Building Block Analysis of ATIII Affinity Fractions of Heparins: Application to the ATIII Binding Capacity of Non-conventional 3-O-Sulfated Sequences.

In heparin, some 3-O-sulfated sequences do not meet the structural requirements of the ATIII binding pentasaccharide. These "non-conventional" sequences are the object of this study. In a previous paper (Mourier P. Heparinase digestion of 3-O-sulfated sequences: selective heparinase II digestion for separation and identification of binding sequences present in ATIII affinity fractions of bovine intestine heparins), we demonstrated that unsaturated 3-O-sulfated disaccharides detected in exhaustive heparin digests were specifically cleaved by heparinase I. Consequently, building blocks analyses of heparins using heparinases I+II+III digestion could be compared with experiments where only heparinase II is used. In these latter conditions of depolymerization, the 3-O-sulfated sequences digested into unsaturated 3-O-sulfated disaccharides with heparinases I+II+III, were heparinase II-resistant on their non-reducing side, resulting in longer new building blocks. These properties were used to study the structural neighborhood of these 3-O-sulfated moieties, which have still-undefined biological functions. In this part, heparinases I+II+III and heparinase II digestions of porcine mucosa, bovine mucosa and bovine lung heparins were compared in six fractions of increasing affinity for ATIII. Tagging of building blocks by reductive amination with sulfanilic acid was used. The distribution of 3-O-sulfated building blocks in the ATIII affinity fractions was used to examine the ATIII binding of these sequences.

Heparinase Digestion of 3-O-Sulfated Sequences: Selective Heparinase II Digestion for Separation and Identification of Binding Sequences Present in ATIII Affinity Fractions of Bovine Intestinal Heparins.

Binding to antithrombin-III (ATIII) determines the anticoagulant activity of heparin. The complexes formed between heparin and ATIII result from a specific pentasaccharide sequence containing a 3-O-sulfated glucosamine in medium position. Building block analysis of heparins, following heparinase digestion, is a critical method in quality control that provides a simple structural characterization of a complex product. Hence, in these applications, study of the digestion of 3-O-sulfated moieties merits special attention. With heparinase II, specific inhibition of cleavage of the non-reducing bond of 3-O-sulfated units is observed. This specificity was erroneously generalized to other heparinases when it was observed that in exhaustive digests of heparins with the heparinase mixture, resistant 3-O-sulfated tetrasaccharides were also obtained from the specific ATIII-binding pentasaccharides. In fact, the detection of unsaturated 3-O-sulfated disaccharides in digests of heparin by heparinases I+II+III, resulting from the cleavage of the 3-O sulfated unit by heparinase I in non-conventional sequences, shows that this inhibition has exceptions. Thus, in experiments where heparinase II is selectively applied, these sequences can only be digested into tetra- or hexasaccharides where the 3-O-sulfated glucosamine is shifted on the reducing end. Heparinase I+II+III and heparinase II digests with additional tagging by reductive amination with sulfanilic acid were used to study the structural neighborhood of 3-O-sulfated disaccharides in bovine mucosal heparin fractions with increasing affinity for ATIII. The 3-O-sulfated disaccharides detected in heparinase I+II+III digests turn into numerous specific 3-O-sulfated tetrasaccharides in heparinase II digests. Additionally, ATIII-binding pentasaccharides with an extra 3-O-sulfate at the reducing glucosamine are detected in fractions of highest affinity as heparinase II-resistant hexasaccharides with two consecutive 3-O-sulfated units.

Specific Non-Reducing Ends in Heparins from Different Animal Origins: Building Blocks Analysis Using Reductive Amination Tagging by Sulfanilic Acid.

