Desensitization of the Platelet Aggregation Response to ADP: Differential Down-regulation of the P2Y1 and P2cyc Receptors

Abstract

SummaryPlatelets activated by ADP become refractory to restimulation, but the mechanism of this process is not well understood. A normal platelet response to ADP requires coactivation of the P2Y1 receptor responsible for shape change and the P2cyc receptor, responsible for completion and amplification of the response. The aim of the present study was to characterize the desensitization of platelets to ADP and to determine whether or not these two receptors are desensitized simultaneously through identical pathways when platelets become refractory to ADP. It was found that full inhibition of platelet aggregation in response to restimulation by ADP required the presence of ADP in the medium or use of a high concentration (1 mM) of its non-hydrolysable analogue ADP β S. Platelets incubated for 1 h at 37° C with 1 mM ADP β S and resuspended in Tyrode’s buffer containing apyrase displayed a stable refractory state characterized by the inability to aggregate or change shape in response to ADP. ADP β S treated platelets loaded with fura2/AM showed complete blockade of the calcium signal in response to ADP, whereas the capacity of ADP to inhibit PGE1 stimulated cAMP accumulation in these platelets was only diminished. Consequently, serotonin was able to promote ADP induced aggregation through activation of the Gq coupled 5HT2A receptor while adrenaline had no such effect. These results suggested that the refractory state of ADP β S treated platelets was entirely due to desensitization of the P2Y1 receptor, the P2cyc receptor remaining functional. Binding studies were performed to determine whether the P2Y1 and/or P2cyc binding sites were modified in refractory platelets. Using selective P2Y1 and P2cyc antagonists (A3P5P and AR-C66096 respectively), we could demonstrate that the decrease in [33P]2MeSADP binding sites on refractory platelets corresponded to disappearance of the P2Y1 sites with no change in the number of P2cyc sites, suggesting internalization of the P2Y1 receptor. This was confirmed by flow cytometric analysis of Jurkat cells expressing an epitope-tagged P2Y1 receptor, where ADP β S treatment resulted in complete loss of the receptor from the cell surface. We conclude that the P2Y1 and P2cyc receptors are differently regulated during platelet activation.

P2 receptors and platelet function

“…The molecular mechanisms of this phenomenon have been studied in detail, but consensus has not been reached, and two different views have not yet been reconciled. On the one hand, it is thought that the phenomenon of platelet refractoriness to ADP is due to selective desensitization and internalization of the P2Y 1 receptor, while the P2Y 12 receptor remains functional with the ability of ADP to induce amplification of the platelet aggregation induced by other agonists [89][90][91]. Desensitization of the P2Y 1 receptor has been shown to be dependent on receptor C-terminal phosphorylation sites, β-arrestin-2 interaction and protein kinase C (PKC) activity [92,93].…”

The plaque lipid lysophosphatidic acid stimulates platelet activation and platelet-monocyte aggregate formation in whole blood: involvement of P2Y1 and P2Y12 receptors

“…In contrast to previous studies of LPA-induced aggregation of isolated platelets, platelets in our study were washed in the presence of prostacyclin and resuspended in buffer containing apyrase to avoid platelet preactivation and desensitization of the ADP-receptor P2Y 1 . 23 In this preparation, 1-acyl-LPA (16:0) induced shape change and subsequent aggregation with an EC 50 of 8 M. A concentration of 1 M 1-acyl-LPA (16:0) only induced shape change, 3M 1-acyl-LPA (16:0) induced shape change, reversible aggregation (10%) with only 1% to 2% serotonin secretion, higher concentrations (10, 30, and 100 M ) 1-acyl-LPA [16:0]) elicited maximal irreversible aggregation with 9%, 19%, and 36% serotonin secretion, respectively (data not shown and Figure 2A). 1-alkyl-LPA (16:0) was again more potent than 1-acyl-LPA (16:0) in inducing shape change and aggregation of washed platelets (data not shown).…”

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).…”