Platelet Ca(2+) responses coupled to glycoprotein VI and Toll-like receptors persist in the presence of endothelial-derived inhibitors: roles for secondary activation of P2X1 receptors and release from intracellular Ca(2+) stores.

Inhibition of Ca(2+) mobilization by cyclic nucleotides is central to the mechanism whereby endothelial-derived prostacyclin and nitric oxide limit platelet activation in the intact circulation. However, we show that ∼ 50% of the Ca(2+) response after stimulation of glycoprotein VI (GPVI) by collagen, or of Toll-like 2/1 receptors by Pam(3)Cys-Ser-(Lys)(4) (Pam(3)CSK(4)), is resistant to prostacyclin. At low agonist concentrations, the prostacyclin-resistant Ca(2+) response was predominantly because of P2X1 receptors activated by ATP release via a phospholipase-C-coupled secretory pathway requiring both protein kinase C and cytosolic Ca(2+) elevation. At higher agonist concentrations, an additional pathway was observed because of intracellular Ca(2+) release that also depended on activation of phospholipase C and, for TLR 2/1, PI3-kinase. Secondary activation of P2X1-dependent Ca(2+) influx also persisted in the presence of nitric oxide, delivered from spermine NONOate, or increased ectonucleotidase levels (apyrase). Surprisingly, apyrase was more effective than prostacyclin and NO at limiting secondary P2X1 activation. Dilution of platelets reduced the average extracellular ATP level without affecting the percentage contribution of P2X1 receptors to collagen-evoked Ca(2+) responses, indicating a highly efficient activation mechanism by local ATP. In conclusion, platelets possess inhibitor-resistant Ca(2+) mobilization pathways, including P2X1 receptors, that may be particularly important during early thrombotic or immune-dependent platelet activation.

Differential sensitivity of human platelet P2X1 and P2Y1 receptors to disruption of lipid rafts.

ATP-stimulated P2X1 and ADP-stimulated P2Y1 receptors play important roles in platelet activation. An increase in intracellular Ca2+ represents a key signalling event coupled to both of these receptors, mediated via direct gating of Ca2+-permeable channels in the case of P2X1 and phospholipase-C-dependent Ca2+ mobilisation for P2Y1. We show that disruption of cholesterol-rich membrane lipid rafts reduces P2X1 receptor-mediated calcium increases by approximately 80%, while P2Y1 receptor-dependent Ca2+ release is unaffected. In contrast to artery, vas deferens, bladder smooth muscle, and recombinant expression in cell lines, where P2X1 receptors show almost exclusive association with lipid rafts, only approximately 20% of platelet P2X1 receptors are co-expressed with the lipid raft marker flotillin-2. We conclude that lipid rafts play a significant role in the regulation of P2X1 but not P2Y1 receptors in human platelets and that a reserve of non-functional P2X1 receptors may exist.

A major role for P2X1 receptors in the early collagen-evoked intracellular Ca2+ responses of human platelets.

In the platelet, ATP-gated P2X1 receptors have been reported to amplify functional responses to collagen, however the relative importance of early CCa2+ mobilisation events is unknown. We now report that selective desensitisation of P2X1 receptor activity leads to a major reduction in the initial intracellular Ca2+ responses to a wide range of collagen concentrations (0.25-2 microg ml(-1)). Peak [Ca2+](i) increases were reduced to 8.5 and 55% of control, and the maximum rate of rise was reduced to 12 and 33% of control, at low and high collagen concentrations, respectively. This P2X1-dependent acceleration and enhancement of collagen-stimulated Ca2+ responses was not observed in the absence of extracellular Ca2+. These results demonstrate a major role for ATP-gated Ca2+ influx in the early collagen-evoked Ca2+ signals and can at least partly explain the important contribution of P2X1 receptors to arterial thrombosis.