Heparins are linear sulfated polysaccharides widely used as anticoagulant drugs. Their nonreducing-end (NRE) has been little investigated due to challenges in their characterization, but is known to be partly generated by enzymatic cleavage with heparanases, resulting in N-sulfated glucosamines at the NRE. Uronic NRE (specifically glucuronic acids) have been isolated from porcine heparin, with GlcA-GlcNS,3S,6S identified as a porcine-specific NRE marker. To further characterize NRE in heparinoids, a building block analysis involving exhaustive heparinase digestion and subsequent reductive amination with sulfanilic acid was performed. This study describes a new method for identifying heparin classical building blocks and novel NRE building blocks using strong anion exchange chromatography on AS11 columns for the assay, and ion-pair liquid chromatography-mass spectrometry for building block identification. Porcine, ovine, and bovine intestine heparins were analyzed. Generally, NRE on these three heparins are highly sulfated moieties, particularly with 3-O sulfates, and the observed composition of the NRE is highly dependent on heparin origin. At the highest level of specificity, the isolated marker was only detected in porcine heparin. However, the proportion of glucosamines in the NRE and the proportion of glucuronic/iduronic configurations in the NRE uronic moieties greatly varied between heparin types.

Oligosaccharide Chromatographic Techniques for Quantitation of Structural Process-Related Impurities in Heparin Resulting From 2-O Desulfation.

Heparin is a widely-used intravenous anticoagulant comprising a complex mixture of highly-sulfated linear polysaccharides of repeating sequences of uronic acids (either iduronic or glucuronic) 1->4 linked to D-glucosamine with specific sulfation patterns. Preparation of crude heparin from mammalian mucosa involves protease digestion with alcalase under basic conditions (pH ≥ 9) and high temperature (>50°C) and also oxidation. Under such conditions, side reactions including the ubiquitous 2-O desulfation occur on the heparin backbone yielding non-endogenous disaccharides within polysaccharide chains. Whatever the process used for its manufacture, some level of corresponding degradation impurities is therefore expected to be found in heparin and the derived Low Molecular Weight Heparins. These impurities should be monitored to control the quality of the final therapeutic product. Two anion exchange chromatography techniques were used to analyze heparin samples exhaustively or partially depolymerized with heparinases and determine the proportions of non-endogenous disaccharides generated by side reactions during the manufacturing process (epoxides and galacturonic moieties). We also present data from a case study of marketed heparin. Current heparin sodium monographs do not directly address process impurities related to modification of the structure of heparin. Although desulfation reduces the overall biological potency, we found that heparin with an average of one modified disaccharide per chain can still comply with the USP or Ph. Eur. heparin sodium monographs requirements. We have implemented disaccharide analysis to monitor the quality of this product on a risk basis.

New Insights in Thrombin Inhibition Structure-Activity Relationships by Characterization of Octadecasaccharides from Low Molecular Weight Heparin.

Low Molecular Weight Heparins (LMWH) are complex anticoagulant drugs that mainly inhibit the blood coagulation cascade through indirect interaction with antithrombin. While inhibition of the factor Xa is well described, little is known about the polysaccharide structure inhibiting thrombin. In fact, a minimal chain length of 18 saccharides units, including an antithrombin (AT) binding pentasaccharide, is mandatory to form the active ternary complex for LMWH obtained by alkaline ß-elimination (e.g., enoxaparin). However, the relationship between structure of octadecasaccharides and their thrombin inhibition has not been yet assessed on natural compounds due to technical hurdles to isolate sufficiently pure material. We report the preparation of five octadecasaccharides by using orthogonal separation methods including size exclusion, AT affinity, ion pairing and strong anion exchange chromatography. Each of these octadecasaccharides possesses two AT binding pentasaccharide sequences located at various positions. After structural elucidation using enzymatic sequencing and NMR, in vitro aFXa and aFIIa were determined. The biological activities reveal the critical role of each pentasaccharide sequence position within the octadecasaccharides and structural requirements to inhibit thrombin. Significant differences in potency, such as the twenty-fold magnitude difference observed between two regioisomers, further highlights the importance of depolymerisation process conditions on LMWH biological activity.

Analytical comparison of a US generic enoxaparin with the originator product: The focus on comparative assessment of antithrombin-binding components.

