Inverted erythrocyte membranes demonstrate β2GPI-antiphospholipid antibody interactions and membrane crosslinking. Issue 146 (October 2016)

• Record Type: Journal Article
• Authors: Bloemen, Saartje; Wu, Xiao Xuan; Devreese, Katrien M.; de Laat, Bas; Rand, Jacob H.; Vasovic, Ljiljana V.
• Is Part Of: Thrombosis research. Issue 146(2016)
• Page Start: 89
• Page End: 94
• Publication Date: 2016-10
• Subjects: Antiphospholipid syndrome -- Antiphospholipid antibody -- Beta 2-glycoprotein I -- Erythrocyte membrane -- Immune complexes -- Steric interactions; Thrombosis -- Periodicals; 616.135
• DOI: 10.1016/j.thromres.2016.09.004
• Languages: English
• ISSNs: 0049-3848

Abstract

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 β2 -glycoprotein I (β2 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 β2 GPI as well as β2 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. Highlights: Inverted erythrocyte membranes (iEMs) expose anionic phospholipids. iEMs are a potential platform for investigating antiphospholipid syndrome (APS). β2 -glycoprotein I and IgG from APS plasmas show increased binding to iEMs. The lupus anticoagulant effect is detected in thrombin generation assays with iEMs.