(Circulation. 1996;94:1719-1725.)
© 1996 American Heart Association, Inc.
Articles |
Antithrombotic Assessment of the Effects of Combination Therapy With the Anticoagulants Efegatran and Heparin and the Glycoprotein IIb-IIIa Platelet Receptor Antagonist 7E3 in a Canine Model of Coronary Artery Thrombosis
the Cardiovascular Research Division, Lilly Research Laboratories, Eli Lilly and Co, Indianapolis, Ind.
Correspondence to Charles V. Jackson, PhD, Research Scientist, Cardiovascular Research Division, Lilly Corporate Center, MC304, Indianapolis, IN 46285-0524.
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Abstract |
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Background There is a paucity of data regarding the antithrombotic pharmacology of the drug-drug interactions between the newer anticoagulant and antiplatelet agents. In this investigation, we have studied the antithrombotic effects of combinations of minimum effective doses of the glycoprotein IIb-IIIa receptor antagonist 7E3 [murine F(ab')2] with both heparin and the novel tripeptide arginal antithrombin efegatran (LY294468) in a canine model of coronary artery thrombosis.
Methods and Results Thrombogenesis was initiated by electrolytic injury of the intimal surface of the left circumflex coronary artery. The groups studied were efegatran (0.25 mg · kg-1 ·h-1), heparin (80 U/kg, single injection, plus 30 U · kg-1 ·h-1), 7E3 (0.4 mg/kg, single injection), 7E3+efegatran, and 7E3+heparin. The combination of 7E3+efegatran was found to maintain better vessel patency (P<.05) at the end of the experiment (4 of 5 vessels) than all other groups (0 of 5, 0 of 4, 1 of 6, 2 of 7, and 1 of 6 for the vehicle-, heparin-, 7E3-, efegatran-, and 7E3+heparin–treated groups, respectively). Bleeding times were increased (P<.05) in both the 7E3+heparin group (fourfold) and the 7E3+efegatran group (threefold). 7E3 alone and both combination treatments produced significant reductions in ADP, arachidonic acid, and thrombin-induced platelet aggregation, whereas efegatran and heparin abolished only thrombin-induced aggregation.
Conclusions The present investigation demonstrates that combination therapy with minimum effective doses of 7E3+efegatran provided enhanced antithrombotic efficacy compared with 7E3+heparin in this model of thrombosis.
Key Words: thrombosis • 7E3 • efegatran • heparin • anticoagulants • arteries • platelet aggregation inhibitors
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Introduction |
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Current clinical antithrombotic treatment of acute coronary syndromes such as acute myocardial infarction, unstable angina, or abrupt closure after PTCA is heparin and aspirin.1 2 3 4 Heparin, while the anticoagulant of choice in the prevention and treatment of these thrombotic disorders, has several disadvantages. When given at a fixed dose, heparin produces varied and unpredictable levels of anticoagulation in patients. This unpredictability necessitates close laboratory monitoring and dose adjustment if sufficient anticoagulant levels are to be achieved without undue risk of bleeding complications. Heparin also is ineffective in inhibiting thrombin that is bound to fibrin5 6 or subendothelial matrix proteins.7 Because clot-bound thrombin is protected from inactivation by fluid-phase inhibitors (ie, antithrombin III and heparin cofactor II), it has the potential to locally amplify coagulation by activating clotting factors V and VIII and to generate more thrombin. This amplification leads to clot growth and cross-linking of fibrin by thrombin-induced activation of factor XIII.8 In addition, thrombin-induced platelet aggregation causes the release of platelet factor 4, which neutralizes heparin.9 10 Thus, heparin has a narrow therapeutic index in that anticoagulation sufficient to produce arterial antithrombotic efficacy is associated with increased bleeding risk.
