Background/Purpose: Neutrophil extracellular traps (NETs) are prothrombotic tangles of chromatin and microbicidal proteins ejected from neutrophils in response to a variety of stimuli.  In antiphospholipid syndrome (APS), recent work has demonstrated high levels of NETs in the blood of APS patients; that antiphospholipid antibodies can engage neutrophils to trigger NET release; and that APS serum degrades NETs poorly.  Here, we hypothesized that APS might be perpetuated by crosstalk between NETs and anti-NET autoantibodies.

Methods: Purified, partially-digested NETs were used to coat ELISA plates.  Levels of anti-NET IgG and IgM (OD at 450 nm) were determined.  A positive cut-off was established as mean OD+2 SD of healthy-control samples.  For a subset of patients, we also measured autoantibody formation with a microarray platform that included 120 potential autoantigens (48 NET-associated antigens and 72 other autoantigens). Univariate logistic regression was performed to determine clinical associations.  Non-parametric Mann-Whitney U-test was used to compare levels of anti-NET autoantibodies and plasma NET remnants (myeloperoxidase-DNA complexes) between groups.

Results: As compared with controls (n=46), primary APS (n=78) was characterized by marked elevations in IgG (0.51 ± 0.36 vs. 0.29 ± 0.09; p< 0.0001) and IgM (0.76 ± 0.50 vs. 0.26 ± 0.23; p< 0.0001) anti-NET autoantibodies.  Importantly, levels of anti-NET antibodies did not correlate with levels of anti-β2GPI (R2 < 0.01).  By univariate logistic regression, positive anti-NET IgG/IgM was significantly associated with arterial thrombosis (P< 0.0001 for IgG / P< 0.0001 for IgM), venous thrombosis (P< 0.0001 for IgG / P< 0.0001 for IgM), and pregnancy morbidity (P=0.02 for IgG / P=0.03 for IgM).  Intriguingly, positive anti-NET IgG/IgM was also associated with non-criteria features such as thrombocytopenia and livedo reticularis (Figure 1).  Higher circulating NET remnants were observed in primary APS patients with venous thrombosis (P=0.0007) and recurrent venous thrombosis (P=0.0003) as compared with healthy controls (Figure 2).  In pursuit of the antigen specificity of anti-NET autoantibodies, 20 of the primary APS samples were analysed by autoantibody microarray and compared with 20 matched controls.  IgG/IgM autoantibodies against the following NET-relevant antigens were significantly elevated in primary APS:  anti-citrullinated-histone H3, anti-chromatin, anti-nucleosome, and anti-bactericidal/permeability-increasing protein (BPI).

Conclusion: Patients with primary APS have high levels of anti-NET autoantibodies.  Anti-NET IgG/IgM is associate with both criteria and some non-criteria APS clinical features. Autoantigen microarray reveals a marked enrichment of various specific citrullinated /non-citrullinated anti-NET autoantibodies in patients with primary APS. Anti-NET activity may associate with specific antigens present in NETs such as citrullinated-histone H3 and BPI.  Interestingly, anti-NET autoantibodies do not correlate with anti-β₂GPI, suggesting their potential as a novel, independent, and clinically-relevant biomarker.

Fig 1

Fig 2

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/anti-neutrophil-extracellular-trap-net-autoantibodies-in-primary-antiphospholipid-syndrome/