Enoxaparin sodium, a low-molecular-weight heparin (LMWH) prepared from porcine intestinal heparin, is widely used for the prevention and treatment of venous thromboembolism. The antithrombotic activity of heparin is mediated mainly through its activation of antithrombin (AT) and subsequent inhibition of coagulation factors. Heparin is a complex heteropolymer and the sulfation pattern of its alternating uronic acid and glucosamine sugar units is a major factor influencing its biological activity. The manufacturing process itself is associated with the introduction of exogenous microheterogeneities that may further affect its biological efficacy. This is important since enoxaparin is prepared by depolymerizing the heparin with the aim of optimizing its biological activity and safety. Changes during its manufacture could thus affect its biological activity and safety. The current study was performed to assess potential differences between the originator enoxaparin and a new generic enoxaparin commercialized by Teva. Heparinase digestion, AT affinity chromatography, gel permeation chromatography, anion exchange chromatography, and nuclear magnetic resonance methodologies were used. The results indicated differences in oligosaccharides related to the cleavage selectivity around the heparin AT-binding sequences of the Teva Enoxaparin Sodium Injection, USP and the originator Sanofi enoxaparin. These differences influence the strength of the AT-binding affinity of the individual oligosaccharides, their ability to activate AT and, therefore, the inhibitory potency on the proteases of the coagulation cascade. This study, together with other published analytical reports, describes specific compositional differences between generics and originator LWMHs. However, it is yet to be established whether such variations might have any clinical relevance.

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.

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.

Isolation of a pure octadecasaccharide with antithrombin activity from an ultra-low-molecular-weight heparin.

Heparin and low-molecular-weight heparins (LMWHs) are anticoagulant drugs that mainly inhibit the coagulation cascade by indirectly interacting with factor Xa and factor IIa (thrombin). Inhibition of factor Xa by antithrombin (AT) requires the activation of AT by specific pentasaccharide sequences containing 3-O-sulfated glucosamine. Activated AT also inhibits thrombin by forming a stable ternary complex of AT, thrombin, and a polysaccharide (requires at least an 18-mer/octadeca-mer polysaccharide). The full structure of any naturally occurring octadecasaccharide sequence has yet to be determined. In the context of the development of LMWH biosimilars, structural data on such important biological mediators could be helpful for better understanding and regulatory handling of these drugs. Here we present the isolation and identification of an octadecasaccharide with very high anti-factor Xa activity (∼3 times higher than USP [U.S. Pharmacopeia] heparin). The octadecasaccharide was purified using five sequential chromatographic methods with orthogonal specificity, including gel permeation, AT affinity, strong anion exchange, and ion-pair chromatography. The structure of the octadecasaccharide was determined by controlled enzymatic sequencing and nuclear magnetic resonance (NMR). The isolated octadecasaccharide contained three consecutive AT-binding sites and was tested in coagulation assays to determine its biological activity. The isolation of this octadecasaccharide provides new insights into the modulation of thrombin activity.

Heparin dodecasaccharide containing two antithrombin-binding pentasaccharides: structural features and biological properties.

The antithrombin (AT) binding properties of heparin and low molecular weight heparins are strongly associated to the presence of the pentasaccharide sequence AGA*IA (A(NAc,6S)-GlcUA-A(NS,3,6S)-I(2S)-A(NS,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.

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.

Heparin sodium compliance to USP monograph: structural elucidation of an atypical 2.18 ppm NMR signal.

The ¹H nuclear magnetic resonance (NMR) acceptance criteria in the new heparin US Pharmacopeia (USP) monograph do not take into account potential structural modifications responsible for any extra signals observed in ¹H NMR spectra, some purified heparins may be non-compliant under the proposed new USP guidelines and incorrectly classified as unsuitable for pharmaceutical use. Heparins from the "ES" source, containing an extra signal at 2.18 ppm, were depolymerized under controlled conditions using heparinases I, II, and III. The oligosaccharides responsible for the 2.18 ppm signal were enriched using orthogonal chromatographic techniques. After multiple purification steps, we obtained an oligosaccharide mixture containing a highly enriched octasaccharide bearing the structural modification responsible for the extra signal. Following heparinase I depolymerization, a pure tetrasaccharide containing the fingerprint structural modification was isolated for full structural determination. Using 1D and 2D ¹H NMR spectroscopy, the structural moiety responsible for the extra signal at 2.18 ppm was identified as an acetyl group on the heparin backbone, most likely resulting from a very minor manufacturing process side reaction that esterifies the uronic acid at position 3. Such analytical peculiarity has always been present in this heparin source and it was used safety over the years.

