radioactivity. Percent lysis over 30 minutes was calculated as the difference in radioactivity in clots incubated with TPA and those not incubated with TPA.
Mechanical Disruption of Clots
To determine whether mechanical disruption of a clot simulating pharmacological clot lysis potentiates procoagulant activity, clots were minced with a scalpel into 1-mm3 pieces. The pieces were incubated in recalcified plasma, and the aliquots were collected for assay of FPA.
Assay for FPA
Assays for FPA were performed with use of a radioimmunoassay previously validated in our laboratory13 in which a polyclonal antiserum (Byck-Sangtek) is used. Plasma samples were treated with bentonite before analysis to remove fibrinogen and large fibrinogen degradation products. We have previously shown with reverse-phase high-pressure liquid chromatography and carboxy terminal–specific antibodies that the assay is specific for FPA.14 15 The lower limit of detection of FPA is 1.0 ng/mL and the linear range is 1.0 to 40.0 ng/mL. Intra-assay variability is 5.7±0.7%.13 Samples with FPA levels >40.0 ng/mL were diluted with pooled plasma containing no detectable FPA.
Values are expressed as mean±SEM. The efficacy of specific inhibitors in attenuating clot-induced procoagulant activity was characterized by comparison of the concentrations of FPA in blood or plasma incubated with clots in the presence of an inhibitor to concentrations induced by incubation of clots alone. Percent inhibition was defined as 1−(FPA with inhibitor/FPA without inhibitor).
Increases in FPA induced by incubation of clots with plasma and blood were compared by ANOVA with a repeated-measures design (statview ii, Abacus Concepts, Inc). Comparisons between inhibitors and different concentrations were analyzed by ANOVA and Scheffé’s test. A value of P<.05 was considered significant.
Procoagulant Activity Induced by Whole Blood Clots
Incubation of whole blood or recalcified plasma at 37°C resulted in only minimal increases in FPA at 10 minutes (lag phase), with a subsequent more rapid increase in FPA between 10 and 15 minutes (Fig 1⇓). In contrast, incubation of nonanticoagulated whole blood or recalcified plasma with whole blood clots resulted in marked acceleration of the rate of increase in FPA over 15 minutes. The increases in FPA observed in whole blood were not statistically significant when compared with those in recalcified plasma; however, four of six whole blood samples clotted by 15 minutes. The more extensive clot-induced activation of the coagulation system in whole blood probably reflects, in part, the contribution of platelets.
Relative Efficacy of Inhibition of Thrombin and Factor Xa on Clot-Induced Procoagulant Activity in Recalcified Plasma
The relative efficacy of specific inhibition of thrombin compared with factor Xa was tested with pharmacologically relevant concentrations of heparin (antithrombin III–dependent thrombin and factor Xa inhibition), hirudin (antithrombin III–independent thrombin inhibition), TAP (factor Xa inhibitor), and TFPI (Xa and tissue factor/VIIa/Xa inhibitor). Concentrations of FPA induced by incubation of whole blood clots in the presence and absence of different amounts of these inhibitors were compared. A concentration of 1.0 U/mL of heparin inhibited 94% of the increase in FPA induced by whole blood clots in recalcified plasma (Fig 2⇓). Concentrations of heparin similar to those achieved by therapeutic anticoagulation (0.3 to 0.5 U/mL) attenuated increases in FPA less markedly (73% with 0.3 U/mL and 83% with 0.5 U/mL of heparin, P<.01 for all doses). Antithrombin III–independent inhibition of thrombin activity with hirudin at concentrations >0.01 μmol/L attenuated increases in FPA by >90%.
Inhibition of factor Xa alone with TAP or TFPI was also effective for inhibiting increases in FPA induced by addition of whole blood clots to plasma (Fig 2⇑). TFPI exhibited nearly equivalent inhibition on a molar basis compared with hirudin, whereas the concentration of TAP required was an order of magnitude higher.
Similar concentrations of inhibitors shown to be effective for inhibiting increases in FPA induced by whole blood clots added to recalcified citrated plasma were effective for attenuating increases in FPA at 15 minutes when clots were incubated in nonanticoagulated whole blood. Thus, 0.1 μmol/L hirudin inhibited increases in FPA by 87% (n=5) in blood compared with 96% (n=3) in recalcified plasma (P=NS), while 1.0 μmol/L TAP and 0.15 μmol/L TFPI inhibited increases in FPA by 87% and 95% (n=5), respectively, in blood compared with 95% and 97% (n=3) in plasma (P=NS).
