Evidence for Two Distinct G-protein-coupled ADP Receptors Mediating Platelet Activation

Abstract

SummaryThe identity of the receptors mediating platelet activation by ADP remains elusive. To distinguish between platelet ADP receptor subtypes, the effects of antagonists on platelet responses and the cloned P2Y1receptor, a putative platelet ADP receptor, have been investigated. 2-methylthio-AMP (2MeSAMP), an inhibitor of ADP-dependent platelet aggregation, antagonized ADP-mediated inhibition of adenylyl cyclase, competed with binding of [3H]2-methylthio-ADP and inhibited the stimulation of [35S]GTP γS binding. 2MeSAMP did not inhibit platelet shape change and was only a weak antagonist of intracellular calcium mobilization in platelets or in cells expressing the cloned human P2Y1receptor. By contrast, the P2Y1receptor antagonist adeno-sine 3’,5’-diphosphate (A3P5P) inhibited ADP-induced platelet aggregation, completely abolished shape change, but did not antagonize ADP effects on cyclic AMP generation or [3H]2-methylthio-ADP binding. However, A3P5P antagonized intracellular calcium mobilization in platelets and cells expressing the cloned P2Y1receptor. Furthermore, using a specific monoclonal antibody and flow cytometry, P2Y1receptor protein was detected on human platelets. These results support the existence of two G protein-coupled ADP receptors mediating platelet aggregation, one of which is coupled to Giproteins and blocked by 2MeSAMP, whereas the second receptor is similar or identical to P2Y1and coupled to Gq.