Desensitization of the platelet aggregation response to ADP: Differential down-regulation of the P2Y(1) and P2cyc receptors

Platelets activated by ADP become refractory to restimulation, but the mech anism of this process is not well understood. A normal platelet response to ADP requires coactivation of the P2Y(1) receptor responsible for shape cha nge and the P2cyc receptor, responsible for completion and amplification of the response. The aim of the present study was to characterize the desensi tization of platelets to ADP and to determine whether or not these two rece ptors are desensitized simultaneously through identical pathways when plate lets become refractory to ADP. It was found that full inhibition of platele t aggregation in response to restimulation by ADP required the presence of ADP in the medium or use of a high concentration (1 mM) of its non-hydrolys able analogue ADP beta S. Platelets incubated for 1 h at 37 degrees C with 1 mM ADP beta S and resuspended in Tyrode's buffer containing apyrase displ ayed a stable refractory state characterized by the inability to aggregate or change shape in response to ADP. ADP beta S treated platelets loaded wit h fura-2/AM showed complete blockade of the calcium signal in response to A DP, whereas the capacity of ADP to inhibit PGE, stimulated cAMP accumulatio n in these platelets was only diminished. Consequently, serotonin was able to promote ADP induced aggregation through activation of the Gq coupled 5HT (2A) receptor while adrenaline had no such effect. These results suggested that the refractory state of ADP beta S treated platelets was entirely due to desensitization of the P2Y(1) receptor, the P2cyc receptor remaining fun ctional. Binding studies were performed to determine whether the P2Y(1) and /or P(2)cyc binding sites were modified in refractory platelets. Using sele ctive P2Y(1) and P2cyc antagonists (A3P5P and AR-C66096 respectively), we c ould demonstrate that the decrease in [P-33]2MeSADP binding sites on refrac tory platelets corresponded to disappearance of the P2Y(1) sites with no ch ange in the number of P2cyc sites, suggesting internalization of the P2Y(1) receptor. This was confirmed by flow cytometric analysis of Jurkat cells e xpressing an epitope-tagged P2Y(1) receptor, where ADP beta S treatment res ulted in complete loss of the receptor from the cell surface. We conclude t hat the P2Y(1) and P2cyc receptors are differently regulated during platele t activation.