Because of these limitations, more potent and selective thrombin inhibitors have been developed that bind directly to the catalytic and/or substrate recognition sites of thrombin. These specific thrombin inhibitors are not bound or inactivated by plasma proteins or platelet factor 4 and can bind clot-bound thrombin. Hirudin, an essentially irreversible thrombin inhibitor that binds both the active site and the fibrinogen recognition site of thrombin, has been compared with heparin as the anticoagulant in several thrombolytic trials: TIMI 9, GUSTO II, and HIT III.11 12 13 Unfortunately, the results of these three phase III studies were not encouraging. The TIMI 9 study was suspended early owing to an increased incidence of intracranial and major spontaneous hemorrhage at noncranial sites in both arms of the study. The study was reconfigured (TIMI 9B) with lower doses of both heparin and hirudin. The findings of the TIMI 9A study were consistent with the observations of the GUSTO IIa and HIT III trials. Both of these trials also encountered increased levels of bleeding in the hirudin arm of the study.
Efegatran, a tripeptide arginal (D-methyl-phenylalanyl-prolyl-arginal, LY294468), active-site, reversible inhibitor of thrombin, is presently in phase II clinical trials for the treatment of acute coronary syndromes. Active-site inhibitors have the ability to inactivate clot-bound thrombin, are resistant to inactivation by heparinase or platelet factor 4, and lack dependence on the cofactor antithrombin III to mediate its effect.14 Efegatran has been shown to have a ratio of antithrombotic efficacy to risk of bleeding of 16:1 in a canine model of coronary artery thrombosis and is resistant to neutralization by platelet factor 4.15
Aspirin, while shown to be beneficial in patients with acute coronary syndromes,16 exerts its actions only through inhibition of the cyclooxygenase pathway,17 inhibiting thromboxane A2 production, a potent platelet agonist and vasoconstrictor. Aspirin does not inhibit the ability of other platelet agonists (ie, thrombin, ADP, or platelet-activating factor) to induce platelet aggregation. The final common pathway for platelet aggregation is activation of the platelet GPIIb-IIIa integrin. On activation, GPIIb-IIIa receptors bind fibrinogen and ultimately make cross bridges between platelets. This integrin has been the major target in two recent clinical trials.18 19 The largest of these studies was the EPIC trial that was designed to assess the efficacy of the chimeric Fab form of the monoclonal antibody 7E3 (c7E3, ReoPro), a GPIIb-IIIa receptor antagonist, in high-risk angioplasty. All patients received aspirin and heparin. The c7E3 bolus+infusion group saw a 35% reduction in ischemic complications resulting from angioplasty or atherectomy compared with the placebo group. The group receiving a single bolus of c7E3 alone also showed a 10% reduction in complications compared with the placebo group. Complications from bleeding were substantially increased in the c7E3 bolus+infusion group, with smaller increases seen in the c7E3 bolus group. The phase III trial EPILOG is now ongoing to confirm the results of the pilot study PROLOG. In PROLOG, it was found that if weight-adjusted heparin was used with the standard dose of c7E3 (0.25 mg/kg bolus+10 µg/min infusion every 12 hours), bleeding risk was reduced compared with the heparin regimen used in the EPIC trial.20
Several recent studies in dogs have examined combinations of the newer, more potent antiplatelet and anticoagulant agents in preclinical models of thrombolysis. Prager et al21 demonstrated that combination therapy with subefficacious doses of aspirin and hirudin was a more effective adjunct to thrombolysis than either agent alone. Similar results were obtained in studies of combination therapy with integrelin plus hirudin22 and ridogrel plus Hirulog23 as adjunctive therapy during tissue plasminogen activator–-induced thrombolysis. We therefore conducted a study to assess the drug-drug interactions between the F(ab')2 fragment of the murine monoclonal antibody 7E3 and efegatran as antithrombotics without the added complication of thrombolysis. The specific aims were twofold: to compare and contrast the antithrombotic profile of combination therapy with 7E3+efegatran to that of 7E3+heparin and to determine the bleeding risk associated with these combination therapies in a canine model of electrolytic injury–induced coronary artery thrombosis.
