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The present study deals with the conformation in solution of two heparin octasaccharides containing the pentasaccharide sequence GlcN(NAc,6S)-GlcA-GlcN(NS,3,6S)-IdoA(2S)-GlcN(NS,6S) [AGA*IA; where GlcN(NAc,6S) is N-acetylated, 6-O-sulfated alpha-D-glucosamine, GlcN(NS,3,6S) is N,3,6-O-trisulfated alpha-D-glucosamine and IdoA(2S) is 2-O-sulfated IdoA (alpha-L-iduronic acid)] located at different positions in the heparin chain and focuses on establishing geometries of IdoA residues (IdoA(2S) and IdoA) both inside and outside the AGA*IA sequence. AGA*IA constitutes the active site for AT (antithrombin) and is essential for the expression of high anticoagulant and antithrombotic activities. Analysis of NMR parameters [NOEs (nuclear Overhauser effects), transferred NOEs and coupling constants] for the two octasaccharides indicated that between the 1C4 and 2S0 conformations present in dynamic equilibrium in the free state for the IdoA(2S) residue within AGA*IA, AT selects the 2S0 form, as previously shown [Hricovini, Guerrini, Bisio, Torri, Petitou and Casu (2001) Biochem. J. 359, 265-272]. Notably, the 2S0 conformation is also adopted by the non-sulfated IdoA residue preceding AGA*IA that, in the absence of AT, adopts predominantly the 1C4 form. These results further support the concept that heparin-binding proteins influence the conformational equilibrium of iduronic acid residues that are directly or indirectly involved in binding and select one of their equi-energetic conformations for best fitting in the complex. The complete reversal of an iduronic acid conformation preferred in the free state is also demonstrated for the first time. Preliminary docking studies provided information on the octasaccharide binding location agreeing most closely with the experimental data. These results suggest a possible biological role for the non-sulfated IdoA residue preceding AGA*IA, previously thought not to influence the AT-binding properties of the pentasaccharide. Thus, for each AT binding sequence longer than AGA*IA, the interactions with the protein could differ and give to each heparin fragment a specific biological response.

Terminal 1,6-anhydro-aminosugars (1,6-anAS) are typical structural moieties of enoxaparin, a low-molecular-weight heparin (LMWH) widely used for prevention and treatment of thrombotic disorders. In the enoxaparin manufacturing process, these modified amino sugars are formed during the β-eliminative cleavage of heparin. To investigate the effect of terminal anAS on antithrombin (AT) binding and on inhibition of factor Xa (FXa), two octasaccharides containing modified AT-binding pentasaccharide sequences were isolated from enoxaparin. The molecular conformation of the octasaccharides terminating with N-sulfo-1,6-anhydro-D-mannosamine and N-sulfo-1,6-anhydro-D-glucosamine, respectively, has been determined both in the absence and presence of AT by NMR experiments and docking simulations. Reduced overall contacts of the terminal anAS residues with the binding region of AT induce a decrease in affinity for AT as well as lower anti-FXa activity. The anti-FXa measured either in buffer or plasma milieu does not show any significant difference, suggesting that the inhibition of anti-FXa remains specific and biologically relevant.