Effect Of Oxidized Low Density Lipoprotein On Platelet Activation And Apoptosis

Background: Platelets are nuclear-free fragments of megakaryocytes and their main physiological functions are involved in hemostasis and thrombosis.Platelet activation and aggregation is an important step in thrombosis.Physiological platelet activation has hemostatic function.Thrombosis may occur due to the abnormal activation of platelets,leading to serious consequences such as myocardial infarction or stroke.Hyperlipidemia is an independent risk factor for cardiovascular disease,especially atherosclerosis(AS).High levels of lipids in the blood can damage the arterial endothelium,and sub-endothelial lipid deposits are atherosclerotic the onset of sclerosis thrombosis.Among them,oxidized low-density lipoprotein(ox-LDL)is the main active ingredient of blood lipid,which has the strongest effect on vascular endothelial injury.Therefore,to explore the impact of hyperlipidemia,especially ox-LDL on platelet function is particularly necessary for studying the mechanism of atherothrombosis.Some studies have shown that ox-LDL can promote platelet activation,but the mechanism is not clear.In vitro experiments focused on the ox-LDL on platelet aggregation,activation and apoptosis and its mechanism,to further elaborate ox-LDL on platelet and its mechanism.Objective: To investigate the effect of ox-LDL on platelet activation and apoptosis under in vitro conditions,and to investigate whether ox-LDL promotes thrombus formation by affecting platelet function.Methods: Blood samples were collected from healthy volunteers for washing platelets.The platelets were incubated with different concentrations of ox-LDL(25?g/ml,50?g/ml and 100?g/ml)for 30 minutes to detect the expression of P-selectin and CD40 L,the extent of reactive oxygen species.The platelet depolarization and phosphatidylserine eversion at different time points(0h,24 h,48h,72 h,96h)were detected by co-incubation of washed platelets with ox-LDL(50?g/ml).Platelets were pre-incubated with different concentrations of ox-LDL(25?g/ml,50?g/ml,100?g/ml)for 5 minutes and platelet aggregation was measured after stimulation with ADP(5?g/ml).Platelets were incubated with various concentrations of ox-LDL(25?g/ml,50?g/ml,100?g/ml)for 30 minutes.The content of PI3 k / AKt signal pathway related proteins in platelets was detected by western blot.Result: 1.Effect of ox-LDL on platelet aggregation induced by ADP: Compared with the control group,ADP-induced platelet aggregation was increased after ox-LDL pretreatment(P < 0.05).It was shown that ox-LDL can increase platelet activity and promote ADP-induced platelet aggregation.However,there was no significant difference in the platelet aggregation rate between the three groups of ox-LDL groups(P > 0.05).2.ox-LDL affects the expression of platelet surfactant: Compared with the control group,the expression of P-selectin and CD40 L on platelet surface after ox-LDL treatment was significantly increased(P < 0.05),indicating that ox-LDL can promote platelet activation.However,there was no significant difference in the expression of platelet surface P-selectin and CD40 L between groups with different concentrations of ox-LDL(P > 0.05).3.Effects of ox-LDL on platelet ROS production: Compared with the control group,ox-LDL promoted the production of ROS in platelets(P < 0.05).At the same time,we found that the production of ROS increased with the increase of ox-LDL concentrations in a dose-dependent manner.There was a statistically significant difference between groups(P < 0.05).4.Effect of ox-LDL on platelet mitochondrial membrane potential(??m)and phosphatidylserine(PS)eversion: compared with control group,ox-LDL and platelet co-incubated at 0h,24 h,48h,the degree of polarization and phosphatidylserine valgus changes were not significant.With the increase of co-incubation time,the results of 72 h and 96 h showed that platelet mitochondrial membrane potential depolarization and PS eversion were significantly increased compared with the control group(P < 0.05).Mitochondrial membrane potential depolarization and phosphatidylserine valgus are indicators of platelets apoptosis,suggesting that the effect of ox-LDL on platelet apoptosis in the short term(within 48 h)is not obvious,and platelet stimulation for a long time can cause apoptosis.5.Effect of ox-LDL on the expression of PI3 k,AKt,and p-AKt: Compared with control group,the expression of p-AKt in platelet was significantly increased after ox-LDL co-incubation with platelets(P < 0.05),and a comparison of different ox-LDL concentrations groups showed that the higher the ox-LDL concentration,the more p-AKt expression.The expression of PI3 k and AKt in the ox-LDL group was not significantly different from that in the control group.Conclusion: In vitro experiments demonstrated that co-incubation of ox-LDL with washed platelets increased the ADP-induced maximum platelet aggregation rate,promoted the expression of platelet surface active substances such as P-selectin and CD40 L,and increased the production of reactive oxygen species in platelets.Ox-LDL can make platelets highly active and induce platelet activation.The experimental results show that in vitro,ox-LDL mainly affects the activation of platelets,while the induction of platelet apoptosis is weaker.Therefore,the expression of p-AKt protein in platelets increases,suggesting that ox-LDL may through the PI3k/AKt signaling pathway,increase AKt phosphorylation to promote platelet activation.