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Methods |
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Instrumentation
The instrumentation has been described previously.24 Briefly, thirty-six 6- to 7-month-old mixed-breed hounds of either sex (17 to 24 kg, Hazelton-LRE, Kalamazoo, Mich, or Butler Farms, Clyde, NY) were anesthetized with sodium pentobarbital (30 mg/kg IV) and ventilated with room air. The carotid artery, jugular vein, and femoral vein were isolated and cannulated for recording of arterial pressure (Millar MPC-500 transducer), blood sampling, and drug administration, respectively. Limb leads were placed subcutaneously for monitoring of lead II ECG. A left thoracotomy was performed, and the heart was suspended in a pericardial cradle. The LCx was isolated and cleaned of all adventitia and fat proximal to the first main branch. An electromagnetic flow probe was placed around the LCx for direct measurement of blood flow. A second MPC-500 Millar transducer was inserted into the ventricle through the left atria for measurement of left ventricular pressure. A stimulating anode (26-gauge needle-tipped, 30-gauge silver-coated/copper wire) was inserted into the LCx distal to the flow probe and in contact with the intimal surface of the artery. The circuit was completed by placing the cathode in a subcutaneous site. A plastic screw occluder was placed around the LCx over the area of the electrode, and a critical stenosis (sufficient to produce a 40% to 50% reduction in the hyperemic response to a 10-second total occlusion of the LCx) was applied. All hemodynamic and ECG measurements were recorded and analyzed with a data acquisition system (M3000, Modular Instruments, Inc).
Thrombus Formation
Thrombogenesis was initiated by applying 100 µA direct current to the anode, producing endothelial cell injury. The current was maintained for 60 minutes and then stopped whether the vessel had occluded or not. Thrombus formation proceeded spontaneously until the LCx was totally occluded (determined as zero coronary blood flow). In the experimental protocol, all compounds were administered 15 minutes before anodal stimulation was begun. Efegatran (dissolved in 10 mL saline) was administered as a 2-hour infusion at a dose of 0.25 mg · kg-1 · h-1 (n=7). Heparin (dissolved in saline) was administered as an initial single injection (10 mL) at 80 U/kg followed by a 2-hour infusion (10 mL) at 30 U · kg-1 · h-1 (n=4). 7E3 (diluted in a final volume of 10 mL saline) was administered as an initial single injection, 0.4 mg/kg (n=6), followed by a 2-hour infusion of 10 mL saline or in conjunction with heparin 80 U/kg+30 U/kg (n=6) or efegatran 0.25 mg · kg-1 · h-1 (n=5). The vehicle-treated group (n=5) received 10 mL saline infused over a 2-hour period. The dogs were monitored for 2 hours after cessation of drug infusion; then they were killed by electric ventricular fibrillation. A 2-cm segment of the LCx containing the thrombus was removed and dissected longitudinally, and the thrombus was removed and weighed.
A pilot study was conducted with 7E3 alone to establish the minimum effective antithrombotic dose to be used in the combination studies. The doses studied were 0.2 (n=6), 0.4 (n=6), 0.6 (n=4), and 0.8 (n=3) mg/kg administered as a single intravenous injection over 5 minutes. A vehicle (10 mL saline) group of three animals was included. Times to occlusion for this vehicle group (56±6 minutes) were not different from those of the vehicle group (61±7 minutes) used for the combination study.
Coagulation Assay, TBT, and Hematology
Whole-blood samples (3.0 mL) were drawn before drug, 60 and 120 minutes during drug administration, and 60 and 120 minutes after drug administration. Clotting time, TT, and aPTT, were determined on nonanticoagulated whole blood by use of a Hemochron 801 (International Technidyne Corp). Clotting time determinations were stopped at 1500 seconds because this was the limit of detection for the Hemochron 801.
TBT tests were performed on the gingiva of the upper and lower jaws of the dogs by use of a Simplate II bleeding time device (Organon Tecknika). TBTs were determined before administration of drug, 60 and 120 minutes during drug administration, and 120 minutes after the cessation of drug infusion.
Whole-blood cell counts and analysis of hemoglobin content were determined on a 40-µL citrated blood sample processed with a hematology analyzer (Cell-Dyn 900, Sequoia-Turner). Samples were taken before drug administration, 60 minutes into the drug infusion, and 2 hours after the cessation of drug infusion.
