Thrombomodulatory Effect of Anti-B2-Glycoprotein I Antibodies on Crystalline Annexin A5 on Phospholipid Bilayers, as Observed by Atomic Force Microscopy

Antibodies against β2-glycoprotein I are a subset of very heterogeneous family of antiphospholipid antibodies. It is well recognised that anti-β2-glycoprotein I antibodies are the main pathogenic players in the autoimmune disease known as antiphospholipid syndrome. Many mechanisms have been proposed through which these autoantibodies could cause microplacental, arterial or venous thrombosis. One of the suggested mechanisms is an antiphospholipid antibody-mediated disruption of annexin A5 protective crystalline shield on negatively charged phospholipid membranes. In current report the study of β2-glycoprotein I, anti-β2-glycoprotein I antibodies and annexin A5 interactions was performed on in vitro model of planar solid-supported phospholipid bilayers and visualized by atomic force microscopy. Planar phospholipid bilayers comprised 30 % L-α-phosphatidylserine and 70 % L-α-phosphatidylcholine. For the study of interactions 10 mg/l annexin A5, 0.15 g/l β2-glycoprotein I, 10 g/l of IgG fraction from healthy blood donor, 10 g/l of IgG fraction from a patient with anti-β2-glycoprotein I antibodies and 0.4 g/l of isolated IgG anti-β2-glycoprotein I antibodies from the same patients in Hepes buffered saline with 1.5 mM Ca2+ were used. We confirmed the clustering of β2-glycoprotein I on planar phospholipid bilayers. We also found that in the presence of annexin A5, β2-glycoprotein I does not bind to planar phospholipid bilayers. However, when adding the anti-β2-glycoprotein I antibodies, the growth of β2-glycoprotein I-anti-β2-glycoprotein I antibodies complexes in the presence of incompletely crystallized annexin A5 on planar phospholipid bilayers was observed. Results confirm the possible thrombomodulatory activity of anti-β2-glycoprotein antibodies through their effect on crystalline annexin A5. In addition, the hypothesis that the presence of possibly pathologic antigen-antibody pair itself is not sufficient to start the pathological process is confirmed and visualized for the first time.