Protease-activated receptor-induced Akt activation--regulation and possible function.

Area of Science:

• Platelet signaling and function
• Thrombin-mediated platelet activation
• Kinase signaling pathways

Background:

• Thrombin activates platelet Akt, a serine/threonine kinase, through phosphorylation at Thr308 and Ser473.
• The precise mechanisms of thrombin-induced Akt phosphorylation and Akt's role in platelet function remain debated.

Purpose of the Study:

• To elucidate how protease-activated receptors (PARs) stimulate Akt phosphorylation in human platelets.
• To determine the functional significance of Akt activation in human platelet responses.

Main Methods:

• Human platelets were stimulated via PAR1 or PAR4.
• Western blotting assessed signaling pathways regulating Akt phosphorylation using specific inhibitors.
• Platelet aggregation, P-selectin expression, and fibrinogen binding were evaluated using aggregometry and flow cytometry to assess the role of activated Akt.

Main Results:

• Phospholipase C (PLC) mediates initial Akt phosphorylation triggered by PAR1 and PAR4 stimulation, independent of secreted ADP and phosphatidylinositol-3-kinase (PI3K).
• Sustained Akt phosphorylation after PAR1 stimulation (approx. 60s) requires the P2Y(12) receptor and PI3K activation; PAR4 sustains phosphorylation for longer (up to 300s) independently of these.
• Inhibition of Akt signaling attenuated P-selectin expression and fibrinogen binding, and delayed platelet aggregation induced by PAR1 or PAR4 stimulation.

Conclusions:

• Platelet PAR activation initiates rapid Akt phosphorylation downstream of PLC.
• Prolonged PAR stimulation necessitates ADP, P2Y(12), and PI3K for sustained Akt phosphorylation.
• Activated Akt plays a regulatory role in platelet secretion and alpha(IIb)beta(3) integrin activation, influencing overall platelet function.