BRADYKININ AND ITS METABOLITE, ARG-PRO-PRO-GLY - PHE, ARE SELECTIVE INHIBITORS OF ALPHA-THROMBIN-INDUCED PLATELET ACTIVATION

Background Plasma kininogens are selective inhibitors of alpha-thrombi n activation of platelets and endothelial cells. In the present study, we localized the alpha-thrombin inhibitory sequence of kininogens and describe its mechanism of action. Methods and Results Bradykinin and an analogue, MKRPPGFSPFRSSRIG, inhibited alpha-thrombin-induced platel et aggregation and secretion with an IC50 of 0.25 and 1 mmol/L and of 0.23 and 0.5 mmol/L: respectively. The minimal inhibitory peptide was RPPGF. Bradykinin and its analogues did not inhibit ADP-, collagen-, U 46619-, or SFLLRN-induced platelet activation or the ability of alpha- thrombin to cleave chromogenic substrates, clot fibrinogen, or block a lpha-thrombin binding to platelets. Bradykinin, MKRPPGFSPFRSSRIG, and RPPGF abolished alpha-thrombin-induced (1 nmol/L) calcium mobilization . On flow cytometry, bradykinin and MKRPPGFSPFRSSRIG blocked alpha-thr ombin from removing the epitope of its cleavage site on the cloned thr ombin receptor. Furthermore, peptide RPPGF or high-molecular-weight ki ninogen prevented alpha-thrombin from cleaving the thrombin receptor p eptide, NATLDPRSFLLR, between arginine and serine. Conclusions These r esults indicate that bradykinin and its metabolites are selective anti thrombins by preventing alpha-thrombin cleavage of the cloned thrombin receptor between arginine-41 and serine-42. These newly recognized an tithrombin peptides, which are termed thrombostatins, contribute to th e cardioprotective nature of kinins.