Platelet Aggregation Tests
Ex vivo platelet aggregation (Bio/Data, PAP-4) was performed to determine the level of platelet inhibition throughout the course of the experiments. Citrated blood samples drawn just before drug administration and 60 minutes into the drug infusion were centrifuged at 150g for 10 minutes to prepare platelet-rich plasma. Platelet aggregation (37°C) was induced by the addition of thrombin (0.1 to 0.35 U/mL), PAF (0.3 µmol/L), ADP (10 µmol/L), or AA (0.625 mmol/L) to 0.45 mL of platelet-rich plasma (total volume, 0.5 mL). A nonaggregating priming concentration of epinephrine (0.8 µmol/L) was used for the ADP- and AA-induced platelet aggregation test.
Drug and Data Analysis
Efegatran was obtained as the sulfate salt from Eli Lilly and Co. The F(ab')2 fragment of the murine monoclonal antibody 7E3 was obtained from Centocor. Heparin (sodium salt), thrombin (bovine), and ADP were obtained from Sigma Chemical Co. PAF (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphoryl-choline) was obtained from Bachem and was prepared according to the method of Jackson et al.25 All data were analyzed by a one-way ANOVA for group comparisons and for repeated measures, followed by a Student-Newman-Keuls post hoc t test to determine the level of significance. Values were determined to be statistically different at a value of P<.05. All values represent the mean±SEM. All studies were conducted according to the Guide for the Care and Use of Laboratory Animals as adopted by the NIH and the Lilly Research Laboratory Animal Care and Use Committee.
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Results |
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7E3 Antithrombotic Assessment
Table 1
summarizes the antithrombotic efficacy and effects of increasing doses of 7E3 on TBT. Time to total thrombotic occlusion was increased dose-dependently. The dogs exposed to a single injection of 0.6 and 0.8 mg/kg 7E3 demonstrated significant (P<.05) prolongations in time to occlusion of their LCx compared with vehicle-treated dogs (195±31 and 225±0 minutes, respectively, versus 56±6 minutes). The lower doses of 7E3 (0.2 and 0.4 mg/kg) demonstrated nonsignificant prolongations in time to occlusion of 
60 minutes versus the vehicle-treated group. Patency rates for the 0.2, 0.4, 0.6, and 0.8 mg/kg 7E3 groups were 2 of 6, 1 of 6, 3 of 4, and 3 of 3 vessels, respectively.
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TBTs were increased dose-dependently with single intravenous injections of 7E3. Changes in TBTs observed 60 minutes after drug administration for the high dose of 7E3 (0.8 mg/kg) were significantly (P<.05) greater than the two lower doses (600±0 seconds versus 307±64 and 340±69 seconds for 0.2 and 0.4 mg/kg, respectively). Effects of 7E3 on TBT were reversible with time. Elevations in response to the low dose (0.2 mg/kg) returned to normal by the end of the experiment (240-minute time point), and there was a graded response with the other doses so that TBT in response to the high dose (0.8 mg/kg) had declined to 404±104 seconds by the end of the experiment.
Anticoagulant and Antiplatelet Effects
Groups receiving anticoagulants (heparin or efegatran) demonstrated significant elevations in TT (Fig 1). Groups treated with heparin alone or 7E3+heparin produced a significant (P<.05) increase in TT ratio after 60 minutes of drug administration (14.1±2.3 and 12.8±2.8, respectively) compared with all other groups. Effects of heparin on TT ratio were lower 120 minutes after drug administration but were significantly elevated compared with baseline (5.7±1.4 and 5.6±0.8, heparin alone and 7E3+heparin, respectively). Groups receiving infusions of efegatran alone or 7E3+efegatran demonstrated a slower time-dependent increase in TT ratio with peak elevations (P<.05) at 120 minutes of drug infusion (5.0±1.2 and 4.1±1.1, respectively). All groups returned to baseline levels within 60 minutes after the cessation of drug administration.
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), 80 U/kg plus 30 U·kg-1·h-1 heparin, n=4; (
), 0.4 mg/kg 7E3, n=6; (
), 0.25 mg·kg-1·h-1 efegatran, n=7; (
), 7E3+heparin, n=6; and (
), 7E3+efegatran, n=5. Each point represents the mean±SEM. *P<.05, heparin-treated vs all other groups. 
P<.05, 7E3 vs all other groups.
