Mechanisms Of Platelet Involved In Venous Thromboembolism Mice Through PDI-GRP94-GPⅡb/Ⅲa Signaling Pathway

BackgroundWith an aging population and changes in lifestyle and dietary habits,the accidence of cardiovascular diseases in China remain high and have surpassed malignant tumours to become the leading cause of death in China,and venous thromboembolism(VTE)is the third most fatal cardiovascular disease in addition to coronary heart disease and stroke.It is now thought that there is a similar pathogenesis between venous and arterial thrombosis,with coagulation and platelet activation being involved in both arterial and venous thrombosis.The main physiological function of platelets is to adhere to the vessel wall in the event of vascular injury,avoiding bleeding and maintaining normal blood flow.As research into the mechanisms of platelet activation has progressed,the importance of antiplatelet therapy in the prevention and control of thrombosis has become better understood.Existing antiplatelet agents,although helpful in reducing thrombotic events,suffer from an increased risk of bleeding and inconsistent efficacy.Therefore,the search for safe and effective new antiplatelet agents remains a hot topic in cardiovascular research.The GPⅡb/Ⅲa receptor is the final pathway for platelet activation,and it has been shown that GPⅡb/Ⅲa activation is regulated by protein disulfide isomerase(PDI),an enzyme that catalyzes the oxidation,.Inhibition of the enzymatic activity of PDI can prevent platelet aggregation and fibrin production,so PDI is a potential target for antithrombotic drug research.Platelet surface GPⅡb/Ⅲa expression is regulated by glucose-regulated protein 94(GRP94),which plays a role in the signaling and activation of GPⅡb/Ⅲa receptors.GRP94 protein relies primarily on C-terminal disulfide bonding to form homodimers for its biological activity,and PDI regulates cellular function by accomplishing disulfide bond transfer and exchange.PDI regulation?No studies are available.However,it is certain that the distribution and changes of PDI and GRP94 in the cell are consistent.Intercellular communication is a fundamental phenomenon in multicellular organisms,and signals are transmitted in a variety of ways,including by secreting microvesicles.Exosomes are cell membrane-derived spherical vesicles that form under conditions of cellular stress and contain active components of parental cells such as proteins.These genetic materials from the parental cells give exosomes their biomarker characteristics.Once attached to the target cell,exosomes can mediate signalling via receptor connectivity,or internalise via endocytosis,or even fuse with the target cell membrane and deliver its contents to the cytoplasm,thereby altering the physiological state of the recipient cell.Vascular endothelial injury is closely associated with the level of endothelial microparticle(EMP)shed by endothelial cell activation.Recent studies have shown that exosomes are significantly elevated in thrombophilia,particularly EMP.The initiation of venous thrombosis involves stagnation of blood flow,a hypercoagulable state of blood and damage to the vascular endothelium.Endothelial cells are in the innermost layer of the blood vessel and provide the surface for thrombosis.The mechanisms involved in thrombosis include the integrity of the endothelial barrier,the interaction between endothelial cells and blood cells,and the imbalance in the release of pro/anti-coagulant substances by endothelial cell activation.Reducing the risk of bleeding during the use of traditional antithrombotic drugs by developing drugs that target endothelial cells and their platelets is a new strategy for antithrombosis.However it is not clear whether platelet activation involves endothelium-derived exosomes.Therefore,the aim of this experiment was to investigate the main mechanisms by which the massive secretion of PDI-carrying endothelium-derived exosomes regulates platelet GRP94 expression and GPⅡb/Ⅲcontent expression and conformational changes under conditions of venous endothelial injury,thereby affecting thrombosis.In this study,we established an animal model of inferior vena cava ligation in C57/BL6 mice to construct an in vivo environment for venous endothelial cell injury;venous endothelial cells were placed under hypoxic conditions to simulate the in vivo environment of venous endothelial injury and to investigate the effect of PDI on platelet activation and thrombosis and its possible mechanisms.Objectives1.To study the mechanisms involved in the development of venous thrombosis by endothelium-derived exosomes carrying PDI.2.To clarify the role of antiplatelet activation targeting the