Binding of CIB1 to the aIIb tail of aIIbβ3 is required for FAK recruitment and activation in platelets

Abstract

Background It is believed that activation of c-Src bound to the integrin β3 subunit initiates outside-in signaling. The involvement of aIIb in outside-in signaling is poorly understood. Objectives We have previously shown that CIB1 specifically interacts with the cytoplasmic domain of aIIb and is required for aIIbβ3 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 aIIb cytoplasmic domain peptide and CIB1-function blocking antibody to inhibit interaction of CIB1 with aIIb subunit as well as Cib1-/-platelets to evaluate the consequence of CIB1 interaction with aIIb on outside-in signaling. Results Fibrinogen binding to aIIbβ3 results in calcium-dependent interaction of CIB1 with aIIb, which is not required for filopodia formation. Dynamic rearrangement of cytoskeleton results in CIB1-dependent recruitment of FAK to the aIIb complex and its activation. Disruption of the association of CIB1 and aIIb by incorporation of aIIb 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-aIIb-FAK complex. Conclusion Our results suggest that interaction of CIB1 with aIIb is one of the early events occurring during outside-in signaling. Furthermore, CIB1 recruits FAK to the aIIbβ3 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.