Glycoprotein IIb/IIIa receptor inhibitor attenuates platelet aggregation induced by thromboxane A2 during in vitro nonpulsatile ventricular assist circulation

A recent development in antithrombotic research allows the inhibition of platelet aggregation via protection of the glycoprotein IIb/IIIa receptor on the platelet membrane. We hypothesized that a GP IIb/IIIa receptor inhibitor would inhibit thromboxane-induced platelet aggregation during circulation in our in vitro ventricular assist device (VAD) circuit and preserve long-term platelet function. Twenty-one in vitro nonpulsatile centrifugal VAD circuits were simulated for 4 days using 450 ml of fresh human whole blood with or without glycoprotein IIb/IIIa receptor inhibitor (tirofiban). Platelet aggregation and degranulation were measured in whole blood induced by ristocetin, collagen, ADP, and thromboxane A2 (TXA2). The tirofiban-treated group preserved the platelet count and tended to exert these beneficial effects by inhibiting pathologic platelet aggregation induced by TXA2, collagen, and ADP as well as degranulation. Tirofiban may be useful in preserving platelet number and function during clinical VAD use.     

Efficacy of FK633, an ultra-short acting glycoprotein IIb/IIIa antagonist on platelet preservation during and after cardiopulmonary bypass.

OBJECTIVE: Temporary pharmacologic inhibition of platelet function during and after cardiopulmonary bypass (CPB) (platelet anesthesia) is an attractive strategy for preserving platelets during CPB. We examined the efficacy of FK633, an ultra-short acting glycoprotein IIb/IIIa antagonist. METHODS: The study was carried out in six mongrel dogs that received an intravenous bolus of 0.1 mg/kg of FK633 at the time of administration of heparin (group F), and six control dogs (group C). All animals underwent 60 min of normothermic CPB followed by a 2-h observation period. Blood samples for platelet count, platelet aggregation to adenosine diphosphate and parameters concerning the coagulation system were obtained at eight time points. Hemodynamics, bleeding time, and postoperative blood loss were assessed serially. Scanning electron micrograph of the oxygenator's membrane was investigated. RESULTS: FK633 significantly protected platelet number (group F, 59+/-10% versus group C, 38+/-15% of the pre-CPB value; P < 0.01), and inhibited platelet aggregation to adenosine diphosphate (group F, 13+/-12% versus group C, 35+/-9% of the pre-CPB value; P < 0.01) during CPB. Postoperative blood loss did not significantly differ between the two groups, but there was a tendency of less bleeding in group F (group F, 73+/-23 ml versus group C, 111+/-44 ml; P = 0.09). In group F, scanning electron micrograph of the oxygenator's membrane showed that its surface was free from platelets. There were no significant differences between the groups in hemodynamics. CONCLUSIONS: An ultra-short acting glycoprotein IIb/IIIa antagonist, FK633, is effective in preventing both platelet aggregation and thrombocytopenia during CPB, and may be effective for minimizing postoperative bleeding.     

The glycoprotein IIb/IIIa antagonist c7E3 inhibits platelet aggregation in the presence of heparin-associated antibodies

Purpose: Heparin-associated antibodies (HAAb), in the presence of heparin, can cause platelet activation and aggregation. The purpose of this study was to assess whether a platelet glycoprotein (GP) IIb/IIIa receptor antagonist, c7E3, would inhibit platelet aggregation in the presence of HAAb. If aggregation is inhibited by c7E3, enzyme-linked immunosorbent assays (ELISA) would be done to determine whether c7E3 interfered with the binding of heparin and the HAAb.Methods: HAAb-positive plasmas from 21 patients (determined by platelet aggregation assays) were studied. Normal donor platelet-rich plasmas (PRP) were incubated (1 minute) with either saline solution or 3 μg/ml of c7E3. Platelet-poor plasma from patients with HAAb and one of three sources of heparin (25 μl, 10 U/ml; porcine heparin, bovine heparin, and low molecular weight heparin [enoxaparin]) were added to the PRP mixture. Aggregation was determined using a platelet aggregometer by measuring time to aggregation, the slope of the aggregation curve, and the percent change in optical density.Results: Platelet aggregation occured in 100%, 100%, and 95% of the saline solution incubations exposed to porcine heparin, bovine heparin, and enoxaparin, respectively. Incubation with c7E3 caused 100% inhibition of platelet aggregation in plasma exposed to porcine heparin, bovine heparin, and enoxaparin. The optical density curves obtained from the ELISA, which were dependent on the binding of HAAb to heparin, were not significantly different when c7E3 was compared to buffer alone.Conclusions: The GP IIb/IIIa receptor antagonist, c7E3, inhibits HAAb-induced platelet aggregation via a mechanism that does not appear to interfere with the binding between heparin and HAAb. Clinical trials are warranted to assess whether GP IIb/IIIa antagonists may allow patients with HAAb to safely receive heparin. (J Vasc Surg 1997;25:124-30.)     

