Migfilin Supports Hemostasis And Thrombosis Through Regulating Platelet αⅡbβ3 Outside-in Signaling

Platelet activation plays an important role in physiological hemostasis and pathological thrombosis.Demonstrating molecular mechanism of platelet activation and elaborating the pathogenesis of blood coagulation disorders or thrombotic diseases could provide new strategies for clinical treatment of these kind of disease.The process of thrombosis can be divided into the following three stages:platelet adhesion,platelet aggregation,extension and stable formation of thrombus.IntegrinαIIbβ3 is a central molecule that controls platelet aggregation,secretion and thrombosis as it characteristically transmits signals bidirectionally across the plasma membrane.Therefore,elucidating the regulation mechanism of integrinαIIbβ3 is key to understand platelet biology and thrombotic diseases.Inside-out signaling transmits the activation signaling from stimulant and results in the high affinity binding ofαIIbβ3 to its ligands,which forms bridges to support platelet-vessel wall adhesion and platelet homotypic aggregation.Outside-in signaling,which is initiated by ligand binding andαIIbβ3clustering,is essential for platelet spreading,the contraction of blood colts and thrombus consolidation.PlateletαIIbβ3 signaling pathway is regulated by the binding of integrinβsubunit cytoplasmic domain with different molecules,such as talin and kindlin which can activateαIIbβ3,and filamin A,which has been suggested to be an inhibitor of integrin activation.At present,how most integrin binding proteins dynamically regulatingαIIbβ3signaling is still unknown.Migfilin is a protein widely expressed in cell-cell and cell-ECM connections.It is involved in cell morphology,motility and differentiation.Migfilin has also been reported to have a strong interaction with filamin A,that competitively dissociates filamin A from theβ3 tail,thus promoting the binding of talin-β3 and the activation of integrin.However,in vivo,the role of migfilin in thrombosis and the dynamic regulation of integrin activation remain unclear.We hypothesized that migfilin can promote platelet activation and thrombosis by influencing the effect of integrinαIIbβ3.In this research,we demonstrated the effects of migfilin on platelet function,hemostasis and thrombosis in vivo by generating migfilin^-/-mice.The main results are as follows:（1）It was observed that the absence of migfilin impaired the hemostatic and thrombotic functions of platelet as migfilin^-/-mice displayed a nearly doubled tail-bleeding time and a prolonged occlusion time in Fecl₃-induced mesenteric arteriolar thrombosis;（2）In vitro,microfluidic whole-blood perfusion assay showed that the thrombi formed on collagen surface significantly decreased in migfilin^-/-platelets and displayed a severely compromised stability under the shear stress of arterial flow rate.（3）Migfilin^-/-platelets displayed impaired capability of aggregation due to hampered dense granule secretion.（4）In the bidirectional signaling transduction process of integrinαIIbβ3,the dysfunction of migfilin^-/-platelets appears to be associated with impaired outside-in signaling,rather than inside-out signaling.（5）A synthesized cell-permeable peptide containing migfilin-filamin A binding sequence rescued the defective function and phosphorylation of signaling molecules of migfilin^-/-platelets.（6）In the aspect of molecular mechanism,we found that migfilin hampered the re-association of filamin A andβ3 during outside-in signaling.While,deficiency of migfilin would lead to the block of outside-in signaling transduction and the decrease in the phosphorylation level of downstream molecules.In conclusion,migfilin supports hemostasis and thrombosis through regulating plateletαIIbβ3 outside-in signaling.