PDI-GRP94-GPⅡb/Ⅲa signalling pathway in the prevention and treatment of VTE.Materials and Methods1.Animal modelingAfter the mice were anesthetized,a 30 G insulin needle was placed in parallel with the inferior vena cava,and then a 7-0 prolene suture was ligated 1 cm below the confluence of the right renal vein into the inferior vena cava,and the needle was withdrawn and sutured off the abdomen.2.Animal groupingInferior vena cava ligation was perfotmed in mice.Ten-week-old male C57/BL6 wild-type mice were randomly divided into sham group(n=7),VTE group(n=9),VTE+PDI inhibitor group(n=8),VTE+GRP94 inhibitor group(n=9).All were subjected to inferior vena cava ligation or sham surgery at 13 weeks and the mice were executed 2 days after surgery.3.Histopathological testingThe thrombus tissue sections were stained with hematoxylin-eosin to observe morphological thrombus;immunohistochemical staining for CD41 to calculate the expression of GPⅡb/Ⅲa per unit area;and two-colour immunofluorescence staining for GRP94 and CD41 to calculate the fluorescence intensity of GRP94 on activated platelets.4.Flow cytometry testingPlatelets were extracted from peripheral blood of mice with spent platelet plasma and stained for CD 144 and PDI by two-colour flow cytometry to observe the expression of PDI on EMP in plasma;platelets were extracted from peripheral blood of mice and stained for CD62P,CD41 and CD61 by three-colour flow cytometry to observe platelet activation and GPⅡb/Ⅲa receptor expression;platelets were co-cultured with venous endothelial cells After co-culture for 10min,trichrome flow cytometry staining for CD62P,CD41 and CD61 was performed to observe platelet activation and GPⅡb/Ⅲa receptor expression.5.Grouping and modelling of cell experimentsHuman umbilical vein endothelial cells were cultured and when the vein endothelial cells were cultured to the 3rd generation,the medium was changed to a special medium for endothelial cells without exosomal serum and the culture dish was placed in a Plexiglas exposure chamber and alternately charged with a hypoxia gas mixture(1%O2,5%CO2 and equilibrium N2,hypoxia,300 seconds)or a normoxic gas mixture(21%O2,5%CO2 and equilibrium N2,oxygenated,600 seconds)Incubation for 1 day.Randomly divided into 4 group:control,hypoxia,hypoxia+PDI inhibitor,and hypoxia+GRP94 inhibitor group.6.Exosome extraction and PKH26 stainingExosomes were extracted from the supernatant of venous endothelial cells by ultracentrifugation;the exosomes were stained with PKH26 and co-incubated with platelets for 10 min,and the endocytosis process of exosomes was observed by laser confocal microscopy.7.Fluorescence resonance energy transfer(FRET)GFP-GPIIb(green fluorescence)and mCherry-GPⅢa(red fluorescence)overexpression plasmids were transfected into 293T cells,co-incubated with exosomes,and the fluorescence energy resonance transfer efficiency N-FRET values were observed by laser confocal microscopy.8.Platelet aggregation assayPlatelets were co-incubated with exosomes for 10min to compare the platelet aggregation rates before and after intervention with PDI inhibitor and GRP94 inhibitor.9.Protein molecular assaysThe expression of platelet PDI content was measured by Western blot technique;the expression of GRP94 dimer content in platelets was measured by non-denaturing electrophoresis technique.Results1.Results of animal experiments2.1 VTE mice Model EstablishmentNo thrombosis was found in the control group.The mould formation rate in the VTE group was 71%and the mean standard deviation of thrombus length was 3.7±3.6 mm.2.2 VTE information positively correlate with the degree of platelet activationThe results of immunohistochemical staining revealed that the platelet activation indicator CD41 was widely distributed on the surface of the thrombus in the VTE group;the results of flow cytometry showed that the expression level of platelet CD62p was significantly higher in the VTE group compared with the control group;the content of CD41 was significantly increased in the VTE group;the results of ELISA showed that the platelet activation indicator CD41 was significantly increased in the peripheral blood of mice in the VTE group compared with the control group The ELISA results showed that the levels of P-selectin was significantly increased in the peripheral blood of the VTE group compared with the control group.This indicates that the degree of platelet activation is increased during thrombosis.2.3 PDI and GRP94 reduce plasma clotting factor levelsCompared to the control group,P-selectin was significantly higher in the VTE group,VTE+PDI inhibitor group and VTE+GRP94 inhibitor group,and P-selectin was significantly lower in the VTE+PDI inhibitor group