CIB1 Positively Regulates Outside-In Signaling through αIIbβ3 by Enhancing FAK and Src Activity.

Integrin α_IIbβ₃, the platelet fibrinogen receptor, mediates outside-in signaling upon fibrinogen binding. Calcium- and integrin-binding protein 1 (CIB1) specifically interacts with the cytoplasmic domain of α_IIb and is involved in platelet spreading. Here we show that CIB1 binding to α_IIb is an essential step in the propagation of outside-in signaling through integrin α_IIbβ₃. Incorporation of BAPTA-AM completely blocked outside-in signaling and CIB-1 binding to a_IIb, suggesting that an intracellular Ca²⁺ rise induced by fibrinogen binding is required for CIB1 interaction with α_IIb. When CIB1-binding to α_IIb was inhibited by introduction of a function blocking antibody or a synthetic peptide corresponding to α_IIb tail, CIB1 localization at the tip of the filopodia, but filopodia formation was not blocked. This result suggests that interaction of CIB1 with α_IIb is not required for filopodia formation, but is needed for CIB1 accumulation at the filopodia, as well as platelet spreading. Immunoprecipitation experiments showed that during outside-in signaling, CIB1 associates with FAK. Although association of FAK and CIB1 does not require dynamic rearrangement of the cytoskeleton, their accumulation at the filopodia and the activation of FAK is dependent on cytoskeletal rearrangement, as treatment with cytochalasin D after the platelets form filopodia affects CIB1 localization. Interestingly, the interaction of CIB1 with α_IIb upon fibrinogen binding is also necessary for FAK and Src activation. However, Src activity is not required for CIB1 accumulation at the filopodia since this accumulation was not stopped by the Src inhibitor PP2, despite blocking platelet spreading. Our results suggest that during outside-in signaling an intracellular Ca²⁺ rise occurs that facilitates CIB1 interaction with α_IIb. CIB1 recruits FAK to this complex and is localized to the filopodia dependent upon dynamic cytoskeletal rearrangement. Furthermore, activation of Src and FAK requires interaction of CIB1 with α_IIb. Although Src activity is not required for the accumulation of CIB1 at the filopodia, it is required for platelet spreading on immobilized fibrinogen. Thus we provide evidence for initial sequence of outside-in signaling in platelets.