Background/Purpose: Beta-2 glycoprotein I (β₂GPI) is a prominent autoantigen in antiphospholipid syndrome (APS).  As an abundant plasma protein, β₂GPI has variably been suggested to have antioxidant, anticoagulant, and scavenging functions, albeit without a clear consensus regarding its defining role.  Many years ago, β₂GPI was shown to be capable of binding cell-free DNA in plasma; however, to our knowledge, this line of investigation was not further pursued.  Here, we tested the hypothesis that β₂GPI might bind to not only negatively-charged DNA, but also neutrophil extracellular traps (NETs)—prothrombotic tangles of chromatin and microbicidal proteins extruded from activated neutrophils.  We also asked whether β₂GPI-DNA complexes might be detectable in vivo, specifically in the blood of patients with APS.

Methods: To induce NET release, control neutrophils were stimulated with either phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS) in the presence of human serum as a source of β₂GPI.  In parallel, APS neutrophils were cultured in the presence of autologous serum and allowed to undergo spontaneous NET release.  To assess a potential direct interaction between β₂GPI and either genomic DNA or purified NETs, we utilized the electrophoretic mobility shift assay (EMSA).  Finally, we developed a novel sandwich ELISA to measure plasma concentrations of β₂GPI-DNA complexes in which the capture antibody was specific for β₂GPI and the detection antibody specific for DNA.

Results: By confocal immunofluorescence microscopy, we observed broad decoration of both anuclear neutrophils remnants and NET strands themselves by β2GPI.  The pattern of β2GPI decoration of NETs was relatively granular, as compared with linear painting of NETs by neutrophil elastase.  β2GPI-decorated NETs were appreciated in the context of PMA-and LPS-stimulated control neutrophils, as well as spontaneous NET release from APS neutrophils.  By agarose-gel EMSA, β2GPI triggered dose-dependent electrophoretic retardation of both neutrophil genomic DNA and purified NETs.  Using a custom ELISA for β2GPI-DNA complexes, we tested 78 primary-APS plasma samples alongside 41 healthy-control samples.  APS plasma demonstrated significantly higher levels of β2GPI-DNA complexes as compared with healthy controls (3.6 µg/ml ± 10 versus 0.57 µg/ml ± 1.6; p< 0.0001 by Mann-Whitney test).  Additional analyses are underway to determine whether β2GPI-DNA complexes are associated with particular APS clinical profiles, and the extent to which these complexes are unique to APS versus other inflammatory states.

Conclusion: β₂GPI appears to directly bind both genomic DNA and NETs.  In neutrophil cultures, β₂GPI decorates anuclear neutrophil remnants, as well as classic NET strands.  Intriguingly, β₂GPI-DNA complexes are on average 6-fold higher in the plasma of patients with primary APS as compared with controls.  Future research should endeavor to determine whether β₂GPI is an endogenous DNA scavenger, and the extent to which the β₂GPI-NET association may play a role as an instigator and/or perpetuator of autoimmunity in APS.

« Back to 2019 ACR/ARP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/beta-2-glycoprotein-i-as-a-dna-and-net-binding-protein/