Preferential recognition of a fragment species of osteoarthritic synovial fluid fibronectin by antibodies to the alternatively spliced EIIIA segment

Objective. To characterize the species of synovial fluid (SF) fibronectin ( FN) bearing the alternatively spliced EIIIA segment. Methods. SF from patients with osteoarthritis (OA) and rheumatoid arthritis (RA), as well as corresponding affinity isolation products, were subjected to I-dimensional and 2-dimensional electrophoresis followed by Western blo t analysis. Results. Regardless of the clinical type of arthritis, a polyclonal antibod y that recognizes antigenic determinants throughout the FN molecule produce d staining of predominantly similar to 200+ and similar to 170-kd species i n reduced 1-dimensional electrophoresis. Despite the overall prevalence of the larger species, 4 monoclonal antibodies (mAb) reactive with sequences l ying near the center of the EIIIA segment exhibited a relative failure to r ecognize the larger of these 2 species in OA, but not RA, SF. The absence o f recognition of EIIIA sequences within the similar to 200+ kd forms of OA SF FN was unrelated to their derivation from dimers, since anti-EIIIA, mAb recognized the smaller fragment species in preference to both monomeric and dimeric forms. The similar to 170-kd EIIIA+ fragments were observed to hav e minimal gelatin-binding capacity and appeared on 2-dimensional electropho resis to extend from the N-terminus of FN through at least the center of th e EIIIA segment. Similar results were obtained for samples obtained by need le aspiration or arthroscopic lavage, suggesting a widespread applicability of these findings. Conclusion. The similar to 170-kd EIIIA+ species of FN could potentially co nstitute a soluble "vehicle" by which chondrocyte-regulating EIIIA sequence s, liberated from inhibitory flanking C-terminal sequences, could reach cel ls in the arthritic joint. Additionally, "FN species-specific" recognition of this segment within OA SF could constitute a marker by which to gauge th e activity of the OA disease process.