CIB1 Regulates Hemostasis through Integrin Outside-in Signaling

On circulating platelets, integrin αIIbβ3, the fibrinogen receptor, is in a low affinity state unable to bind soluble ligands. Agonist-dependent inside-out signaling is necessary for activation of the integrin αIIbβ3 in order to bind ligands such as fibrinogen (Fg). Ligand binding induces a cascade of signaling that culminates in platelet aggregation, leading to hemostatic plug formation and clot retraction. Although it is well known that signaling through integrin αIIbβ3 is necessary, the mechanism of clot retraction is not well understood. Previously, we have identified a calcium- and integrin-binding protein 1 (CIB1) that specifically interacts with integrin αIIbβ3. One study implicated CIB1 as an endogenous inhibitor of integrin αIIbβ3 activation, while another study suggested CIB1 to be an activator of inside-out signaling. Using a novel technique to inhibit interaction of CIB1 with integrin in vivo, we have shown that CIB1 regulates outside-in signaling through integrin αIIbβ3. Definitive proof for the role of CIB1 in platelets remained absent, however. Here, using Cib1 null platelets, we show for the first time that CIB1 is a key regulator of hemostasis. Genetic ablation of CIB1 had no effect on agonist-induced platelet aggregation, excluding the role for CIB1 in inside-out signaling. However, deletion of the Cib1 gene significantly increased mouse bleeding times. More than 50% of the mice also showed a rebleeding phenotype, suggesting a defect in clot stability. Incomplete and significantly delayed clot retraction was observed in Cib1 null platelets compared to wildtype littermates. The absence of CIB1 also affects the ability of platelets to spread on immobilized Fg. Platelet adhesion and subsequent filopodia formation was not affected by the absence of CIB1. Platelet spreading can be restored by addition of exogenous ADP, suggesting that the defect is not in the ability of the platelet to spread. These in vivo and in vitro results clearly show that CIB1 regulates hemostasis by supporting transition of platelets from filopodial stage to fully spread morphology during outside-in signaling through integrin αIIbβ3.