© 1996 American Heart Association, Inc.
Articles |
Sustained Inhibition of Whole-Blood Clot Procoagulant Activity by Inhibition of Thrombus-Associated Factor Xa
the Washington University School of Medicine, St Louis, Mo.
Correspondence to Paul R. Eisenberg, MD, MPH, Washington University School of Medicine, Cardiovascular Division, Box 8086, 660 S Euclid Ave, St Louis, MO 63110.
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Abstract |
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Progression of arterial thrombosis partly depends on thrombus-associated thrombin and activated factor X (Xa) activity. However, whether Xa or thrombin is the most appropriate target for inhibition of recurrent thrombosis is unknown. This study was designed to determine whether inhibition of Xa results in more sustained attenuation of thrombus-associated procoagulant activity than does inhibition of thrombin. Clots prepared ex vivo from human whole blood and pathological arterial thrombi from patients were preincubated in citrated plasma containing no inhibitor, 0.5 to 1 U/mL heparin, 0.5 to 1 µmol/L hirudin, 5 to 10 µmol/L tick anticoagulant peptide (TAP), 0.15 to 3 µmol/L tissue factor pathway inhibitor (TFPI), or a combination of 1 µmol/L hirudin and 10 µmol/L TAP for 2 hours. After preincubation the clots were removed from first-stage plasma, extensively washed in phosphate-buffered saline, and added to nonanticoagulated whole blood. Clots preincubated in plasma without inhibitors induced marked activation of the coagulation system in whole blood, as characterized by greater increases in the concentration of fibrinopeptide A (FPA) over 7 minutes than in blood without added clots (1522±568 compared with 117±170 ng/mL, P<.01). Preincubation of clots with heparin or hirudin did not attenuate the increases in FPA in whole blood. In contrast, compared with incubation without an inhibitor, preincubation of clots with TAP or TFPI markedly attenuated the increases in FPA when clots were added to whole blood (551±316 and 508±208 ng/mL, respectively, P<.01). Similar results were obtained with arterial thrombi from patients. Inhibition of Xa but not of thrombin results in sustained attenuation of thrombus-associated procoagulant activity. Uninhibited thrombus-associated Xa activity may account for the increases in thrombin activity that are commonly observed in clinical trials after discontinuation of thrombin inhibitors.
Key Words: thrombin • thrombosis • factor X • tissue factor pathway inhibitor • tick anticoagulant peptide
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Introduction |
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Platelet-rich arterial thrombosis plays a critical role in the pathophysiology of unstable coronary syndromes and myocardial infarction.1 2 The progression of arterial thrombosis depends in part on thrombus-associated thrombin and Xa activity.3 4 5 Xa forms a complex with Va on the surface of platelets, whereas thrombin binds to fibrin.3 4 5 6 When associated with thrombi, both Xa and thrombin are resistant to inhibition by ATIII,3 4 5 6 7 which has been suggested as a mechanism for recurrent arterial thrombosis despite anticoagulation with heparin.3 This hypothesis is supported by data that show that ATIII-independent inhibitors of Xa (eg, [TAP]) and thrombin (eg, hirudin) are more effective than heparin in inhibiting thrombosis in experimental preparations of coronary thrombolysis.7 8 9 10 11 However, whether thrombus-associated thrombin or Xa/Va activity is the more appropriate target for anticoagulant therapy has not been well characterized.
Thrombin and Xa are both involved in thrombogenesis. Thrombin activates platelets and coagulation cofactors V and VIII, which promote formation of the Xa/Va complex and thus elaboration of more thrombin.12 In addition, thrombin-mediated activation of factors VIII and XI may promote IXa/VIIIa activation of X.13 Thrombus-associated Xa activity may have even greater significance than the activity of fibrin-bound thrombin, because the Xa/Va complex induces prothrombin activation and thus, markedly increases local thrombin activity.3 We have recently shown that Xa inhibitors are as effective as hirudin in attenuating the procoagulant activity of whole-blood clots in nonanticoagulated whole blood in an ex vivo assay.4 Hirudin inhibits increases in fibrin formation induced by clots ex vivo4 5 and prevents recurrent thrombosis after thrombolysis in vivo,9 10 but whether this reflects inhibition of free thrombin or of clot-associated thrombin is unclear. Furthermore, thrombin inhibitors would not be expected to inhibit thrombus-associated Xa activity. The persistence of thrombus-associated Xa/Va activity may account in part for the recurrent thrombin activity that has been observed in clinical trials after discontinuation of direct-acting thrombin inhibitors.14 15 In contrast, inhibition of Xa in experimental animals has been associated with sustained inhibition of arterial thrombosis after coronary thrombolysis.7 16 These data are consistent with the hypothesis that local procoagulant activity in arterial thrombosis depends on the extent of Xa/Va activity associated with the platelet-rich thrombus.3
This study was designed to determine the relative importance of thrombus-associated Xa and thrombin to activation of the coagulation system in whole blood and plasma. To accomplish this, we preincubated whole-blood clots prepared in vitro and arterial thrombi recovered from patients with hirudin to inhibit thrombin or with rTAP or TFPI to inhibit Xa. Preincubated clots and thrombi were extensively washed to remove unbound inhibitor and then incubated in nonanticoagulated whole blood to determine the extent to which inhibition of thrombin compared with that of Xa resulted in sustained attenuation of thrombus-induced activation of the coagulation system.