than in the VTE group.Compared to the control group,von willebrand factor(vWF)was elevated in the VTE group,while vWF was significantly lower in the VTE+GRP94 inhibitor group.In addition,fibrinogen(Fib)levels were significantly lower in the VTE group than in the control group,and Fib levels were increased in the VTE+GRP94 inhibitor group and the VTE+PDI inhibitor group compared to the VTE group,but the differences were not statistically significant.Outcome 1.2 and Outcome 1.3 showed that the PDI inhibitor and GRP94 inhibitor excluded the effect of coagulation levels on thrombosis,making thrombosis only related to platelet activation.2.4 PDI and GRP94 positively correlate with the degree of platelet activation on the surface of the thrombusImmunohistochemical staining revealed that the mean optical density expression of GPⅡb/Ⅲa was significantly lower in the VTE+PDI inhibitor group and the VTE+GRP94 inhibitor group compared to the VTE group,indicating that inhibition of PDI and GRP94 resulted in reduced platelet activation.2.5 PDI and GRP94 correlate with blood-derived platelet GPⅡb/Ⅲa levelsFlow cytometry results showed that platelet CD61 expression was decreased in the VTE+PDI inhibitor group and the VTE+GRP94 inhibitor group compared to the VTE group.2.6 PDI and GRP94 positively correlate with platelet GRP94 levels on thrombus surfacesCD41,GRP94 immunodouble fluorescence staining revealed that GRP94 fluorescence intensity was significantly suppressed in thrombus of VTE+PDI inhibitor group and VTE+GRP94 inhibitor group compared to control group,indicating that PDI inhibitor and GRP94 inhibitor significantly inhibited GRP94 expression during platelet activation and thrombosis.2.7 EMP carries a significantly higher level of PDIThe PDI content on EMP was significantly increased in the VTE group compared to the control group,and the PDI content on EMP was significantly increased in the VTE+PDI inhibitor group compared to the VTE group,while the PDI content on EMP was not significantly downregulated in the VTE+GRP94 inhibitor group.The PDI content on EMP increased after inferior vena cava ligation,and the PDI inhibitor could inhibit the increase in PDI content,but was not affected by the GRP94 inhibitor.2.Results of cellular experiments2.1 EMP can be taken up by plateletsAfter 10 min incubation of PKH26-labelled EMP with platelets,red,scattered fluorescent dots are visible in the platelets,indicating that the EMP can be taken up by the platelets.2.2 PDI induces platelet GRP94 homodimer formationThe endothelial cells and platelets were co-cultured for 10 min.The PDI content of platelets in the hypoxia group was significantly higher,and that the PDI content of platelets in the hypoxia+PDI inhibitor group was significantly lower than that in the hypoxia group.The non-denaturing electrophoresis results showed that the GRP94 dimer content was significantly higher in the hypoxia group compared to the control group,and the GRP94 dimer content was significantly lower in the hypoxia+PDI inhibitor group and the hypoxia+GRP94 inhibitor group compared to the hypoxia group,all these results indicated that the platelet GRP94 homodimer was regulated by the PDI content.2.3 PDI and GRP94 positively correlate with platelet aggregation levelsExosomes in the hypoxia group significantly increased platelet aggregation rates compared to the control group;platelet aggregation rates were significantly lower in the PDI inhibitor group and GRP94 inhibitor group compared to the control group,indicating a positive correlation between PDI and GRP94 and platelet aggregation levels.2.4 PDI regulates the conformational changes of GPⅡb/Ⅲa on the platelet surfaceN-FRET was reduced in 293T cells with the addition of hypoxia-derived EMP compared to controls,which,combined with the results of animal experiment 1.5,suggests that PDI affects the spatial conformational changes of GPⅡb/Ⅲa.Conclusions1.The involvement of platelet activation in the process of venous thromboembolism formation;2.Activation of platelets by PDI-carrying exosomes released by endothelial cell injury following hypoxia to promote venous thromboembolism formation;3.EMP-PDI,when taken up by platelets,promotes the formation of GRP94 homodimers within platelets,increases the expression of GPⅡb/Ⅲa on the platelet surface and thus activates platelets;4.PDI catalyzes the formation of disulfide bonds between GRP94 and the GPⅡb/Ⅲa receptor,leading to the formation of a dimer of GRP94 and a change in the GPⅡb/Ⅲa spatial conformation.5.The PDI-GRP94-GPⅡb/Ⅲa receptor signalling pathway plays an important role in platelet activation for venous thromboembolism formation and may be targeted to prevent venous thrombosis.