Groups receiving heparin or efegatran alone produced significant increases in the aPTT ratio after 60 minutes of drug administration (1.39±0.07 and 1.15±0.03, respectively; Fig 2). Administration of 7E3 to dogs receiving heparin or efegatran had no effect on aPTT values. At the 120-minute time point, all the above-mentioned groups except that receiving heparin alone continued to produce significant increases in aPTT. All groups returned to baseline values within 60 minutes after the cessation of drug administration.
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Table 2 demonstrates that administration of efegatran or heparin alone selectively and completely abolished ex vivo thrombin-induced platelet aggregation. The addition of 7E3 to both anticoagulant dose regimens provided additional platelet inhibition not only of thrombin but also of ADP-, PAF-, and AA- induced platelet aggregation (80%, 40%, and 60%, respectively). The degree of ex vivo platelet inhibition observed with 7E3+heparin or 7E3+efegatran was not different from that observed with a single intravenous injection of 0.4 mg/kg 7E3 alone.
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Antithrombotic Efficacy
Minimum effective antithrombotic doses of heparin, efegatran, or 7E3 alone were used in this study. Fig 3 illustrates that times to occlusion were elevated in response to 80 U/kg+30 U · kg-1 · h-1 heparin, 0.25 mg · kg-1 ·h-1 efegatran, and 0.4 mg/kg 7E3 (94±20, 125±27, and 124±25 minutes, respectively, versus 56±6 minutes for vehicle). A significant (P<.05) prolongation in time to occlusion was observed in the 7E3+efegatran–treated group (223±2 minutes). There was a small additive but insignificant prolongation of time to occlusion of the LCx in the dogs receiving 7E3+heparin (147±20 minutes). Quantification of coronary blood flow throughout the experiments demonstrated that the group receiving 7E3+efegatran maintained significantly (P<.05) higher coronary blood flow than the other groups (Fig 4). The 7E3+efegatran group maintained coronary blood flow to the level observed before initiation of vessel injury throughout the 2-hour efegatran infusion. After cessation of the efegatran infusion, coronary blood flow for the 7E3+efegatran group began to decline in the later stages of the experiment. There also were significantly (P<.05) more patent vessels at the end of the experiment in the 7E3+efegatran–treated group than in any other group (4 of 5 versus 0 of 5 [vehicle], 0 of 4 [heparin], 2 of 7 [efegatran], 1 of 6 [7E3], and 1 of 6 [7E3+heparin]). Analysis of CFVs in these preparations demonstrated that in the 7E3+efegatran–treated group, only 1 of the 5 vessels demonstrated CFVs (2.7 CFV/h for the vessel that occluded before the end of the experiment; noted above). In the other groups, CFV frequency was similar, averaging 2 to 4 CFV/h before total occlusion of the LCx. The 7E3+heparin–treated group demonstrated 3.1±1.6 CFV/h (all vessels had CFVs).
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), 80 U/kg plus 30 U·kg-1·h-1 heparin, n=4; (
), 0.4 mg/kg 7E3, n=6; (
), 0.25 mg·kg-1·h-1 efegatran, n=7; (
), 7E3+heparin, n=6; and (
), 7E3+efegatran, n=5. Each bar represents the mean±SEM. *P<.05 vs vehicle group. Numbers below bars indicate vessel patency ratios.
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), 80 U/kg+30 U·kg-1·h-1 heparin, n=4; (
The minimum effective antithrombotic doses used had no significant effect on thrombus mass as determined at the end of the experiment (Fig 5). Combination therapy with 7E3+efegatran, however, produced a significant (P<.05) reduction in thrombus mass compared with the 7E3+heparin–treated group (5.1±1.7 versus 11.9±2.6 mg, respectively).