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Methods |
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Materials
Heparin was purchased from LyphoMed. rDesulfatohirudin was provided by CIBA-GEIGY; rTAP by Merck, Sharpe & Dohme; and full-length rhTFPI by Monsanto/Searle. The chromogenic substrates used were S-2238 (H-D-Phe-L-piperzine-L-Arg-p-nitroanaline HCl) and S-2222 (N-benzoyl-L-Leu-L-Gly-L-Gly-L-Arg-p-nitroanaline HCl) for thrombin and Xa, respectively; both substrates were purchased from Chromogenix. Human prothrombin was isolated and purified from pooled citrated plasma as previously described,17 which was obtained from the American Red Cross.
Plasma was depleted of vitamin K–dependent factors by addition of 100 mmol/L BaCl2 to pooled citrated plasma at 4°C for 60 minutes followed by centrifugation to separate the precipitate. The supernatant was recovered and additional BaCl2 precipitate was allowed to form. The plasma was again centrifuged and the supernatant was collected. The supernatant plasma was dialyzed exhaustively against 0.15 mol/L NaCl and 0.012 mol/L sodium citrate, pH 6.0. The barium citrate–adsorbed plasma (barium-adsorbed plasma) was stored in 1.0-mL aliquots at -70°C and thawed at 37°C immediately before use.
Preparation of Whole-Blood Clots
Whole blood was obtained from healthy volunteers with previously documented platelet counts of 1.5 to 3.0x106 who had not taken aspirin or aspirin-containing medication in the preceding 72 hours. After informed consent was obtained as part of a protocol approved by the Washington University Human Studies Committee, venipuncture was performed with a 19-gauge winged infusion set (Terumo). Blood was drawn into a polypropylene syringe and then transferred in 1-mL aliquots to lengths of tygon tubing (Fischer Scientific Products). The tygon tubing was connected to form a closed loop and the ends were apposed with latex tubing. The rings were rotated at 21 rpm for 90 minutes on a tube rotator incubated at 37°C. Clots were prepared by this method to mimic arterial thrombi with a platelet-rich head and a fibrin tail.18 The clots were then removed and washed extensively in 1-mL aliquots of 0.02 mol/L Na2PO4–0.15 mol/L NaCl (PBS) at pH 7.4. We had determined in preliminary studies that thrombus-associated Xa/Va activity was similar after the washing procedure whether or not 2 mmol/L CaCl2 was added to the washing buffer.19
To confirm that the procedure for washing whole-blood clots was sufficient to remove trapped FPA, concentrations of FPA were measured in the last aliquot of buffer from selected clots and were always <8 ng/mL (n=4). In addition, aliquots of the final wash buffer were assayed for thrombin and Xa activity by measurement of amidolytic activity against S-2238 and S-2222, respectively. There was no hydrolysis of either substrate with aliquots of the last wash buffer, thereby indicating that the procedure for washing the clots was sufficient to remove thrombin and Xa that were not tightly bound. Using purified human thrombin and Xa, we determined that 0.1 nmol/L of either factor could be detected in the wash buffer with the procedures used to measure amidolytic activity toward the chromogenic substrate.
Pathological Thrombi
Arterial thrombi were obtained at the time of surgery according to a protocol approved by the Washington University Human Studies Committee. Immediately after acquisition the thrombi were placed in PBS and washed extensively, as described for in vitro clots. The thrombi were cut to prepare multiple segments of dimensions similar to those of whole-blood thrombi formed ex vivo (two segments from each thrombus for each condition).
