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Inhibition of Ca2+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 Ca2+response after stimulation of glycoprotein VI (GPVI) by collagen, or of Toll-like 2/1 receptors by Pam3Cys-Ser-(Lys)4(Pam3CSK4), is resistant to prostacyclin. At low agonist concentrations, the prostacyclin-resistant Ca2+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 Ca2+elevation. At higher agonist concentrations, an additional pathway was observed because of intracellular Ca2+release that also depended on activation of phospholipase C and, for TLR 2/1, PI3-kinase. Secondary activation of P2X1-dependent Ca2+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 Ca2+responses, indicating a highly efficient activation mechanism by local ATP. In conclusion, platelets possess inhibitor-resistant Ca2+mobilization pathways, including P2X1 receptors, that may be particularly important during early thrombotic or immune-dependent platelet activation.

The difficulty of conducting electrophysiologic recordings from the platelet has restricted investigations into the role of ion channels in thrombosis and hemostasis. We now demonstrate that the well-established synergy between P2Y1and P2Y12receptors during adenosine diphosphate (ADP)–dependent activation of the platelet αIIbβ3integrin also exists in murine marrow megakaryocytes, further supporting the progenitor cell as a bona fide model of platelet P2 receptor signaling. In patch clamp recordings, ADP (30 μM) stimulated a transient inward current at –70 mV, which was carried by Na+and Ca2+and was amplified by phenylarsine oxide, a potentiator of certain transient receptor potential (TRP) ion channels by phosphatidylinositol 4,5-bisphosphate depletion. This initial current decayed to a sustained phase, upon which repetitive transient inward cation currents with pre-dominantly P2X1-like kinetics were super-imposed. Abolishing P2X1-receptor activity prevented most of the repetitive currents, consistent with their activation by secreted adenosine triphosphate (ATP). Recordings in P2Y1-receptor–deficient megakaryocytes demonstrated an essential requirement of this receptor for activation of all ADP-evoked inward currents. However, P2Y12receptors, through the activation of PI3-kinase, played a synergistic role in both P2Y1and P2X1-receptor–dependent currents. Thus, direct stimulation of P2Y1and P2Y12receptors, together with autocrine P2X1activation, is responsible for the activation of nonselective cation currents by the platelet agonistADP.

Purine nucleotides acting through P2 receptors play key roles in platelet signaling. The P2X1receptor is an adenosine triphosphate (ATP)-gated ion channel that mediates a rapid calcium influx signal, but can also synergize with subsequent adenosine diphosphate (ADP)-evoked P2Y1receptor-mediated responses and thus may contribute to platelet activation during hemostasis. Recent studies have shown that P2X1receptors contribute to the formation of platelet thrombi, particularly under conditions of high shear stress. Based on intracellular Ca2+measurements a previous report has suggested that a splice variant of the P2X1receptor, P2X1del, is expressed in platelets and, in contrast to the full-length P2X1WT receptor, is activated by ADP. In the present study we show that the P2X1delreceptor fails to form functional ion channels and is below the limit of detection in human platelets. Furthermore, ADP does not contribute to the rapid ionotropic P2X receptor-mediated response in platelets. These results support the notion that ATP is the principal physiologic agonist at P2X1receptors and that it plays a role in the activation of platelets. (Blood. 2003;102:3646-3651)