The effect of increasing the sulfation level of chondroitin sulfate on anticoagulant specific activity and activation of the kinin system.

Oversulfated chondroitin sulfate (OSCS) was identified as a contaminant in certain heparin preparations as the cause of adverse reactions in patients. OSCS was found to possess both plasma anticoagulant activity and the ability to activate prekallikrein to kallikrein. Differentially sulfated chondroitin sulfates were prepared by synthetic modification of chondroitin sulfate and were compared to the activity of OSCS purified from contaminated heparin. Whilst chondroitin sulfate was found to have minimal anticoagulant activity, increasing sulfation levels produced an anticoagulant response which we directly show for the first time is mediated through heparin cofactor II. However, the tetra-sulfated preparations did not possess any higher anticoagulant activity than several tri-sulfated variants, and also had lower heparin cofactor II mediated activity. Activation of prekallikrein was concentration dependent for all samples, and broadly increased with the degree of sulfation, though the di-sulfated preparation was able to form more kallikrein than some of the tri-sulfated preparations. The ability of the samples to activate the kinin system, as measured by bradykinin, was observed to be through kallikrein generation. These results show that whilst an increase in sulfation of chondroitin sulfate did cause an increase in anticoagulant activity and activation of the kinin system, there may be subtler structural interactions other than sulfation at play given the different responses observed.

Cyr61 is a target for heparin in reducing MV3 melanoma cell adhesion and migration via the integrin VLA-4.

The integrin VLA-4 is important for the metastatic dissemination of melanoma cells. We could recently show that heparin can block VLA-4 binding, which contributes, next to blocking P- and L-selectin, to the understanding of antimetastatic activities of heparin. The matricellular ligand Cyr61, secreted by numerous tumours, is responsible for increased tumourigenicity and metastasis. This has been attributed to Cyr61 binding to, and thus activating integrins. However, a VLA-4/Cyr61 axis has not yet been reported. Since Cyr61 possesses heparin binding capabilities, Cyr61 can be supposed as potential target for heparin to indirectly interfere with integrin functions. The present in vitro studies address (i) the existence of a Cyr61/VLA-4 axis and (ii) the functional relevance of heparin interference via Cyr61. The C-terminal module III of Cyr61 could be exposed as nanomolar affine binding site for VLA-4. A shRNA-based knockdown of Cyr61 in MV3 human melanoma cells reduced VLA-4-mediated cell binding to VCAM-1, migration on fibronectin, and integrin signalling functions significantly. Using a biosensor approach we provide insight into heparin interference with this process. The low-molecular-weight heparin tinzaparin, but not the pentasaccharide fondaparinux, binds module IV of Cyr61 with micromolar affinity. But tinzaparin cannot interfere with Cyr61 accumulation onto syndecan-4, indicating different Cyr61 binding sites for heparin and other GAGs. Nonetheless, tinzaparin affects the VLA-4 binding and signalling functions selectively via Cyr61 already at very low concentration most likely by blocking the cellular secreted free Cyr61. This study emphasises Cyr61 as promising, and hitherto not considered target for heparin to selectively influence integrin functions.

Generation of anti-factor Xa active, 3-O-sulfated glucosamine-rich sequences by controlled desulfation of oversulfated heparins.

In the framework of a project aimed at generating heparin-like sulfation patterns and biological activities in biotechnological glycosaminoglycans, different approaches have been considered for simulating the alpha(1-->4)-linked 2-O-sulfated L-iduronic acid (IdoA2SO(3))-->N,6-O-sulfated D-glucosamine (GlcNSO(3)6SO(3)) disaccharide sequences prevalent in mammalian heparins. Since the direct approach of sulfating totally O-desulfated heparins, taken as model compounds for C-5-epimerized sulfaminoheparosan (N-deacetylated, N-sulfated Escherichia coli K5 polysaccharide), preferentially afforded heparins O-sulfated at C-3 instead than at C-2 of the iduronate residues, leading to products with low anticoagulant activities, the problem of re-generating a substantial proportion of the original IdoA2SO(3) residues was circumvented by performing controlled solvolytic desulfation (with 9:1 v/v DMSO-MeOH) of extensively sulfated heparins. The order of desulfation of major residues of heparin GlcN and IdoA and of the minor one D-glucuronic acid was: GlcNSO(3)>GlcN6SO(3)>IdoA3SO(3) congruent with GlcA2SO(3) congruent with GlcN3SO(3)>IdoA2SO(3) congruent with GlcA3SO(3). Starting from a 'supersulfated' low-molecular weight heparin, we obtained products with up to 40% of iduronate residues O-sulfated exclusively at C-2 and up to 40% of their glucosamine residues O-sulfated at both C-6 and C-3. Upon re-N-sulfation, these products displayed an in vitro antithrombotic activity (expressed as anti-factor Xa units) comparable with those of current low-molecular weight heparins.

