Non-redundant Roles of Phosphoinositide 3-Kinase Isoforms α and β in Glycoprotein VI-induced Platelet Signaling and Thrombus Formation

Platelets are activated by adhesion to vascular collagen via the immunoglobulin receptor, glycoprotein VI ( GPVI). This causes potent signaling toward activation of phospholipase C gamma 2, which bears similarity to the signaling pathway evoked by T- and B-cell receptors. Phosphoinositide 3-kinase (PI3K) plays an important role in collagen-induced platelet activation, because this activity modulates the autocrine effects of secreted ADP. Here, we identified the PI3K isoforms directly downstream of GPVI in human and mouse platelets and determined their role in GPVI-dependent thrombus formation. The targeting of platelet PI3K alpha or -beta strongly and selectively suppressed GPVI-induced Ca2+ mobilization and inositol 1,4,5-triphosphate production, thus demonstrating enhancement of phospholipase C gamma 2 by PI3K alpha/beta. That PI3K alpha and -beta have a non-redundant function in GPVI-induced platelet activation and thrombus formation was concluded from measurements of: (i) serine phosphorylation of Akt, (ii) dense granule secretion, (iii) intracellular Ca2+ increases and surface expression of phosphatidylserine under flow, and (iv) thrombus formation, under conditions where PI3K alpha/beta was blocked or p85 alpha was deficient. In contrast, GPVI-induced platelet activation was insensitive to inhibition or deficiency of PI3K alpha or -gamma. Furthermore, PI3K alpha/beta, but not PI3K gamma, contributed to GPVI-induced Rap1b activation and, surprisingly, also to Rap1b-independent platelet activation via GPVI. Together, these findings demonstrate that both PI3K alpha and -beta isoforms are required for full GPVI-dependent platelet Ca2+ signaling and thrombus formation, partly independently of Rap1b. This provides a new mechanistic explanation for the anti-thrombotic effect of PI3K inhibition and makes PI3K alpha an interesting new target for anti-platelet therapy.