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.

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 ß-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.

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 (DeltaU). 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 oligosaccharide-AT complexes.

Isolation and characterization of a heparin with low antithrombin activity from the body of Styela plicata (Chordata-Tunicata). Distinct effects on venous and arterial models of thrombosis.

INTRODUCTION: A heparin preparation with low antithrombin activity and different disaccharide composition than mammalian heparin was isolated from the body of the ascidian Styela plicata (Chordata-Tunicata). The disaccharide composition and the effect of the invertebrate glycan on venous and arterial models of thrombosis was investigated. METHODS AND RESULTS: High performance liquid chromatography of the products formed by a mixture of heparin lyases showed that the ascidian heparin is composed mainly by delta UA(2SO4)-1-->4-beta-d-GlcN(SO4) (47.5%), delta UA(2SO4)-1-->4-beta-d-GlcN(SO4)(6SO4) (38.3%) disaccharides and smaller amounts of the disaccharides delta UA(2SO4)-1-->4-beta-d-GlcN(SO4)(3SO4)(6SO4) (2.8%) and delta UA(2SO4)-1-->4-beta-d-GlcN(SO4)(3SO4) (8.0%). The invertebrate heparin has an aPTT activity of 18 IU/mg and an antithrombin-mediated antithrombin and anti-factor Xa activities 10-fold lower than that of mammalian heparin. In a venous model of thrombosis in the vena cava, S. plicata heparin inhibits only 80% of thrombosis at a dose 10-fold higher than that of the mammalian heparin that inhibits 100% of thrombosis. However, in an arterio-shunt model of arterial thrombosis, both S. plicata and mammalian heparin possess equivalent antithrombotic activities. It is also shown that at equivalent doses, ascidian heparin has a lower bleeding effect than mammalian heparin. CONCLUSION: The antithrombin-mediated anticoagulant activity of heparin polymers is not directly related to antithrombotic potency in the arterio-venous shunt. The results of the present work suggest that heparin preparations obtained from the body of S. plicata may have a safer therapeutic action in the treatment of arterial thrombosis than mammalian heparin.

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 ( approximately 60%) and galactose ( approximately 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 DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4)) (39.7%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(6SO(4)) (38.2%). Small amounts of the 3-O-sulfated disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4)) (9.8%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4))(6SO(4)) (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.

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.

Chromatographic analysis and sequencing approach of heparin oligosaccharides using cetyltrimethylammonium dynamically coated stationary phases.

C(18) and C(8) bonded stationary phases dynamically coated with cetyltrimethylammonium (CTA) and strong anion exchange (SAX) were developed to obtain separations of oligosaccharide mixtures resulting from chemical or enzymatic depolymerization of heparin. With this method, the retention of sulfated oligosaccharides is directly adjustable depending on the amount of CTA adsorbed into the column. Oligosaccharides containing up to 20 sulfates were separated with a resolving power superior to that of conventional SAX analysis. The stability of the column coating enables hundreds of injections. Using ammonium methane sulfonate aqueous solutions as ultraviolet transparent mobile phases, it was possible to set up double detection, including selective detection of acetylated oligosaccharides. Analytical gel permeation chromatography was directly coupled to CTA-SAX, obtaining a two-dimensional profile of analyzed oligosaccharidic mixtures. A sequencing method of heparin oligosaccharides using partial depolymerization by heparinases according to their size and sulfation pattern and digest analysis by CTA-SAX was developed. A direct application of this method to the analysis of oligosaccharide mixtures obtained by complete digestion of heparins by heparinases I, II, and III was done. It allowed a reliable quantification of heparin building blocks. We also focused our attention on di- and tetrasaccharidic species containing the 3-O-sulfated glucosamines taken as markers of the active sites for antithrombin III. The method was also applied to more complex mixtures resulting from porcine heparin partially depolymerized with heparinase I. The specificity of the reaction was studied up to decasaccharidic fractions.