| BackgroundCardiovascular disease and diabetes is the main cause of death and disease burden in the world currently,China has the largest number of people with diabetes mellitus(DM)in the world.DM is the main death cause in patients with atherosclerosis,and the mortality of cardiovascular disease in DM is 2～4 times of non-diabetes,meanwhile the vast majority of acute cardiovascular events are associated with thrombosis.Platelet activation is the start factors in the process of thrombosis.GPⅡb/Ⅲa activation is the final common step of platelet activation,and it is closely related with thrombosis and platelet aggregation.However,the fundamental mechanism that underlies platelet activation in DM remains incompletely understood,and platelet activation and the endothelial cells injury may play an important role.Antiplatelet therapy could reduce the incidence of cardiovascular events in DM patients significantly,but even under the dual antiplatelet therapy,platelet aggregation and activation of DM patients are still increasing,so it is necessary to find the best antiplatelet therapy to reduce the risk of cardiovascular disease in DM patients,so as to reduce mortality.The recent studies emphasized that the role of protein disulfide isomerase(PDI)and microparticles in the progress of platelet activation.PDI can regulate the rearrangement of disulfide bonds to make GPⅡb/Ⅲa turn to a high affinity from a low affinity.When the spatial configuration changed,in which condition GPⅡb/Ⅲa could bind to fibrinogen and promote thrombosis.Under the condition of DM,endothelial cells were injured,and released more endothelial microparticles(EMP).Then the platelet was activated,and it released more platelet derived microparticles(PMP)in which PDI is carried.We speculate that under the condition of DM,endothelial cells released more EMP,which carrying PDI.And PDI can combine with platelet by GPⅡb/Ⅲa which is on the platelet surface.Then the platelet was activated,and released more PMP carrying PDI,to realize platelet cascade amplifying signal conduction.Meanwhile,the platelet microparticle-associated PDI weaken the role of insulin in anti-platelet activation.Therefore,the aggregation and adhesion of platelet significantly increased under the condition of DM.Objectives(1)To study platelet activity of DM mice;(2)To investigate the changes of blood coagulation status of DM mice;(3)To explore the changes of EMP and EMP-PDI in plasma when the endothelial cells were damaged in DM mice.(4)To explore mechanism of EMP-PDI promoting platelet activation of DM mice.Methods and materials1.Animal experiment:Sixty 4-week-old male ApoE-/-mice were randomized into 2 groups:chow group(n=30)and diabetes group(n=30)after intraperitoneal glucose tolerance test(IPGTT).The mice were fed on chow diet or high fat and sugar diet respectively for 6 weeks.IPGTT was performed to ensure the appearance of insulin resistance(IR).Those IR mice were injected once with low dose of STZ(intraperitoneal at 85mg-90mg/kg).After 2 weeks,random blood glucose of the mice more than 11.1mmol/1 were regarded as type 2 diabetic mellitus mouse model.Then the mice were randomized into 4 groups:the chow,the chow + rutin,the DM,the DM + rutin.The chow group and the DM group were given saline by gavage once a day for 8 weeks,and the chow + rutin group and the DM + rutin group were given rutin(80mg/kg)by gavage once a day for 8 weeks.We obtained plasma and platelet of mice.2.Monitor body weight of mice every week.3.Isolate plasma.Draw blood from the heart of mice to anticoagulation tubes with sodium citrate.Isolate plasma by centrifugation at 3000rpm for 10 min at room temperature to get platelet-poor plasma(PPP).4.Isolate mice platelet.Draw blood from the heart of mice to anticoagulation tubes with sodium citrate.Platelet-rich plasma(PRP)was prepared by centrifugation at 800rpm for 10 min at room temperature,and platelets were isolated from PRP after centrifugation at 800g for 10 min.Then platelets were washed and resuspended in modified Tyrode’s buffer.Platelet concentration was adjusted to 1×106/mL.5.Flow cytometry.Add appropriate amount of fluorescent antibody of tested factors and sample in a 100μL system based on PBS to BD tube.Incubate antibody and sample in the dark at room temperature for 15-30 min.Fixed by 200μL 4%paraformaldehyde and detect.6.Enzyme linked immunosorbent assay(ELISA).The quantity of PDI,soluble P-selectin,vWF,fibrinogen in plasma were tested step by step according to the manual.Results1.Results of intraperitoneal glucose tolerance test(IPGTT)of mice.At the age of 4 weeks,no significant difference was detected between the two groups.After high fat diet for 6 weeks,diabetic mice showed significant increased blood glucose(P<0.05).After administration of STZ,diabetic mice showed significant higher blood glucose than chow-diet mice(P<0.05).No significant difference were detected among the chow-diet mice with different ages(P>0.05).Significant differences were detected