Sol Sherry Thrombosis Research Center and the Department of Cardiovascular Sciences, Lewis Katz School of Medicine, Temple University, Philadelphia, PA, USA.
Platelets. 2022 Nov 17;33(8):1301-1306. doi: 10.1080/09537104.2022.2071855. Epub 2022 May 6.
Platelet activation by adenosine diphosphate (ADP) is mediated through two G-protein-coupled receptors, P2Y1 and P2Y12, which signal through Gq and Gi, respectively. P2Y1 stimulation leads to phospholipase C activation and an increase in cytosolic calcium necessary for CalDAG-GEF1 activation. Engagement of P2Y12 inhibits adenylate cyclase, which reduces cAMP, and activation of PI3-kinase, which inhibits RASA3 resulting in sustained activated Rap1b. In this study we activated human platelets with 2-MeSADP in the presence of LY294002, a PI3-kinase inhibitor, AR-C69931MX, a P2Y12 antagonist or MRS2179, a P2Y1 antagonist. We measured the phosphorylation of Akt on Ser473 as an indicator of PI3-kinase activity. As previously shown, LY294002 and ARC69931MX abolished 2MeSADP-induced Akt phosphorylation. MRS2179 reduced ADP-induced Akt phosphorylation but did not abolish it. Rap1b activation, however, was only reduced, but not ablated, using LY294002 and was completely inhibited by ARC69931MX or MRS2179. Furthermore, 2MeSADP-induced Rap1b activation was abolished in either P2Y1 or P2Y12 null platelets. These data suggest that ADP-induced Rap1b activation requires both P2Y1 and P2Y12. In addition, although stimulation of P2Y12 results in PI3-kinase activation leading to Akt phosphorylation and Rap1b activation, Rap1b activation can occur independently of PI3-kinase downstream of P2Y12. Thus, we propose that the P2Y12 receptor can regulate Rap1b, possibly through RASA3, in a pathway independent of PI3-kinase.
血小板通过二磷酸腺苷（ADP）的激活是通过两个 G 蛋白偶联受体（P2Y1 和 P2Y12）介导的，它们分别通过 Gq 和 Gi 信号转导。P2Y1 的刺激导致磷脂酶 C 的激活和细胞内钙离子的增加，这是 CalDAG-GEF1 激活所必需的。P2Y12 的结合抑制腺苷酸环化酶，降低 cAMP，激活 PI3-激酶，抑制 RASA3，导致持续激活 Rap1b。在这项研究中，我们在 LY294002（一种 PI3-激酶抑制剂）、AR-C69931MX（一种 P2Y12 拮抗剂）或 MRS2179（一种 P2Y1 拮抗剂）存在的情况下，用 2-MeSADP 激活人血小板。我们测量了 Akt 丝氨酸 473 的磷酸化作为 PI3-激酶活性的指标。如前所述，LY294002 和 ARC69931MX 消除了 2-MeSADP 诱导的 Akt 磷酸化。MRS2179 降低了 ADP 诱导的 Akt 磷酸化，但并未完全消除。然而，Rap1b 的激活仅被 LY294002 和 ARC69931MX 部分抑制，而被完全抑制。此外，在 P2Y1 或 P2Y12 缺失血小板中，2-MeSADP 诱导的 Rap1b 激活被完全消除。这些数据表明，ADP 诱导的 Rap1b 激活需要 P2Y1 和 P2Y12。此外，尽管 P2Y12 的刺激导致 PI3-激酶的激活，导致 Akt 的磷酸化和 Rap1b 的激活，但 Rap1b 的激活可以独立于 P2Y12 的下游 PI3-激酶发生。因此，我们提出 P2Y12 受体可以通过 RASA3 调节 Rap1b，可能是通过独立于 PI3-激酶的途径。