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TBT, Hematology, Blood Pressure, and Heart Rate
Fig 6 illustrates the effect of the anticoagulants, 7E3, and combination therapies on TBT. Baseline TBT values for each group were 162±3, 142±10, 149±21, 147±20, and 190±32 seconds for heparin, efegatran, 7E3, 7E3+heparin, and 7E3+efegatran, respectively. The minimum effective dose of each agent alone produced small but insignificant increases in TBT. The combination of 7E3+heparin produced a significant (P<.05) increase in TBT at 60 minutes of drug administration (572±20 seconds), whereas the combination of 7E3+efegatran produced a significant (P<.05) increase at 120 minutes of drug administration (551±44 seconds). After the cessation of drug infusions (heparin and efegatran), TBT began to return to normal; only the 7E3+efegatran–treated group had not completely returned to normal by the end of the experiment.
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Throughout the course of these experiments, there were no observable effects on red blood cell count, hemoglobin, or platelet counts (Table 3). Table 4 summarizes the effects of the various groups on heart rate and mean arterial blood pressure. No significant effects on heart rate were observed in any group. The vehicle, heparin, and efegatran groups demonstrated significant (P<.05) reductions in mean arterial blood pressure by the end of the experiment. There were no significant effects on mean arterial blood pressure in the groups receiving 7E3 alone or combinations (7E3+heparin and 7E3+efegatran).
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Discussion |
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Presently, aspirin and heparin are the standards for antiplatelet and anticoagulant therapy, respectively, in the treatment of acute coronary syndromes. Recently, a new and very potent antiplatelet GPIIb/IIIa receptor antagonist, ReoPro, was approved for use in high-risk patients undergoing PTCA. ReoPro administered conjunctively with aspirin and heparin was found to significantly reduce the incidence of ischemic complications after PTCA.19 In addition, new anticoagulant agents (eg, hirudin and efegatran) are being studied in clinical trials as replacements for heparin in the setting of acute coronary syndromes.
There is a paucity of data regarding the antithrombotic pharmacology of the drug-drug interactions between the newer antiplatelet (eg, ReoPro and integrelin) and anticoagulant (eg, hirudin and efegatran) agents. Because of the potential for increased bleeding risk associated with the newer agents, we designed a study to address the interactions of minimum effective antithrombotic doses (ie, doses of minimum antithrombotic efficacy and low bleeding risk) of the murine F(ab')2 fragment of 7E3 in combination with efegatran compared with 7E3+heparin. We observed that the combination therapy of 7E3+efegatran was a more effective antithrombotic combination than 7E3+heparin.
Minimum effective antithrombotic doses for efegatran and heparin were chosen on the basis of results from a previous study by Jackson et al.15 Previous antithrombotic studies conducted with 7E3 in the dog did not establish a minimum effective dose.26 27 28 In these studies, a dose of 0.8 mg/kg was used for optimal antithrombotic activity. A pilot study was therefore conducted to determine the minimum effective antithrombotic dose for 7E3 in this canine model of coronary artery thrombosis (Table 1
). The dose of 0.4 mg/kg 7E3 was chosen as the minimum effective dose because it provided a suboptimal effect on time to occlusion (124±25 versus >225 minutes for 0.8 mg/kg 7E3). In addition, 0.4 mg/kg 7E3 produced significantly more platelet inhibition than the 0.2 mg/kg dose of 7E3 (Table 2) but not as complete an inhibition profile as observed with 0.6 and 0.8 mg/kg 7E3. In the present study, 0.25 mg · kg-1 · h-1 efegatran produced similar antithrombotic activity (time to occlusion, 125±27 minutes) as did 0.4 mg/kg 7E3 (Fig 3); however, only thrombin-induced platelet aggregation was abolished (Table 2). The dose of heparin chosen (80 U/kg+30 U · kg-1 · h-1) produced similar levels of antithrombotic and antiplatelet activity as efegatran (time to occlusion, 94±20 minutes, with 100% inhibition of thrombin-induced platelet aggregation). Levels of anticoagulation for efegatran and heparin were similar to those observed in the previous report15 ; a 14- and 5-fold increase in TT and a 1.4- to 1.2-fold increase in aPTT, respectively (Figs 1 and 2). Antithrombotic therapy with 0.4 mg/kg 7E3 had no effect on whole-blood clotting times.
