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To cite this article: Resé ndiz JC, Kroll MH, Lassila R. Protease-activated receptor-induced Akt activation -regulation and possible function. Summary. Background: Thrombin induces the activation of the platelet serine/threonine kinase Akt. Akt activation is dependent on its phosphorylation at Thr308 and Ser473. The mechanism by which thrombin induces Akt phosphorylation is controversial, as is the role of Akt in platelet function. Objectives: To investigate how protease-activated receptors (PARs) stimulate Akt and the role that Akt plays in human platelet function. Methods: Platelets were stimulated through PAR1 or PAR4. Specific inhibitors were used to evaluate, by Western blotting, signaling pathways regulating Akt phosphorylation, and the role of activated Akt was evaluated by aggregometry and flow cytometry. Results: Phospholipase C (PLC) controls Akt phosphorylation elicited by PARs. Stimulation of PAR1 or PAR4 resulted in rapid Akt phosphorylation, independently of secreted ADP and phosphatidylinositol-3-kinase (PI3K) activation. Akt phosphorylation 60 s after PAR1 stimulation became entirely dependent on the purinergic receptor P2Y 12 and the activation of PI3K. In contrast, PAR4 partially sustained Akt phosphorylation independently of P2Y 12 and PI3K for up to 300 s. Pharmacologic inhibition of Akt reduced P-selectin expression and fibrinogen binding in platelets stimulated through PAR1, and delayed platelet aggregation in response to submaximal PAR1 or PAR4 stimulation, although aggregation at 300 s was unaffected. Conclusions: Platelet PAR stimulation causes rapid Akt phosphorylation downstream of PLC, whereas with continuous stimulation, ADP and PI3K are required for maintaining Akt phosphorylation. Activated Akt regulates platelet function by modulating secretion and a IIb b 3 activation.

Pathologically elevated shear stress triggers aspirin-insensitive platelet thrombosis. Signaling mechanisms involved in shear-induced platelet thrombosis are not well understood. To investigate these, we examined the hypothesis that functionally important platelet phosphatidylinositol 3-kinase (PI3-K) activity is stimulated by an in vitro shear stress of 120 dynes/cm 2 (shear rate of 6000 sec Ϫ1 ). Phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) production was examined in washed human platelets subjected to pathological shear stress in a cone-plate viscometer. PIP 3 production peaks 30 s after shear begins and is initiated by von Willebrand factor (VWF) binding to the glycoprotein (Gp) Ib-IX-V complex. Inhibiting PI3-K with wortmannin or 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) results in the inhibition of shear-induced platelet aggregation. In resting platelets, class IA PI3-K associates with the tyrosine kinase Syk. Within 30 s of beginning shear, PI3-K-associated Syk becomes tyrosine phosphorylated. Inhibiting Syk activation with piceatannol results in the inhibition of PIP 3 production and aggregation. Selective blockade of the P2Y 12 receptor results in the inhibition of Syk phosphorylation, PIP 3 production, and aggregation. These results indicate that shear-induced VWF binding to platelet GpIb-IX-V stimulates functionally important PI3-K activity. PI3-K activation is signaled by rapid feedback amplification that involves P2Y 12 receptormediated activation of Syk.