| Objective: Hypercholesterolemia is associated with a high incidence of atherosclerosis and thrombotic complications. Increasing evidence is accumulating, suggesting that the main determinant of the prothrombotic state associated with hypercholesterolemia is enhanced platelet activation. Elevation of low-density lipoprotein(LDL) level affects platelet function and increases the sensitivity of platelets to several natural agonists involving various mechanisms. Statins are the most widely used cholesterol-lowering drugs, and have beneficial effects in the primary and secondary prevention of coronary artery disease. Statins inhibit the activity of a key enzyme in cholesterol synthesis, 3-hydroxymethyl-3-methylglutaryl coenzyme A(HMG-Co A) reductase, and thus reduce cholesterol formation. In recent years, studies of the pleiotropic effects of statins reveal that these drugs have many additional anti-atherogenic effects, including antithrombotic effects. Many studies have indicated that statins can influence platelet funtion, as well as circulating coagulation factors or cofactors, and these effects have been attributed to direct or indirect effects of the reduction in cholesterol level by statins. In addition, some studies have suggested that statins exert their inhibitory effects on platelet activation beyond their cholesterol-lowering activities. However, the antiplatelet activity of statins has not been sufficiently explored.PPARs are ligand-activated transcription factors, belonging to the nuclear hormone receptor superfamily. Recent reports have suggested that these nuclear recetptors are involved in many biological processes, including lipid metabolism and atherogenesis. Although, platelets are anucleated cells released from megakaryocytes, they also contain PPARs. So far, three PPAR isoforms(PPARα, PPARβ/δ, and PPARγ) have been found in human platelets, and up-regulation of PPARs may inhibit platelet activation through a nongenomic mechanism. Several studies demonstrate that drugs that affect the activation of PPARs may exert antiplatelet activities, and this would, at least partly, explain in the inhibitory effects of statins, such as simvastatin, on platelets activation. The detailed mechanisms, involving PPARs, by which simvastatin inhibits platelet activation have not yet been completely elucidated. In this study, we examined the role of PPAR activation on the inhibition by simvastatin of collagen-stimulated platelet activation. We demonstrated that PPARγ activation modulates multiple cellular signaling events, including exposure of CD62 and PAC-1, c AMP elevation, intracellular Ca2+ mobilization, Akt and mitogen activated protein kinases(MAPKs) phosphorylation associated with platelet activation stimulated by collagen, which contribute to the antiplatelet activity of simvastatin.Methods: This study was approved by the Institutional Review Board of Hebei Medical University and conformed to the principles outlined in the Helsinki Declaration. Blood samples were donated from healthy volunteers who had not taken any drugs known to interfere with platelet function in the past 2 weeks before venipuncture and informed consent was obtained. Human platelet suspensions were also prepared as previously described. The impedance aggregometry was carried out by measuring changes in electrical impedance. And the exposure of CD62 and PAC-1 was measured by flow cytometric analysis. Furthermore, Elisa and spectrophotometer were also used to measure c AMP elevation and intracellular Ca2+ mobilization. Akt and mitogen activated protein kinases(MAPKs) phosphorylation associated with platelet activation stimulated by collagen were measured by western blot and immunoprecipitation.Results: The results of our study showed that simvastatin inhibited aggregation of washed platelets induced by collagen in a concentration-dependent manner and simvastatin dose-dependently increased PPARα and PPARγ activity but did not affect PPARβ activity. When PPARs antagonists were used, it was found that PPARγ antagonist(GW9662) did, but not PPARα antagonist(GW6471), cause significant suppression of the simvastatin-inhibition of collagen stimulated platelet aggregation. These data indicate that PPARγ activation is involved in mediating the antiplatelet activity of simvastatin. Furthermore, the results of flow cytometry indicated that while collagen markedly increased the expressions of CD62 and PAC-1 on washed