Christian Viskov

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 (GlcNNAc/NS,6S-GlcA-GlcNNS,3,6S-IdoUA2S-GlcNNS,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 (ΔU). In two octasaccharides, AGA*IA was elongated at the reducing end by units IdoUA2S-GlcNNS,6S (OCTA-1) or IdoUA-GlcNNAc,6S (OCTA-2). In the other two octasaccharides (OCTA-3 and OCTA-4), AGA*IA was elongated at the nonreducing side by units GlcNNS,6S-IdoUA and GlcNNS,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 oligosaccharide-AT complexes.

Effects on Molecular Conformation and Anticoagulant Activities of 1,6-Anhydrosugars at the Reducing Terminal of Antithrombin-Binding Octasaccharides Isolated from Low-Molecular-Weight Heparin Enoxaparin

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.

The Hemolymph of the Ascidian Styela plicata (Chordata-Tunicata) Contains Heparin inside Basophil-like Cells and a Unique Sulfated Galactoglucan in the Plasma

The hemolymph of ascidians (Chordata-Tunicata) contains different types of hemocytes embedded in a liquid plasma. In the present study, heparin and a sulfated heteropolysaccharide were purified from the hemolymph of the ascidian Styela plicata. The heteropolysaccharide occurs free in the plasma, is composed of glucose (∼60%) and galactose (∼40%), and is highly sulfated. Heparin, on the other hand, occurs in the hemocytes, and high performance liquid chromatography of the products formed by degradation with specific lyases revealed that it is composed mainly by the disaccharides ΔUA(2SO4)-1→4-β-d-GlcN(SO4) (39.7%) and ΔUA(2SO4)-1→4-β-d-GlcN(SO4)(6SO4) (38.2%). Small amounts of the 3-O-sulfated disaccharides ΔUA(2SO4)-1→4-β-d-GlcN(SO4)(3SO4) (9.8%) and ΔUA(2SO4)-1→4-β-d-GlcN(SO4)(3SO4)(6SO4) (3.8%) were also detected. These 3-O-sulfated disaccharides were demonstrated to be essential for the binding of the hemocyte heparin to antithrombin III. Electron microscopy techniques were used to characterize the ultrastructure of the hemocytes and to localize heparin and histamine in these cells. At least five cell types were recognized and classified as univacuolated and multivacuolated cells, amebocytes, hemoblasts, and granulocytes. Immunocytochemistry showed that heparin and histamine co-localize in intracellular granules of only one type of hemocyte, the granulocyte. These results show for the first time that in ascidians, a sulfated galactoglucan circulates free in the plasma, and heparin occurs as an intracellular product of a circulating basophil-like cell.

An unusual antithrombin-binding heparin octasaccharide with an additional 3-O-sulfated glucosamine in the active pentasaccharide sequence

The 3-O-sulfation of N-sulfated glucosamine is the last event in the biosynthesis of heparin/heparan sulfate, giving rise to the antithrombin-binding pentasaccharide sequence AGA*IA, which is largely associated with the antithrombotic activity of these molecules. The aim of the present study was the structural and biochemical characterization of a previously unreported AGA*IA*-containing octasaccharide isolated from the very-low-molecular-mass heparin semuloparin, in which both glucosamine residues of the pentasaccharide moiety located at the non-reducing end bear 3-O-sulfate groups. Two-dimensional and STD (saturation transfer difference) NMR experiments clearly confirmed its structure and identified its ligand epitope binding to antithrombin. The molecular conformation of the octasaccharide-antithrombin complex has been determined by NMR experiments and docking/energy minimization. The presence of the second 3-O-sulfated glucosamine in the octasaccharide induced more than one order of magnitude increase in affinity to antithrombin compared to the pentasaccharide AGA*IA.

Conformational transitions induced in heparin octasaccharides by binding with antithrombin III

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 Dodecasaccharide Containing Two Antithrombin-binding Pentasaccharides STRUCTURAL FEATURES AND BIOLOGICAL PROPERTIES

Background: Heparin is a linear sulfated polysaccharide used clinically as an anticoagulant. Results: A heparin dodecasaccharide, containing two contiguous antithrombin-binding sequences, has been described and characterized for the first time. Conclusion: The dodecasaccharide binds antithrombin in two different molecular assemblies enhancing the probability of the binding and the affinity. Significance: The discovery of this dodecasaccharide improves the knowledge of heparin structure. The antithrombin (AT) binding properties of heparin and low molecular weight heparins are strongly associated to the presence of the pentasaccharide sequence AGA*IA (ANAc,6S-GlcUA-ANS,3,6S-I2S-ANS,6S). By using the highly chemoselective depolymerization to prepare new ultra low molecular weight heparin and coupling it with the original separation techniques, it was possible to isolate a polysaccharide with a biosynthetically unexpected structure and excellent antithrombotic properties. It consisted of a dodecasaccharide containing an unsaturated uronate unit at the nonreducing end and two contiguous AT-binding sequences separated by a nonsulfated iduronate residue. This novel oligosaccharide was characterized by NMR spectroscopy, and its binding with AT was determined by fluorescence titration, NMR, and LC-MS. The dodecasaccharide displayed a significantly increased anti-FXa activity compared with those of the pentasaccharide, fondaparinux, and low molecular weight heparin enoxaparin.

