Binding of CIB1 to the α(IIb) tail of α(IIb)β(3) is required for FAK recruitment and activation in platelets

BACKGROUND: It is believed that activation of c-Src bound to the integrin β(3) subunit initiates outside-in signaling. The involvement of α(IIb) in outside-in signaling is poorly understood. OBJECTIVES: We have previously shown that CIB1 specifically interacts with the cytoplasmic domain of α(IIb) and is required for α(IIb)β(3) outside-in signaling. Here we evaluated the role of CIB1 in regulating outside-in signaling in the absence of inside-out signaling. METHODS: We used α(IIb) cytoplasmic domain peptide and CIB1-function blocking antibody to inhibit interaction of CIB1 with α(IIb) subunit as well as Cib1(-/-) platelets to evaluate the consequence of CIB1 interaction with α(IIb) on outside-in signaling. RESULTS: Fibrinogen binding to α(IIb)β(3) results in calcium-dependent interaction of CIB1 with α(IIb), which is not required for filopodia formation. Dynamic rearrangement of cytoskeleton results in CIB1-dependent recruitment of FAK to the α(IIb) complex and its activation. Disruption of the association of CIB1 and α(IIb) by incorporation of α(IIb) peptide or anti-CIB1 inhibited both FAK association and activation. Furthermore, FAK recruitment to the integrin complex was required for c-Src activation. Inhibition of c-Src had no effect on CIB1 accumulation with the integrin at the filopodia, suggesting that c-Src activity is not required for the formation of CIB1-α(IIb)-FAK complex. CONCLUSION: Our results suggest that interaction of CIB1 with α(IIb) is one of the early events occurring during outside-in signaling. Furthermore, CIB1 recruits FAK to the α(IIb)β(3) complex at the filopodia where FAK is activated, which in turn activates c-Src, resulting in propagation of outside-in signaling leading to platelet spreading.