Lack of evidence for functional ADP-activated human P2X1 receptors supports a role for ATP during hemostasis and thrombosis

Abstract

Purine nucleotides acting through P2 receptors play key roles in platelet signaling. The P2X 1 receptor 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 P2Y 1 receptormediated responses and thus may contribute to platelet activation during hemostasis. Recent studies have shown that P2X 1 receptors contribute to the formation of platelet thrombi, particularly under conditions of high shear stress. Based on intracellular Ca 2؉ measurements a previous report has suggested that a splice variant of the P2X 1 receptor, P2X 1del , is expressed in platelets and, in contrast to the full-length P2X 1 WT receptor, is activated by ADP. In the present study we show that the P2X 1del receptor 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 P2X 1 receptors and that it plays a role in the activation of platelets. IntroductionPurine nucleotides acting through P2 receptors play key roles in platelet signaling and hemostasis. P2 receptors can be subdivided into P2X ionotropic and P2Y metabotropic receptors, and transgenic mice models have now firmly established roles of P2X 1 , P2Y 1 , and P2Y 12 receptors in platelet function. 1-6 P2X 1 receptors for adenosine triphosphate (ATP) mediate rapid transient increases in calcium concentration that can synergize with the larger, more sustained response through the adenosine diphosphate (ADP)-dependent P2Y 1 receptor, leading to speeding and amplification of the calcium increase. 4 In addition, P2X 1 receptor-deficient mice have recently been shown to have a decreased aggregation response and to exhibit protection against thromboembolism. 7 A major problem that has complicated the understanding and characterization of P2 receptors is the use of agonists to define P2 receptors because impurity or interconversion of the agonist has led to the pharmacologic misclassification of receptors. For example uridine diphosphate (UDP) appears as a weak agonist at the uridine triphosphate (UTP)-sensitive P2Y 4 receptor; however, when contaminating UTP is removed with hexokinase, agonist activity is essentially abolished. 8 The same problem has also influenced the study of P2 receptors in platelets and characterization of the specificity of action of different purines at individual receptors. For the P2X 1 receptor we have shown that the apparent activity of ADP at the receptor can be accounted for by ATP contamination of commercially available ADP. 9,10 This has important implications for the physiologic activation of platelet purinoceptors because ATP acts as a competitive antagonist rather than an agonist at platelet P2Y 1 receptors as a result of their low level of expression. 11,12 Thus, in platelets ATP and ADP are selective stimuli at P2X 1 an...

Historical perspective on ADP-induced platelet activation

“…Vial et al [109] have suggested that in vivo, these ATP-induced reactions could synergize with the platelet response to ADP through the P2Y 1 receptor and amplify the increase in intracellular calcium. Oury et al [110,111] obtained evidence for the importance in hemostasis of a synergy between ADP and ATP released from platelets stimulated by adherence to collagen.…”

Human Plasma ATP Concentration

“…ATP can affect thromboregulation and stimulate the immune cells responsible for asthma attacks (6,7 ). ATP has also been proposed as a mediator of vasodilation during ischemia, hypoxia, and exercise.…”

Activators, receptors and signal transduction pathways of blood platelets

“…В случае полного удаления ATP, восстановление этого рецептора происходит примерно за 5 мин [80]. Активация рецептора P2X 1 приводит к изменению формы тромбоцита, быстрой и обратимой централизации его секреторных гранул, однако агрегации тромбоцитов при этом не наблюдается [81,82]. Этот рецептор играет вспомогательную роль при активации тромбоцитов от коллагена, тромбина, ADP и TXA 2 .…”