Platelet glycoprotein IIb/IIIa receptor antagonist (abciximab) inhibited platelet activation and promoted skin flap survival after ischemia/reperfusion injury

BACKGROUND: Evidence has shown that platelets play an important role in the pathogenesis of flap failure. Employing a rat inferior epigastric artery skin flap as a flap reperfusion injury model, we investigated whether platelet activation was involved in the skin flap failure and whether administration of abciximab (ReoPro, chimeric 7E3 Fab) could decrease platelet activation/aggregation and promote flap survival. METHODS: Normal saline and abciximab (0.06 mg/kg; 0.2 mg/kg; 1 mg/kg) were injected intravenously into skin flaps 30 min before reperfusion and 1 h after reperfusion (each subgroup n = 6). Platelet activation as demonstrated by P-selectin (CD62P) was analyzed by flow cytometry. P-selectin expression on flap vessels was detected by immunohistochemical staining. Platelet aggregation was induced with adenosine diphosphate (ADP). Laser Doppler flowmetry monitored tissue perfusion. The surviving area was evaluated 7 days postoperatively. RESULTS: CD62P progressively increased after reperfusion. The peak CD62P occurred after reperfusion for 12 h. Immunohistochemical staining showed CD62P significantly deposited on the endothelium after reperfusion. Administration of abciximab (1 mg/kg) effectively improved flap survival rate (P = 0.003), significantly decreased ADP-induced platelet aggregation (P < 0.001), and suppressed CD62P expression on blood platelets (P = 0.002) and its deposition on the flap vessels. CONCLUSION: Abciximab promotion of skin flap survival is due to blocked platelet activation/aggregation and decreased activated-platelet deposition on the vascular endothelium. Thus, administration of a platelet glycoprotein IIb/IIIa receptor antagonist such as abciximab may save the skin flap from reperfusion injury after a long period of ischemia.     

Effect of platelet glycoprotein IIb/IIIa receptor antagonist on survival of epigastric island flaps in rats with total venous occlusions.

We evaluated the effect of tirofiban hydrochloride on the survival of epigastric island flaps in rats that had had all the veins occluded. Male Wistar Albino rats were randomly assigned to control (treated with sterile saline) and experimental (treated with tirofiban hydrochloride 1 mg/kg intravenously) groups. An epigastric island skin flap 3x6 cm was raised in each rat. All veins that drained the flap were ligated to give total venous occlusion. Blood flow was recorded by laser Doppler preoperatively (baseline), immediately after the flap had been sutured back to its original position (acute) and on postoperative days 1 and 3. The degree of necrosis was evaluated on day 3. Mean percentage necrosis and minimum laser Doppler values were compared in the two groups. Total necrosis was evident on day 1 in the control group and on day 3 in the experimental group. Macroscopic evidence was confirmed by histopathological examination. There were appreciable differences in blood flow and in the necrotic area of the flap in the experimental group compared with the control group on both days 1 and 3. Tirofiban hydrochloride might be effective in this flap model.     

Emergency coronary artery bypass surgery in the era of glycoprotein IIb/IIIa receptor antagonist use

The use of intravenous glycoprotein (GP) IIb/IIIa platelet receptor antagonists in the management of patients with acute coronary syndrome or those undergoing percutaneous coronary intervention (PCI) has become increasingly common in recent years. There are three GP IIb/IIIa receptor antagonists currently available for clinical use. Patients on GP IIb/IIIa receptor antagonists who require emergency surgical revascularization may be at increased risk for excessive peri- and postoperative bleeding. The duration of action of eptifibatide and tirofiban are short because they bind reversibly to the GP IIb/IIIa receptor and have a short half-life. Therefore, within a relatively short time after discontinuation of these agents, surgery can be performed with little or no increased risk of bleeding and without the need for additional hemostatic measures. Abciximab has a short plasma half-life but a long duration of action due to its high-affinity binding of GP IIb/IIIa receptors. Early retrospective studies demonstrated a higher incidence of major bleeding and requirement for blood transfusion, especially in those undergoing surgery within 12 hours of the discontinuation of abciximab. However, platelet transfusion has been shown to successfully reduce the incidence of these complications. The current evidence therefore indicates that, with appropriate measures, urgent surgical revascularization can be safely performed in patients who have received a GP IIb/IIIa receptor antagonist with little added risk. The benefits of these agents in the treatment of patients with an acute coronary syndrome or undergoing PCI are not obviated by the need for emergency bypass surgery.     

