CIB1 Regulates Thrombosis through Integrin αaIIbβ3 Outside-in Signaling, but Not Inside-out Signaling.

Abstract 2992

Poster Board II-969

Platelet aggregation plays an important role in physiological hemostasis and pathological thrombosis. Platelet agonists induce a series of signaling events called inside-out that leads to the activation of integrin αaIIbβ3. Upon ligand binding to integrin αaIIbβ3, a cascade of signaling known as outside-in signaling is induced through the integrin that regulates platelet aggregation and clot retraction. The regulation of these signaling events is not well understood. Previously, we had identified a calcium- and integrin-binding protein (CIB1) that specifically interacts with integrin αaIIbβ3. Using a novel technique to inhibit interaction of CIB1 with integrin αaIIbβ3 in intact platelets, we have shown that CIB1 regulates outside-in signaling through integrin αaIIbβ3. Recently, using Cib1-/- mice, we confirmed that CIB1 is a key regulator of hemostasis. FeCl3-induced carotid artery thrombosis, a well recognized in vivo model of thrombosis showed a significantly extended time of occlusion in Cib1-/- mice compared to wild type (WT) mice. Since CIB1 is expressed both in the endothelium and in platelets, the observed defect could arise from the lack of CIB1 in either cell types. To identify the specific involvement of platelets, we performed a collagen-epinephrine-induced pulmonary thromboembolism assay. In this assay, pulmonary vessel occlusion occurs due to platelet thrombus formation without injury to the endothelium. We found no difference in the number of Cib1-/- mice and WT mice that died within two minutes (n=20). When analyzed for the extent of pulmonary vascular occlusion by Evans blue exclusion as well as histochemical analysis, no difference between Cib1-/- and WT mice was observed. Consistent with this finding, agonist-induced platelet aggregation and secretion was normal in Cib1-/- platelets. Furthermore, expression or activation of integrin αaIIbβ3 was also not affected by Cib1 deficiency. When we analyzed the rate of clot retraction, we found that incomplete and significantly (P<0.001) delayed clot retraction was observed in Cib1-/- platelets compared to WT littermates. To delineate the molecular mechanism regulated by CIB1, we analyzed the known signaling cascade involved in clot retraction. We found that outside-in signaling-induced activation of ERK1 and p38 MAP kinase was significantly reduced in Cib1-/- null platelets. Furthermore, phosphorylation of the myosin light chain during outside-in signaling was blocked in Cib1-/- platelets. Interestingly, agonist-induced tyrosine phosphorylation of integrin β3 chain was unaffected in Cib1-/- platelets, but Mn2+-induced outside-in signaling-dependent tyrosine phosphorylation of β3 was greatly reduced. When analyzed for the candidate tyrosine kinase responsible for β3 phosphorylation, both Src and FAK activation was significantly reduced in Cib1-/- platelets. 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 αaIIbβ3.