Adhesion of 3-carboxymethyl-cellulose-6-sulfate to extra domain A-containing fibronectin: development of ligands for cryogel removal

Abstract

Rheumatoid arthritis (RA) patients, in whom cryogelation occurs in the presence of heparin (HP), ex-hibit abnormally high concentrations of extra domain A-containing fibronectin [EDA(+)FN] in their plasma. RA-associated cryogel is a complex of EDA(+)FN, plasma FN, fibrinogen, and HP in patient plasma. We believe that removal of EDA(+)FN will result in effective inhibition of cryogelation. Although HP has a high affinity for FN and selectivity for EDA(+)FN over plasma FN, HP also binds antithrombin III (which is a normal plasma protein). In this study, we synthesized the following three artificial ligands, which do not bind antithrombin III, for the purpose of removing EDA(+)FN from plasma: cellulose-6-sulfate (C-6S), 3-carboxymethyl cellulose-6-sulfate (3CM-C-6S), and 2-carboxymethyl cellulose-6-sulfate (2CM-C-6S). We evaluated interaction between these artificial ligands and EDA(+)FN by determining affinity constants (KAs). The KA of 3CM-C-6S for binding to EDA(+)FN (7.86 × 108 M−1) was higher than that of other molecules: C-6S, 3.03 × 108 M−1; 2CM-C-6S, 3.57 × 108 M−1. Furthermore, selectivity of adsorption (S = KAEDA(+)FN/KAEDA(−)FN) of 3CM-C-6S for EDA(+)FN over EDA(−)FN (S = 6.25) was higher than that of HP (S = 2.52). These results suggest 3CM-C-6S is a selective ligand suitable for removal of EDA(+)FN from plasma, as a method of inhibition of cryogelation.