Background/Purpose: Peptidylarginine deiminases (PADs) have emerged as key participants in the pathogenesis of rheumatoid arthritis (RA) due to their expression in inflamed RA synovium and ability to citrullinate autoantigens. In addition, PAD4 autoantibodies are present in 35% of RA patients. The unexpected expression of PAD3 in neutrophils prompted us to investigate PAD3 as a potential autoantigen in RA.

Methods: Anti-PAD3 antibodies were detected by immunoprecipitation of [³⁵S]Methionine-labeled PAD3. Sera were obtained from 36 healthy controls, 44 RA patients from a convenience sample, and 194 patients from a longitudinal cohort study of subclinical cardiovascular disease in RA (ESCAPE RA).  In ESCAPE RA, disease activity and severity were assessed at baseline and two additional time points, with the final visit occurring an average of 39±4 months post-baseline.  Radiographs of the hands and feet were obtained at the first and third visits and scored according to the Sharp-van der Heijde (SvdH) method. Recombinant human PAD3 and PAD4 were used for anti-PAD competition assays. The effect of autoantibodies on PAD4 enzymatic activity was accessed by measuring citrullination of benzoyl-arginine ethyl ester (BAEE) and histone H3 following pre-incubation with patient or control IgG.

Results: Anti-PAD3 autoantibodies were present in 18% of RA sera in the convenience sample, 12% of ESCAPE RA sera, and 0% of healthy controls.  Anti-PAD3 antibodies were only detected in anti-PAD4 positive sera and were found through competition experiments to be PAD3/PAD4 cross-reactive autoantibodies. Analysis of clinical features revealed that patients with PAD3/PAD4 antibodies had a  higher baseline SvdH score compared to anti-PAD4 only patients (2.5 fold; p=0.039) and anti-PAD negative patients (4.5 fold; p<0.001), even after adjusting for cohort-specific indicators of radiographic damage.  Furthermore, patients with anti-PAD3/PAD4 antibodies had a 50% higher rate of radiographic progression (i.e. any longitudinal increase in SvdH) compared to PAD antibody negative individuals (p=0.004), despite equivalent therapeutic intervention. Strikingly,  PAD3/PAD4 autoantibodies increased the catalytic efficiency of PAD4 (160-fold at 0.2mM Ca²⁺) by dramatically decreasing the enzyme’s requirement for calcium from a K_0.5 of 3.0 mM to a K_0.5 of 0.5mM, rendering the enzyme responsive to calcium concentrations now within the physiological range (0.2-1mM) . This effect was observed with the macromolecular substrate histone H3 but not with the small molecule BAEE, indicating that anti-PAD3/PAD4 antibodies do not affect the fundamental catalytic properties of PAD4. These autoantibodies influence the binding and catalysis of histone H3, and achieve activities of PAD4 which normally require supraphysiologic calcium concentrations. It remains to be determined how broadly this property extends to other macromolecular substrates.

Conclusion: These studies describe a novel biomarker associated with erosive joint damage in RA.  PAD3/PAD4 cross-reactive antibodies may contribute to disease pathogenesis by activating PAD4 enzymatic function and identify a patient subgroup in whom PAD inhibition may be particularly beneficial therapeutically.   

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/anti-peptidylarginine-deiminase-34-cross-reactive-antibodies-a-novel-biomarker-with-clinical-and-mechanistic-implications-in-rheumatoid-arthritis/