Toward a biotechnological heparin through combined chemical and enzymatic modification of the Escherichia coli K5 polysaccharide.

A process to generate glycosaminoglycans with heparin- and heparan sulfate-like sequences from the Escherichia coli K5 capsular polysaccharide is described. This polymer has the same structure as N-acetylheparosan, the precursor in heparin/ heparan sulfate biosynthesis. The process involves chemical N-deacetylation and N-sulfation, enzymatic conversion of up to 60% of the D-glucuronic acid to L-iduronic acid residues, and chemical O-sulfation. Because direct sulfation afforded unwanted 3-O-sulfated (instead of 2-O-sulfated) iduronic acid residues, a strategy involving graded solvolytic desulfation of chemically oversulfated C5-epimerized sulfaminoheparosans was assessed using persulfated heparin and heparan sulfate as model compounds. The O-desulfation process was shown to increase the anti-factor Xa activity of oversulfated heparin.

MALDI mass spectrometry as a tool for characterizing glycosaminoglycan oligosaccharides and their interaction with proteins.

Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry (MS) has emerged as a powerful, sensitive technique for structural analysis of glycosaminoglycans (GAGs) and their fractions and fragments. Whereas the molecular size of low sulfated or nonsulfated species (such as low-molecular weight [LMW] K5 polysaccharides) can be directly determined up to molecular weights (MWs) of 12 kD, polysulfated species require complexing with a basic polypeptide and at present can be characterized (in terms of both MW and end residues) up to the size of a decasaccharide, even in complex mixtures. MALDI spectra of GAG oligosaccharides in the presence of a complexing protein permit to assess binding to the protein and the presence of multimeric complexes.

Effects of sulfation on antithrombin-thrombin/factor Xa interactions in semisynthetic low molecular weight heparins.

Most of the biological effects of heparin and low molecular weight (LMW) heparins are related to their ability to bind to many different proteins. To gain insight into structure-activity relationships, we investigated quantitatively the interactions of a series of sulfated LMW heparins of similar molecular weights (derived from statistical desulfation of a supersulfated heparin) with the target enzymes human antithrombin (AT) and thrombin (T). In addition, we analyzed the activation of the protease inhibitor against T and factor Xa (FXa). A nonlinear correlation between the strength of the AT-heparin complex and the degree of sulfation of the LMW heparins was observed, whereas only a modest modulation of T binding to heparin occurred. The efficiency of the heparin derivatives in activating AT toward the proteases is generally high for derivatives exhibiting a low dissociation constant. Only the supersulfated LMW heparin showed serpin activation ability higher than expected from the affinity studies. These results indicate that chemical modification of the sulfation pattern of LMW heparin can be used to efficiently modulate binding affinity and activity toward biological targets.

Influence of glucose on production and N-sulfation of heparan sulfate in cultured adipocyte cells.

Altered lipoprotein lipase regulation associated with diabetes leading to the development of hypertriglyceridemia might be attributed to possible changes in content and the fine structure of heparan sulfate and its associated lipoprotein lipase. Adipocyte cell surface is the primary site of synthesis of lipoprotein lipase and the enzyme is bound to cell surface heparan sulfate proteoglycans via heparan sulfate side chains. In this study, the effect of diabetes on the production of adipocyte heparan sulfate and its sulfation (especially N-sulfation) were examined. Mouse 3T3-L1 adipocytes were exposed to high glucose (25 mM) and low glucose (5.55 mM) in the medium and cell-associated heparan sulfate was isolated and characterized. A significant decrease in total content of heparan sulfate was observed in adipocytes cultured under high glucose as compared to low glucose conditions. The degree of N-sulfation was-assessed through oligosaccharide mapping of heparan sulfate after chemical cleavages involving low pH (1.5) nitrous acid and hydrazinolysis/high pH (4.0) nitrous acid treatments; N-sulfation was found to be comparable between the adipocyte heparan sulfates produced under these glucose conditions. The activity and message levels for N-deacetylase/N-sulfotransferase, the enzyme responsible for N-sulfation in the biosynthesis of heparan sulfate, did not vary in adipocytes whether they were exposed to low or high glucose. While most cells or tissues in diabetic situations produce heparan sulfate with low-charge density concomitant with a decrease in N-sulfation, adipocyte cell system is an exception in this regard. Heparan sulfate from adipocytes cultured in low glucose conditions binds to lipoprotein lipase by the same order of magnitude as that derived from high glucose conditions. It is apparent that adipocytes cultured under high glucose conditions produce diminished levels of heparan sulfate (without significant changes in N-sulfation). In conclusion, it is possible that the reduction in heparan sulfate in diabetes could contribute to the decreased levels of heparan sulfate associated lipoprotein lipase, leading to diabetic hypertriglyceridemia.