Characterization of Clot-Associated Procoagulant Activity
To characterize the extent to which inhibition of clot-associated procoagulants resulted in sustained inhibition of procoagulant activity, a two-stage plasma system was designed. Whole-blood clots or pathological thrombi were incubated during the first stage in citrated plasma (0.012 mol/L) containing no inhibitors, 0.5 to 1.0 U/mL heparin, 0.5 to 1 µmol/L hirudin, 5 to 10 µmol/L TAP, 0.15 to 3 µmol/L TFPI, or a combination of 1 µmol/L hirudin and 10 µmol/L TAP for 2 hours. After incubation with or without anticoagulants, the clots were removed from the first-stage plasma and extensively washed in 1-mL aliquots of PBS. The clots were then added in a second stage to citrated plasma, which was recalcified with CaCl2 to a final concentration of 25 mmol/L, or to nonanticoagulated whole blood freshly collected from healthy volunteers. All incubations were carried out at 37°C in polypropylene tubes. Aliquots of incubated plasma or blood were obtained at 3, 5, and 7 minutes and immediately inhibited with a solution containing 5 mmol/L EDTA, 1000 KIU/mL aprotinin, and D-Phe-Pro-Arg-chloromethyl ketone (FPA anticoagulant, Byk-Sangtec).
To confirm that washing the clots after the first stage was sufficient to remove unbound inhibitors, aliquots of the last wash buffer after incubations with hirudin or heparin were incubated with thrombin, and aliquots after incubations with TAP or TFPI were incubated with Xa. In each instance there was no evidence of thrombin- or Xa-inhibiting activity in the last wash buffer, as judged by the lack of attenuation of thrombin or Xa activity toward the appropriate chromogenic substrate. We determined by incubation of 0.5 to 10 nmol/L hirudin with 1.0 nmol/L thrombin and 0.5 to 10 nmol/L TAP and of 0.5 to 10 nmol/L TFPI with 4.0 nmol/L Xa that the lower limit of detection with this procedure was 1.0 nmol/L for each inhibitor.
Characterization of Inhibition of Clot-Associated Thrombin and Xa Activity With Specific Inhibitors
To characterize the extent to which clot-associated thrombin activity was inhibited by preincubation with heparin or hirudin, FPA concentrations were measured in the first-stage citrated plasma after 2 hours' incubation. Because citrated plasma does not contain calcium, the only mechanism for fibrin formation, and hence increases in FPA, is the activity of fibrin-bound thrombin in the clots.
Inhibition of Xa by preincubation with TAP or TFPI was characterized by recovering the clots after incubation with these inhibitors in first-stage plasma, washing the clots as described, and measuring the extent to which preincubated clots induced prothrombin activation when incubated at 37°C in recalcified barium-adsorbed plasma containing 0.9 µmol/L prothrombin, as previously described.3
Characterization of Clot-Associated TF Activity
To determine whether whole-blood clots induced TF-mediated activation of the coagulation system, clots were incubated in first-stage plasma with a monoclonal antibody to human TF that recognizes the TF/VIIa complex.20 Clots were then washed, and procoagulant activity was characterized in second-stage recalcified citrated plasma, as described above.
Radioimmunoassay of FPA
FPA was analyzed with a polyclonal antiserum–based radioimmunoassay (Byk-Sangtec) that had been previously validated in our laboratory.21 Cross-reacting fibrinogen and fibrinogen degradation products in plasma were adsorbed with bentonite before FPA assay.22
Statistical Methods
Values are expressed as mean±SEM. The efficacy of specific inhibitors in attenuating clot-induced procoagulant activity was characterized by comparison of FPA concentrations in blood or plasma incubated with clots that had been exposed to inhibitors with FPA concentrations in blood or plasma incubated with untreated clots. Time-dependent 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 Fisher's test. A value of P<.05 was considered significant.
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Results |
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All whole-blood clots used in this study were prepared from 1 mL nonanticoagulated blood by the method of Chandler18 and allowed to form for 90 minutes before each experiment. We have previously shown that the thrombin and Xa/Va activities of whole-blood clots prepared in this manner are identical to those of arterial thrombi induced by electrical injury in vivo in dogs.3 Whole-blood clots preincubated for 2 hours in citrated plasma without inhibitors induced marked activation of the coagulation system when incubated in second-stage plasma with nonanticoagulated whole blood, as characterized by more marked increases in FPA concentration over 7 minutes than those in spontaneously clotting whole blood without clots (Fig 1
). The procoagulant activity of clots preincubated in plasma without anticoagulants was similar to that of clots added directly to nonanticoagulated blood without preincubation (data not shown). Similar results were obtained with preincubated whole-blood clots added to second-stage recalcified citrated plasma (FPA at 7 minutes, 2913±425 ng/mL compared with 73±17 ng/mL in recalcified citrated plasma alone; P<.01). Thus, the procoagulant activity of whole-blood clots is not attenuated by incubation in citrated plasma for 2 hours.
