Frédéric Chaubet

Further data on the structure of brown seaweed fucans: relationships with anticoagulant activity.

The composition, molecular weight (MW), anticoagulant activity and nuclear magnetic resonance spectra of various low-molecular-weight fucans (LMWFs) obtained by partial hydrolysis or radical depolymerization of a crude fucoidan extracted from the brown seaweed Ascophyllum nodosum are compared. Fucose units were found mainly sulfated at O-2, to a lesser extent at O-3, and only slightly at O-4, contrary to previously published results for fucoidans from other brown seaweeds, and fucose 2, 3-O-disulfate residues were observed for the first time. As the sulfation pattern excluded an alpha-(1-->2)-linked fucose backbone and a high proportion of alpha-(1-->4) linkages was found, it would appear that the concept of fucoidan structure needs to be revised. Anticoagulant activity is apparently related not only to MW and sulfation content, as previously determined, but also (and more precisely) to 2-O-sulfation and 2,3-O-disulfation levels.

Fabrication of porous polysaccharide-based scaffolds using a combined freeze-drying/cross-linking process.

Biocompatible three-dimensional (3-D) porous scaffolds are of great interest for tissue engineering applications. We here present a novel combined freeze-drying/cross-linking process to prepare porous polysaccharide-based scaffolds. This process does not require an organic solvent or porogen agent. We unexpectedly found that cross-linking of biomacromolecules such as pullulan and dextran with sodium trimetaphosphate could be performed during freeze-drying. We have demonstrated that the freeze-drying pressure modulates the degree of porosity. High freeze-drying pressure scaffolds presented pores with a mean diameter of 55 +/- 4 microm and a porosity of 33 +/- 12%, whereas low freeze-drying pressure scaffolds contained larger pores with a mean diameter of 243 +/- 14 microm and a porosity of 68 +/- 3%. Porous scaffolds of the desired shape could be easily obtained and were stable in culture medium for weeks. In vitro viable mesenchymal stem cells were found associated with porous scaffolds in higher proportions than with non-porous scaffolds. Moreover, cells penetrated deeper into scaffolds with larger pores. This novel combined freeze-drying/cross-linking processing of polysaccharides enabled the fabrication of biocompatible scaffolds with controlled porosity and architectures suitable for 3-D in vitro culture and biomedical applications.

Relationships between chemical characteristics and anticomplementary activity of fucans.

We have shown previously that a low-molecular-weight fucan extracted from the brown seaweed Ascophylum nodosum strongly inhibited human complement activation in vitro and its mechanism of action was largely elucidated. We further investigated the influence of molecular weight and chemical composition of fucan on its anticomplementary activity. The capacity of 12 fragments of fucan (ranging from a molecular weight of 4100 to 214,000) to prevent complement-mediated haemolysis of sheep erythrocytes (classical pathway) and of rabbit erythrocytes (alternative pathway) increased with increasing molecular weight, and reached a plateau for 40,000 and 13,500, respectively. The most potent fucan fractions were 40-fold more active than heparin in inhibiting the classical pathway. They were, however, as active as heparin in inhibiting the alternative pathway. In addition, we have developed a haemolytic test based on the CH50 protocol, which allows discrimination between activators and inhibitors of complement proteins. Although the mannose content within the different fucan fragments did not vary, the galactose and glucuronic acid contents increased with increasing activity, suggesting that these residues should be essential for full anticomplementary activity. Meanwhile, sulphate groups appeared to be necessary, but were clearly not a sufficient requirement for anticomplementary activity of fucans. Taken together, these data illustrate the prospects for the use of fucans as potential anti-inflammatory agents.

Affinity of low molecular weight fucoidan for P-selectin triggers its binding to activated human platelets.

