Thrombin binding to platelets and their activation in plasma

Summary. The interactions of α-thrombin with platelets are critical in haemostasis and arterial thrombosis. This study established methods for characterizing the binding of α-thrombin to platelets and some of its consequences in platelet-rich plasma. The binding of α-thrombin to platelets and the subsequent platelet activation were quantified by flow cytometry, using affinity purified polyclonal antibodies to human α-thrombin and a monoclonal antibody to GMP-140, respectively. Dose-dependent binding of α-thrombin to platelets and their activation occurred in parallel, both reaching the maxima for each enzyme concentration within 10 s after → 1.0 n m α-thrombin was added to recalcified PRP containing 1 μ m recombinant tick anticoagulant peptide. The tick anticoagulant peptide abrogated prothrombin activation in the platelet-rich plasma. α-Thrombin binding to platelets, and their activation, were abrogated by a monoclonal antibody to the hirudin tail-like domain of the seven transmembrane thrombin receptor on platelets. Therefore this receptor represents an important site for α-thrombin binding to platelets suspended in plasma. d -Phe-Pro-ArgCH₂-α-thrombin only bound to platelets when its concentration was → 100 n m , and it did so without inhibiting platelet activation by α-thrombin. Whereas concentrations of hirudin equimolar to those of α-thrombin failed to abrogate α-thrombin-mediated activation of platelets, a 10-fold molar excesses of hirudin over α-thrombin abrogated α-thrombin binding to platelets. The demonstration that → 1.0 n m α-thrombin can bind to platelets and initiate their activation raises the possibility that the levels of thrombin generated in venous and arterial thrombosis contribute to platelet activation in vivo .