Heparin has been used clinically as an anticoagulant for over 60 years. Typically isolated from porcine intestine, heparin is a mixture of dimeric glycosidic sequences generating complex polysaccharide glycosaminoglycan chains. Recently, certain lots of heparin have been associated with an acute, rapid onset of significant side effects indicative of an allergic-type reaction. To identify potential causes for this serious rise in side effects, we examined lots of heparin that correlated with adverse events using orthogonal high resolution analytical techniques. Through comparison of these results with those obtained on reference lots, suspect lots were found to contain a highly sulfated chondroitin sulfate contaminant. Through detailed structural analysis, the contaminant was found to contain a disaccharide repeat unit of glucuronic acid linked β1→3 to a β-galactosamine. Surprisingly, the disaccharide unit contains an unusual sulfation pattern and is sulfated at the 2-O and 3-O positions of the glucuronic acid as well as at the 4-O and 6-O positions of the galactosamine. The presence of such a contaminant could elicit a biological response as highly sulfated polysaccharides, such as dextran sulfate, are known to be potent mediators of the immune system. Given the nature of the contaminant, traditional screening tests -such as those present as part of the current United States Pharmacopeia heparin monograph -cannot differentiate between affected and unaffected lots. Our analysis suggests effective screening methods that can be employed to determine whether or not heparin lots contain the contaminants reported here.

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Individual low molecular weight heparins (LMWHs) exhibit distinct pharmacological and biochemical profiles because of manufacturing differences. Correlation of biological properties with particular structural motifs is a major challenge in the design of new LMWHs as well as in the development of generic versions of proprietary LMWHs. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy permits identification and quantification of structural peculiarities of LMWH preparations. In this article, heteronuclear single quantum coherence spectroscopy, previously used to determine variously substituted monosaccharide components of heparan sulfate (HS) and HS-like glycosaminoglycan mimics, has been applied to the structural characterization of three commercially available LMWHs (enoxaparin, dalteparin, and tinzaparin). Relevant residues belonging to the parent heparin, as well as minor residues generated by each depolymerization procedure, have been characterized and quantified. The use of a high-sensitivity NMR spectrometer (600 MHz equipped with cryoprobe) allowed the accurate quantification of residues with sensitivity better than 1 to 2%.

Tumor neovascularization (angiogenesis) is regarded as a promising target for anticancer drugs. Heparin binds to fibroblast growth factor-2 (FGF2) and promotes the formation of ternary complexes with endothelial cell surface receptors, inducing an angiogenic response. As a novel strategy to generate antiangiogenic substances exploiting binding to FGF2 while preventing FGF receptor (FGFR) activation, sulfation gaps were generated along the heparin chains by controlled alkali-catalyzed removal of sulfate groups of iduronic acid 2-O-sulfate residues, giving rise to the corresponding epoxide derivatives. A new class of heparin derivatives was then obtained by opening the epoxide rings followed by oxidative glycol-splitting of the newly formed (and the preexisting) nonsulfated uronic acid residues. In vitro these heparin derivatives prevent the formation of FGFR/FGF2/heparan sulfate proteoglycan ternary complexes and inhibit FGF2-stimulated endothelial cell proliferation. They exert an antiangiogenic activity in the chick embryo chorioallantoic membrane assay, where the parent heparin is inactive. Low and very low molecular weight derivatives of a prototype compound, as well as its glycine and taurine derivatives obtained by reductive amination of glycol-split residues, retained the angiostatic activity. A significant relationship was found between the extent of glycol-splitting and the FGF2-antagonist/angiostatic activities of these heparin derivatives. Molecular dynamics calculations support the assumption that glycol-split residues act as flexible joints that, while favoring 1:1 binding to FGF2, disrupt the linearity of heparin chains necessary for formation of active complexes with FGFRs.

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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.

Heparin and heparan sulfate (HS) glycosaminoglycans (HSGAGs) are sulfated polysaccharidesthat play important roles in fundamental biological processes by binding to proteins. The prototypic exampleof HSGAG-protein interactions is that with the fibroblast growth factors (FGFs), specifically FGF1 andFGF2. Structural and biochemical studies have shown that the chain length, sulfation pattern, andconformation of HSGAGs play a critical role in FGF binding and activity. Previously, we showed that atetrasaccharide of the form ANS,6X-I2S-ANS,6X-I2S-OPr (where X is OH or O-sulfate and Pr is propyl) withat least one of the ANS,6X residues having a 6-O sulfate group was the minimum binding motif for FGF1[Guerrini, M., Agulles, T., Bisio, A., Hricovini, M., Lay, L., Naggi, A., Poletti, L., Sturiale, L., Torri, G.,and Casu, B. (2002) Biochem. Biophys. Res. Commun. 292, 222-230]. We report NMR structural analysisusing two-dimensional NOE spectroscopy (2D-NOESY) and transferred NOESY (trNOESY) on a non-6-O-sulfated synthetic tetrasaccharide TETRA (ANS-I2S-ANS-I2S-OPr) both in its free state and bound toFGF2. This tetrasaccharide comprises both the structural trisaccharide motif ANS-I2S-ANS that forms "kinks"in longer heparin chains induced by FGF binding [Raman, R., Venkataraman, G., Ernst, S., Sasisekharan,V., and Sasisekharan, R. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 2357-2362] and the common bindingmotif I2S-ANS-I2S present in octasaccharides that exhibited strong FGF2 binding [Kreuger, J., Salmivirta,M., Sturiale, L., Gimenez-Gallego, G., and Lindahl, U. (2001) J. Biol. Chem. 276, 30744-30752]. Thesedata suggest that TETRA could be the shortest HSGAG oligosaccharide that binds to FGF2. Furthermore,our study confirms that both the IdoA residues in TETRA adopt the chair 1C4 conformation upon FGF2binding to provide the best molecular fit in contrast to an analogous 6-O-sulfated tetrasaccharide motifobserved in the FGF2-HSGAG cocrystal structure where one of the IdoAs adopts skew-boat 2SOconformation. Thus, our study highlights the fact that the conformational plurality of IdoA is able toaccommodate the changes in the sulfation pattern to provide the necessary specificity for protein binding.

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