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


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 (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.


RESULTS
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.


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.
Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, Cornell University, New York, NY, USA. Electronic address: s.bloemen@thrombin.com.
Saartje
S
Bloemen
Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, Cornell University, New York, NY, USA.
Xiao Xuan
XX
Wu
Coagulation Laboratory, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent, Belgium.
Katrien M
KM
Devreese
Synapse Research Institute, Maastricht University Medical Center, Maastricht, The Netherlands.
Bas
B
de Laat
Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, Cornell University, New York, NY, USA.
Jacob H
JH
Rand
Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, Cornell University, New York, NY, USA.
Ljiljana V
LV
Vasovic
D017152:Antibodies, Antiphospholipid / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000378:metabolism*; D016736:Antiphospholipid Syndrome / Q000097:blood*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D011485:Protein Binding; D053482:beta 2-Glycoprotein I / Q000276:immunology*
D017152:Antibodies, Antiphospholipid; D000936:Antigen-Antibody Complex; D053482:beta 2-Glycoprotein I
Antiphospholipid antibody; Antiphospholipid syndrome; Beta 2-glycoprotein I; Erythrocyte membrane; Immune complexes; Steric interactions