Critical Role Of Peroxisome Proliferator-activated Receptor α In Hyperlipidemia-related Platelet Activation And Thrombosis

Platelets play an important role in hemostasis and thrombosis.Platelet dysfunction can affect thrombus formation.Impaired platelet function has been associated with an increased propensity for hemorrhage,and excessive or abnormal activation of platelets can lead to some thrombotic diseases.Therefore,in-depth study of target molecules and molecule mechanism of platelet activation and elucidating the pathogenesis of thrombotic diseases and hemorrhage will provide a theoretical basis for treatment of those diseases.Hyperlipidemia can lead to platelets hyperreactivity and an increased risk of thrombosis.Peroxisome proliferator-activated receptor（PPAR）αis a ligand-dependent nuclear hormone receptor and an important regulator in lipid metabolism.It has been reported that PPARαis involved in hemostasis,and PPARαligands inhibit platelet activation.However,the role and molecular mechanism of PPARαin physiological hemostasis and thrombosis,as well as hyperlipidemia-related platelet activation and thrombosis are still unclear.In this study,with PPARα-deficient(PPARα^-/-)mice,we first investigate the role of PPARαin platelet function,hemostasis and thrombus formation.The main physiological effects of PPARαcan be summarized as follows:（1）In the tail-bleeding assay,PPARα^-/-mice exhibited a longer bleeding time than wild-type（WT）mice,which is consistent with previous report,indicating that PPARαis involved in hemostasis.（2）In a mouse model of the mesenteric artery thrombosis induced by ferric chloride,the arterial vessel occlusion time of in PPARα^-/-mice was significantly prolonger than WT mice.Thrombus formation on a collagen matrix under arterial shear conditions was significantly reduced in PPARα^-/-platelets,indicating that PPARαpositively regulates thrombus by regulating platelet function.（3）PPARαknockout does not affect the adhesion of platelets to collagen and the synthesis of thromboxane A₂.PPARα^-/-platelets exhibited impaired aggregation and dense granule secretion in response to low doses of collagen and thrombin.Moreover,PPARα^-/-platelets showed decreased spreading on fibrinogen and impaired clot retraction.（4）When ADP was hydrolyzed by apyrase,the difference in platelet aggregation and spreading between WT and PPARα^-/-platelets was disappeared.And the defective aggregation and spreading of PPARα^-/-platelets can be rescued by exogenous ADP.These data indicate that impaired ADP secretion was responsible for the defective aggregation of PPARα^-/-platelet.（5）The effect of PPARαis critically dependent on platelet dense granule secretion,and is contributed by p38MAPK/Akt,fatty acidβ-oxidation,and NADPH Oxidase pathways.PPARαregulates ROS generation derived from NADPH oxidase pathway and mitochondria.More importantly,the phenotype of PPARα^+/-is between WT and PPARα^-/-mice,suggesting that PPARαexhibited a gene-dosage effect in platelet activation.Second,the role and molecular mechanism of PPARαin platelet activation and thrombosis induced by hyperlipidemia were demonstrated.（1）The occlusion time of mesenteric artery were significantly accelerated in Apo E^-/-mice fed with high-fat diet,and PPARαdeficiency significantly prolonged occlusion time of arterial vessels,indicating that PPARαpromoted thrombus formation in hyperlipidemic mice.（2）PPARαwas involved in platelet activation and ROS generation induced by oxidized low-density lipoprotein（ox LDL）.We found that the activation of Src/NADPH/ROS/ERK5,p38 and Akt were impaired in PPARα^-/-platelet induced by ox LDL.（3）Compared with the control group mice fed with normal diet,platelet activation of mice fed with high-fat diet was significantly increased,accompanied with increased PPARαprotein expression.Platelet reactivities are positively correlated with the expression levels of PPARα,as evidenced by data from WT,PPARα^+/-and PPARα^-/-mice.This linear correlation is recapitulated in platelets from healthy volunteers and hyperlipidemic patients.Finally,to investigate the mechanism of increased PPARαprotein expression in platelets,human platelets and megakaryocytes treated with lipids such as fatty acid,cholesterol and ox LDL were performed.We found that human platelets treated with lipids for 12 and 24 hours did not exhibit an alteration of the expression of PPARαprotein,indicating that increased PPARαprotein expression may be inherited from megakaryocytes,the precursor cells of platelets.Compared to control group,PPARαprotein and m RNA in megakaryocytes treated with fatty acids,cholesterol and ox LDL for 24 hours were significantly increased.In contrast,m RNA and protein of PPAR in megakaryocyte were significantly increased after 24 hours incubation with lipids.We further demonstrate that lipids upregulate PPARαexpression via activation of ROS-NF-κB signaling pathway and promoting p65 bind to the promoter of PPARα.In summary,PPARαwas involved in platelet activation,hemostasis and thrombosis,and platelet PPARαis responsible for increased platelet activity and thrombosis potential by hyperlipidemia.