Effect of substitution pattern on 1H, 13C NMR chemical shifts and 1J(CH) coupling constants in heparin derivatives.

1H, 13C NMR chemical shifts and 1J(CH) coupling constants were measured for derivatives of heparin containing various sulfation patterns. 1H and 13C chemical shifts varied considerably after introducing electronegative sulfate groups. Chemical shifts of protons linked to carbons changed by up to 1 ppm on substitution with O- and N-sulfate or acetyl groups. Differences up to 10 ppm were detected for 13C chemical shifts in substituted glucosamine, but a less clear dependence was found in iduronate. 1J(CH) values formed two groups, corresponding to either sulfation or non-sulfation at positions 2 and 3 of glucosamine. O-sulfation caused increases up to 6 Hz in 1J(CH) and N-sulfation decreases up to 4 Hz. N-acetylation gave similar 1J(CH) values to N-sulfation. At positions 2 and 3 of iduronate the trend was less marked; 1J(CH) for O-sulfated positions usually increasing. Introduction of sulfate groups influences chemical shift and 1J(CH) values at the position of substitution, but also at more remote positions. 1J(CH) at the glycosidic linkage positions varied between free-amino and N-sulfated compounds, by up to 9 Hz. These results and changes in chemical shift values suggest that iduronate residues and the glycosidic linkages are affected, indicating overall conformational change. This may have important implications for biological activities.

New insights on the specificity of heparin and heparan sulfate lyases from Flavobacterium heparinum revealed by the use of synthetic derivatives of K5 polysaccharide from E. coli and 2-O-desulfated heparin.

The capsular polysaccharide from E. Coli, strain K5 composed of ...-->4)beta-D-GlcA(1-->4)alpha-D-GlcNAc(1-->4)beta-D-GlcA (1-->..., chemically modified K5 polysaccharides, bearing sulfates at C-2 and C-6 of the hexosamine moiety and at the C-2 of the glucuronic acid residues as well as 2-O desulfated heparin were used as substrates to study the specificity of heparitinases I and II and heparinase from Flavobacterium heparinum. The natural K5 polysaccharide was susceptible only to heparitinase I forming deltaU-GlcNAc. N-deacetylated, N-sulfated K5 became susceptible to both heparitinases I and II producing deltaU-GlcNS. The K5 polysaccharides containing sulfate at the C-2 and C-6 positions of the hexosamine moiety and C-2 position of the glucuronic acid residues were susceptible only to heparitinase II producing deltaU-GlcNS,6S and deltaU,2S-GlcNS,6S respectively. These combined results led to the conclusion that the sulfate at C-6 position of the glucosamine is impeditive for the action of heparitinase I and that heparitinase II requires at least a C-2 or a C-6 sulfate in the glucosamine residues of the substrate for its activity. Iduronic acid-2-O-desulfated heparin was susceptible only to heparitinase II producing deltaU-GlcNS,6S. All the modified K5 polysaccharides as well as the desulfated heparin were not substrates for heparinase. This led to the conclusion that heparitinase II acts upon linkages containing non-sulfated iduronic acid residues and that heparinase requires C-2 sulfated iduronic acid residues for its activity.

"Linkage region" sequences of heparins and heparan sulfates: detection and quantification by nuclear magnetic resonance spectroscopy.

The (13)C NMR spectra of most heparin and heparan sulfate preparations display minor signals not attributable to the glycosaminoglycan chains of these polysaccharides. These signals have been "concentrated" in oligosaccharides isolated from an acid hydrolyzate of heparin and shown to arise from the sequence GlcA-Gal-Gal-Xyl of the "linkage region" (LR) connecting the carbohydrate chains to the peptide chains in the original proteoglycans. Mono- and two-dimensional (1)H and (13)C NMR analysis of the major oligosaccharide (LR-OLIGO) indicated the prevalent structure GlcA-GlcNAc-GlcA-Gal-Gal-Xyl, where GlcNAc is partially 6-O-sulfated. (13)C NMR signals at 84.6 and 85.0 ppm, arising from C-3 of the two Gal residues, lend themselves to easy detection and quantification of the linkage region in heparins and heparan sulfates and can be used to assess the importance of the LR in the modulation of various biological activities of these glycosaminoglycans.

