Shp2and P2X₁p38Mediated Platelet Activation Mechanism

Shp2is a ubiquitous tyrosine phosphatase containing two Src Homology2domains. It plays major biological functions in response to various growth factors, hormones or cytokines. Previous research had demonstrated that Shp2can associated with the phosphorylated ITIM motifs of PEC AM-1after cross-linking, and inhibit GPVI signaling pathway. However, the role of Shp2in platelet activation remains unclear. In this work, we have investigated the effects and the mechanisms of Shp2engagement in platelet activation. Our study confirmed that inhibition of Shp2attenuated low doses collagen and ADP induced platelet aggregation and secretion. Under arterial sheer rate (1000s’1), PHPSi dramatically reduced platelet adhesion on collagen surface. Shp2inhibitor PHPSi also prevented platelet spreading on fibrinogen surface, which is mediated by the early phage outside-in signaling, but didn’t affect thrombin induced platelet clot retraction which is mediated by the late phage outside-in signaling. We also confirmed that, Shp2associated with Syk in a integrin αⅡbβ3independent manner upon GPVI activation. Shp2inhibitor PHPS1can dissociated Shp2-Syk complex in low dose collagen but not high dose collagen induced platelet activation. Shp2-Syk association might in turn regulate the phosphorylation of the downstream effectors Akt and Erk. ADP-induced platelet activation was also attenuated by PHPS1. This effect is likely mediated by Akt/Erk dephosphorylation via the P2Y12receptor. P2X1is the only ATP receptor identified on platelets. Previous researches have demonstrated that ATP released by activated platelets can promote Erk phosphorylation to enhance low dose collagen induced platelet activation. However, the other functions and mechanisms of P2X1in platelet are not very clear. Our research confirmed that P2X1served as positive feedback machinery via p38phosphorylation to amplify Thromboxane A2(TXA2) analogue U46619induced platelet aggregation and secretion. P2X1inhibition by NF449or pre-desensitisation inhibited platelet P-selectin expression and aggregation induced by a low concentration of U46619. αβ-Me-ATP caused p38phosphorylation in dose and time dependent manners. P38inhibition by SB203580, but not Erk inhibition by U0126, elicited a similar inhibition by NF499. The combination of NF449and SB203580provided, however, no additive effects, suggested that P2X1promote U46619induced platelet activation possibly through p38phosphorylation. Pre-desensitisation or inhibition P2X1, U46619induced p38phosphorylation was significantly decreased at different time points, whereas Erk phosphorylation was not affected. P38inhibitor SB203580did not influence Erk phosphorylation and Mek inhibitor U0126had no effect on p38phosphorylation in U46619induced platelet activation, which suggested Erk was not involved in P2X1signaling of U46619. However, the P2X1-p38pathway mainly enhanced mild platelet activation by U46619, because αβ-Me-ATP supplementation or p38blockade had no effect on intense platelet activation induced by a higher concentration of U46619.