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 ϳ 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 prostacyclinresistant 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 collagenevoked Ca 2؉ responses, indicating a highly efficient activation mechanism by local ATP. In conclusion, platelets possess inhibitor-resistant Ca 2؉ IntroductionPlatelet activation is the initial required event during the physiologic process of hemostasis. With increasing age, the onset of atherosclerosis lowers the threshold for platelet reactivity and consequently the risk of thrombosis. In the intact circulation, there are inhibitors of platelet reactivity that serve to maintain blood fluidity. These inhibitors include endothelial-derived prostacyclin (PGI 2 ) and nitric oxide (NO) that elevate intraplatelet cyclic AMP (cAMP) and cyclic GMP (cGMP), respectively. 1 cAMP-and cGMP-dependent kinases target several platelet signaling pathways, particularly mobilization of the key second messenger Ca 2ϩ . 1,2 Degradation of nucleotides by enzymes in the plasma and on the surface of endothelium and leukocytes represents a further important inhibitory influence. The dominant ectonucleotidase CD39 converts the platelet agonists ATP and ADP to inert AMP, and AMP is subsequently degraded by CD73 to adenosine and therefore further inhibits platelets through stimulation of G scoupled A 2a and A 2b receptors. [3][4][5] After breach of vascular integrity, disruption of the endothelial layer leads to local loss of these inhibitory pathways that will facilitate platelet activation by adhesive macromolecules such as collagen and diffusible agonists such as ADP, thromboxane A 2 (TxA 2 ), and thrombin. 6 In identifying potential strategies to manage thrombosis, it is therefore important to consider the relative effectiveness of natural platelet inhibitory influences on various platelet activation pathways.Platelets possess multiple mechanisms whereby agonists can generate an incre...