INHIBITION OF COLLAGEN-INDUCED PLATELET-AGGREGATION AS THE RESULT OF CLEAVAGE OF ALPHA(2)BETA(1)-INTEGRIN BY THE SNAKE-VENOM METALLOPROTEINASE JARARHAGIN

Jararhagin is a high-molecular-mass (52 kDa) haemorrhagic metalloprote inase from Bothrops jararaca venom and a member of the metalloproteina se/disintegrin/cysterin-rich protein family. The disintegrin domain of jararhagin has been implicated in the inhibition of platelet response s to collagen by a mechanism that is not entirely known. The present i nvestigation demonstrates that both active and 1,10-phenanthroline-ina ctivated jararhagin inhibit platelet aggregation by collagen with an I C50 of 40 and 140 nM respectively. The apparently higher inhibitory ef fect of the active enzyme clearly indicates that, in addition to the d isintegrin region, the metalloproteinase domain of jararhagin also par ticipates in this inhibition. As collagen interacts with platelets via alpha(2) beta(1)-integrin, we investigated the effects of jararhagin on this integrin using selected function-blocking monoclonal antibodie s against both of its subunits. Flow cytometry of platelets treated wi th native jararhagin and immunoprecipitation of platelet surface glyco proteins from lysates after jararhagin treatment showed an apparently selective reduction of alpha(2) beta(1)- integrin immunoreactivity wit h both anti-alpha(2) and anti-beta(1) monoclonal antibodies. The loss of immunoreactivity was not due to integrin internalization, since it also took place in cytochalasin D-treated platelets. Here we show that jararhagin cleaved isolated alpha(2) beta(1)-integrin resulting in th e generation of a 115 kDa beta(1) fragment. We therefore propose that the inhibition by jararhagin of platelet response to collagen is media ted through the binding of jararhagin to platelet alpha(2)-subunit via the disintegrin domain, followed by proteolysis of the beta(1)-subuni t with loss of the integrin structure (conformation) necessary for the binding of macromolecular ligands.