Platelet Activation Pathways Controlling Reversible Integrin aIIbß3 Activation

Agonist-induced platelet activation, with the integrin aIIbß3 conformational change, is required for fibrinogen binding. This is considered reversible under specific conditions, allowing a second phase of platelet aggregation. The signaling pathways that differentiate between a permanent or transient activation state of platelets are poorly elucidated. To explore platelet signaling mechanisms induced by the collagen receptor glycoprotein VI (GPVI) or by protease-activated receptors (PAR) for thrombin that regulate time-dependent aIIbß3 activation. Platelets were activated with collagen-related peptide (CRP, stimulating GPVI), thrombin receptor-activating peptides, or thrombin (stimulating PAR1 and/or 4). Integrin aIIbß3 activation and P-selectin expression was assessed by two-color flow cytometry. Signaling pathway inhibitors were applied before or after agonist addition. Reversibility of platelet spreading was studied by microscopy. Platelet pretreatment with pharmacological inhibitors decreased GPVI- and PAR-induced integrin aIIbß3 activation and P-selectin expression in the target order of protein kinase C (PKC) > glycogen synthase kinase 3 > ß-arrestin > phosphatidylinositol-3-kinase. Posttreatment revealed secondary aIIbß3 inactivation (not P-selectin expression), in the same order, but this reversibility was confined to CRP and PAR1 agonist. Combined inhibition of conventional and novel PKC isoforms was most effective for integrin closure. Pre- and posttreatment with ticagrelor, blocking the P2Y adenosine diphosphate (ADP) receptor, enhanced aIIbß3 inactivation. Spreading assays showed that PKC or P2Y inhibition provoked a partial conversion from filopodia to a more discoid platelet shape. PKC and autocrine ADP signaling contribute to persistent integrin aIIbß3 activation in the order of PAR1/GPVI > PAR4 stimulation and hence to stabilized platelet aggregation. These findings are relevant for optimization of effective antiplatelet treatment.