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

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.

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 β₂-glycoprotein I (β₂GPI)-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.

iEMs readily precipitated purified β₂GPI as well as β₂GPI 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.

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.