Rapid resensitization of purinergic receptor function in human platelets

Abstract

Summary. Background: Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), the P2Y 1 and P2Y 12 purinergic receptors. Recently, we demonstrated that both receptors desensitize and internalize in human platelets by differential kinase-dependent mechanisms. Objectives: To demonstrate whether responses to P2Y 1 and P2Y 12 purinergic receptors resensitize in human platelets and determine the role of receptor traffic in this process. Methods: These studies were undertaken either in human platelets or in cells stably expressing epitope-tagged P2Y 1 and P2Y 12 purinergic receptor constructs. Results: In this study we show for the first time that responses to both of these receptors can rapidly resensitize following agonistdependent desensitization in human platelets. Further, we show that in human platelets or in 1321N1 cells stably expressing receptor constructs, the disruption of receptor internalization, dephosphorylation or subsequent receptor recycling is sufficient to block resensitization of purinergic receptor responses. We also show that, in platelets, internalization of both these receptors is dependent upon dynamin, and that this process is required for resensitization of responses. Conclusions: This study is therefore the first to show that both P2Y 1 and P2Y 12 receptor activities are rapidly and reversibly modulated in human platelets, and it reveals that the underlying mechanism requires receptor trafficking as an essential part of this process.

An intact PDZ motif is essential for correct P2Y12 purinoceptor traffic in human platelets

“…3 Recent work from our laboratory has shown that P2Y 12 receptor (P2Y 12 R) responsiveness is rapidly and reversibly modulated in human platelets. 4 On prolonged exposure to agonist, the responsiveness of P2Y 12 R, in human platelets, decreases because of receptor desensitization 5 and subsequent internalization. 6 In cell lines P2Y 12 Rs are regulated in a G protein-receptor coupled kinase (GRK)-dependent manner, 5 leading to their rapid recruitment to clathrin-coated pits (CCPs) in an arrestin-dependent manner.…”

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).…”

“…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). Following removal of ADP there is a rapid resensitization of P2Y 12 receptor responsiveness in human platelets (11). In order to resensitize, the P2Y 12 receptor internalizes following agonist addition and then recycles back to the cell membrane following removal of ADP (11).…”

“…Following removal of ADP there is a rapid resensitization of P2Y 12 receptor responsiveness in human platelets (11). In order to resensitize, the P2Y 12 receptor internalizes following agonist addition and then recycles back to the cell membrane following removal of ADP (11). The P2Y 12 receptor, similarly to P2Y 1 , internalizes via clathrin-and dynaminmediated endocytosis (14).…”

“…Rapid recycling of the P2Y 12 receptor had previously been described in human platelets although the mechanism at that time was unclear (8). Blockade of receptor internalization or subsequent recycling results in attenuation of receptor resensitization in human platelets (11). Our understanding of this process has been further advanced by the study of patients with rare genetic variations in the platelet P2Y 12 receptor.…”