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Inhibition of Clot-Associated Thrombin and Xa/Va Activity
To inhibit clot-associated thrombin activity, clots were preincubated in first-stage citrated plasma that contained concentrations of heparin and hirudin similar to the higher range of plasma concentrations that are achieved pharmacologically in vivo. Under these conditions and without calcium, FPA cleavage from fibrinogen can only be due to preformed thrombin associated with the clots. Increases in FPA attributable to fibrin-bound thrombin in whole-blood clots were relatively modest, consistent with our previous results3 and those of Weitz et al.5 Both heparin and hirudin significantly attenuated FPA generation at 2 hours compared with results from clots preincubated with citrated plasma without inhibitors (P=.001 by ANOVA; Fig 2). Hirudin was slightly more effective than heparin, but the difference did not reach statistical significance.
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To inhibit clot-associated Xa/Va activity, whole-blood clots were preincubated in citrated plasma containing either TAP or TFPI at concentrations that have been shown to be effective in inhibiting Xa in the Xa/Va complex.4 To characterize the extent of Xa/Va activity after preincubation with TAP or TFPI, clots were removed from first-stage citrated plasma, washed, and incubated with recalcified (final concentration, 25 mmol/L CaCl2) barium-adsorbed plasma replete with 0.9 µmol/L prothrombin. We have previously shown that increases in FPA concentrations in barium-adsorbed plasma under these conditions are attributable to activation of prothrombin by thrombus-associated Xa/Va because Xa cannot be formed in barium-adsorbed plasma, which is deficient in all of the vitamin K–dependent factors.3 As shown in Fig 3, whole-blood clots induced marked activation of prothrombin, generation of FPA, and clotting of plasma when clot-associated Xa was not inhibited by preincubation with TAP or TFPI. In contrast, preincubation with TAP or TFPI at pharmacologically relevant concentrations markedly attenuated activation of prothrombin and generation of FPA (P=.001). Inhibition of clot-associated Xa activity was significantly greater with TAP than with TFPI. The minimal increases in FPA induced by clots incubated with Xa inhibitors is attributable to fibrin formation induced by clot-associated thrombin (see Fig 2
).
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Procoagulant Activity of Thrombin-Inhibited Clots
The procoagulant activity of clots that had been preincubated with either heparin or hirudin in citrated plasma (thrombin-inhibited clots) was characterized by incubating the extensively washed clots with nonanticoagulated blood in the second-stage assay. Despite inhibition of clot-associated thrombin activity by heparin and hirudin in the first stage, procoagulant activity of preincubated clots in second-stage nonanticoagulated whole blood was similar to that induced by whole-blood clots preincubated in citrated plasma without anticoagulants (P=NS; Fig 4) Similar results were obtained when recalcified citrated plasma was substituted for whole blood in the second stage (data not shown).
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Procoagulant Activity of Xa-Inhibited Clots
To characterize the procoagulant activity of clots after preincubation with TAP or TFPI in citrated plasma (Xa-inhibited clots), clots were incubated in either second-stage recalcified citrated plasma or nonanticoagulated whole blood. Fibrin formation, as evidenced by the change in FPA concentration, was markedly attenuated whether Xa-inhibited clots were incubated in recalcified plasma (P<.01; Fig 5A) or nonanticoagulated whole blood (P<.01; Fig 5B). Preincubation of clots with TFPI was associated with greater attenuation of FPA generation in recalcified plasma or nonanticoagulated blood at 7 minutes compared with preincubation with TAP (P<.05), even though TFPI appeared less effective in inhibiting clot-associated Xa activity (see Fig 3).
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Combined Inhibition of Clot-Associated Thrombin and Xa
Whole-blood clots were also preincubated in citrated plasma with high concentrations of hirudin (1 µmol/L) and TAP (10 µmol/L) to characterize the extent to which combined inhibition of thrombin and Xa attenuated clot-associated procoagulant activity. Combined inhibition of clot-associated thrombin and Xa did not enhance the extent to which increases in FPA were attenuated in second-stage nonanticoagulated whole blood compared with preincubation with TAP alone (data not shown).
Inhibition of Procoagulant Activity of Pathological Thrombi
Arterial thrombi were obtained from 3 patients: 2 with chronic renal failure and clotted arteriovenous fistulas and 1 with acute occlusion of a femoral artery. The duration of clinically evident thrombosis was <24 hours in each patient. The thrombi were cut to prepare multiple segments with dimensions similar to those of whole-blood thrombi formed ex vivo (two segments from each thrombus for each incubation condition). Thrombus segments were extensively washed in PBS and preincubated in first-stage citrated plasma with either 1 µmol/L hirudin, 0.3 µmol/L TFPI, 10 µmol/L TAP, or a combination of 1 µmol/L hirudin and 10 µmol/L TAP. Increases in FPA induced by thrombus segments in second-stage nonanticoagulated whole blood were significantly attenuated by preincubation with TAP or TFPI (P<.05), but not hirudin, compared with increases in FPA induced by thrombus segments preincubated in plasma alone (Fig 6). Combined inhibition of thrombin and Xa/Va with 1 µmol/L hirudin and 10 µmol/L TAP attenuated increases in FPA to a slightly greater extent than did TAP alone, but the difference was not statistically significant.
