The Effect Of Leptin On Platelet Function And The Underlying Mechanism

Part ? The effect of leptin on platelet functionObjective: Platelets in normal circulation are in a nonadherent “resting” state and become activated at sites of vascular injury after exposure to immobilized adhesive proteins,and then formed the thrombus to participate the physiological hemostasis.However,in patients with pathological conditions such as obesity,which is a chronic low-grade inflammation environment,associated with abnormal platelets,leading tovenous thromboembolism,myocardial infarction,stroke and so on.Therefore,it is an urgent task to study the relationship between obesity and platelets,to find out the cause of platelet activation and its mechanism.Leptin is derived from the adipocyte and maintains the balance of energy metabolism.However,previous studies have shown that leptin levels are higher in obese compared with normal,and leptin promotes platelets aggregation induced by ADP.In vivo,leptin promotes the formation of venous and arterial thrombosis.While the reason why leptin have positive effect on platelets is still unclear.In this study,we focused on the effect of leptin on platelets and tried to find its mechanism,so as to provide a theoretical basis for the clinical treatment of cardiovascular disease in obese patients.Methods: Platelet aggregometer is used to test platelet aggregation and release in the presence of collagen.P-selectin expression and integrin-?IIb?3 activation were measured by flow cytometry.Besides,we used fluorescence microscope to analyze platelets spreading on immobilized fibrinogen.Clot retraction assay was carried out by adding thrombin into solution containing fibrinogen to observe the transformation of platelets.Results:(1)Leptin exhibited a promoting effect on platelet aggregation stimulated by ADP in platelet-rich plasma.Besides,pretreatment of washed platelet with leptin also showed a promoting effect on aggregation by the agonist of collagen and thrombin,(2)P-selectin expression induced by collagen was no difference between controls and leptin pretreatment,while the ATP release induced by collagen was significantly increased by leptin,(3)Leptin promoted the expression of JON/A,(4)Pretreatment of platelet with leptin markedly increased the single platelet spreading area on immobilized fibrinogen,and also(5)reduced clot retraction.Conclusion: Leptin promoted the platelets aggregation on the stimulation of agonists,and made platelets in a hyperactivity state by increasing the “inside-out ”and “outside-in ” signaling.Part ? The effect of leptin on the platelet activation signaling pathwayObjective: In first part,we found that leptin promote platelets aggregation and release induced by collagen,promote platelets spreading on the condition of fibrinogen and clot retraction.However,the specific mechanism of leptin on platelets activation is still unclear.In order to further explain the effect of leptin on platelets,we detected the protein phosphorylation on certain signal pathway.Plasminogen activator inhibitor-1(PAI-1)is a major inhibitor of plasminogen and is the most important regulator of fibrinolysis and coagulation.PAI-1 is synthesized by a number of different tissues,including hepatic,vascular,and adipose,and is stored in platelets to provide high local concentrations for clot stabilization.Clinical studies have revealed an association between elevated plasma PAI-1 levels and the incidence of thrombosis and atherosclerosis.Therefore,we detected the change of platelets release PAI-1 under leptin incubation,looking for the potential target of leptin to promote thrombosis.Methods: After incubation with leptin to mouse washed platelet,platelets activated by collagen were lysed by using the Western Blot technique to detect PLC?2(Tyr1217),Akt(Ser473/474),GSK3?(S9)and MAPKs phosphorylation level.Besides,the reactive oxygen species(ROS)generation on platelet was detected byflow cytometry.Furthermore,the release of PAI-1 in platelet was detected by the Western Blot technique in the time-course.Results: Collagen-stimulated PLC?2(Tyr1217)and its downstream Akt(Ser473/474)and GSK3?(S9)phosphorylation was significant increased by the pretreatment of leptin.Besides,the same tendency with phosphorylation of ERK1/2(E-4)and human p38 MAPK(Thr180/Tyr182)phosphorylation,but the effect of JNK(Thr183/Tyr185)phosphorylation was slightly.Reactive oxygen species generation was increased pretreated with leptin which induced by Collagen Related-peptide(CRP)stimulation.On the condition of collagen,leptin could promote platelets to release PAI-1 in a short time,which was increased compared with wildtype mice platelets,but leptin itself could not induce PAI-1 release.Conclusion: This study demonstrates that leptin regulates platelet activity,which may be mediated by promoting the PI3K-Akt and MAPKs cascades,thus promoting “inside-out” and “outside-in” signaling pathway.Furthermore,leptin have a positive effect on platelet activity by promoting the release of PAI-1.