The Antithrombotic Effect of Atorvastatin Is Mediated by Decreased Plasma Membrane Exposure of Phosphatidylserine.

Abstract

HMGCoA reductase inhibitors (statins) exhibit poorly understood antithrombotic properties that are independent of reductions in circulating LDL cholesterol. The activities of the vitamin K-dependent proteolytic complexes such as factor VIIa/tissue factor (fVIIa/TF) and the prothrombinase complex are sensitive to the membrane environment. The plasma membrane is marked by lateral segregation of cholesterol-rich lipid rafts and by transbilayer segregation of phosphatidylserine to the internal membrane leaflet. We hypothesized that the antithrombotic properties of statins are mediated by their impact on cholesterol and phospholipid metabolism. We utilized the immortal cell line EA.hy926 to clarify the mechanism whereby statins alter thrombosis. The endothelial characteristics of these cells include expression of tissue factor pathway inhibitor, nitric oxide synthase, and von Willebrand factor. EA.hy926 cells were incubated in the presence of physiologic concentrations of atorvastatin (1 μM) supplemented with 10% fetal bovine serum (FBS) or 10% of a serum mixture consisting of FBS and lipid-depleted FBS (1:3 v/v) to restrict cellular access to exogenous sterol. Cells cultured in the presence of 1 μM atorvastatin and lipid depleted serum grew at identical rates over 72 hours to cells that were incubated in the absence of atorvastatin. Following 72 hours of treatment with atorvastatin and lipid restriction, cells were harvested and membrane lipids examined by tandem mass spectrometry. Lipid restriction alone had no effect on cell lipid composition but when atorvastatin was included, phosphatidylserine, sphingomyelin, and cholesterol were reduced by 50% while ceramide content decreased by 70% (normalized to lipid phosphate). The changes in lipid compostion did not alter the association of CD59 with detergent-resistant buoyant membrane domains indicating that lipid rafts remained intact. The functional impact of atorvastatin treatment on EA.hy926 cells was assessed by measuring fVIIa/TF activity. Basal and decrypted fVIIa/TF activities were reduced by 60% and 75%, respectively, compared to cells treated with no atorvastatin, atorvastatin alone, or lipid restriction alone. Expression of TF was not altered by atorvastatin and lipid restriction as assessed by both ELISA and western blot, suggesting that changes in fVIIa/TF were mediated by reduced exposure of phosphatidylserine. To confirm the role of phosphatidylserine in diminished fVIIa/TF activity, the prothrombinase complex was reconstituted in the presence of cells treated identically with atorvastatin and lipid restriction. Prothrombinase activity was reduced by 50% compared to control cells, an extent similar to the observed decrease in basal fVIIa/TF. The inclusion of 200 μM mevalonic acid to the culture media partially reversed the observed changes in lipid content and prothrombinase activity induced by atorvastation. We conclude that atorvastatin combined with a reduction in exogenous sterol limits exposure of phosphatidylserine at the cell surface and restricts the activity of proteolytic enzyme complexes that propagate the coagulation cascade.