Atorvastatin Reduced Platelet Accumulation in Arteriolar Thrombi and Increases Platelet S-Nitrosylated N-Ethylmaleimide-Sensitive Factor.
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AbstractBackground: Statins are well recognized to effect a lowering of plasma lipoproteins and this may be beneficial in cardiovascular disease.Objectives: We investigated whether statins may reduce the burden of atherothrombotic cardiovascular disease independently of their known effects on plasma lipoproteins.Methods: Wild type C57BL/6 mice were fed atorvastatin, at a dose of 33 mg of atorvastatin per kg of standard chow. Controls received the same chow, but without the drug. After 14 days, we induced thrombi within arterioles of the cremaster muscle by laser injury and, using intravital microscopy, quantified platelet accumulation within each thrombus by measuring the fluorescence from anti-CD41antibody with a fluorescent-tagged secondary antibody.Results: Mean platelet accumulation in thrombi was slightly, but significantly, lower at early time points in atrovastatin-fed mice compared with thrombi generated in untreated control mice i.e. one minute after arteriolar injury, area under the curve analysis revealed atorvastatin-fed mice to have 15% less platelet accumulation within thrombi compared to controls (Mann-Whitney test: P = 0.02); thereafter, no difference was observed. To determine whether this effect may be directly related to altered platelet function, we devised a novel method to study platelet accumulation within laser-induced arteriolar thrombi: Platelets were isolated from two mice using differential centrifugation and gel chromatography. The platelets from one mouse were labeled with DiI, while those from the other mouse were labeled with DiO. An equal proportion of labeled platelets were simultaneously transfused into a third mouse. The arrival and departure of individual green-fluorescent (DiI) and red-fluorescent (DiO) platelets within each thrombus were analyzed. This dual-population dual-label technique revealed that platelets from atrovastatin-treated mice accumulated in thrombi half as much as control platelets; this occurred in thrombi generated in both atorvastatin-treated and control mice. Because analysis is independent of the degree of injury, which is variable in the laser-induced model, this technique represents a sensitive new method to compare platelet accumulation among different platelet populations. Some of atorvastatin’s beneficial effects may involve the nitric oxide pathway. S-nitrosylation of N-ethylmaleimide-Sensitive Factor (NSF) inhibits platelet granule secretion, which may reduce platelet accumulation in a thrombus. Accordingly, we compared the amount of S-nitrosylation of NSF in platelets from atorvastatin-treated versus untreated control mice. Platelet proteins were immunoprecipitated with a rat polyclonal anti-conjugated NO-L-cysteine antibody. Western blot testing with anti-NSF revealed more product (normalized for the amount of platelet protein)in platelets from atorvastatin-treated mice than in control platelets. Hence, treatment of mice with atorvastatin may yield platelets that express higher amounts of S-nitrosylated NSF, impairing their ability to accumulate within thrombi.Conclusions: These findings enhance our understanding of other possible mechanisms by which statins effect a rapid reduction in the progression of atherothrombosis.
