Background: Plasma kininogens are selective inhibitors of alpha-thrombin 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 platelet 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-, U46619-, or SFLLRN-induced platelet activation or the ability of alpha-thrombin to cleave chromogenic substrates, clot fibrinogen, or block alpha-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-thrombin from removing the epitope of its cleavage site on the cloned thrombin receptor. Furthermore, peptide RPPGF or high-molecular-weight kininogen prevented alpha-thrombin from cleaving the thrombin receptor peptide, NATLDPRSFLLR, between arginine and serine.
Conclusions: These results indicate that bradykinin and its metabolites are selective antithrombins by preventing alpha-thrombin cleavage of the cloned thrombin receptor between arginine-41 and serine-42. These newly recognized antithrombin peptides, which are termed thrombostatins, contribute to the cardioprotective nature of kinins.