among the diabetic mice with different ages(P<0.05).2.Comparison of body weight of mice.At the age of 4 weeks,no significant difference was detected between the two groups.After high fat diet for 4 weeks,diabetic mice show significant increased body weight(P<0.05).However,no significant difference was detected between the two groups at two weeks after administration of STZ(P>0.05).At week 20,body weight of diabetic mice increased than chow-diet mice(P<0.05).3.Biochemical results of mice.Compared with the DM group,the level of total cholesterol(TC),triglyceride(TG)and low density lipoprotein cholesterol(LDL-C)in chow group and chow + rutin group decreased(P<0.05～0.001).The difference of high density lipoprotein cholesterol(HDL-C)level of in these groups didn’t reach a statistical significance(P<0.05).4.The quantity of fibrinogen in all groups had no difference,and the quantity of vWF in DM group increased.ELISA detected the quantity of fibrinogen and vWF in mice plasma.The difference of fibrinogen quantity of in these groups didn’t reach a statistical significance.Compared with the DM group,the quantity of vWF in plasma in the other groups decreased(P<0.05～0.01).The difference between the other groups didn’t reach a statistical significance.5.The platelet activated more in DM group.The level of platelet P-selectin and GPIIb/IIIa in DM group increased(P<0.05～0.001),the level of platelet GPIIb/IIIa of chow + rutin group decreased(P<0.05).Compared with the DM group,the level of platelet P-selectin and GPIIb/IIIa of DM + rutin group decreased(P<0.01-0.001).Compared with DM group,the level of PMA and PNA of chow + rutin group and DM + rutin decreased(P<0.05～0.001).Compared with the chow group the level of PMA and PNA in chow + rutin group decreased(P<0.05～0.01)6.The quantity of sP-sel in DM group increased.ELISA detected the quantity of sP-selectin in mice plasma.Compared with the DM group,the quantity of sP-selectin in plasma in the other groups decreased(P<0.05-0.001).The difference between the other groups didn’t reach a statistical significance.7.The quantity of CD144+ EMP in DM group increased.PDI was carried by EMP,and the quantity of EMP-PDI in DM group increasedCompared with DM group,quantity of CD144+EMP in the other groups decreased(P<0.05),while difference between the other groups didn’t reach a statistical significance.Bicolor flow cytometry of CD144-APC and PDI-FITC confirmed EMP carrying PDI,and compared with DM group,quantity of EMP-PDI in the other groups decreased(P<0.05～0.01).8.The quantity of PDI in DM group increased.Enzyme linked immunosorbentassay(ELISA)detected the quantity of’ PDI in mice plasma.Compared with the DM group,the quantity of PDI in plasma in the other groups decreased(P<0.05-0.001).And compared with the chow + rutin group,the quantity of PDI in the other groups increased(P<0.05～0.001).9.Isolate and identification of EMPCD144 positive represented endothelial cell derivation microparticles.We used magnetic activated cell sorting and ultracentrifugation to isolate CD 144+ EMP.Define gate of microparticles using standard fluorescent microspheres with 100nm and 1000nm.Transmission electron microscope(TEM)images demonstrated intact EMP of various sizes,and the diameter was bigger than 100nm.10.EMP activated platelet.RL90 and rutin inhibited PDI-dependent platelet activation induced by EMP.Stimulate platelet of WT mice with EMP in different conditions to detect platelet activation.Compared with EMP group,the level of platelet GPⅡb/Ⅲa and P-selectin of other groups decreased(P<0.05～0.001).11.EMP of DM group increased platelet activation.Stimulate platelet of WT mice with EMP in different group to detect platelet activation.Compared with ADP + EMP of DM group,the level of platelet GPⅡb/Ⅲa of chow group increased.Compared with EMP of DM group,the level of platelet GPⅡb/Ⅲa and P-selectin of other groups decreased(P<0.05～0.001).12.Proximity Ligation Assay(PLA)confirmed that EMP-PDI can combine with platelet GPⅡb/Ⅲa.Isolate mice platelet and EMP.Then they were incubated by antibody and PLA probe,ligated,amplificated and mounted step by step according to the manual.Then the staining results were observed with fluorescence microscope.We detected the red light to confirm that EMP-PDI can combine with platelet GPⅡb/Ⅲa.Conclusions1.The level of platelet GPⅡb/Ⅲa and P-selectin increased,and the level of PNA in plasma increased,platelet activation increased in DM mice.2.The level of TC,TG,LDL-C,sP-sel and vWF in DM mice plasma increased,the blood was on high coagulation state.3.EMP,PDI and EMP-PDI in DM mice plasma increased,the vascular endothelium was damaged.4.Endothelial cells released EMP when they were damaged,and EMP interacts with GPⅡb/Ⅲa which is on the surface of platelet to activate platelet.This process was enhanced in DM mice.5.Rutin inhibited PDI-dependent EMP-induced platelet activation partly,and this process was enhanced in DM mice. |
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