Role of G protein–gated inwardly rectifying potassium channels in P2Y12 receptor–mediated platelet functional responses

Abstract

The role of the G i -coupled platelet P2Y 12 receptor in platelet function has been well established. However, the functional effector or effectors contributing directly to ␣IIb␤3 activation in human platelets has not been delineated. As the P2Y 12 receptor has been shown to activate G protein-gated, inwardly rectifying potassium (GIRK) channels, we investigated whether GIRK channels mediate any of the functional responses of the platelet P2Y 12 receptor. Western blot analysis revealed that platelets express GIRK1, GIRK2, and GIRK4. In aspirin-treated and washed human platelets, 2 structurally distinct GIRK inhibitors, SCH23390 (R(؉)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride) and U50488H (trans-(؎)-3,4-dichloro-N-methyl-N-[2-(pyrrolidinyl)cyclohexyl] benzeneacetamide methanesulfonate), inhibited adenosine diphosphate (ADP)-, 2-methylthioADP (2-MeSADP)-, U46619-, and low-dose thrombin-mediated platelet aggregation. However, the GIRK channel inhibitors did not affect platelet aggregation induced by high concentrations of thrombin, AYPGKF, or convulxin. Furthermore, the GIRK channel inhibitors reversed SFLLRN-induced platelet aggregation, inhibited the P2Y 12 -mediated potentiation of dense granule secretion and Akt phosphorylation, and did not affect the agonist-induced G qmediated platelet shape change and intracellular calcium mobilization. Unlike AR-C 69931MX, a P2Y 12 receptor-selective antagonist, the GIRK channel blockers did not affect the ADP-induced adenlylyl cyclase inhibition, indicating that they do not directly antagonize the P2Y 12 receptor. We conclude that GIRK channels are important functional effectors of the P2Y 12 receptor in human platelets. IntroductionPlatelets play an important role in normal hemostasis and abnormal activation of platelets leads to thrombosis. During vascular injury or conditions of high shear, exposure of the collagen-rich subendothelium activates the platelets and results in the formation of a stable thrombus due to the combined action of adenosine diphosphate (ADP) secreted from platelet-dense granules and generated thrombin. 1 Any perturbations in this system can have pathologic cardiovascular implications such as intravascular thrombus formation and vascular occlusion.ADP is an important component of the platelet-dense granules and also an important platelet agonist that stimulates platelets by acting on the G q -coupled P2Y 1 receptor and G i -coupled P2Y 12 receptor. 2,3 Once released, it amplifies the primary responses of other agonists such as collagen and thrombin, leading to enhanced platelet aggregation and stability of the thrombus. 4,5 It is now well established that concomitant signaling through G q -coupled P2Y 1 and G i -coupled P2Y 12 is necessary and sufficient for the integrin GPIIb/IIIa activation. 3 Furthermore, selective G i activation, when coupled with selective G 12/13 stimulation, also results in platelet aggregation. 6,7 Notably, the P2Y 12 -coupled G i signaling is also crucial for potentiating dense...

C-type natriuretic peptide inhibits leukocyte recruitment and platelet-leukocyte interactions via suppression of P-selectin expression

“…Yet, if G-protein coupling is responsible for transducing the signals conveyed by NPR-C activation (as appears to be the case in the mesenteric and coronary vasculature; refs. 11 and 12), one might expect CNP to promote platelet aggregation because activation of G i signaling pathways is now well established to be a prerequisite for maximal platelet aggregation (34)(35)(36), including a role for GIRKs (37). However, previous reports suggest that only NPR-C (not NPR-A or NPR-B) are expressed on human platelets (38), intimating that an NPR-B-mediated increase in platelet cGMP (the second messenger invoked by NO to inhibit platelet reactivity) does not underlie the antiaggregatory effects of CNP.…”

Contribution of Kir2 potassium channels to ATP-induced cell death in brain capillary endothelial cells and reconstructed HEK293 cell model

“…Additionally, it is known that G i -coupled P2Y 12 receptor modulated GIRK channel activation (41). The possibility that ATP-induced anomalous hyperpolarization in t-BBEC 117 might be due to activation of GIRK was also examined.…”

Central role of the P2Y12 receptor in platelet activation

“…Mice lacking PI3K-γ show aberrations in platelet function only when low doses of ADP are used, but are provided protection from thromboembolism (27). Recent studies indicate that Rap1b, Akt, and potassium channels are important functional effectors downstream of P2Y 12 receptor stimulation (28)(29)(30)(31) (Figure 3). …”