When comparing combination therapy with 7E3+anticoagulants, we observed that 7E3+efegatran produced an enhanced antithrombotic effect (Fig 3). Time to total thrombotic occlusion for this group was 223±2 minutes, with 4 of the 5 vessels patent at the end of the experiment and no CFVs demonstrated throughout the experiments for the 5 patent vessels. The fifth vessel, which occluded at 215 minutes (10 minutes before the end of the experimental protocol), had a CFV frequency of 2.7 CFV/h. These data are similar to those observed in the present study (Table 1) with the high dose of 7E3 alone (0.8 mg/kg) and in other investigations.27 28 The optimal antithrombotic dose of 0.8 mg/kg 7E3 provided for patency of all exposed vessels and no observable CFVs. Therefore, the addition of efegatran to an antithrombotic regimen of 7E3 allowed the dose of 7E3 to be reduced by 50%. Combination therapy of 7E3+efegatran also appeared to reduce the bleeding risk, at least in the context of these experiments. Peak TBTs were <10 minutes (Fig 5) in the 7E3+efegatran–treated group. This indicates a potential reduced bleeding risk compared with single high-dose therapy with 7E3 alone (0.8 mg/kg); peak TBTs for that therapy were >10 (present study) and >30 minutes.26 Enhanced antithrombotic efficacy was not observed when 7E3 was combined with heparin; a small, nonsignificant additive antithrombotic effect was observed (time to occlusion, 147±20 minutes). Only one vessel in the 7E3+heparin group remained patent at the end of the experiment; however, all vessels in this group demonstrated CFVs. This raises a question about the dose of heparin selected and what would have happened if the dose had been increased. From the previous work of Jackson et al,15 a higher-dose regimen (120 U/kg+50 U/kg) of heparin produced more antithrombotic efficacy than the 80 U/kg+30 U · kg-1 · h-1 regimen; however, the penalty was a further increase in bleeding compared with the lower dose of heparin.
Anticoagulant levels were higher in the 7E3+heparin group compared with the 7E3+efegatran group for both TT and aPTT (Figs 1 and 2). Therefore, the level of systemic anticoagulation between the combination groups does not explain the difference observed in antithrombotic efficacy. However, local thrombus-associated neutralization of heparin versus efegatran may contribute to the difference. Heparin is neutralized by platelet factor 4 as it is released from activated platelets, thereby decreasing the local concentration of free heparin that could complex with thrombin, whereas efegatran is not neutralized by platelet factor 4.15 In addition, active-site inhibitors (eg, hirudin, PPACK, and Ro 46-6240) also possess a greater ability than heparin to inactivate clot-bound thrombin5 6 29 through less steric henderance to clot diffusion.
The difference in antithrombotic efficacy observed between the combination groups does not appear to be due to differences in ex vivo platelet aggregation. Efegatran and heparin alone both selectively and completely inhibited thrombin-induced platelet aggregation. 7E3 alone produced inhibition of ADP, AA, and thrombin (72±8%, 83±12%, and 100%, respectively) and inhibited PAF to a lesser extent (43±6%). Combinations of antithrombin and antiplatelet therapy produced levels of inhibition of platelet aggregation similar to that observed with 7E3 alone. Inhibition of platelet agonists ranged from 37±9% to 94±13% and 40±7% to 100% for 7E3+efegatran and 7E3+heparin, respectively (Table 2).
The results of the present study demonstrate that the combination therapy of 7E3+efegatran provided superior antithrombotic efficacy compared with the 7E3+heparin combination in this canine model of coronary artery thrombosis. Combining efegatran with 7E3 allowed the dose of 7E3 to be reduced by 50%. Newer anticoagulants like efegatran also may provide safer alternatives to heparin while providing optimal antithrombotic efficacy. In conclusion, the data suggest the potential for additive antithrombotic pharmacology when 7E3 is combined with efegatran in acute coronary syndromes.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This investigation is a partial fulfillment for an MS degree in pharmaceutical sciences for T.J. Shetler under the guidance of Drs James Burger (Butler University, Indianapolis, Ind) and Jackson (Lilly Research Laboratories, Indianapolis, Ind). We would like to thank Drs Robert Jordan and Mark Needleman (Centocor) for the generous supply of the murine F(ab')2 7E3 fragment used in the study.
Received January 12, 1996; revision received April 14, 1996; accepted April 23, 1996.
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