Solid state NMR spectroscopy study of molecular motion in cyclomaltoheptaose (beta-cyclodextrin) crosslinked with epichlorohydrin.

Dry and hydrated insoluble cyclomaltoheptaose (beta-cyclodextrin, beta-CD) polymers have been investigated by solid state 13C NMR spectroscopy techniques such as cross polarization/magic angle spinning with dipolar decoupling (CP/MAS), magic angle spinning both with (DD-MAS) and without (MAS) dipolar decoupling and CP/MAS dipolar dephasing (dd-CP/MAS) to allow the assignment of the main 13C signals. In the solid state, the presence of water in the samples resulted in a better resolution reflecting increased mobility. Two distinct components (crosslinked beta-CD and polymerized epichlorohydrin) have been found. The molecular mobility of these two components has been analyzed in terms of relaxation parameters such as 13C spin lattice relaxation (T1) and 1H spin lattice relaxation in the rotating frame (T1 rho). The T1 values of the polymers show that the beta-CD trapped inside the polymers does not seem to undergo changes in its mobility whatever the amount of epichlorohydrin. The addition of water to beta-CD significantly increases the T1 values reflecting strong interaction between beta-CD and the solvent. The T1P values obtained reflect the homogeneous nature of the materials.

Synthesis and biological effects of N-alkylamine-labeled low-molecular-mass dermatan sulfate.

Dermatan sulfate (DS) is a component of connective tissue and catalyzes the heparin cofactor II-mediated inhibition of thrombin. Low-molecular-mass dermatan sulfates (LMMDS) are produced to prolong the antithrombotic activity of this substance. Cleavage of DS by nitrous acid leads to an LMMDS with a terminal 2,5-anhydrotalose (At) group at the reducing end which can react with primary amines. Tyramine (Tyr) was bound to the terminal At of LMMDS using reductive amination. LMMDS-tyr is produced using DS. LMMDS desacetglated were produced using totally deaminated DS. These compounds were employed as a model for the characterization of DS using NMR spectroscopy. The purity of the compounds was checked using capillary electrophoresis. The structure of the products was proven by 1H- and 13C-NMR spectroscopy. LMMDS-Tyr was radiolabeled with 125I for use in a radioimmunoassay. The anti-Xa activity and antithrombin activity of the tyramine-labeled DS are very low. The clotting assays Heptest, aPTT, thrombin time, and ecarin time indicate a highly anticoagulant-active substance. The heparin cofactor II-mediated inhibition of thrombin is similar to the parent compound. LMMDS were labeled "endpoint-attached." They are a new tool to understand the actions of DS in biologic systems.

Electrostatic interactions between human leukocyte elastase and sulfated glycosaminoglycans: physiological implications.

The influence of ionic strength and composition on the binding and inhibition of human leukocyte elastase by glycosaminoglycans with variable degree and position of sulfation was investigated. The kinetic mechanism of inhibition had a hyperbolic, mixed-type character with a competitive component that was promoted by low ionic strength, reduced by phosphate ions, and which also depended on the substrate and glycosaminoglycan structure. Enzyme binding was a cooperative phenomenon that varied with ionic strength and composition. The inhibition patterns correlated with the cationic character of elastase and with the distribution of arginines on its molecular surface, most notably with residues located in the vicinity of the substrate binding region. The order of affinity for elastase binding was chondroitin 4-sulfate < chondroitin 6-sulfate < dermatan sulfate, iduronate-containing derivatives being superior with respect to the glucuronate-containing counterparts. Additional sulfation at both the 4- and 6- positions or at the N- and 4-positions of the N-acetylgalactosamine moiety decidedly improved the inhibitory efficiency. The results highlight a fundamental physiological role of enzyme-glycosaminoglycan interactions. In the azurophil granule of the human polymorphonuclear neutrophil, elastase and other enzymes are bound to a matrix of chondroitin 4-sulfate because this is the only glycosaminoglycan that simultaneously offers good binding for enzyme compartmentalization together with prompt release from the bound state at the onset of phagocytosis.

1H and 13C NMR spectral assignments of the major sequences of twelve systematically modified heparin derivatives.

The complete 1H and 13C NMR spectral assignments are described for the most prevalent patterns of sulfation and acetylation which can be found in polymeric heparin or can be obtained by standard chemical modifications. These include a number of novel structures containing unsubstituted or acetylated amino groups and the first complete NMR assignments of many of the other derivatives. Beef lung heparin was chosen as a model system and studies were carried out using conditions to control the influences on the chemical shift positions in heparin samples of divalent cations and variations in pH and temperature.

