Ni-mediated inhibition of human platelet adenylate cyclase by thrombin

Abstract

The influence of the proteolytic enzyme, thrombin, was studied on adenylate cyclase activity in human platelet membranes. Thrombin had a biphasic inhibitory effect on the enzyme. Up to a concentration of about 1 U/ml, thrombin inhibited the enzyme in a strictly GTP-dependent manner by maximal 60-70%, with half-maximal inhibition occurring at about 0.005 U/ml thrombin. At higher concentrations, thrombin-induced inhibition of platelet adenylate cyclase was independent of GTP. The inhibitory effect of thrombin observed at low concentrations was further evaluated. The inhibition was half-maximal and maximal at about 0.1 and 3 microM GTP, respectively, occurred without major lag phase and was observed with the unstimulated and the forskolin or prostaglandin E1-stimulated platelet adenylate cyclase. Additionally, thrombin accelerated and potentiated the enzyme inhibition caused by the stable GTP analog, guanosine 5'-(gamma-thio)triphosphate. Furthermore, at identical concentrations causing adenylate cyclase inhibition, thrombin effectively stimulated GTP hydrolysis in platelet membranes. Finally, the thrombin inhibition of human platelet adenylate cyclase was impaired or abolished by Mn2+ and by treatment of the platelet membranes with N-ethylmaleimide or pertussis toxin. All of these data indicate that thrombin at low concentrations inhibits platelet adenylate cyclase by a process involving the inhibitory guanine nucleotide-binding regulatory component, Ni, in a manner similar to hormonal factors. However, in contrast to typical hormonal inhibition, thrombin inhibition of the platelet enzyme was not reversed by washing of the platelet membranes or by subsequent addition of the thrombin inactivator, hirudin, which prevented the inhibition when added together with thrombin. These data suggest that thrombin by its proteolytic capacity causes a persistent activation of its receptor site which leads to a persistent activation of Ni with subsequent persistent adenylate cyclase inhibition.