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Role of TF in the Procoagulant Activity of Clots
The efficacy of TFPI in inhibiting clot- and thrombus-associated procoagulant activity despite incomplete inhibition of Xa activity suggests that TF-mediated activation of the coagulation system plays a role in the procoagulant activity of clots and thrombi under the conditions described. To test this hypothesis, clots were preincubated in first-stage plasma with a monoclonal antibody that inhibits human TF and TF/VIIa activity, washed extensively, and then incubated in recalcified citrated second-stage plasma. Although clots preincubated with antibody induced activation of the coagulation system, the extent of FPA generation was significantly attenuated compared with that induced by clots not preincubated with antibody (1519±340 versus 3858±925 ng/mL, respectively, at 7 minutes; P=.03 by ANOVA for repeated measures; Fig 7). The extent of inhibition of increases in FPA induced by clot preincubation with the anti-TF antibody was less than that observed by preincubation with TAP or TFPI (Fig 5).
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Discussion |
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Thrombus-associated procoagulants are thought to play a critical role in thrombosis progression and in thrombosis recurrence after discontinuation of antithrombotic interventions. Although fibrin-bound thrombin was initially thought to be the primary procoagulant associated with thrombi,5 6 22 we have shown that thrombus-associated Xa/Va activity may be more important.3 4 We have also shown that whole-blood clots prepared ex vivo by the method described by Chandler18 model the thrombin and Xa/Va activities of arterial thrombi induced by electrical injury in vivo.3 In a previous study fibrin formation induced by whole-blood clots in nonanticoagulated blood was inhibited equally well by hirudin and Xa inhibitors.4 In a variety of animal models, Xa inhibitors inhibit arterial thrombosis as well as hirudin does.7 11 Whether these results reflect inhibition of thrombus-associated procoagulants has not been well defined. Accordingly, the present study was designed to specifically characterize the effects of inhibition of thrombus-associated procoagulants in contrast to previous studies that characterized inhibition of thrombus-induced activation of coagulation in the presence of specific anticoagulants. Our results suggest that inhibition of thrombus-associated Xa activity may be a more effective strategy for persistent inhibition of thrombus-associated procoagulant activity. Preincubation of whole-blood clots prepared in vitro to mimic arterial thrombi or of arterial thrombi recovered from patients with Xa inhibitors markedly attenuated procoagulant activity, even though the activity of fibrin-bound thrombin in the thrombus was not inhibited. Thus, thrombus-associated procoagulant activity primarily depends on de novo elaboration of thrombin induced by the Xa/Va complex, which is likely associated with platelets in the thrombus. These results provide a mechanism for the sustained anticoagulant effects of Xa inhibitors after coronary thrombolysis in dogs.7 11
An unexpected finding in the current study was the greater efficacy of TFPI compared with TAP in attenuating thrombus-associated procoagulant activity (Figs 5 and 6), despite incomplete inhibition of Xa by the former (Fig 3). A potential explanation for this finding is that activity of the TF/VIIa complex contributed to the increases in procoagulant activity observed in our two-stage assay. Attenuation of fibrin formation in the presence of anti-TF antibody when whole-blood clots were incubated with recalcified citrated plasma is consistent with this hypothesis. Whether similar effects would be observed in vivo is unclear, because small amounts of TF activity in blood may result from venipuncture. However, these results are consistent with the finding in rabbits that an antibody against TF prevents thrombosis after carotid artery injury in the presence of a stenosis that promotes platelet aggregation.23 TF has also been found in blood and plasma.24 25 26 27 In experimental preparations of arterial fibrinolysis, TFPI was effective in attenuating recurrent thrombosis,16 28 although not to the extent that it was attenuated by inhibition of thrombin with hirudin or of Xa with TAP in other studies.10 11 Whether these in vivo results reflect differences in local concentrations of TFPI compared with those in our in vitro preparation or are due to procoagulant factors that cannot be modeled in vitro will need to be clarified.
