Immunochemical properties and pathological relevance of anti-ß2-glycoprotein I antibodies of different avidity.

Despite available treatment, there is still significant morbidity and mortality present among patients with the autoimmune thrombophilic condition termed 'antiphospholipid syndrome' (Espinosa, G. and Cervera, R. 2009. Morbidity and mortality in the antiphospholipid syndrome. Curr. Opin. Pulm. Med. 15:413.). High-avidity (HAv) anti-ß(2)-glycoprotein I (anti-ß(2)GPI) antibodies, shown to correlate with thrombotic events in patients, could represent the much needed improved prognostic marker. By studying their effect on crystalline annexin A5 shield on phospholipid surfaces (one of proposed pathogenic mechanisms), with the use of atomic force microscopy, the pathogenic potential of HAv anti-ß(2)GPI antibodies was confirmed. Furthermore, by using surface plasmon resonance and enzyme-linked immunosorbent assays, unique binding characteristics of HAv antibodies in comparison with low avidity antibodies were established. HAv anti-ß(2)GPI were confirmed to (i) recognize ß(2)-glycoprotein I in a solution, (ii) interact predominantly monovalently (much lower dependency on the antigen density) and (iii) form more stable complexes with the antigen. Since enzyme-linked immunosorbent assays currently used in routine diagnostics detect anti-ß(2)GPI antibodies of unknown avidity, our observations are potentially useful for the development of improved diagnostic tests capable of detecting clinically relevant antibodies.

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

Antibodies against ß2-glycoprotein I are a subset of very heterogeneous family of antiphospholipid antibodies. It is well recognised that anti-ß2-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 ß2-glycoprotein I, anti-ß2-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 ß2-glycoprotein I, 10 g/l of IgG fraction from healthy blood donor, 10 g/l of IgG fraction from a patient with anti-ß2-glycoprotein I antibodies and 0.4 g/l of isolated IgG anti-ß2-glycoprotein I antibodies from the same patients in Hepes buffered saline with 1.5 mM Ca2+ were used. We confirmed the clustering of ß2-glycoprotein I on planar phospholipid bilayers. We also found that in the presence of annexin A5, ß2-glycoprotein I does not bind to planar phospholipid bilayers. However, when adding the anti-ß2-glycoprotein I antibodies, the growth of ß2-glycoprotein I-anti-ß2-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-ß2-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.

The use of atomic force microscopy to study the pathologic effects of anti-annexin autoantibodies.

Patients with recurrent pregnancy loss and a history of thrombotic events have often been noted to have autoantibodies directed at annexin A5. However, the relationship of these autoantibodies to immunopathology is still unknown, although it has been proposed that they have a direct effect on the function of annexin A5. Annexin A5 may be a significant immunological target with pathologic implications. Essentially, annexin A5 is an anticoagulant protein that crystallizes over negatively charged phospholipid surfaces and thereby blocks them from availability for coagulation reactions. To address this issue, we have taken advantage of our expertise with atomic force microscopy and studied anti-annexin A5 autoantibodies isolated from patients and focused on the ability of these antibodies to influence annexin A5 crystallization on planar mica-supported phospholipid bilayers. We report herein that such antibodies from patients, but not controls, produced a significant disruption of incomplete annexin A5 crystalline shield on phospholipid bilayer. In addition, the IgG fraction isolated from such patients significantly decreased the velocity of annexin A5 crystallization. Atomic force microscopy is a powerful tool to study the pathologic mechanisms of autoantibodies and the data herein reflect the potential of anti-annexin A5 antibodies that produce pathology in a number of varied but overlapping clinical conditions, including autoimmune thrombosis and antiphospholipid syndrome.

In vitro model of annexin A5 crystallization on natural phospholipid bilayers observed by atomic force microscopy.

Annexin A5 is a potent anticoagulant protein with a thrombomodulatory function. It is frequently mentioned with systemic inflammatory autoimmune disease, which share higher vulnerability to cardiovascular diseases. The protein has the ability to bind to membranes containing negatively charged phospholipids in a calcium-dependent manner. The potent anticoagulant properties of the protein are a consequence of this crystallization, which forms the lattice of annexin A5 over phospholipid surface, blocking its availability for coagulation reactions. Crystallization of annexin A5 has been proven on homogeneous synthetic phospholipids. However, the crystallization of annexin A5 on inhomogeneous, naturally derived phospholipid surfaces, in p3 and p6 crystal form, has now been reported for the first time. Atomic force microscopy was chosen for the observation of the crystallization of annexin A5 on different solid supported phospholipid bilayers. In this study model, the optimal results were obtained by using: 0.5 mg/ml lipid vesicles suspension (70% phosphatidylcholine, 30% phosphatidylserine) in HEPES buffer saline (HBS) with 2 mM CaCl(2), large unilamellar vesicles with sizes around 200 nm, 41 degrees C of phase transition temperature and 21 microg/ml of native annexin A5 in HBS with 2 or 20 mM CaCl(2). Results were evaluated by imaging and force measurements. Demonstration that native annexin A5 is able to spontaneously crystallize on naturally derived, inhomogeneous phospholipids is supporting the putative role of annexin A5 crystal structures as possible antithrombotic shield. This in vitro system is probably more appropriate for studying the pathogenetic role of antiphospholipid antibodies.

Beta2-glycoprotein I and annexin A5 phospholipid interactions: artificial and cell membranes.

The aim of this report was an overview of beta2-glycoprotein I (beta2-GPI)- and annexin A5 (AnxA5)-phospholipid interactions including candidate beta2-GPI receptors, and their relevance to the investigation of physiological/pathological processes. Both beta2-GPI and AnxA5 have thrombomodulatory functions in vivo and their antigenicity is particularly important for thrombotic manifestations and pregnancy complications in antiphospholipid syndrome. Specific elements of beta2-GPI- and AnxA5-phospholipid interactions are different. The crucial elements for beta2-GPI are conformational change and dimerization, both of which are also required and necessary for receptor signaling, leading to the prothombotic state. AnxA5 differs in its ability to crystallize into a protective shield, the disruption of which seems to be the major prothrombotic mechanism. These differences may explain some of the functional consequences of both molecules seen in the pathological conditions. Future alternative therapies of antiphospholipid syndrome are proposed to be based on the expanding knowledge of beta2-GPI- and AnxA5-phospholipid interactions, specifically antagonizing beta2-GPI receptors, as well as inhibiting their signaling pathways.

Antibodies against annexin A5: detection pitfalls and clinical associations.

Antiphospholipid syndrome (APS) has been defined as a clinical and laboratory entity. Laboratory criteria include the presence of anticardiolipin antibodies (aCL) and/or lupus anticoagulant (LA), collectively termed as antiphospholipid antibodies (aPL). However, there has been a rising interest in antibodies against so-called protein cofactors, particularly in beta(2)-glycoprotein I. In the early 90s, annexins were considered as target antigens for aPL, but at present the exact role of antibodies against annexins (aANX) remains puzzling. This review is concerned with annexin V or annexin A5 (ANXA5), a widespread member of the annexin family, and antibodies directed towards it. We have endeavoured to summarise essential information about the detection of anti-annexin V antibodies (aANXA5) and their clinical relevance. This review has also brought together some relevant published data concerning the structure, physiological role and therapeutic potential of ANXA5.