Dissecting the Molecular Basis of the Antiphospholipid Syndrome
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The antiphospholipid syndrome (APS) is an autoimmune disorder characterized by circulating antiphospholipid antibodies (aPL), thrombotic events, recurrent pregnancy loss, and increased risk of coronary artery disease. The endothelium is a critical direct target of aPL, which cause increases in adhesion molecule expression and procoagulant activity. However, the molecular mechanisms underlying aPL actions on endothelium are unknown. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) prevents leukocyte adhesion and thrombosis. In the present study we determined if aPL-induced alterations in endothelial cell phenotype are mediated by aPL actions on eNOS. Normal human IgG (NHIgG) and human IgG containing polyclonal aPL were obtained from healthy individuals and APS patients, respectively, and purified by protein G-sepharose chromatography. We found that aPL prevents VEGF-mediated attenuation of monocyte adhesion to cultured endothelial cells and this is reversed by an NO donor, indicating a role for eNOS antagonism. In contrast, NHIgG has no effect on adhesion. Whereas NHIgG does not alter eNOS activation, stimulation of eNOS by VEGF and other agonists is fully antagonized by aPL. In intact mice, NO-dependent, acetylcholine-induced increases in carotid vascular conductance are unchanged by NHIgG treatment but impaired following aPL, indicating that these processes are operative in vivo. Additional studies in cultured endothelial cells demonstrated that aPL attenuates eNOS activation by inhibiting Ser1179 phosphorylation. We further found that deprivation of the cell surface protein beta-2 glycoprotein I from endothelial cells prevents aPL inhibition of eNOS, and that FC1, a monoclonal antibody against beta-2 glycoprotein I, mimics the effects of aPL. Receptor-associated protein or RAP, an antagonist of the LDL receptor (LDLR) family, fully prevents aPL antagonism of eNOS in endothelial cells. Moreover, eNOS antagonism by aPL as indicated in vivo in carotid vascular conductance studies is absent in apoER2 -/- mice. Thus, aPL-induced changes in endothelial cell phenotype are mediated by eNOS antagonism, which is due to impaired Ser1179 phosphorylation and requires beta-2 glycoprotein I and apoER2. eNOS antagonism through these mechanisms represent a novel, potentially critical proximal process in the pathogenesis of thrombosis and cardiovascular disease in APS.