Inverted erythrocyte membranes demonstrate β2GPI-antiphospholipid antibody interactions and membrane crosslinking

Abstract Introduction The antiphospholipid syndrome (APS) is an acquired autoimmune disorder predisposing patients to thrombosis or pregnancy complications. Since inverted erythrocyte membranes (iEMs) might provide a physiologically relevant source of anionic phospholipids, we studied the interactions of phospholipid-binding proteins and APS antibodies using iEMs. Materials & methods iEMs were prepared from packed erythrocytes by hypotonic lysis. Phosphatidylserine (PS) exposure was confirmed by annexin A5 (A5) binding using fluorescence microscopy and flow cytometry. Binding of β 2 -glycoprotein I (β 2 GPI)–IgG immune complexes to iEMs was investigated with gel electrophoresis, western blot and flow cytometry. Functional involvement in coagulation was documented in the thrombin generation assay. Results iEMs readily precipitated purified β 2 GPI as well as β 2 GPI from normal plasma and APS plasma. The plasma of APS patients provided higher levels of IgG binding to iEMs relative to healthy controls. Thrombin generation increased with increasing concentrations of iEMs, documenting that coagulation proteins bound to the exposed phospholipids. The LA effect was also distinguished in thrombin generation when comparing APS patients, as indicated by an increased lag time. Agglutination was observed after incubation with APS patient plasma and this was augmented by anti-human globulin. Conclusions In conclusion, iEMs can provide a more physiological approach than phospholipid vesicle-based tests for investigating APS and are more amenable to standardization than platelet membranes.