Effect of Fibrinolysis on Procoagulant Activity Induced by Clots and Its Inhibition
To characterize the effects of fibrinolysis on clot-associated procoagulant activity, whole blood clots were incubated with 2.5 μg/mL TPA in either citrated plasma or blood for 30 minutes. After preincubation with TPA, the plasma or citrated whole blood was recalcified with 25 mmol/L CaCl2, and the change in concentration of FPA was measured as described above to characterize clot-induced activation of the coagulation system and elaboration of thrombin activity. Incubation of whole blood clots with 2.5 μg/mL of TPA induced 14.4±0.4% (n=6) clot lysis after 30 minutes, as characterized by release of 125I-labeled fibrinogen into the plasma. Partial fibrinolysis markedly potentiated clot-associated procoagulant activity in recalcified citrated blood and plasma (Fig 3⇓). In plasma, concentrations of FPA increased more rapidly and were somewhat higher than those observed in blood; however, more rapid clotting occurred in blood than in plasma (Fig 3⇓).
To determine whether increased exposure of procoagulants as a consequence of partial clot lysis accounted for increases in clot-associated procoagulant activity, whole blood clots were extensively minced and incubated in recalcified citrated plasma, and concentrations of FPA were measured by assay. All of the samples incubated with minced clots were clotted by 15 minutes, and in clots preincubated with TPA, FPA increased to 25 145±7064 ng/mL. In contrast, FPA increased to 4583±726 ng/mL in recalcified plasma incubated with clots not exposed to TPA (P<.01 compared with clots preincubated with TPA). Thus, increases in procoagulant activity appeared to be attributable, at least in part, to increased exposure of clot-associated procoagulants to plasma.
To characterize the efficacy of thrombin and Xa inhibition on attenuating clot-associated procoagulant activity after partial fibrinolysis, heparin, hirudin, TAP, and TFPI were added to recalcified citrated plasma preincubated with whole blood clots in TPA for 30 minutes, as described above. Procoagulant activity was markedly attenuated by each of the inhibitors after fibrinolysis at concentrations lower than those required to inhibit procoagulant activity induced by clots that had not been preincubated with TPA (Fig 4⇓). Thus, fibrinolysis induced by preincubation with TPA potentiated the inhibition of procoagulant activity achieved by addition of specific inhibitors of thrombin and factor Xa. At the concentrations tested in whole blood, there was a trend toward greater inhibition of clot-associated procoagulant activity with hirudin and TFPI after partial fibrinolysis (Fig 5⇓).
To verify the dependence of clot-associated procoagulant activity on serine proteases, whole blood clots were incubated with d-phe-l-pro-l-arg-chloromethylketone (PPACK) in recalcified citrated plasma. After 15 minutes, FPA concentration was increased minimally with concentrations of 1, 5, and 10 μmol/L PPACK (68, 51, and 54 ng/mL, respectively, n=2).
Procoagulant activity of residual thrombi, which can promote rethrombosis and limit the efficacy of fibrinolytic agents, has been attributed to preexisting thrombin bound to fibrin that is reexposed during fibrinolysis.3 Our results showing concentration-dependent and nearly equivalent attenuation of FPA generation with inhibitors of either thrombin or factor Xa in recalcified plasma or blood incubated with whole blood clots (Fig 2⇑) indicate that the procoagulant activity of clots is dependent on de novo activation of prothrombin mediated by factor Xa/Va and not primarily preexisting clot-bound thrombin. Complete inhibition of clot procoagulant activity by incubation in plasma-containing PPACK verified its dependence on serine proteases. Results in vitro are consistent with those in experimental animals and preliminary results in patients showing marked attenuation of thrombotic reocclusion after coronary fibrinolysis by conjunctive administration of either direct inhibitors of thrombin such as hirudin,4 5 6 Hirulog,16 and argatroban17 18 or inhibitors of prothrombin activation including TAP,8 TFPI,19 and low molecular weight heparin.20 Further studies will be necessary to determine the relative importance of factor Xa compared with clot-associated thrombin in the progression of thrombosis in vivo. The presence of clot-associated factor Xa implies that inhibition of factor Xa in combination with inhibition of thrombin during fibrinolysis may be most efficacious for suppression of procoagulant activity and may therefore facilitate prompt and persistent coronary recanalization.
