Background/Purpose: Most patients with rheumatoid arthritis (RA) harbor antibodies to citrullinated autoantigens, such as citrullinated fibrinogen. Two isoforms of peptidylarginine deiminase (PAD), PAD2 and PAD4, which catalyze citrullination with different substrate specificities, can be detected in rheumatoid arthritis (RA) synovium. This study was undertaken to determine whether RA patient antibodies preferentially bind PAD2 or PAD4 citrullinated fibrinogen.

Methods: RA patient and normal donor plasma was tested for binding to PAD2 or PAD4 citrullinated fibrinogen, native fibrinogen, or citrullinated fibrinogen peptides in various dilutions by ELISA and Western blot. Bands corresponding to masses demonstrating RA patient reactivity by Western blot were excised and analyzed by mass spectrometry.

Results: As expected, at low titers neither normal donors nor RA patients harbored antibodies to unreactive (native) plasma derived fibrinogen while RA patients harbored significantly elevated antibody to both PAD2 and PAD4 citrullinated fibrinogen. When plasma was further diluted to 1:250 and 1:1000, the OD of RA patient plasma binding of PAD4 citrullinated fibrinogen was significantly greater than PAD2 citrullinated fibrinogen (p<0.01). The ratio of ELISA OD of PAD4 citrullinated fibrinogen divided by PAD2 citrullinated fibrinogen was calculated for each patient. The mean ratios of PAD4/PAD2 citrullinated fibrinogen at the 1:250 and 1:1000 dilutions were 1.26 (95% confidence interval 1.18-1.35) and 1.39 (95% confidence interval 1.24-1.53) respectively, indicating preferential binding of PAD4 citrullinated fibrinogen. RA patient plasma also bound PAD4 citrullinated fibrinogen more than PAD2 citrullinated fibrinogen on Western blot at both 56kD and 76kD (p<0.01 for both bands). Increasing antibody titer associated with increasing avidity (p<0.0001) but there was no difference in avidity for PAD2 or PAD4 citrullinated fibrinogen at low or high titer. Mass spectrometry based analysis of bands corresponding to masses demonstrating RA patient reactivity on western blot identified a hotspot of citrullination of the beta chain of fibrinogen between arginine residues 44 and 74. This hotspot region was modified more heavily than any other site. Both enzymes hypercitrullinated fibrinogen, but PAD4 citrullinates arginines more intermittently, generating peptides with a mix of citrullinated and nonmodifed arginines.

Conclusion: At high titer, RA patient antibodies preferentially bind fibrinogen with a combination of both citrullinated and non-citrullinated arginines.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/high-titer-rheumatoid-arthritis-antibodies-distinguish-between-pad2-and-pad4-citrullinated-fibrinogen/