Angiographic evaluation of middle cerebral artery reperfusion caused by platelet glycoprotein IIb/IIIa receptor complex antagonist murine 7E3 F(ab')2 in a model of focal cerebral ischemia in rats

OBJECT: Antagonists of the glycoprotein IIb/IIIa (GPIIb/IIIa) receptor complex are currently used for the treatment of acute coronary syndromes. The platelet GPIIb/IIIa mediates platelet aggregation, and blocking this receptor complex can reduce or prevent arterial thrombosis. To study the recanalization efficacy of a GPIIb/IIIa antagonist in treating cerebral ischemia, we investigated the therapeutic effects of murine 7E3 F(ab'), in a focal embolic cerebral ischemia model in rats. METHODS: Focal cerebral ischemia was produced by introducing an autologous thrombus into the right side of the middle cerebral artery (MCA). Thirty male Wistar rats were randomly divided into three groups of 10 rats each: control, 7E3 F(ab')2 administered 1 hour postischemia, and 7E3 F(ab')2 administered 3 hours postischemia. Animals in the therapeutic groups received intravenous infusion of 6 mg/kg 7E3 F(ab')2 at 1 or 3 hours following cerebral embolization. Brain infarct volume, neurobehavioral scores, duration of bleeding, and findings on angiograms of the MCA (before and after infusion) were assessed in all animals. Angiographic evaluation revealed full MCA recanalization in three of 10 animals in each 7E3 F(ab')2 treatment group. Animals in these groups exhibited a significant reduction in infarct volume when compared with animals in the control group: 1) infarct volume 1 hour postischemia, 22 +/- 13.9% (p = 0.005); 2) infarct volume 3 hours postischemia, 22.1 +/- 14.8% (p = 0.008); and 3) infarct volume in control animals, 42.4 +/- 16%. Postischemia treatment with 7E3 F(ab')2 also improved the animal's neurobehavioral performance. The duration of bleeding significantly increased by more than two times, but there was no associated increase in intracerebral hemorrhage in any group. CONCLUSIONS: On the basis of their findings, the authors conclude that murine 7E3 F(ab'), is a potent and safe antiplatelet agent in this experimental focal embolic cerebral ischemia model. Neuronal lesions were significantly reduced when the treatment was delayed up to 3 hours.     

Clinical significance of PlA polymorphism of platelet GP IIb/IIIa receptors during long-term VAD support

BACKGROUND: Although bleeding and thromboembolism remain major complications after implantation of ventricular assist devices (VADs), no standard anticoagulation protocols are available. Genetic polymorphism of platelet glycoprotein IIb/IIIa may contribute to the development of complications. The present study demonstrates a relationship between the PlA genotype and postoperative complications in patients implanted with pulsatile and axial flow VADs. METHODS: The PlA genotype was determined in 41 consecutive patients treated with a VAD who received anticoagulation with phenprocoumon and aspirin. Pulsatile Novacor (Novacor Corp, Oakland, CA) and Berlin Heart VADs (Berlin Heart, Berlin, Germany) were implanted in 28 patients and the axial flow MicroMed DeBakey VAD (MicroMed Technology, Inc, Houston, TX) in 13. The relationship between the PlA genotype, the anticoagulation regime, and bleeding and thromboembolic events was analyzed. RESULTS: There were no differences between patients with the A1A1 and A1A2 genotype regarding demographic characteristics, weight, or infection episodes. The international normalized ratio (INR), platelet activation tests, and doses of aspirin and dipyridamol before events were similar in both groups. Patients with the A1A1 genotype developed more bleeding complications (39% vs 0%, p = 0.021), while patients with the A1A2 genotype showed a tendency toward more thromboembolic events (13% vs 30%, p = 0.33). With regard to different types of VAD, patients with the axial flow DeBakey VAD and the A1A1 genotype developed significantly more bleeding complications (70% vs 0%, p = 0.033). CONCLUSIONS: In patients with a long-term VAD determination of PlA polymorphism and subsequent adjustment of the anticoagulation regime may lead to a reduction of bleeding and thromboembolic complications.     

