Inhibition of acute vascular thrombosis in chimpanzees by an anti-human tissue factor antibody targeting the factor X binding site.

Tissue factor (TF) antagonists targeting the factor VII (FVII) binding domain have been shown to interrupt acute vascular thrombus formation without impairing haemostasis in non-human primates. In this study, we evaluate whether a human/mouse chimeric monoclonal antibody (ALT-836, formerly known as Sunol-cH36) blocking the factor X/factor IX (FX/FIX) binding site of tissue factor could achieve similar clinical benefits in an arterial thrombosis model induced by surgical endarterectomy in chimpanzees. In this model, sequential surgical endarterectomies on right and left superficial femoral arteries were performed 30 days apart in five chimpanzees. A bolus (1 mg/kg) of ALT-836 was injected intravenously immediately preceding the restoration of flow in the endarterectomised femoral artery. Pre-surgical labelling of autologous platelets using (111)In-Oxine and post-surgical gamma camera imaging of (111)In-platelet deposition at endarterectomy sites was performed. The manipulated arterial segments were harvested for patency analysis 30 days following surgery. The results indicate that ALT-836 was highly effective at reducing acute vascular thrombosis, with no significant variations in surgical blood loss and template-bleeding time in the treated group compared to the control animals. These data suggest that ALT-836 is an effective and safe antithrombotic agent in preventing TF-initiated vascular thrombogenesis without compromising haemostasis.

Soluble thrombomodulin is antithrombotic in the presence of neutralising antibodies to protein C and reduces circulating activated protein C levels in primates.

We studied whether there was a relationship between the anticoagulant effects of recombinant human soluble thrombomodulin (rhsTM) and activation of protein C in a primate model of acute vascular graft thrombosis in 11 baboons (Papio species). Baboons were pretreated with 0.1, 1 and 5 mg/kg of rhsTM, with or without co-injection of a neutralising monoclonal antibody to protein C (HPC4) in the 1 mg/kg rhsTM group. Subsequently, thrombogenic polyester grafts were deployed for 3 h into chronic exteriorised arteriovenous shunts. Thrombus growth in the graft, plasma-activated protein C (APC) levels, coagulation and thrombosis markers were determined. In untreated baboons, baseline circulating APC levels more than doubled and graft thrombi propagated until reaching equilibrium in about 1 h. Treatment with rhsTM reduced thrombus propagation rates, prolonged the clotting and bleeding times, decreased thrombin-antithrombin complex, beta-thromboglobulin and fibrinopeptide A levels, and, surprisingly, also decreased systemic APC levels, in a dose-dependent manner. In the presence of HPC4 antibody to inhibit APC generation, the acute antithrombotic activity of rhsTM on graft thromboses was not attenuated for up to 80 min, but sustained thrombus accumulation was observed over a 180-min period. These findings suggest that, in contrast to the prevailing hypotheses, the primary antithrombotic activity of rhsTM is independent of protein C, at least in this primate model. Direct inhibition of thrombin's prothrombotic activity upon complex formation with rhsTM might explain the molecular mechanism of the observed antithrombotic effect.

In vivo platelet redistribution and acute transient thrombocytopenia after eptifibatide injection in baboons.

BACKGROUND: The occurrence of thrombocytopenia has been reported during clinical eptifibatide (Integrilin) therapy, but the exact mechanism is not yet established to explain the varied duration and severity of thrombocytopenia associated with glycoprotein (GP) IIb/IIIa inhibitors. We assessed the redistribution of platelets in juvenile baboons during acute transient thrombocytopenia that was observed after eptifibatide injection. METHODS: Eptifibatide was administered intravenously to eight baboons by infusion at 20 microg/kg/min or a bolus injection of 10 mg. Platelet distribution was measured with a gamma scintillation camera using 111In-labeled autologous platelets. Platelet function and GP IIb/IIIa receptor inhibition were evaluated using the Plateletworks system. The effects of pretreatment with abciximab (0.4 mg/kg) or human immunoglobulin concentrate (0.75 g/kg) were also investigated. RESULTS: Eptifibatide, administered as an infusion or a bolus, caused transient thrombocytopenia with uptake of platelets predominantly by the liver. The recovery of platelet aggregation was associated with the re-entry of platelets from the liver into the systemic circulation. Pretreatment with either abciximab (0.4 mg/kg) or human intravenous immunoglobulin (IVIG, 0.75 g/kg) attenuated eptifibatide-induced thrombocytopenia and the hepatic uptake of radiolabeled platelets. CONCLUSION: Acute thrombocytopenia after eptifibatide injection was caused by the transient redistribution of platelets to the liver. Attenuation of the decrease in platelet count and hepatic sequestration by abciximab and IVIG suggests that thrombocytopenia may have been caused by ligand-induced binding site antigen induction and recognition by the reticuloendothelial system.

Clopidogrel (Plavix) and cardiac surgical patients: implications for platelet function monitoring and postoperative bleeding.

The use of clopidogrel (Plavix), an inhibitor of adenosine diphosphate (ADP)-induced platelet aggregation, has been proven to reduce ischemic events in cardiovascular patients, but little information is available for optimal monitoring of platelet function in patients receiving the drug preoperatively. In the first part of the study we compared different testing modalities (thrombelastography (TEG), platelet aggregometry, and whole blood aggregation) to assess platelet ADP receptor inhibition. Because clopidogrel is a pro-drug, we used an in vitro model of ADP inhibition with 5'-p-fluorosulfonylbenzoyladenosine (FSBA). FSBA at final concentration of 80 microM completely inhibited platelet aggregation but had no effect on TEG maximum amplitude (MA). In the second part of the study, antiplatelet effects of clopidogrel were clinically assessed and correlated to postoperative bleeding in 18 coronary bypass surgery patients. Preoperative TEG results were normal or hypercoagulable in clopidogrel-treated patients, although platelet aggregation responses to ADP were inhibited. Clopidogrel-treated patients who underwent cardiopulmonary bypass had a high incidence (84.6%) of platelet transfusion therapy due to increased chest tube drainage. In conclusion, we have demonstrated that normal preoperative TEG-MA does not preclude clopidogrel-induced ADP receptor blockade; however, TEG can be a reliable monitor for CPB-induced platelet dysfunction related to GPIIb/IIIa. For monitoring clopidogrel, it is necessary to perform more specific platelet function tests (aggregometry or platelet count ratio) using ADP as an activator.