platelets, these increases were significantly reduced by simvastatin, in a concentration-dependent manner, suggesting that the incubation of platelets with simvastatin reduced collagen-stimulated platelet activation. When using antagonists, we observed that the reduced expression of CD62 and PAC-1 by simvastatin was reversed by GW9662, but not GW6471. The same result was found when the intracellular calcium concentration in human platelets was assessed by the Fura 2/AM loading method.CAMP is regarded as an endogenous negative regulator of platelet activation that operates through inhibition of platelet aggregation, adhesion, and granule secretion. So we investigated the influence of simvastatin on basal levels of c AMP in platelets. It was found that simvastatin increased the c AMP production in a concentration-dependent manner and treatment with GW9662 markedly inhibited the increase in c AMP induced by simvastatin. This effect was supported by testing VASP Ser157 phosphorylation which was induced by intracellular c AMP elevation in platelets. In our study, simvastatin markedly induced VASP Ser157 phosphorylation. When PPARs antagonists were used, GW9662 significantly reversed VASP Ser157 phosphorylation induced by simvastatin. These results indicate that simvastatin inhibits platelet activation, at least in part, via a c AMP-dependent pathway and that PPARγ activation participates in the antiplatelet activity of simvastatin.Collagen binding to the platelet receptor glycoprotein VI(GPVI) results in recruiting phosphoinositide 3-kinase(PI3-K) and, through the generation of phosphatidylinositol(3, 4, 5)-trisphosphate, influences the liberation of the second messengers 1,2-diacylglycerol and inositol 1,4,5-trisphosphate. The formation of these molecules is responsible for the activation of Akt, which is important for platelet function and thrombus formation. In our study, simvastatin markedly abolished the phosphorylation of Akt stimulated by collagen. Moreover, the suppression of Akt phosphorylation caused by simvastatin was significantly reversed by GW9662. It suggests that the treatment of platelets with simvastatin results in suppression of PI3-K/Akt signaling. In addition, PPARγ activation is certainly contributed to the inhibition by simvastatin of the PI3-k/Akt pathway.Furthermore we detected MAPK signaling molecules and revealed that simvastatin significantly and dose dependently suppressed collagen-induced p38 MAPK and ERK phosphorylations, but not JNK activation. When we used GW9662 and SQ22536(adenylate cyclase inhibitor), both the two inhibitors reversed inhibition of simvastatin on p38 MAPK and ERK activations induced by collagen. Taken together, these results indicate that c AMP-dependent pathway involves in mediating MAPKs phosphorylations inhibited by simvastatin and PPARγ activation participates in simvastatin-antiplatelet activity via inhibition of MAPKs activations. In order to detect how PPARγ acted on the MAPKs signaling pathway, we studied the interaction between PPARγ and p38 MAPK or ERK. Our results showed that in collagen-activated platelets, simvastatin enhanced association of PPARγ with p38 MAPK and ERK in a concentration-dependent manner. When we used SQ22536 and GW9662, only GW9662 reduced the association of PPARγ with p38 MAPK and ERK activations induced by collagen. These results indicated that there are direct-connections between PPARγ and components of MAPK signaling, which could contribute to the mechanisms by which simvastatin affects platelet function.Conclusion:1 This study suggests that PPARγ activation, but not PPARα activation, plays a critical role in the inhibition by simvastatin of platelet aggregation induced by collagen.2 The mechanisms may involve inhibition of Akt phosphorylation, increasing the level of c AMP and VASP phosphorylation, and inhibition of p38 MAPK and ERK phosphorylations.3 Furthermore, we demonstrated for the first time a direct interaction with PPARγ and MAPKs, which contributes to inhibition of PPARγ on MAPKs phosphorylations and involves the inhibitory effect of simvastatin on platelet activation induced by collagen. This study provides new insight into the antiplatelet mechanisms of simvastatin involving PPAR activation. The details need further investigation, but our results could help in explaining the protective effect of simvastatin in coronary artery disease. |
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