A critical question not answered by our study is the extent to which thrombus-associated Xa/Va activity persists over time. Previous studies have suggested that the Xa/Va complex is resistant to ATIII-mediated inhibition.3 29 Therefore, in vivo inhibition of the Xa/Va complex activity may depend on inhibition of Va by protein C. In experimental animals administration of activated protein C prevents platelet-rich arterial thrombosis,24 25 26 but the extent to which this physiological inhibitor regulates coronary thrombosis is unknown. In our study pathological thrombi acquired from patients with arterial thrombosis consistently demonstrated Xa/Va activity; this also appears to be true when thrombi are acquired days or weeks after the clinical thrombotic event (P.R.E., unpublished data, 1993). Long-term increases in prothrombin fragment 1.2 concentration have also been noted in patients with unstable angina, consistent with persistent Xa/Va activity.30 Thus, it is possible that thrombus-associated Xa activity is not inhibited by physiological anticoagulants but becomes isolated from the blood as plasma proteins coat and eventually "passivate" the thrombogenic surface. Our results with pathological thrombi suggest that thrombus disruption could reexpose active Xa/Va and induce marked prothrombin activation. This mechanism may be critically important to recurrent heparin-resistant thrombosis after vascular interventions.
An important consideration in the interpretation of our results is the extent to which preincubation of thrombi with anticoagulants results in carryover of the anticoagulant to the second stage of the assay and inhibition of procoagulant activity. The presence of anticoagulants in the second stage would confound findings of sustained effects of a specific anticoagulant. To account for this possibility, thrombi were extensively washed in buffer, and aliquots of the last wash buffer were assayed for anti-Xa or antithrombin activity, as appropriate. This procedure was sufficient to exclude the presence of >1 nmol/L hirudin, TAP, or TFPI in the last wash buffer. However, we cannot exclude the possibility that adherence of small amounts of anticoagulant to the thrombus rather than inhibition of the targeted thrombus-associated procoagulant partially accounted for the attenuation of procoagulant activity in the second stage. Even small amounts of a Xa inhibitor would be expected to markedly attenuate the activation rate of the coagulation system attributable to clot-bound thrombin or to Xa elaborated by the TF/VIIa associated with the thrombus. Nonetheless, our results suggest that a relatively brief incubation of thrombi with inhibitors of Xa/Va is a more effective strategy for sustained inhibition of thrombus-associated procoagulant activity than is preincubation with thrombin inhibitors.
Considerable clinical data suggest that procoagulant activity of coronary arterial thrombi is responsible for recurrent thrombosis after thrombolysis or mechanical interventions.31 32 33 34 35 36 37 38 39 Furthermore, persistent thrombosis appears to accelerate the rate of progression of coronary arterial lesions, as defined by angiographic criteria.40 41 Accordingly, it is reasonable to target inhibition of thrombus-associated procoagulants such as thrombin and Xa. Our results show that thrombus-associated Xa/Va activity is the principal determinant of procoagulant activity of arterial thrombi. This observation is consistent with previous reports3 4 19 and has important clinical consequences because use of specific thrombin inhibitors would not be expected to attenuate thrombus-associated Xa/Va activity. Not surprisingly, several clinical studies have shown that prothrombin activation persists in patients treated with specific thrombin inhibitors such as hirudin.42 43 44 Consequently, its persistence may account for the rebound increases in thrombin activity that have been observed clinically after discontinuation of thrombin inhibitors such as argatroban.14 Furthermore, our results suggest that anticoagulant strategies targeted toward inhibition of thrombus-associated Xa/Va activity may have a sustained inhibitory effect on the progression or recurrence of thrombosis. One means of accomplishing this would be to directly inhibit thrombus-associated Xa/Va activity, perhaps with a local drug-delivery device. If Xa/Va activity were completely inhibited, it could abolish local de novo thrombin generation.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by a National Heart, Lung, and Blood Institute/Specialized Center of Research grant in Coronary and Vascular Diseases (HL 17646 to Dr Eisenberg) and a Monsanto/Washington University Biomedical Research Grant (to Dr Abendschein).
Received November 24, 1995;
revision received July 9, 1996;
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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1. Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndromes. N Engl J Med. 1992;326:310-318.[Medline] [Order article via Infotrieve]
2. Eisenberg PR. Thrombosis and fibrinolysis in acute myocardial infarction. Alcoholism Clin Exp Res. 1994;18:97-104.[Medline] [Order article via Infotrieve]
3. 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.
4.
Prager NA, Abendschein DR, McKenzie CR, Eisenberg PR. Role of thrombin compared with factor Xa in the procoagulant activity of whole blood clots. Circulation. 1995;92:962-967.
5. 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.
6.
Liu CY, Nossel HL, Kaplan K. The binding of thrombin by fibrin. J Biol Chem. 1979;254:10421-10425.
7.
