Primary and secondary agonists can use P2X receptors as a major pathway to increase intracellular Ca in the human platelet

In the platelet, it is well established that many G-protein- and tyrosine kinase-coupled receptors stimulate phospholipase-C-dependent Ca[superscript: 2+] mobilization; however, the extent to which secondary activation of adenosine 5′-triphosphate (ATP)-gated P2X[subscript: 1] receptors contributes to intracellular Ca[superscript: 2+] responses remains unclear. We now show that selective inhibition of P2X1 receptors substantially reduces the [Ca[superscript: 2+]]i increase evoked by several important agonists in human platelets; for collagen, thromboxane A2, 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[subscript: 1] to the secondary Ca[superscript: 2+] response was far greater than that of either P2Y receptors activated by co-released ADP, or via synergistic P2X[subscript: 1]:P2Y interactions. The relative contribution of P2X[subscript: 1] to the peak Ca[superscript: 2+] 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[subscript: 1] contributed equally at both low and high levels of stimulation of thromboxane A2 receptors. In contrast, only strong stimulation of P2Y receptors resulted in significant P2X[subscript: 1] receptor activation. ATP release was detected by soluble luciferin:luciferase in response to all agonists that stimulated secondary P2X[subscript: 1] receptor activation. However, P2X[subscript: 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 [Ca[superscript: 2+]]i increases in platelet activation, these studies indicate that ATP should be considered alongside ADP and thromboxane A[subscript: 2] as a significant secondary platelet agonist.