Characterization of sulfation patterns of beef and pig mucosal heparins by nuclear magnetic resonance spectroscopy.

Though differing only slightly in their degrees of sulfation, heparin preparations from pig mucosa and those from beef mucosa have consistently different 13C- and 1H-NMR spectra, which provide useful fingerprints for distinguishing the two types of heparin. Integrated areas of NMR signals associated with minor, undersulfated sequences (assigned by comparison with mono-dimensional spectra of selectively desulfated heparins and by analysis of two-dimensional spectra of heparins prepared from pig and beef mucosa) permit quantitation of differences in sulfation patterns. Undersulfation of pig mucosal heparins at position 6 of the hexosamine units, determined by 13C-NMR and expressed as percent glucosamines nonsulfated at C6 referred to total glucosamines, is substantially lower for pig mucosal heparins than for beef mucosal heparins (16.9-21.7% vs 36.7-40.7%; average values: 18.6% vs 40.3%). By contrast, undersulfation at position 2 of the iduronic acid units, determined by 1H-NMR and expressed as percent nonsulfated iduronic acid referred to total (sulfated + nonsulfated) iduronic acid is significantly higher for pig mucosal preparations (9.6-13.5% vs 2.1-2.7%; average values: 12.7% vs 2.3%). Pig mucosal heparins also have a significantly higher content of 3-O-sulfated glucosamine units, which are markers for the active site of heparin for antithrombin-III.

Structural and functional properties of heparin analogues obtained by chemical sulphation of Escherichia coli K5 capsular polysaccharide.

Capsular polysaccharide from Escherichia coli K5, with the basic structure (GlcA beta 1-4GlcNAc alpha 1-4)n, was chemically modified through N-deacetylation, N-sulphation and O-sulphation [Casu, Grazioli, Razi, Guerrini, Naggi, Torri, Oreste, Tursi, Zoppetti and Lindahl (1994) Carbohydr. Res. 263, 271-284]. Depending on the reaction conditions, the products showed different proportions of components with high affinity for antithrombin (AT). A high-affinity subfraction, M(r) approx. 36,000, was shown by near-UV CD, UV-absorption difference spectroscopy and fluorescence to cause conformational changes in the AT molecule very similar to those induced by high-affinity heparin. Fluorescence titrations demonstrated about two AT-binding sites per polysaccharide chain, each with a Kd of approx. 200 nM. The anti-(Factor Xa) activity was 170 units/mg, similar to that of the IIId international heparin standard and markedly higher than activities of previously described heparin analogues. Another preparation, M(r) approx. 13,000, of higher overall O-sulphate content, exhibited a single binding site per chain, with Kd approx. 1 microM, and an anti-(Factor Xa) activity of 70 units/mg. Compositional analysis of polysaccharide fractions revealed a correlation between the contents of -GlcA-GlcNSO3(3,6-di-OSO3)- disaccharide units and affinity for AT; the 3-O-sulphated GlcN unit has previously been identified as a marker component of the AT-binding pentasaccharide sequence in heparin. The abundance of the implicated disaccharide unit approximately equalled that of AT-binding sites in the 36,000-M(r) polysaccharide fraction, and approached one per high-affinity oligosaccharide (predominantly 10-12 monosaccharide units) isolated after partial depolymerization of AT-binding polysaccharide. These findings suggest that the modified bacterial polysaccharide interacts with AT and promotes its anticoagulant action in a manner similar to that of heparin.

Heparin-like compounds prepared by chemical modification of capsular polysaccharide from E. coli K5.

O-Sulfation of sulfaminoheparosan SAH, a glycosaminoglucuronan with the structure-->4)-beta-D-GlcA(1-->4)-beta-D-GlcNSO3(-)-(1-->, obtained by N-deacetylation and N-sulfation of the capsular polysaccharide from E. coli K5, was investigated in order to characterize the sulfation pattern eliciting heparin-like activities. SAH was reacted (as the tributylammonium salt in N,N-dimethylformamide) with pyridine-sulfur trioxide under systematically different experimental conditions. The structure of O-sulfated products (SAHS), as determined by mono- and two-dimensional 1H and 13C NMR, varied with variation of reaction parameters. Sulfation of SAH preferentially occurred at O-6 of the GlcNSO3- residues. Further sulfation occurred either at O-3 or at O-2 of the GlcA residues, depending on the experimental conditions. Products with significantly high affinity for antithrombin and antifactor Xa activity were obtained under well-defined conditions. These products contained the trisulfated aminosugar GlcNSO3-3,6SO3-, which is a marker component of the pentasaccharide sequence through which heparin binds to antithrombin.