Inhibition of cryogelation by the novel synthetic peptide (Gly–Arg–Lys–Lys–Thr): recognition site of extra domain A containing fibronectin for heparin in cryogelation
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文摘
Cryogel is a physical gel formed by the heterophilic aggregation of extra domain A (EDA) containing fibronectin [EDA(+)FN], plasma fibronectin (pFN), fibrinogen (Fbg) and heparin (Hep) in the blood of rheumatoid arthritis (RA) patients. In cryogelation EDA(+)FN cross-links to form an interaggregate of cryogel with Hep. In the present study, we determined the recognition structure of Hep for EDA(+)FN by using oligo- and desulfonated-Hep. The affinity constant (KA) (1.2×108 per M) of oligo-Hep for EDA(+)FN did not change with a decrease in number-average molecular weight (4.9×104→6.0×103). The KA-value of desulfonated-Hep for EDA(+)FN decreased from 3.2×108 to 1.0×107 per M with a decrease in the sulfonation ratio (7.0→4.3 % ). We also determined the recognition structure of EDA(+)FN for Hep by an inhibition experiment on the heparin binding domain II (HepII) in EDA(+)FN with the synthetic peptides, Arg–Arg–Ala–Arg (RRAR), Asp–Gln–Ala–Arg (DNAR), Ile–Lys–Tyr–Glu–Lys (IKYEK), and Gly–Arg–Lys–Lys–Try (GRKKT). The GRKKT sequence clearly inhibited bonding between EDA(+)FN and Heps containing oligo- and desulfonated-Hep. The amount of cryogel formed in the RA-patient model plasma corresponded to the EDA(+)FN concentration in cryogel (36.7 % ) normalized by the EDA(+)FN concentration in plasma. When GRKKT was added to plasma, the EDA(+)FN concentration fell to 10.5 % . These results demonstrated that inhibition of cryogelation in plasma could progress to a novel treatment for RA.