The Study Of Miltirone On Antiplatelet And Antithrombotic Effect

Chapter 1 Effects of miltirone on platelet functionAims: The increasing risk of thrombotic diseases requires more appropriate anti-platelet therapies,and there are many deficiencies in currently available drugs.Excessive activation of platelets is an important factor in thrombosis,searching for anti-platelet ingredients from traditional Chinese medicine is a great breakthrough.In the previous experiments,we found an anti-platelet active ingredient from Salvia miltiorrhiza,but its function and mechanism are still unclear.This experiment mainly studied the effects of miltirone on platelet function in mice and studied its possible mechanism.Method: Evaluation of the effects of miltirone on platelet aggregation induced by different agonists(collagen,CRP,thrombin,ADP,and arachidonic acid);evaluation of the effects of miltirone on platelet releasewhich was induced by collagen and thrombin;Flow cytometry was used to evaluate the expression of p-selectin and JON/A on platelets which were incubated with or without miltirone and induced with thrombin;the microfluorescence technique was used to detect the effect of miltirone on the adhesion of platelets on the collagen surface and the spreading on fibrinogen.To observe the effect of miltirone on platelet by using thrombin-induced clot retraction.Flow cytometry was used to evaluate the effects of miltirone on platelets ROS induced by different agonists and intracellular Ca2+ levels in platelets.The effect of miltirone on the platelet-related signaling pathway was detected by Western blotting.Results:: Miltirone at a final concentration of 2 ?M,4 ?M and 8 ?M could inhibit platelet aggregation induced by collagen,CRP,and thrombin in a concentration-dependent manner,whereas miltirone showed no effect on platelets which were induced by ADP(4 ?M,10 ?M)and arachidonic acid(50 ?M,100 ?M).Miltirone can inhibit the release of platelet ATP caused by collagen and thrombin and the expression of platelet p-selectin and JON/A induced by collagen.Miltirone can reduce the adhesion of platelets on the surface of collagen and the spreading on fibrinogen surface.Miltirone could significantly inhibit the retraction of platelets but did not affect intracellular calcium release.Western blotting results showed that miltirone could inhibit the phosphorylation of PLC?2,PKC,Akt,GSK3?,ERK1/2 and other proteins downstream of the collagen receptor.Miltirone could reduce the phosphorylation of Src and FAK in the integrin ?IIb?3 mediated outside-in signaling,however,it did not inhibit the ?3 phosphorylation.Conclusion: Miltirone may inhibit platelet "inside-out" and "outside-in" signal pathways by influencing platelet PLC?2/PKC/ERK1/2,PI3K/Akt,Src/FAK signaling,thus inhibiting platelet activation and aggregation.Due to these factors,miltirone exhibited antiplatelet effects.Chapter 2 Effects of miltirone on thrombosis Aim: The previous chapter showed that miltirone possessed great anti-platelet activity,even the onset concentration is lower.However,frequently,after the active ingredients are metabolized in the body,their effects are significantly different from the in vitro experiments.Therefore,we studied the anti-platelet effect of miltirone in vivo and observed the anti-thrombosis effect in vivo.Method: To detect the influence of miltirone on platelet aggregation induced by different agonists in vivo.To establishe a mouse model of pulmonary embolism by combining collagen and adrenaline,and then to observe the effect of miltirone on the thrombosis of the model after tail vein administration.To establish ferric chloride-induced mouse model of carotid artery thrombosis and observe the changes in occlusion time after administration of miltirone.To establish deep vein thrombosis model by the method of inferior vena cava stenosis,then observe the thrombosis rate,the size of thrombus,and the length of thrombus after the administration of miltirione.To observe the effect of miltirone on the bleeding time of mice.Results: After 0.5h of tail vein intravenous administration of miltirone,the platelet aggregation induced by collagen and thrombin was significantly decreased compared with the control group.Miltirone could reduce the formation of microthrombi in the lungs of mice caused by collagen and epinephrine.Miltirone could prolong the occlusion time and inhibit the length of venous thrombi in DVT mice.Also,it could slightly prolong the bleeding time.Conclusion: Miltirone could inhibit thrombosis in experimental mice model and slightly prolonged the bleeding time.Chapter 3 The effect of platelet microparticles(PMPs)onthe formation of deep vein thrombosis(DVT)Aim:The pathogenesis of venous thrombosis is significantly different from that of arterial thrombosis,and platelets may play different roles in these two thrombotic processes.PMPs with procoagulant ability may be associated with platelets and deep vein thrombosis and play an important role in the development of venous thrombosis.To further investigate the effect of miltirone on deep vein thrombosis,we use PMPs as pointcut.Here,we investigated the possible link between platelets and deep vein thrombosis by studying the effect of PMPs on deep vein thrombosis.Method: To establish the assay of PMPs by flow cytometry;To investigate platelet aggregation of PMPs inducing platelets using turbidimetric study;Flow cytometry was used to detect PMPs in peripheral blood of deep venous thrombosis model.Flow cytometry was used to study the effect of PMPs on the activation of neutrophils in peripheral blood of mice.The effect of PMPs on NETs in deep vein thrombosis in mice was studied by immunofluorescence.Result: PMPs do not cause platelet aggregation by themselves,but they can promote collagen-induced platelet aggregation.PMPs can promote deep vein thrombosis caused by inferior vena cava stenosis.The levels of PMPs in peripheral blood of DVT mice were increased.Mice treated with PMPs got an Increased expression of NETs in deep vein thrombosis.Conclusion: PMPs may promote the formation of deep vein thrombosis by activating neutrophils,promoting their release of NETs.The following project will study the effects of miltirone on the production of PMPs and the formation of neutrophil NETs.