Lynch JJ Jr, Sitko GR, Mellott MJ, Nutt EM, Lehman ED, Friedman PA, Dunwiddie CT, Vlasuk GP. Maintenance of canine coronary artery patency following thrombolysis with front loaded plus low dose maintenance conjunctive therapy: a comparison of factor Xa versus thrombin inhibition. Cardiovasc Res. 1994;28:78-85.
8.
Heras M, Chesebro JH, Penny WJ, Bailey KR, Badimon L, Fuster V. Effects of thrombin inhibition on the development of acute platelet-thrombus deposition during angioplasty in pigs: heparin versus recombinant hirudin, a specific thrombin inhibitor. Circulation. 1989;79:657-665.
9. Cannon CP, McCabe CH, Henry TD, Schweiger MJ, Gibson RS, Mueller HS, Becker RC, Kleiman NS, Haugland JM, Anderson JL, et al. 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.[Abstract]
10.
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.
11.
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: comparison to hirudin and heparin in a canine model of acute coronary artery thrombosis. Circulation. 1992;85:805-815.
12. Mann KG. The coagulation explosion. Ann N Y Acad Sci. 1994;714:265-269.[Medline] [Order article via Infotrieve]
13.
Gailani D, Broze GJ. Factor XI activation in a revised model of blood coagulation. Science. 1991;253:909-912.
14. Gold HK, Torres FW, Garabedian HD, Werner W, Jang I, Khan A, Hagstrom JN, Yasuda T, Leinbach RC, Newell JB, Bovill EG, Stump DC, Collen D. Evidence for a rebound coagulation phenomenon after cessation of a 4-hour infusion of a specific thrombin inhibitor in patients with unstable angina pectoris. J Am Coll Cardiol. 1993;21:1039-1047.[Abstract]
15. Theroux P, Waters D, Lam J, Juneau M, McCans J. Reactivation of unstable angina after the discontinuation of heparin. N Engl J Med. 1992;327:141-145.[Abstract]
16.
Abendschein D, Meng YY, Torr-Brown S, Sobel BE. Maintenance of coronary patency after fibrinolysis with tissue factor pathway inhibitor. Circulation. 1995;92:944-949.
17. Miletich JP, Broze GH, Majerus PW. The synthesis of sulfated dextran beads for isolation of human plasma coagulation factors II, IX, X. Anal Biochem. 1980;105:304-310.[Medline] [Order article via Infotrieve]
18. Chandler AB. In vitro thrombotic coagulation of the blood. Lab Invest. 1958;7:110-114.[Medline] [Order article via Infotrieve]
19. Eisenberg PR, Lee J, Miletich JP. Importance of platelets in arterial clot-associated procoagulant activity. Circulation. 1995;92(suppl I):I-803. Abstract.
20. Ruf W, Edgington TS. An anti-tissue factor monoclonal antibody which inhibits TF.VIIa complex is a potent anticoagulant in plasma. Thromb Haemost. 1991;66:529-533.[Medline] [Order article via Infotrieve]
21.
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.
22. Kochum C, Frebelius S. Rapid radioimmunoassay of human fibrinopeptide A-removal of cross-reacting fibrinogen with bentonite. Thromb Res. 1980;19:589-598.[Medline] [Order article via Infotrieve]
23.
Pawashe AB, Golino P, Ambrosio G, Migliaccio F, Ragni M, Pasucci I, Chiariello M, Bach R, Garen A, Konigsberg WK, Ezekowitz MD. A monoclonal antibody against rabbit tissue factor inhibits thrombus formation in stenotic injured rabbit carotid arteries. Circ Res. 1994;74:56-63.
24. Albrecht S, Luther T, Grossman H, Flossel C, Kotzsch M, Muller M. An ELISA for tissue factor using monoclonal antibodies. Blood Coagulation Fibrinol. 1992;3:263-270.[Medline] [Order article via Infotrieve]
25.
Fukuda S, Iijima K, Nakamura K. Measuring tissue factor (factor III) activity in plasma. Clin Chem. 1989;35:1897-1900.
26. Iijima K, Fukuda C, Nakamura K. Measurements of tissue factor-like activity in plasma of patients with DIC. Thromb Res. 1991;61:29-38.[Medline] [Order article via Infotrieve]
27.
Leatham EW, Bath PMW, Tooze JA, Camm AJ. Increased monocyte tissue factor expression in coronary disease. Br Heart J. 1995;73:10-13.
28.
Haskel EJ, Torr SR, Day KC, Palmier M, Wun TC, Sobel BE, Abendschein D. Prevention of arterial reocclusion after thrombolysis with lipoprotein associated coagulation inhibitor (LACI). Circulation. 1991;84:821-827.