Generation of FPA in recalcified plasma and blood containing clot was increased more than fourfold by preincubation of the clots with TPA (Fig 3⇑). Potentiation of procoagulant activity was not mediated by either plasmin or TPA-induced cleavage of FPA from fibrinogen; it was not observed in plasma containing TPA but no added clot (data not shown). Results of experiments showing similar potentiation when clots were minced before incubation with plasma in the absence of TPA suggest that increased procoagulant activity attributable to incubation with TPA is due to the increased surface area of clots induced by partial fibrinolysis.
Of interest, and contrary to a previous report,21 we found that inhibition of the procoagulant activity associated with whole blood clots was facilitated by partial fibrinolysis (Figs 4⇑ and 5⇑). This may have resulted from the release of factor Xa and/or thrombin from the clot, making them more accessible to inhibitors, or from the more efficient penetration of inhibitors into the clot after partial fibrinolysis.
Concentrations of hirudin, TAP, and TFPI found to suppress procoagulant activity of clots in vitro were analogous to those shown to reduce reocclusion after fibrinolysis in vivo.4 5 8 19 Comparable results were obtained with inhibitors in either plasma or whole blood. The concentrations of hirudin and TFPI required to inhibit the procoagulant activity of clots were quite similar (Fig 2⇑), consistent with the one-to-one stoichiometry of prothrombin activation to thrombin by factor Xa. The concentrations of TAP required to achieve comparable inhibition of thrombin generation were nearly 10-fold higher, possibly because of the slow kinetics of binding of TAP to factor Xa.9 Although the physiological action of TFPI in vivo is believed to be the inhibition of factor Xa mediated by the complex of tissue factor and factor VIIa,22 our data with whole blood clots that presumably lack tissue factor show a marked, direct inhibitory effect of TFPI on factor Xa activity, confirming an earlier report.12 It is unlikely that TFPI inhibits the activity of the factor IXa/VIIIa complex because TFPI does not bind or interact with factor IXa in purified systems (personal communication, Dr J. Miletich). Thus, in vivo, pharmacological concentrations of TFPI may significantly attenuate both the activity of Xa and its generation by the tissue factor/VIIa complex.
Pharmacological fibrinolysis has become an established intervention after acute myocardial infarction.23 However, its success is often limited by failure to achieve adequate coronary recanalization in as many as 50% of treated patients and by early reocclusion in 8% to 15% of recanalized vessels despite administration of heparin.24 Furthermore, early withdrawal of heparin after fibrinolysis is frequently associated with recurrent ischemia, which is indicative of persistent thrombosis.25 Impaired fibrinolysis and persistent thrombosis appear to result from competing procoagulant activity, attributable in part to the free plasmin generated by fibrinolytic agents that can activate prothrombin as well as factors V and XII.14 15 26 27 28 Procoagulant activity has been attributed also to thrombin bound to fibrin that not only induces continued activation of fibrinogen but activation of platelets and factors V and VIII, which increases the activity of factor IXa/VIIIa and factor Xa/Va complexes.29 The limited efficacy of heparin observed both clinically and in experimental animals appears to reflect relative protection of clot-bound thrombin and factor Xa to inhibition by heparin–antithrombin III. The results of this in vitro study show that thrombin elaboration induced by incubation of clots with recalcified plasma or whole blood is due to clot-associated factor Xa activity. Thus, in vivo inhibition of thrombin alone may not be effective once antithrombin agents are discontinued because clot-associated Xa activity may not be inhibited. Accordingly, inhibitors of factor Xa alone or a combination of direct potent inhibitors of thrombin and factor Xa may be particularly effective for facilitating fibrinolysis and warrant testing in vivo.
This study was supported in part by NHLBI SCOR in Coronary and Vascular Diseases HL-17646. We acknowledge the invaluable technical assistance of Mary Jane Eichenseer, Debbie Tinker, John Girotto, Evan Wasserman, and Brad Reznick. We thank CIBA-GEIGY for providing recombinant hirudin, Merck Sharp and Dohme for providing recombinant TAP, and Monsanto for providing recombinant TFPI. We also thank Ava Ysaguirre and Barbara Donnelly for typing the manuscript.
- Received August 15, 1994.
- Revision received February 2, 1995.
- Accepted February 20, 1995.
- Copyright © 1995 by American Heart Association
Badimon L, Lassila R, Badimon J, Vallabhajosula S, Chesebro JH, Fuster V. Residual thrombosis is more thrombogenic than severely damaged vessel wall. Circulation. 1988;78(suppl II):II-119. Abstract.
