Integrin αⅡbβ3-mediated Outside-in Signaling Activates A Negative Feedback Pathway To Suppress Platelet Activation

Integrin αⅡbβ3-mediated outside-in signaling is widely accepted as an amplifier of platelet activation, accumulating evidence suggests that outside-in signaling can, under certain conditions, also function as an inhibitor of platelet activation. The role of integrin αⅡbβ3-mediated outside-in signaling in platelet activation is disputable. We employed flow cytometry, aggregometry, immunoprecipitation and immunoblotting to investigate the role of Integrin αⅡbβ3-mediated outside-in signaling 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 dephosphorylation, leading to inactivation of PI3K/Akt signaling. Furthermore,3AC (SHIP-1 inhibitor) inhibits platelet disaggregation, and restores ADP-and TRAP-induced platelet dense granule secretion. Upon ADP stimulation, Talin and Kindlin-3 are recruited to αⅡbβ3, and they are dissociated from αⅡbβ3 when platelets disaggregate. In addition, treatment with RGD-peptide, an inhibitor of αⅡbβ3, which blocks αⅡbβ3-mediated outside-in signaling, can markedly prevent the dissociation of Talin and Kindlin-3 from integrin. These results suggest that integrin αⅡbβ3-mediated outside-in signaling can serve as a brake to restrict unnecessary platelet activation by promoting integrin inactivation and blocking PI3K/Akt signaling to restrict platelet ATP secretion.