The Role of Akt in Platelet Activation

Platelet activation is critical for the maintenance of hemostasis. Under pathological conditions, platelets also play a critical role in thrombosis, a leading cause of heart attack and stroke. Platelets are activated by adhesive proteins and soluble agonists. This initiates a signal transduction cascade leading to platelet adhesion, spreading, secretion, and aggregation, which are important for the formation of a platelet plug. Therefore, understanding the mechanisms of platelet activation may aid in the development of novel anti-thrombotic agents.;PI3 Kinases (PI3Ks) have been shown to be a common and key signal mediator in platelet activation. The most well known effector of PI3Ks is Akt. Akt is a family of serine threonine kinases with 3 isoforms: Akt1, Akt2, and Akt3. Previous studies from our lab and others have shown that Akt1 and Akt2 have important roles in stimulating platelet activation. In particular, Akt1 and Akt2 have been shown to be important in stimulating the second wave of secretion dependent platelet activation induced by thrombin, thromboxane (TXA2 ), and von willebrand factor (vWF), and a major downstream mechanism for the role of Akt1 and Akt2 is mediated by the NO-cGMP-PKG signaling pathway. Although Akt3 was not previously identified in platelets in some previous studies, we present data in Chapter IV A that Akt3 is expressed in substantial amounts in human and mouse platelets. Using platelets isolated from Akt3 knockout mice, to assess the role of Akt3 in platelet function, we show that Akt3 is important in stimulating platelet aggregation and secretion in response to low concentrations of thrombin receptor agonists and thromboxane A2 (TXA2), but not collagen or VWF. This is in contrast to Akt1 and Akt2, which are important is stimulating platelet activation induced by thrombin, TXA2, and vWF, while Akt1 is also important in stimulating platelet aggregation in response to collagen. Thus, our study revealed that there are differences among Akt isoforms in specific platelet signaling pathways. Importantly, we have found that Akt3 also promotes the formation of occlusive arterial thrombi in an in vivo FeCl3-induced carotid artery thrombosis model. Thus, we show that Akt3 plays an important and distinct role in platelet activation and in thrombosis.;In identifying a mechanism responsible for the role of Akt3 in platelet activation, we demonstrated that Akt3 is important in thrombin-induced phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) at Ser9, which is known to inhibit GSK-3beta function. Inhibition of GSK-3beta reversed the inhibitory effect of Akt3 knockout on platelet aggregation and secretion in response to low dose thrombin. Thus, Akt3-mediated phosphorylation and inhibition of GSK-3beta may be a mechanism by which Akt3 stimulates platelet activation.;Integrins mediate platelet adhesion and transmit "outside in" signals, leading to platelet spreading. 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 hypothesized that downstream from PI3K, Akt plays a stimulatory role in integrin outside-in signaling and platelet spreading. In testing this hypothesis in Chapter III B, we demonstrate that Akt is important in integrin outside-in signaling and platelet spreading on fibrinogen using platelets treated with an inhibitor of Akt and platelets from Akt3 knockout mice. Our studies revealed that Akt isoforms are also important in integrin-dependent phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) and the role of PI3K and Akt on platelet spreading is at least partially mediated by inhibition of GSK-3beta. We have determined that integrin-dependent activation of the PI3K-Akt pathway requires Src Family Kinase (SFK). In addition, integrin-dependent activation of the PI3K-Akt pathway is also promoted by ADP secretion signaling through P2Y1 and P2Y12 ADP receptors during spreading. Thus, we have characterized an integrin outside-in signaling pathway involving SFK-dependent and P2Y12/P2Y1-facilitated activation of the PI3K-Akt pathway, which inhibits GSK-3beta and promotes platelet spreading.