The Role Of TRAF3 And VPS33B In Platelet Activation And Thrombosis And Its Mechanism

Part I Preparation of the TRAF3 and VPS33B platelet-specific knock out miceAims:We established the TRAF3 and VPS33B platelets specific knock out mice, to study the effect of TRAF3 and VPS33B on platelet function.Method:The VPSSSBfl/fl mouse, TRAFSfl/fl mouse and Pf4 Cre mouse was previously constructed in our lab. The results of RT-PCR testing show that Cre genes were only expressed in the platelets. The Pf4 Cre mouse were bred with VPSSSBfl/fl mouse or TRAF3fl/fl mouse. Then we hired the PCR and western blotting to test the VPS33B, TRAF3 and Cre gene and protein.Results:Through several generations of breeding, we have established a reliable and TRAF3 or VPS33B platelet-specific knockout mice model, and in subsequent experiments, take the mouse's tail to detect gene knockout mice and their heterozygous control by genomic PCR.Conclusions:In summary, conditional gene knockout mediated by LoxP/Cre system could overcome some drawbacks of conventional gene knockout technology, such as the high lethality caused by TRAF3 or VPS33B gene knockout. We successfully established TRAF3 or VPS33B platelet-specific knockout mouse for further study.Part? The Effect of TRAF3 knockout on platelet activation and thrombosis and its mechanismAims:CD40 ligand (CD40L), a member of the tumor necrosis factor family (TNF) superfamily, can bind to CD40 and potentiate platelet activation and thrombus formation. A family of proteins called TNF receptor associated factors (TRAFs) plays key roles in mediating CD40 ligand (CD40L)-CD40 signaling. Here, we investigated the role of TRAF3 in platelet activation and thrombus formation using a platelet-specific knock out mice.Method:We hired different agonists, such as collagen, thrombin and Par4, to eaxaminate platelets aggregation and secretion of TRAF3"7" mice and wildtype mice. Then we test the platelets aggregation and secretion of TRAF3V'mice and wildtype mice with additional CD40L. We detect the expression of integrin 03 and GPVI of TRAF3-/- mice and wildtype mice by By flow cytometry and western blotting. Detection the tail bleedingtime and thrombosis formation changes over time of TRAF3-/- mice and wildtype mice.Results:Here we show that platelet also express TRAF3, which plays a negative role in regulating platelet activation. Thrombin-or collagen-induced platelet aggregation and secretion are increased in TRAF3 knockout mice. The expression level of collagen receptor GPVI, integrin ??b?3, and TRAF2 in platelets was not affected by deletion of TRAF3, suggesting that increased platelet activation in the TRAF3 knockout mice was not due to increased expression level of the platelet receptors or TRAF2. Time to the formation of thrombi in a FeC13-induced arterial thrombosis model was significantly shortened in the TRAF3 knockout mice. However, mouse tail-bleeding times were not affected by deletion of TRAF3.Conclusions:Thus, TRAF3 plays a negative role in platelet activation and in thrombus formation in vivo.Part ? The Effect of VPS33B knockout on platelet activation and thrombosis and its mechanismAims:Integrins are heterodimeric (?/?) membrane proteins that play fundamental roles in many biological processes, for example, cell adhesion and spreading, which are important for platelet function and hemostasis. VPS33B, a member of the Secl/Munc18 family, is a novel integrin-binding protein. However, the role of VPS33B in platelet activation and thrombosis are not completely understood. Here, we investigate the role of VPS33B in platelet activation and thrombosis using VPS33B platelet-specific knock out mice.Methods:We hired different agonists, such as collagen, thrombin, to eaxaminate platelets aggregation and secretion of VPS33B-/- mice and wildtype mice. Then we detected the expression of integrin ?3, pf4, VWF, fibronectin of VPS33B-/- mice and wildtype mice by by flow cytometry or western blotting. We also examined the platelet adhesion, platelet spreading to immobilized fibrinogen and clot retraction of VPS33B-/-mice and wildtype mice.We detected the tail bleedingtime and thrombosis formation changes over time of VPS33B-/- mice and wildtype mice. We used the confocal microscope to colocalize ?IIb?3 and VPS33B in CHO cells and Co-immunoprecipitation to identify the interaction of VPS33B protein and integrin protein.Results:We show that VPS33B, a member of the Secl/Munc18 family, binds directly to the integrin?subunit. Overexpression of VPS33B in Chinese hamster ovary cells potentiated allb?3 outside-in signaling but not inside-out signaling. Platelets, from megakaryocyte- and platelet-specific VPS33B conditional knockout mice, had normal morphology, yet their spreading on fibrinogen was impaired and they failed to support clot retraction. Platelet aggregation and ATP secretion in response to low-dose agonists were reduced in the VPS33B knockout mice. ?llb?3-mediated endocytosis of fibrinogen was also defective. Tail bleeding times and times to occlusion in FeC13-induced thrombosis model were prolonged in the VPS33B knockout mice. Furthermore, VPS33B acted upstream of the RhoAROCK-MLC and Racl-dependent pathways that lead to clot retraction and cell spreading, respectively.Conclusions:Our work demonstrates that vesicular trafficking complexes, containing VPS33B, are a novel class of modifiers of integrin function. Our data also provide insights into the molecular mechanism and treatment of arthrogryposis, renal dysfunction, and cholestasis syndrome.