Primary and secondary agonists can use P2X1 receptors as a major pathway to increase intracellular Ca2+ in the human platelet

In the platelet, it is well established that many G-protein- and tyrosine kinase-coupled receptors stimulate phospholipase-C-dependent Ca2+ mobilization; however, the extent to which secondary activation of adenosine 5'-triphosphate (ATP)-gated P2X(1) receptors contributes to intracellular Ca2+ responses remains unclear. We now show that selective inhibition of P2X(1) receptors substantially reduces the [Ca2+](i) increase evoked by several important agonists in human platelets; for collagen, thromboxane A(2), thrombin, and adenosine 5'-diphoshate (ADP) the maximal effect was a reduction to 18%, 34%, 52%, and 69% of control, respectively. The direct contribution of P2X(1) to the secondary Ca2+ response was far greater than that of either P2Y receptors activated by co-released ADP, or via synergistic P2X(1):P2Y interactions. The relative contribution of P2X(1) to the peak Ca2+ increase varied with the strength of the initial stimulus, being greater at low compared to high levels of stimulation for both glycoprotein VI and PAR-1, whereas P2X(1) contributed equally at both low and high levels of stimulation of thromboxane A(2) receptors. In contrast, only strong stimulation of P2Y receptors resulted in significant P2X(1) receptor activation. ATP release was detected by soluble luciferin:luciferase in response to all agonists that stimulated secondary P2X(1) receptor activation. However, P2X(1) receptors were stimulated earlier and to a greater extent than predicted from the average ATP release, which can be accounted for by a predominantly autocrine mechanism of activation. Given the central role of [Ca2+](i) increases in platelet activation, these studies indicate that ATP should be considered alongside ADP and thromboxane A(2) as a significant secondary platelet agonist.