Integrin-αIIbβ3-mediated outside-in signalling activates a negative feedback pathway to suppress platelet activation

Abstract

SummaryIntegrin-αIIbβ3-mediated outside-in signalling is widely accepted as an amplifier of platelet activation; accumulating evidence suggests that outside-in signalling can, under certain conditions, also function as an inhibitor of platelet activation. The role of integrin-αIIbβ3-mediated outside-in signalling in platelet activation is disputable. We employed flow cytometry, aggregometry, immunoprecipitation, and immunoblotting to investigate the role of integrin-αIIbβ3-mediated outside-in signalling in platelet activation. Integrin αIIbβ3 inhibition enhances agonist-induced platelet ATP secretion. Human platelets lacking expression of αIIbβ3 exhibited more platelet ATP secretion than their wild-type counterparts. Moreover, integrin-αIIbβ3-mediated outside-in signals activate SHIP-1, which in turn mediates p-Akt dep-hosphorylation, leading to inactivation of PI3K/Akt signalling. Furthermore, 3AC (SHIP-1 inhibitor) inhibits platelet disaggregation, and promotes platelet ATP secretion. Upon ADP stimulation, Talin is recruited to αIIbβ3, and it is dissociated from αIIbβ3 when platelets disaggregate. In addition, treatment with RUC2, an inhibitor of αIIbβ3, which blocks αIIbβ3-mediated outside-in signalling, can markedly prevent the dissociation of talin from integrin. SHIP1 Inhibitor 3AC inhibits the dissociation of talin from integrin-β3. These results suggest that integrin-αIIbβ3-mediated outside-in signalling can serve as a brake to restrict unnecessary platelet activation by activated SHIP-1, which mediated the disassociation of talin from β3, leading to integrin inactivation and blocking of PI3K/Akt signalling to restrict platelet ATP secretion.