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 struc ture (GlcA beta 1-4GlcNAc alpha 1-4)(n), was chemically modified throu gh N-deacetylation, N-sulphation and O-sulphation [Casu, Grazioli, Raz i, Guerrini, Naggi, Torri, Oreste, Tursi, Zoppetti and Lindahl (1994) Carbohydr. Res, 263, 271-284]. Depending on the reaction conditions, t he products showed different proportions of components with high affin ity for antithrombin (AT), A high-affinity subfraction, M(r) approx. 3 6000, was shown by near-UV CD, UV-absorption difference spectroscopy a nd fluorescence to cause conformational changes in the AT molecule ver y similar to those induced by high-affinity heparin. Fluorescence titr ations demonstrated about two AT-binding sites per polysaccharide chai n, each with a K-d of approx. 200 nM. The anti-(Factor Xa) activity wa s 170 units/mg, similar to that of the IIId international heparin stan dard and markedly higher than activities of previously described hepar in analogues. Another preparation, M(r) approx. 13000, of higher overa ll 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 correlat ion 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 pentasacchari de sequence in heparin. The abundance of the implicated disaccharide u nit approximately equalled that of AT-binding sites in the 36000-M(r) polysaccharide fraction, and approached one per high-affinity oligosac charide (predominantly 10-12 monosaccharide units) isolated after part ial depolymerization of AT-binding polysaccharide. These findings sugg est that the modified bacterial polysaccharide interacts with AT and p romotes its anticoagulant action in a manner similar to that of hepari n.