Antithrombin-binding octasaccharides and role of extensions of the active pentasaccharide sequence in the specificity and strength of interaction -: Evidence for very high affinity induced by an unusual Glucuronic acid residue

The antithrombotic activity of low molecular weight heparins (LMWHs) is largely associated with the antithrombin ( AT)binding pentasaccharide sequence AGA*IA (GlcN(NAc/NS),(6S)-GlcA-GlcN(NS),(3,6S)-IdoUA(2S)-GlcN(NS,6S)). The location of the AGA*IA sequences along the LMWH chains is also expected to influence binding to AT. This study was aimed at investigating the role of the structure and molecular conformation of different disaccharide extensions on both sides of the AGA*IA sequence in modulating the affinity for AT. Four high purity octasaccharides isolated by size exclusion chromatography, high pressure liquid chromatography, and AT-affinity chromatography from the LMWH enoxaparin were selected for the study. All the four octasaccharides terminate at their nonreducing end with 4,5-unsaturated uronic acid residues (Delta U). In two octasaccharides, AGA*IA was elongated at the reducing end by units IdoUA(2S)-GlcN(NS,6S) (OCTA- 1) or IdoUA-GlcN(NAc),(6S) (OCTA-2). In the other two octasaccharides (OCTA-3 and OCTA-4), AGA*IA was elongated at the nonreducing side by units GlcN(NS),(6S)-IdoUA and GlcN(NS),(6S)-GlcA, respectively. Extensions increased the affinity for AT of octasaccharides with respect to pentasaccharide AGA*IA, as also confirmed by fluorescence titration. Two- dimensional NMR and docking studies clearly indicated that, although elongation of the AGA*IA sequence does not substantially modify the bound conformation of the AGA*IA segment, extensions promote additional contacts with the protein. It should be noted that, as not previously reported, the unusual GlcA residue that precedes the AGA*IA sequence in OCTA-4 induced an unexpected 1 order of magnitude increase in the affinity to AT with respect to its IdoUA- containing homolog OCTA-3. Such a residue was found to orientate its two hydroxyl groups at close distance to residues of the protein. Besides the well established ionic interactions, nonionic interactions may thus contribute to strengthen oligosaccharideAT complexes.