Dual Regulation of Glycogen Synthase Kinase 3 (GSK3)α/β by Protein Kinase C (PKC)α and Akt Promotes Thrombin-mediated Integrin αIIbβ3 Activation and Granule Secretion in Platelets

Glycogen synthase kinase-3 is a Ser/Thr kinase, tonically active in resting cells but inhibited by phosphorylation of an N-terminal Ser residue (Ser 21 in GSK3α and Ser 9 in GSK3β) in response to varied external stimuli. Recent work suggests that GSK3 functions as a negative regulator of platelet function, but how GSK3 is regulated in platelets has not been examined in detail. Here, we show that early thrombin-mediated GSK3 phosphorylation (0–30 s) was blocked by PKC inhibitors and largely absent in platelets from PKCα knock-out mice. In contrast, late (2–5 min) GSK3 phosphorylation was dependent on the PI3K/Akt pathway. Similarly, early thrombin-mediated inhibition of GSK3 activity was blocked in PKCα knock-out platelets, whereas the Akt inhibitor MK2206 reduced late thrombin-mediated GSK3 inhibition and largely prevented GSK3 inhibition in PKCα knock-out platelets. More importantly, GSK3 phosphorylation contributes to platelet function as knock-in mice where GSK3α Ser 21 and GSK3β Ser 9 were mutated to Ala showed a significant reduction in PAR4-mediated platelet aggregation, fibrinogen binding, and P-selectin expression, whereas the GSK3 inhibitor CHIR99021 enhanced these responses. Together, these results demonstrate that PKCα and Akt modulate platelet function by phosphorylating and inhibiting GSK3α/β, thereby relieving the negative effect of GSK3α/β on thrombin-mediated platelet activation. Background: The constitutively active kinase GSK3β is a negative regulator of thrombin-stimulated platelet function. Results: Interfering with PKCα and Akt blocked thrombin-mediated GSK3α/β phosphorylation and reduced platelet function. Conclusion: PKCα and Akt phosphorylate GSK3α/β resulting in reduced GSK3α/β activity and increased thrombin-mediated platelet function. Significance: This study shows a novel mechanism by which GSK3α/β is regulated and contributes to platelet function.