| Recently, it has been recognized thatanucleate platelets are able to undergo programmed cell death(apoptosis). Platelet apoptosis is also triggered by various stimuli including chemical agonists(e.g., thrombin, collagen, ADP, etc.), oxidative stress-induced pathological conditions(e.g., hyperlipidemia, type 2 diabetes and uremia) and physical factors(e.g., hyperthermia, shear stress). The platelet apoptosis, may play a key role in the major platelet-dependent processes, haemostasis and thrombosis, and in the regulation of the platelet lifespan. Atherosclerosis and microangiopathy are common complications of type 2 diabetes(T2DM). Several factors are responsible for the atherosclerotic process, including hyperglycemia, dyslipidemia and lipid peroxides, inflammatory factors, as well as platelet hyperactivity. Increased platelet aggregation and activation have been verified in T2 DM patients, but the underlying mechanisms remain unclear. Excessive synthesis of thromboxane A2(TXA2), lipid peroxides, and increased platelet activation factor might correlate with the platelet dysfunction of T2 DM. At present, the role of platelet apoptosis in type 2 diabetes remains unclear. Moreover, diabetic hyperlipidemia is a common complication of diabetes mellitus, which is caused by the disorder of lipid metabolism in diabetic patients. The statin family of drugs, widely used in the control of hyper-cholesterolemia, is a first-line treatment of coronary artery diseaseand atherosclerosis. Statins are also routinely used in type 2 diabetes and other disorders associated with hyperlipidemia. Statins decrease choles-terol production by inhibiting hydroxymethyl glutaryl coenzyme A(HMG-Co A) reductase, which is responsible for the conversionof HMG-Co A to mevalonate, a rate-limiting step in synthesis ofcholesterol. Independent from its cholesterol-dependent effects,the benefits of statins are pleiotropic. Moreover,statins have been reported to induce the tumor cell apoptosis andhave been trialed in cancer treatments. However, like other medications, statinshave many potential side effects. Thrombocytopenia appears to be one of side effect of statins. At present, the effect of statins on the platelet apoptosis is still unclear.In this study, we will focus on the biological process of platelet apoptosis, and clarify its mechanisms in patients with type 2 diabetes and in the statin family of drugs by means of molecular cells.Part I Platelet Apoptosis in patients with Type 2 diabetes and its related mechanismObjective: The aim of this study was to investigate the platelet apoptosis in patients with type 2 diabetes and explore its related mechanism.Methods: This study included 25 diagnosis confirmed type 2 diabetes and 11 age matched healthy control subjects. The clinical data and the venous blood was collected. The plasma level of advanced glycation end products(AGEs) was determined and platelet rich plasmafrom patients and control subjects was isolated by centrifugation methods. Flow cytometry was employed to evaluate the phosphatidylserine exposureafter calcium Ionophore A23187 stimulation and depolarization of mitchondrial inner transmembrane potential. The changes of apoptosis protein markers caspase-3, Bcl-2 and Bcl-XL were detected by Western-blot. Furthermore, a serious concentration(50-400 ?g/ml) of AGEs was used to incubate with the wash platelet from the healthy subjects and the phosphatidylserine exposure, depolarization of mitchondrial inner transmembrane potential and the changes of apoptois protein markers caspase-3 and Baxwere respectively examined by flow cytometry and Western-blot analysis.Results: Our results showed that significantly increased level of AGEs in patients with type 2 diabetes compared to healthy control subjects(p<0.05). Moreover, significantly decreased depolarization of mitchondrial inner transmembrane potential in patients with type 2 diabetes. Compared to the control subjects, increased level of PS exposure was found in the A23187 stimulated platelets in type 2 diabetes.Decreased level pro-caspase-3, Bcl-2 and Bcl-XL, and upregulated level of active caspase-3were found in the platelets in type 2 diabetes. Decreased depolarization of mitchondrial inner transmembrane potential was also found in the normal platelets stimulated with AGEs, which showed a dose dependent manner. High concentration of AGEs could induce the PS exposure of platelets, and immunoblot results showed that upregulated active caspase-3 and Bax was found in these normal platelets stimulated with AGEs, which showed a dose dependent manner.Conclusions: These data demonstrated that increased platelet apoptosis was found in pateints with type 2 diabetes, and we proposed that increased level of AGEs in the plasma of diabetes patients may be the cause of the increased apoptosis of platelet.Part II Lovstatin induced platelet apoptosis and its related mechanismObjective: The aim of this study was to investigate the role of lovstatin in platelet apoptosis and its related mechanism.Methods: Human venous blood was collected and wash platelet were isolated by centrifugation method. After the platelets were stimulated with a series concentration of lovastatin(6.25 ?M-25 ?M), the flow cytometry was employed to examine the depolarization of mitchondrial inner transmembrane potential to investigate the platelet apoptosis. Western-blot was employed to examine the apoptosis related Bak, Bcl-XL and active form of caspase-3/9/8. The phosphatidylserine exposure was also detemined by flow cytometry after a serious stimulation with lovstatin.Through P-selectin and PAC-1 binding, we observed the effect of lovastatin on the platelet activation. On mechanism exploring, Chinese hamster ovary(CHO) cells were employed as the cell model and the integrin ?IIb?3 antagonist, RGDS, was employed to explore the effect of integrin on platelet apoptosis. An in vivo mouse model was also established to examine the effect of intraperitoneal injections with lovastatin on the number of circulating platelets.Results: We show that lovastatindose-dependently induces depolarization of mitochondrial inner transmembrane potential, leading toup-regulation of Bak, down-regulation of Bcl-XL, and activation of caspase-3/8/9. Lovastatin treatment didnot increase the platelet surface expression of P-selectin or PAC-1 binding. The integrin ?IIb?3 antagonist, RGDS, inhibited lovastatin-induced apoptosis in human platelets. Compared to the wild type,the apoptosis induced by lovastatin was increased significantly in Chinese hamster ovary cells which stably expressed integrin ?IIb?3. The number of circulating platelets in mice was significantly reduced after intraperitoneal injections with lovastatin.Conclusions:These data indicated that lovastatin induced caspase-dependentplatelet apoptosis. Lovastatin did not incur platelet activation, whereasreduced circulating platelets in vivo, suggesting the possible pathogenesis of the side effects in patients treated with statins. |
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