Isolation and Characterization of Contaminants in Recalled Unfractionated Heparin and Low-Molecular-Weight Heparin

Recently, a contaminant was found in some clinically used unfractionated heparin (UFH) preparations. Administration of this UFH was associated with an increased risk of developing a wide range of adverse effects including death. To further investigate the chemical profile of the contaminant, contaminated batches of UFH were treated by exhaustive nitrous acid depolymerization followed by methanol precipitation to remove heparin oligosaccharides. Because contaminated heparins may have been used as starting material in the production of low-molecular-weight heparins (LMWHs), a similar procedure was carried out using an experimental batch of enoxaparin prepared from contaminated heparin. While high-pressure liquid chromatography (HPLC) analysis of contaminated heparin did not distinguish the presence of the contaminant, it could readily be observed as a high-molecular weight shoulder in the elution profile of contaminated enoxaparin. Digesting contaminated heparin with heparinase-I prior to HPLC analysis showed the presence of a nondigestible component (15%-30% of the mixture). This contaminant was also resistant to degradation by chondroitinases A, B, and C. Proton nuclear magnetic resonance (NMR) indicated that the contaminant was oversulfated chondroitin sulfate (OSCS). Size-exclusion chromatography indicated that the mean molecular weight of the OSCS was 16.8 kD, comparable to that of a synthetic porcine cartilage OSCS preparation that was used as a reference material (17.2 kD). While varying degrees of high-molecular weight dermatan sulfate and other minor impurities were detected, OSCS appeared to be the major contaminant in these preparations. The process involved in the production of enoxaparin does not significantly degrade OSCS.

Heparin sodium compliance to the new proposed USP monograph: Elucidation of a minor structural modification responsible for a process dependent 2.10 ppm NMR signal

Heparin is a highly sulfated hetero polysaccharide mixture found and extracted from mammalian tissues. It has been widely used as an anticoagulant drug during the past decades. In the new proposed USP heparin monograph, the ¹H NMR acceptance criteria to prevent contamination by over sulfated chondroitin sulfate (OSCS), or other persulfated glycosaminoglycans, specifies that no unidentified signals greater than 4% of the mean of signal height of 1 and 2 should be present in the following ranges: 0.10-2.00, 2.10-3.20, and 5.70-8.00 ppm. However, those criteria do not take into account the impact of potential structural modifications generated by the heparin manufacturing processes. In fact, starting from pig mucosa, heparin purification involves oxidizing reagents such as sodium peroxide, potassium permanganate and peracetic acid. In the present work, we demonstrate that potassium permanganate treated heparins show a small but characteristic extra signal at 2.10 ppm. Controlled heparinase I depolymerisation is used to target and excise the oligosaccharide responsible for this extra signal from the polysaccharide backbone. By using orthogonal chromatographic techniques, the fingerprint oligosaccharide was isolated and its structure elucidated. Without the identification of this structural moiety, such purified heparins may have been considered as non-compliant drug substance and not suitable for pharmaceutical use.

Antithrombin-binding oligosaccharides: structural diversities in a unique function?

Heparin-antithrombin interaction is one of the most documented examples of heparin/protein complexes. The specific heparin sequence responsible for the binding corresponds to a pentasaccharide sequence with an internal 3-O-sulfated glucosamine residue. Moreover, the position of the pentasaccharide along the chain as well as the structure of the neighbor units affects the affinity to antithrombin. The development of separation and purification techniques, in conjunction with physico-chemical approaches (mostly NMR), allowed to characterize several structural variants of antithrombin-binding oligosaccharides, both in the free state and in complex with antithrombin. The article provides an overview of the studies that lead to the elucidation of the mechanism of interaction as well as acquiring new knowledge in heparin biosynthesis.

Analytical and statistical comparability of generic enoxaparins from the US market with the originator product.

Low-molecular-weight heparins (LMWHs) are complex anticoagulant drugs, made from heparin porcine mucosa starting material. Enoxaparin sodium manufactured by Sanofi is one of the most widely prescribed LMWHs and has been used since 1993 in the USA. In 2010, US Food and Drug Administration approval for supplying generic enoxaparin was granted to Sandoz and subsequently to Amphastar. Little is known, however, of the differences in composition of these preparations. In this study, samples from several batches of generic enoxaparins were purchased on the US market and analyzed with state of the art methodologies, including disaccharide building blocks quantification, nuclear magnetic resonance (NMR), and a combination of orthogonal separation techniques. Direct high-performance liquid chromatography analysis of the different enoxaparin batches revealed distinct process fingerprints associated with each manufacturer. Disaccharide building block analysis showed differences in the degree of sulfation, the presence of glycoserine derivatives, as well as in proportions of disaccharides. Results were compared by statistical approaches using multivariate analysis with a partial least squares discriminant analysis methodology. The variations were statistically significant and allowed a clear distinction to be made between the enoxaparin batches according to their manufacturer. These results were further confirmed by orthogonal analytical techniques, including NMR, which revealed compositional differences of oligosaccharides both in low- and high-affinity antithrombin fractions of enoxaparin.