29. Teitel JM, Rosenberg RD. Protection of factor Xa from neutralization by heparin-antithrombin complex. J Clin Invest. 1983;71:1383-1391.
30.
Merlini PA, Bauer KA, Oltrona L, Ardissino D, Cattaneo M, Belli C, Mannucci PM, Rosenberg RD. Persistent activation of coagulation mechanism in unstable angina and myocardial infarction. Circulation. 1994;90:61-68.
31. Chesebro JH, Badimon L, Fuster V. Importance of antithrombin therapy during coronary angioplasty. J Am Coll Cardiol. 1991;17(6 suppl B):96B-100B.
32. Mabin TA, Holmes DR, Smith HC. Intracoronary thrombus role in coronary occlusion complicating PTCA. J Am Coll Cardiol. 1985;5:198-202.[Abstract]
33.
Topol EJ, Fuster V, Harrington RA, Califf RM, Kleiman NS, Kereiakes DJ, Cohen M, Chapekis A, Gold HK, Tannenbaum MA, et al. Recombinant hirudin for unstable angina pectoris: a multicenter, randomized angiographic trial. Circulation. 1994;89:1557-1566.
34. Eisenberg PR, Sherman LA, Rich M, Schwartz D, Schectman K, Geltman EM, Sobel BE, Jaffe AS. Importance of continued activation of thrombin reflected by fibrinopeptide A to the efficacy of thrombolysis. J Am Coll Cardiol. 1986;7:1255-1262.[Abstract]
35.
Rapold HJ, de Bono D, Arnold AER, Arnout J, De Cock F, Collen D, Verstraete M. Plasma fibrinopeptide A levels in patients with acute myocardial infarction treated with alteplase. Circulation. 1992;85:928-934.
36. Hsia J, Hamilton WP, Kleiman NS, Roberts R, Chaitman BR, Ross AM. A comparison between heparin and low-dose aspirin as adjunctive therapy with tissue plasminogen activator for acute myocardial infarction. N Engl J Med. 1990;323:1433-1437.[Abstract]
37.
de Bono DP, Simoons ML, Tijssen J, Arnold AER, Betriu A, Burgersdijk C, Lopez Bescos L, Mueller E, Pfisterer M, Van de Werf F, Zijlstra F, Verstraete M, for the European Cooperative Study Group. Early intravenous heparin improves coronary patency in thrombolysis with recombinant tissue-type plasminogen activator. Br Heart J. 1992;67:122-128.
38. Bleich SD, Schumacher RR, Cooke DH, Tate DA, Teichman SL. Effect of heparin on coronary arterial patency after thrombolysis with tissue plasminogen activator in acute myocardial infarction. Am J Cardiol. 1990;66:1412-1417.[Medline] [Order article via Infotrieve]
39.
Braunwald E, Jones RH, Mark DB, Brown J, Brown L, Cheitlin MD, Concannon CA, Cowan M, Edwards C, Fuster V, Goldman L, Green LA, Grines CL, Lytle BW, McCauley KM, Mushlin AI, Rose GC, Smith EE, Swain JA, Topol EJ, Willerson JT. Diagnosing and managing unstable angina. Circulation. 1994;90:613-622.
40. Nagatoma Y, Nakagawa S, Koiwaya Y, Tanaka K. Coronary angiographic ruptured atheromatous plaque as a predictor of future progression of stenosis. Am Heart J. 1990;119:1244-1253.[Medline] [Order article via Infotrieve]
41.
Lijia C, Chester MR, Redwood S, Huang J, Leatham E, Kaski JC. Angiographic stenosis progression and coronary events in patient's with stabilized unstable angina. Circulation. 1995;91:2319-2324.
42. Ginsberg JS, Nurmohamed MT, Gent M, MacKinnon B, Stevens P, Weitz J, Maraganore J, Hirsh J. Effects on thrombin generation of single injections of Hirulog in patients with calf vein thrombosis. Thromb Haemost. 1994;72(4):523-525.
43. Scharfstein JS, Abendschein DR, Eisenberg PR, George D, Cannon CP, Becker RC, Sobel BE, Cupples LA, Braunwald E, Loscalzo J, TIMI-5 Investigators. Fibrino(geno)lytic and procoagulant markers during thrombolytic therapy with rt-PA and adjunctive antithrombotic therapy predict clinical outcomes. Circulation. 1996. In press.
44.
Zoldhelyi P, Bichler J, Owen WG, Grill DE, Fuster V, Mruk JS, Chesebro JH. Persistent thrombin generation in humans during specific thrombin inhibition with hirudin. Circulation. 1994;90:2671-2678.
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