A pharmacological characterization of ADP receptors on human platelets

1. The platelet responses to adenosine 5'-diphosphate (ADP) and ADP-induced signal transduction involved in mediating the platelet responses are reviewed. The current knowledge of P2 purinoceptor classification is summarised and the current model of platelet activation involving multiple ADP receptors is discussed. 2. The structure-activity relationships for several analogues of adenine nucleotides in causing aggregation and shape change were compared in washed platelets using an aggregometer. In general, the structure-activity relationships for both responses were similar, but for some analogues differences were observed. Adenosine 5'-O- (1-thiodiphosphate) (ADPbetaS) and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) were much more potent agonists relative to ADP for shape change than for aggregation and indeed ADPbetaS antagonized ADP-induced aggregation. 2-Methylthioadenosine 5'-triphosphate (2-MeSATP) also had different effects in aggregation and shape change, being a much higher affinity antagonist of aggregation than of shape change. These results support the suggestion that these two responses are mediated by multiple ADP receptors on human platelets, and are consistent with shape change being mediated via one receptor (the P2Y1 receptor) with aggregation requiring the activation of two receptors (the P2Y1 and the P2YAC receptors). 3. The effects of adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS) which has been proposed to be a selective antagonist at P2Y1 receptors, were tested on the responses of human platelets to ADP. A3P5PS selectively antagonised in an apparently competitive manner ADP-induced platelet aggregation, as well as the ability of ADP to cause shape change and increases in [Ca+2]i in platelets, but had no effect on the inhibition of stimulated adenylate cyclase by ADP, confirming suggestions that this response is mediated by means of a different receptor subtype. 4. These studies using washed platelets indicated different effects of ADPbetaS, ADPbetaS and 2-MeSATP on aggregation between washed platelets in buffer and unwashed platelets in plasma. To see whether the different responses between washed platelets and platelets in plasma is caused by some factors in plasma, or by the process of washing resulting in receptor desensitization, washed platelets were resuspended in plasma and the effects of ADPbetaS, ADPbetaS and 2-MeSATP were simultaneously compared both in unwashed platelets and in washed platelets in plasma. The extent of aggregation induced by ADPbetaS or ADPbetaS, which act as agonists preferentially on the P2Y1 receptor, was not affected by the presence of plasma, but was decreased by the washing procedure, suggesting that the P2Y1 receptor is desensitized by washing. Unlike ADPbetaS and ADPbetaS, the extent of response to ADP in the presence of 2-MeSATP, which acts preferentially on the P2YAC receptor, appeared to be affected by the presence of plasma rather than by the process of washing as if the plasma restores the ability of P2Y1 receptor to induce some aggregation alone. 5. The effects of platelets on cultured human vascular smooth muscle cells were investigated using explant cultures of human saphenous vein vascular smooth muscle as the experimental material. Released products from activated and nonactivated platelets were tested for their effects on [3H]-thymidine incorporation into the human smooth muscle cells to see their effects on the proliferation of smooth muscle cells, and phosphoinositide hydrolysis was measured to see if the effects on smooth muscle cell proliferation involve the phosphoinositide signaling pathway. No marked effects on smooth muscle cell proliferation were shown by ADP or by adenosine 5'-triphosphate (ATP) alone, but ADP and ATP increased smooth muscle cell proliferation in synergy with platelet-derived growth factor (PDGF). Aggregated and non-aggregated platelets caused increases in the proliferation of vascular smooth muscle cells with aggregated platelets showing a greater effect than non-aggregated platelets although the stimulatory effects were markedly decreased by an increased density of platelets. The effect of platelet-derived products on smooth muscle cells was not observed for phosphoinositide hydrolysis, suggesting that the effect of aggregated platelets on smooth muscle cell proliferation is not due to phosphoinositide turnover.