Multi-phased kinetics and interaction of protein kinase signaling in glycoprotein VI-induced platelet aIIbß3 integrin activation and degranulation

BACKGROUND: Platelet glycoprotein VI (GPVI) stimulation activates the tyrosine kinases Syk and Btk, and the effector proteins phospholipase C? 2 (PLC?2) and protein kinase C (PKC). Here, the activation sequence, crosstalk and downstream effects of this Syk-Btk-PKC signalosome in human platelets was analyzed. METHODS AND RESULTS: Using immunoblotting, we quantified 14 regulated phospho-sites in platelets stimulated by convulxin with and without inhibition of Syk, Btk or PKC. Convulxin induced fast, reversible tyrosine phosphorylation (pY) of Syk, Btk, LAT and PLC?2, followed by reversible serine/threonine phosphorylation (pS/T) of Syk, Btk and downstream kinases MEK1/2, Erk1/2, p38 and Akt. Syk inhibition by PRT-060318 abolished all phosphorylations, except Syk pY352. Btk inhibition by acalabrutinib strongly decreased Btk pY223/pS180, Syk pS297, PLC?2 pY759/Y1217, MEK1/2 pS217/221, Erk1/2 pT202/Y204, p38 pT180/Y182 and Akt pT308/S473. PKC inhibition by GF109203X abolished most pS/T phosphorylations except p38 pT180/Y182 and Akt pT308, but enhanced most Y-phosphorylations. Acalabrutinib,but not GF109203X, suppressed convulxin-induced intracellular Ca mobilization, whereas all three protein kinase inhibitors abolished degranulation and aIIbß3 integrin activation assessed by flow cytometry. Inhibition of autocrine ADP effects by AR-C669931 partly diminished convulxin-triggered degranulation. CONCLUSION: Kinetic analysis of GPVI-initiated multisite protein phosphorylation in human platelets demonstrates multiple phases and interactions of tyrosine and serine/threonine kinases with activation-altering feedforward and feedback loops partly involving PKC. The protein kinase inhibitor effects on multisite protein phosphorylation and functional readouts reveal that the signaling network of Syk, Btk and PKC controls platelet granule exocytosis and aIIbß3 integrin activation.