Weitz JI, Hudoba M, Massel D, Maraganore J, Hirsh J. Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors. J Clin Invest. 1990;86:385-391.
Haskel EJ, Prager NA, Sobel BE, Abendschein DR. Relative efficacy of antithrombin compared with antiplatelet agents in accelerating coronary thrombolysis and preventing early reocclusion. Circulation. 1991;83:1048-1056.
Cannon CP, McCabe CH, Henry TD, Schweiger MJ, Gibson RS, Mueller HS, Becher RC, Kleiman NS, Haugland JM, Anderson JL, Sharaf BL, Edwards SJ, Rogers WJ, Williams DO, Braunwald E, TIMI 5 Investigators. A pilot trial of recombinant desulfatohirudin compared with heparin in conjunction with tissue-type plasminogen activator and aspirin for acute myocardial infarction: results of the Thrombolysis in Myocardial Infarction (TIMI) 5 Trial. J Am Coll Cardiol. 1994;23:993-1003.
Eisenberg PR, Siegel JE, Abendschein DR, Miletich JP. Importance of factor Xa in determining the procoagulant activity of whole-blood clots. J Clin Invest. 1993;91:1877-1883.
Sitko GR, Ramjit DR, Stabilito II, Lehman D, Lynch JJ, Vlasuk GP. Conjunctive enhancement of enzymatic thrombolysis and prevention of thrombotic reocclusion with the selective factor Xa inhibitor, tick anticoagulant peptide. Circulation. 1992;85:805-815.
Waxman L, Smith DE, Arcuri KE, Vlasuk GP. Tick anticoagulant peptide (TAP) is a novel inhibitor of blood coagulation factor Xa. Science. 1990;248:593-596.
Broze GJ Jr, Warren LA, Novotny WF, Higuchi DA, Girard JJ, Miletich JP. The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: insight into its possible mechanism of action. Blood. 1988;71:335-343.
Yao S-K, Ober JC, Ferguson JJ, Anderson HV, Maraganore J, Buja LM, Willerson JT. Combination of inhibition of thrombin and blockade of thromboxane A2 synthetase and receptors enhances thrombolysis and delays reocclusion in canine coronary arteries. Circulation. 1992;86:1993-1999.
Fitzgerald DJ, Fitzgerald GA. Role of thrombin and thromboxane A2 in reocclusion following coronary thrombolysis with tissue-type plasminogen activator. Proc Natl Acad Sci U S A. 1989;86:7585-7589.
Yasuda T, Gold HK, Yaoita H, Leinbach RC, Guerrero JL, Jang I-K, Holt R, Fallon JT, Collen D. Comparative effects of aspirin, a synthetic thrombin inhibitor and a monoclonal antiplatelet glycoprotein IIb/IIIa antibody on coronary artery reperfusion, reocclusion and bleeding with recombinant tissue-type plasminogen activator in a canine preparation. J Am Coll Cardiol. 1990;16:714-722.
Haskel EJ, Torr SR, Day KC, Palmier MO, Wun T-C, Sobel BE, Abendschein DR. Prevention of arterial reocclusion after thrombolysis with recombinant lipoprotein-associated coagulation inhibitor. Circulation. 1991;84:821-827.
Mirshahi M, Soria J, Soria C, Faivre R, Lu H, Courtney M, Roitsch C, Tripier D, Coen JP. Evaluation of the inhibition by heparin and hirudin of coagulation activation during r-tPA-induced thrombolysis. Blood. 1989;74:1025-1030.
Tiefenbrunn AJ, Sobel BE. Thrombolysis and myocardial infarction. Fibrinolysis. 1991;5:1-15.
Collen D. Coronary thrombolysis: streptokinase or recombinant tissue-type plasminogen activator? Ann Intern Med. 1990;112:529-538.
Eisenberg PR, Sherman LA, Schectman K, Perez J, Sobel BE, Jaffe AS. Fibrinopeptide A: a marker of acute coronary thrombosis. Circulation. 1985;71:912-918.
Lee CD, Mann KG. Activation/inactivation of human factor V by plasmin. Blood. 1989;73:185-190.
Ewald GA, Eisenberg PR. Increased procoagulant activity in response to fibrinolytic agents due to plasmin-mediated activation of the contact system. Thromb Haemost. 1993;69:823a. Abstract.
Pieters J, Lindhout T, Hemker HC. In situ-generated thrombin is the only enzyme that effectively activates factor VIII and factor V in thromboplastin-activated plasma. Blood. 1989;74:1021-1024.