P2Y1 and P2Y12 receptors for ADP desensitize by distinct kinase-dependent mechanisms

Abstract

Adenosine 5-diphosphate (ADP) plays a central role in regulating platelet function by the activation of the G protein-coupled receptors P2Y 1 and P2Y 12 . Although it is well established that aggregation responses of platelets to ADP desensitize, the underlying mechanisms involved remain unclear. In this study we demonstrate that P2Y 1 -and P2Y 12 -mediated platelet responses desensitize rapidly. Furthermore, we have established that these receptors desensitize by different kinase-dependent mechanisms. G protein-coupled receptor kinase (GRK) 2 and GRK6 are both endogenously expressed in platelets. Transient overexpression of dominant-negative mutants of these kinases or reductions in endogenous GRK expression by the use of specific siRNAs in 1321N1 cells showed that P2Y 12 , but not P2Y 1 , desensitization is mediated by GRKs. In contrast, desensitization of P2Y 1 , but not P2Y 12 , is largely dependent on protein kinase C activity. This study is the first to show that both P2Y 1 and P2Y 12 desensitize in human platelets, and it reveals ways in which their sensitivity to ADP may be differentially and independently altered. IntroductionPlatelets are activated by a variety of extracellular stimuli and are an essential component of the normal response to vascular injury. Central among these stimuli is adenosine 5Ј-diphosphate (ADP), which induces multiple platelet responses and potentiates platelet aggregation to other agonists (for reviews, see Gachet 1 and Kunapuli et al 2 ). Indeed, since it was recognized 40 years ago, ADP has been regarded as a central mediator of hemostasis and thrombosis by providing a positive feedback mechanism for the activation of platelets by multiple agonists. For example, both thrombin and collagen promote ADP release from platelet-dense granules; ADP subsequently acts on purinergic receptors to reinforce platelet aggregation responses 1,2 and thrombus formation.ADP acts on 2 G protein-coupled receptors (GPCRs), P2Y 1 and P2Y 12 . 1,2 The P2Y 1 purinergic receptor was the first of the ADP receptors to be cloned. [3][4][5] This receptor is widely expressed throughout the body and couples to G q , leading to the activation of phospholipase C␤, a subsequent increase in cytosolic calcium, and the activation of protein kinase C (PKC). The P2Y 12 receptor was only recently identified 6 and is the target of the clinically effective antithrombotic drugs clopidogrel and ticlopidine. P2Y 12 couples through G i to the inhibition of adenylyl cyclase and the activation of PI3-kinase. On the basis of pharmacologic and genetic studies, it is now accepted that P2Y 1 is required for platelet activation by ADP, whereas P2Y 12 is important in synergizing with P2Y 1 or other G q -coupled receptors to induce platelet activation by ADP and other agonists playing a major role in stabilizing platelet thrombi in vivo.Given the established crucial role of ADP in platelet activation, it is likely that the responsiveness of platelets to ADP is tightly regulated, and knowledge of the mechanisms responsible for t...

P2 receptors and platelet function

“…The in vivo consequence is that under conditions of platelets refractory to stimulation by ADP, the P2Y 12 receptor remains functional and able to promote their reactivity at sites of injury, thus preventing loss of haemostatic function. On the other hand, it is reported that both P2Y 1 and P2Y 12 receptors undergo desensitization, and that P2Y 12 desensitization is mediated by G proteincoupled receptor kinases (GRK) [93,94]. Further studies are required to solve the apparent contradiction of these reports.…”

Platelets at the vascular interface

“…PAR4 binds thrombin with much lower affinity, but it also internalizes more slowly and thus generates a steady and sustained integrin activation signal[16–18]. Similarly, the ADP receptor P2Y1, which is not very abundant on the platelet surface[19], is rapidly desensitized[20,21]. Thus, it can only cause a rapid but short-lived rise of the cytosolic calcium concentration and needs co-signaling through the P2Y12 receptor to support platelet aggregation[22,23].…”

Pathophysiological consequences of receptor mistraffic: Tales from the platelet P2Y 12 receptor

“…In order to minimize inappropriate platelet recruitment and activation at the site of a growing thrombus, platelet ADP responsiveness is tightly regulated in human platelets (8)(9)(10)(11)(12). P2Y 12 receptor responsiveness rapidly desensitizes in human platelets (13).…”

“…P2Y 12 receptor responsiveness rapidly desensitizes in human platelets (13). Previous studies in cell lines have identified that G protein-coupled receptor kinase (GRK)2-and GRK6-dependent phosphorylation of the P2Y 12 receptor is required for arrestin association and desensitization (9).…”

“…So far coding polymorphisms with significant functional impact have only been discovered in the visual arrestin-1 gene, which lead to visual disorders (e.g., Oguchi's disease(91), night blindness or retinitis pigmentosa (92)). Given both the physiological importance of the arrestin family in the regulation of the majority of GPCRs and that there are only two isoforms of the non-visual M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 13 arrestins, perhaps it is not surprising that evolution may not have allowed for high levels of genetic variation of these proteins seen in GPCRs.…”