ADP-Stimulated Activation of Akt During Integrin Outside-In Signaling Promotes Platelet Spreading by Inhibiting Glycogen Synthase Kinase-3β

Objective-Integrins mediate platelet adhesion and transmit outside-in signals leading to platelet spreading. Phosphoinositide 3-kinases (PI3Ks) play a critical role in outside-in signaling and platelet spreading; however, the mechanisms of PI3K activation and function in outside-in signaling are unclear. We sought to determine the role of the Akt family of serine/threonine kinases and activation mechanisms of the PI3K/Akt pathway in outside-in signaling. Methods and Results-Akt inhibitors and Akt3 knockout inhibited platelet spreading on fibrinogen, indicating that Akt is important in integrin outside-in signaling. Akt inhibitors and Akt3 knockout also diminished integrin-dependent phosphorylation of glycogen synthase kinase-3 beta. Inhibition of glycogen synthase kinase-3 beta reversed the inhibitory effects of Akt3 knockout and inhibitors of Akt or PI3K on platelet spreading, indicating that glycogen synthase kinase-3 beta is a downstream target of Akt in outside-in signaling. Integrin-dependent activation of the PI3K-Akt pathway requires Src family kinase. Akt phosphorylation is also significantly inhibited in ADP receptor P2Y12 knockout platelets and further inhibited in P2Y12 knockout platelets treated with a P2Y1 antagonist. Consistently, P2Y12 knockout and P2Y1 inhibition together reduced platelet spreading. Conclusion-These results demonstrate that integrin outside-in signaling and platelet spreading requires Src family kinase-dependent and ADP receptor-amplified activation of the PI3K-Akt-GSK-3 beta pathway. (Arterioscler Thromb Vasc Biol. 2012;32:2232-2240.)