Affinity-Purified Anti-Beta-2 Glycoprotein-1 Antibodies from a Patient with Lupus Anticoagulant-Associated Thrombosis Amplify Thrombus Formation in the Living Mouse.
Authors: Arad Ariela , Furie Richard A , Furie Barbara C , Furie Bruce 
Published: October 2019

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.

Disclosures:

No relevant conflicts of interest to declare.