Affinity-Purified Anti-Beta-2 Glycoprotein-1 Antibodies from a Patient with Lupus Anticoagulant-Associated Thrombosis Amplify Thrombus Formation in the Living Mouse.

Abstract 146

Antiphospholipid syndrome (APS) is characterized by thrombosis, recurrent fetal loss and the presence of the lupus anticoagulant, anticardiolipin antibodies or anti-beta-2 glycoprotein 1 antibodies. Sera from patients with APS contain polyclonal antibodies that bind to various plasma proteins including beta-2 glycoprotein 1. Although beta-2 glycoprotein 1 antibodies have been well-documented as a biomarker for the diagnosis of APS, their direct role in the pathogenesis of thrombosis is unknown. Here, we have demonstrated using intravital microscopy that purified anti-beta-2 glycoprotein 1 antibodies isolated from the serum of a patient with APS greatly amplify thrombus size following laser-induced vessel wall injury in live mice. A patient with systemic lupus and APS complicated by pulmonary embolism was studied. IgG was isolated from serum by affinity chromatography using a Protein A/G column. Anti-beta-2 glycoprotein 1 antibodies in the IgG fraction were affinity-purified using homogeneous beta-2 glycoprotein 1 covalently bound to CNBr-activated agarose beads. Purified anti-beta-2 glycoprotein 1 antibodies were eluted at low pH. Patient IgG depleted of anti-beta-2 glycoprotein 1 antibodies was obtained by repeated chromatography over the beta-2 glycoprotein 1 column. The effects of (a) purified anti-beta-2 glycoprotein 1 antibodies, (b) anti-beta-2 glycoprotein 1 antibody-depleted patient IgG, and (c) IgG from normal human sera on thrombus formation were studied quantitatively in the live mouse. Intravital microscopy was performed using the cremaster muscle as a vascular window, and thrombus formation was initiated by laser injury to the arteriolar wall. Five minutes prior to vessel wall injury, purified anti-beta-2 glycoprotein 1 antibodies, anti-beta-2 glycoprotein 1 antibody-depleted patient IgG, or normal human IgG were infused via a jugular catheter. Platelet thrombus size was determined by widefield microscopy and Alexa 647-conjugated Fab fragments of an anti-CD 41 monoclonal antibody. Up to 10 thrombi were generated per mouse, and the median integrated fluorescence for 25-30 thrombi determined. Infusion of anti-beta-2 glycoprotein 1 antibodies increased thrombus size in a dose-dependent manner. Infusion of purified anti-beta-2 glycoprotein 1 antibodies at 0.12 μg/g mouse and 0.40 μg/g mouse increased thrombus size by about 18-fold and 122-fold respectively over thrombi formed in untreated mice. However, anti-beta-2 glycoprotein 1 antibody-depleted patient IgG and normal human IgG did not affect platelet thrombus size. These results indicate that the anti-beta-2 glycoprotein 1 antibodies isolated from APS patient serum are responsible for markedly increased thrombus size in this thrombosis model. The target cellular antigen of the anti-beta-2 glycoprotein 1 antibodies and the mechanism of enhanced thrombus formation remain unknown. However, these results provide evidence that anti-beta-2 glycoprotein 1 antibodies are not only a marker but are directly involved in the pathogenesis of thrombosis. This in vivo animal model offers an approach to identifying inhibitors of anti-beta-2 glycoprotein 1-mediated thrombosis.