THROMBIN BINDING TO PLATELETS AND THEIR ACTIVATION IN PLASMA

The interactions of alpha-thrombin with platelets are critical in haem ostasis and arterial thrombosis. This study established methods for ch aracterizing the binding of alpha-thrombin to platelets and some of it s consequences in platelet-rich plasma. The binding of alpha-thrombin to platelets and the subsequent platelet activation were quantified by now cytometry, using affinity purified polyclonal antibodies to human alpha-thrombin and a monoclonal antibody to GMP-140, respectively. Do se-dependent binding of alpha-thrombin to platelets and their activati on occurred in parallel, both reaching the maxima for each enzyme conc entration within 10 s after greater than or equal to 1.0 nM alpha-thro mbin was added to recalcified PRP containing 1 mu M recombinant tick a nticoagulant peptide. The tick anticoagulant peptide abrogated prothro mbin activation in the platelet-rich plasma. alpha-Thrombin binding to platelets, and their activation, were abrogated by a monoclonal antib ody to the hirudin tail-like domain of the seven transmembrane thrombi n receptor on platelets. Therefore this receptor represents an importa nt site for alpha-thrombin binding to platelets suspended in plasma. D -Phe-Pro-ArgCH(2)-alpha-thrombin only bound to platelets when its conc entration was greater than or equal to 100 nM, and it did so without i nhibiting platelet activation by alpha-thrombin. Whereas concentration s of hirudin equimolar to those of alpha-thrombin failed to abrogate a lpha-thrombin-mediated activation of platelets, a 10-fold molar excess es of hirudin over alpha-thrombin abrogated alpha-thrombin binding to platelets. The demonstration that greater than or equal to 1.0 nM alph a-thrombin can bind to platelets and initiate their activation raises the possibility that the levels of thrombin generated in venous and ar terial thrombosis contribute to platelet activation in vivo.