Flow cytometric evaluation of the effect of various thrombin inhibitors on platelet activation in whole blood

In an in vitro study the effect of various thrombin inhibitors (argatroban, efegatran, DuP 714, recombinant hirudin and PEG-hirudin) on platelet activation in whole blood was investigated. Blood was drawn from normal human volunteers using the double syringe technique without use of a tourniquet to avoid autoaggregation of platelets. Blood was anticoagulated with either argatroban, efegatran, DuP 714, hirudin or PEG-hirudin at final concentrations of 10 mu g/ml. Blood samples were then incubated at 37 degrees C either with saline, r-tissue factor, arachidonic acid, adenosine diphosphate or collagen. At definite times (1, 2.5, 5, 10 min) aliquots were taken and after various steps of fixative procedure the percentage of platelet activation was measured using fluorescent monoclonal antibodies to platelet surface receptors GPIIIa (CD-61) and P-selectin (CD-62). Flow cytometric analysis showed a platelet activation after all agonists used. All thrombin inhibitors studied caused a nearly complete inhibition of r-tissue factor-mediated platelet activation. In contrast, after activation with the other agonists an increased percent CD-62 expression was found with a maximum after 2.5 to 5 min. The results show that in whole blood thrombin inhibitors are effective in preventing platelet activation induced by r-tissue factor. The formation of active serine proteases including thrombin may be effectively inhibited by these agents. The observations further suggest that while thrombin inhibitors may control serine proteases, these agents do not inhibit the activation of platelets mediated by other agonists.