STRUCTURAL AND FUNCTIONAL-PROPERTIES OF HEPARIN ANALOGS OBTAINED BY CHEMICAL SULFATION OF ESCHERICHIA-COLI K5 CAPSULAR POLYSACCHARIDE

Capsular polysaccharide from Escherichia coli K5, with the basic structure (GlcA beta 1-4GlcNAc alpha 1-4)(n), was chemically modified through N-deacetylation, N-sulphation and O-sulphation [Casu, Grazioli, Razi, Guerrini, Naggi, Torri, Oreste, Tursi, Zoppetti and Lindahl (1994) Carbohydr. Res, 263, 271-284]. Depending on the reaction conditions, the products showed different proportions of components with high affinity for antithrombin (AT), A high-affinity subfraction, M(r) approx. 36000, was shown by near-UV CD, UV-absorption difference spectroscopy and fluorescence to cause conformational changes in the AT molecule very similar to those induced by high-affinity heparin. Fluorescence titrations demonstrated about two AT-binding sites per polysaccharide chain, each with a K-d of approx. 200 nM. The anti-(Factor Xa) activity was 170 units/mg, similar to that of the IIId international heparin standard and markedly higher than activities of previously described heparin analogues. Another preparation, M(r) approx. 13000, of higher overall O-sulphate content, exhibited a single binding site per chain, with K-d approx. 1 mu M, and an anti-(Factor Xa) activity of 70 units/mg. Compositional analysis of polysaccharide fractions revealed a correlation between the contents of -GlcA-GlcNSO(3)(3, 6-di-OSO3)-disaccharide units and affinity for AT; the 3-O-sulphated GlcN unit has previously been identified as a marker component of the AT-binding pentasaccharide sequence in heparin. The abundance of the implicated disaccharide unit approximately equalled that of AT-binding sites in the 36000-M(r) polysaccharide fraction, and approached one per high-affinity oligosaccharide (predominantly 10-12 monosaccharide units) isolated after partial depolymerization of AT-binding polysaccharide. These findings suggest that the modified bacterial polysaccharide interacts with AT and promotes its anticoagulant action in a manner similar to that of heparin.