Local glycoprotein IIb/IIIa receptor inhibitor delivery from the pump surface attenuates platelet adhesion in continuous flow ventricular assist devices

Shear-induced platelet activation (SIPA) has been identified to induce platelet adhesion and thrombus formation in continuous flow left ventricular assist devices (LVAD). Platelet glycoprotein (GP) IIb/IIIa receptor inhibitors are effective to prevent SIPA. However, systemic GP IIb/IIIa receptor inhibitor application is associated with severe bleeding complications. The aim of the study was to evaluate (i) the feasibility of absorption and elution of the GP IIb/IIIa receptor blocker TAK-029 from the Ti6Al4V surface of the pump; and (ii) the effect of local GP IIb/IIIa receptor blocker delivery regarding platelet adhesion on the surface of a continuous flow VAD model. Saturating concentrations of TAK-029 were adsorbed on the surface of a centrifugal pump. Whole human blood was perfused in circulatory mock loops using untreated (control), albumin-coated, or TAK-029-coated pumps. Peripheral resistance of the circulatory systems were adjusted accordingly to generate 5 L flow per min with impeller rotational speeds of 3500 (high-shear group) and 1500 rpm (low-shear group), respectively. Platelet adhesions on the respective impellers were quantified by ELISA and scanning electron microscopy (SEM). TAK-029 elution and half-life time were determined by ELISA. Compared with control, albumin-coated pumps showed 64 and 20% less platelet adhesions in the high- and low-shear group, respectively. TAK-029 coated pumps reduced platelet adhesion by additional 33 and 65%, respectively, compared with the albumin group. Elution of TAK 029 was initially very rapid and continued slowly. The results show that it is possible to adsorb and elute a small molecular weight GP IIb/IIIa receptor blocker from the pump surface. This drug elution reduced platelet adhesion on the pump significantly. Further studies are necessary to find a suitable drug bonding that will prolong the antiplatelet effect and preclude any bleeding complication caused by this procedure.     

Prohemorrhagic and bleeding time activities of recombinant tissue plasminogen activator, heparin, aspirin, and a glycoprotein IIb/IIIa antagonist

Intracerebral hemorrhage (ICH) is the most serious side effect of antithrombotic agents, especially in cases of cerebrovascular disease. In the present study, we compared the exacerbation of ICH and prolongation of bleeding time (BT) in guinea pigs with recombinant tissue plasminogen activator (rt-PA), heparin, aspirin, and FK419, a novel nonpeptide platelet glycoprotein (GP) IIb/IIIa receptor antagonist. ICH was induced by injection of bacterial collagenase into the caudate nucleus; BT was measured with a Simplate R device. Neither heparin nor aspirin prolonged BT. In contrast, rt-PA at the highest dose used in the study did prolong BT, and FK419 caused a dose-dependent prolongation of BT. Moreover, rt-PA and heparin increased the degree of ICH in a dose-dependent manner, leading to death in more than half of the animals treated with higher doses of these drugs. These findings show that the prohemorrhagic mechanisms underlying the prolongation of BT differ from those in collagenase-induced ICH, and that the risk of an agent with antithrombotic effects potentiating hemorrhage in the collagenase-induced model of ICH more closely parallels that in stroke patients than does the effect of the agent on BT. The findings also suggest that antiplatelet agents, including FK419, may be safer than thrombolytic or anticoagulant agents for use in patients at risk for ICH, such as those with stroke or cerebral aneurysm.     

Assessment of prosthetic vascular graft thrombogenicity using the technetium-99m labeled glycoprotein IIb/IIIa receptor antagonist DMP444 in a dog model

INTRODUCTION: Prosthetic graft patency greatly depends on graft thrombogenicity. The concept of graft thrombogenicity is poorly understood and difficult to measure or quantify. In a study we tested the experimental radiopharmaceutical DMP444 and developed a suitable dog model. This agent is a radiolabelled ((99m)Technetium) glycoprotein IIb/IIIa receptor antagonist with a high affinity for activated platelets. It binds to platelets that are intimately involved in thrombus formation. The agent does not affect thrombocyte function, when used in a dose necessary for imaging. DMP444 does not require platelet harvesting and processing. Early imaging of thrombocyte aggregation sites such as vascular prostheses is possible within 4 hours after injection. MATERIAL AND METHODS: Adult Beagle dogs weighing 12-15 kg were used for the experiments. In 16 dogs a prosthetic patch was sewn onto the abdominal aorta (Bovine pericard: n=4, Dacron: n=6, Human Umbilical Vein: n=6). Imaging cycles after injection of (99m)Technetium-labelled DMP444 were performed on days 1, 7, 14 and 28 after surgery. RESULTS: We noticed differences in thrombus formation on the tested graft materials. The bovine pericard patches (n=4) showed a relatively high rate of thrombocyte aggregation. In the Dacron patches (n=6) aggregation was not seen. In 1 of 6 cases of human umbilical vein patches a measurable focal aggregation was recorded. CONCLUSION: The method outlined in this study is a relatively simple and reproducable method to visualize thrombocyte aggregation.