High‐Titer Rheumatoid Arthritis Antibodies Preferentially Bind Fibrinogen Citrullinated by Peptidylarginine Deiminase 4

Objective Most patients with rheumatoid arthritis (RA) harbor antibodies to citrullinated autoantigens such as citrullinated fibrinogen. Two isoforms of peptidylarginine deiminase (PAD), PAD type 2 (PAD2) and PAD4, which catalyze citrullination with different substrate specificities, can be detected in the synovium of RA patients. This study was undertaken to determine whether RA antibodies preferentially bind PAD2‐ or PAD4‐citrullinated fibrinogen. Methods RA patient and normal donor plasma specimens were tested for binding to PAD2‐ or PAD4‐citrullinated fibrinogen, native fibrinogen, or citrullinated fibrinogen peptides in various dilutions by enzyme‐linked immunosorbent assay (ELISA) and Western blotting. Bands corresponding to masses demonstrating RA antibody reactivity by Western blotting were excised and analyzed by mass spectrometry. Results At low antibody titers (1:40 and 1:100), there was no significant difference between RA antibody reactivity to PAD2‐ and PAD4‐citrullinated fibrinogen. When plasma was further diluted to 1:250 and 1:1,000, RA patient plasma bound PAD4‐citrullinated fibrinogen significantly more than PAD2‐citrullinated fibrinogen, as measured by ELISA and Western blotting. An increased antibody titer was associated with increased avidity for both PAD2‐ and PAD4‐citrullinated fibrinogen. Both enzymes hypercitrullinated fibrinogen, but PAD4 citrullinated arginines more intermittently, generating a mix of citrullinated and noncitrullinated arginines. Peptide ELISA and preadsorption assays confirmed that the region of intermittent citrullination accounts for the majority of RA antibody binding to the β‐chain of citrullinated fibrinogen. Conclusion At high titers, RA antibodies preferentially bind fibrinogen modified by PAD4, because intermittent citrullination offers a more diverse assortment of citrullinated epitopes.