Preparation of gellan sulfate as an artificial ligand for removal of extra domain A containing fibronectin

The extra domain A containing fibronectin (EDA(+)FN) concentration in plasma of rheumatoid arthritis (RA) is abnormally higher than the normal level. We synthesized various gellan-sulfate (GS) candidates as artificial ligands for removing EDA(+)FN from plasma. The interaction between these artificial ligands and EDA(+)FN was evaluated using affinity constants (KA), which were determined by surface plasmon resonance measurement. The KA (3.6×10⁸ per M) of GS-25 [degree of substitution for sulfonation (DS)=25%] with EDA(+)FN was higher than those of other molecules: GS-16 (DS=16%) at 8.3×10⁷ per M, and GS-35 (DS=35%) at 1.7×10⁸ per M. Furthermore, GSs displayed selectivity of EDA(+)FN for binding with plasma FN (KA_EDA(+)FN/KA_{plasma FN}>2). The removal ratio in plasma was measured by using GS-immobilized gel. Removals of 66, 11, 7.7, 6.2, 6.9, and 12% for EDA(+)FN, plasma FN, fibrinogen, albumin, immunoglobulin G (IgG) and antithrombin III from the patient-model plasma were, respectively, achieved with GS-25-immobilized gel. These results suggest that GS may be used as a selective artificial ligand for EDA(+)FN removal from plasma in RA treatment.