Integrin αIIb cytoplasmic Tail Is Essential for Integrin Outside-in Signaling and Regulation of in Vivo Thrombosis

Platelet aggregation plays an important role in physiological hemostasis and pathological thrombosis. Platelet agonists induce a series of events called inside-out signaling that lead to the activation of integrin αIIbβ3. Fibrinogen binding to the activated integrin relay signals termed as outside-in signaling that regulate thrombus growth and stability. Talin and kindlin bind to the integrin β3 cytoplasmic tail to induce inside-out signaling. Although, integrin αIIb cytoplasmic domain also bind to a number of proteins, its importance in hemostasis or thrombosis is not well understood. Previously, we have identified a calcium- and integrin-binding protein (CIB1) that specifically interacts with the integrin αIIb cytoplasmic tail. Using a novel technique to inhibit interaction of CIB1 with integrin αIIb in intact human platelets, we have shown that CIB1 regulates outside-in signaling through integrin αIIbβ3. Recently, using Cib1-/- mice, we showed that CIB1 is a key regulator of thrombosis in vivo . Interestingly, agonist-induced platelet aggregation and secretion was normal in Cib1-/- platelets. Furthermore, expression or activation of integrin αIIbβ3 was also not affected by Cib1 deficiency, suggesting that integrin inside-out signaling is not affected in Cib1-/- platelets. However, adhesion and spreading on immobilized fibrinogen (Fg) was severely affected in Cib1-/- platelets. When we analyzed the rate of clot retraction, we found that significantly ( P <0.001) delayed clot retraction was observed in Cib1-/- platelets compared to WT littermates, suggesting that integrin outside-in signaling is impaired in the absence of Cib1 . To delineate the molecular mechanism regulated by CIB1 during platelet spreading and clot retraction, we analyzed the known signaling events activated during outside-in signaling. We found that Fg-dependent activation of ERK1 and p38 MAP kinase was significantly reduced in Cib1-/- null platelets. Furthermore, phosphorylation of the myosin light chain was also blocked in Cib1-/- platelets adhered to immobilized Fg. Furthermore, outside-in signaling-dependent tyrosine phosphorylation of β3 was greatly reduced in Cib1-/- platelets. When analyzed for the candidate tyrosine kinase responsible for reduced β3 phosphorylation, both Src and FAK activation was significantly reduced in Cib1-/- platelets. Furthermore, downstream signaling events such as activation of PAK1, PI3K, PDK1, as well as Akt were significantly affected in Cib1-/- platelets adhered to immobilized Fg. To test if impaired inhibition of GSK3-β is the cause of defective outside in signaling in Cib1-/- platelets we treated Cib1-/- platelets with SB216763, a specific GSK3-β inhibitor. We found that inhibition of GSK3-β rescued defective platelet adhesion and clot retraction observed in Cib1-/- platelets. It also rescued activation of p38 and Erk2 activation as well as MLC phosphorylation. However, activation of FAK, Src, PAK1, PI3K, PDK1, and Akt was not rescued, suggesting that these are upstream of GSK3-β. Furthermore, we found that outside-in dependent recruitment of FAK to the integrin-c-Src complex is greatly reduced in the absence of Cib1 suggesting that integrin αIIb cytoplasmic domain serves as a docking site for CIB1 so that it can recruit FAK to the integrin-c-Src complex and propagate outside-in signaling leading to GSK3-β inhibition, which is crucial for thrombus growth and stability. These in vivo and in vitro results clearly show that CIB1 regulates thrombosis by regulating outside-in signaling without affecting inside-out signaling through integrin αIIbβ3. Our results highlight an essential function to integrin αIIb cytoplasmic tail in regulating integrin outside-in signaling and thus thrombus growth and stability. Disclosures No relevant conflicts of interest to declare.