BACKGROUND P-selectin is an adhesion receptor expressed on activated platelets and endothelial cells. Its natural ligand, P-selectin glycoprotein ligand-1, is expressed on leucocytes and the P-selectin/PSGL-1 interaction is involved in leukocyte rolling. We have compared the interaction of P-selectin with several low molecular weight polysaccharides: fucoidan, heparin and dextran sulfate. METHODS Binding assays were obtained from the interaction of the polysaccharides with Sialyl Lewis X and PSGL-1 based constructs onto microtiter plates coated with P-selectin. SELDI TOF mass spectrometry was performed with anionic chips arrays coated with P-selectin in the absence or in the presence of polysaccharides. Kd were obtained from surface plasmon resonance experiments with immobilized P-selectin constructs, polysaccharides being injected in the mobile phase. Human whole blood flow cytometry experiments were performed with fluorescein isothiocyanate labelled polysaccharides with or without platelets activators. RESULTS The fucoidan prevented P-selectin binding to Sialyl Lewis X with an IC(50) of 20 nM as compared to 400 nM for heparin and <25000 nM for dextran sulfate. It exhibited the highest affinity for immobilized P-selectin with a KD of 1.2 nM, two orders of magnitude greater than the K(D) of the other polysaccharides. Mass spectrometry evidenced the formation of a complex between P-selectin and fucoidan. The intensity of the fucoidan binding to platelets was dependent on the level of platelet activation. Competition between fucoidan and an anti P-selectin antibody demonstrated the specificity of the interaction. GENERAL SIGNIFICANCE Low molecular weight fucoidan is a promising therapeutic agent of natural origin for biomedical applications.

Anticoagulant properties of dextranmethylcarboxylate benzylamide sulfate (DMCBSu); a new generation of bioactive functionalized dextran.

Dextranmethylcarboxylate benzylamide sulfate (DMCBSu), a functionalized dextran, exhibits anticoagulant properties. Its synthesis involves three steps: a carboxymethylation with monochloroacetic acid in alkaline water-iso-propanol, a benzylamidification of some of the methylcarboxylate groups with benzylamine in the presence of a water soluble carbodiimide and a partial sulfation of the remaining hydroxyl groups with SO3-pyridine in dimethylformamide. This procedure yields reproducibly DMCBSu with degrees of substitution in methylcarboxylate (MC), benzylamide (B) and sulfate (Su) groups, respectively, up to 1.61, 0.35 and 1.5, each obtained in one step. For a degree of substitution of methylcarboxylate ca. 1, the presence of sulfate groups is absolutely necessary to confer anticoagulant activities to the samples. In addition, the anticoagulant ability is higher for derivatives bearing benzylamide groups. The anticoagulant ability of DMCBSu increases with the degree of sulfation, reaching 20% of heparin activity for a degree of substitution of Su groups about 1.3.

Anticoagulant activity of a sulfated polysaccharide from the green alga Arthrospira platensis.

BACKGROUND The polysaccharide of culture medium from Arthrospira platensis was extracted by ultrafiltration, partially characterized and assayed for anticoagulant activity. METHODS The crude polysaccharidic fraction was fractionated by anion exchange chromatography on DEAE-cellulose, subjected to acetate cellulose electrophoresis and characterized by physicochemical procedures. The anticoagulant effect of the ultrafiltrated polysaccharide was checked by several coagulation tests. RESULTS Anion exchange chromatography revealed in the whole ultrafiltrated polysaccharidic fraction the occurrence of a sulfated spirulan-like component designated PUF2. The average molecular weight of PUF2 was determined by size exclusion chromatography combined with multi-angle light scattering (SEC-MALS) and viscosimetry and was 199 kDa and the sulfate content was 20% weight/dry weight. The physicochemical characterization indicated the occurrence of rhamnose (49.7%), galacturonic and glucuronic acid (32% of total sugar). The anticoagulant effect of this sulfated polysaccharide was mainly due to the potentiation of thrombin inhibition by heparin cofactor II and was 4-times higher than that of the porcine dermatan sulfate whereas it had no effect on anti-Xa activity. CONCLUSIONS An ultrafiltrated sulfated polysaccharide, likely a calcium spirulan was obtained from the culture medium of A. platensis and showed an anticoagulant activity mediated by heparin cofactor II. GENERAL SIGNIFICANCE Old culture medium of A. platensis may represent an important source for the spirulan-like PUF2 which was endowed with potentially useful anticoagulant properties whereas its obtention by ultrafiltration may represent an extraction procedure of interest.

Synthesis and structure—anticoagulant property relationships of functionalized dextrans: CMDBS

The CMDBS compounds (carboxymethyl dextran benzylamide sulfonate) are prepared from native dextran (average molecular weight of 40,000 gmol) after three consecutive reactions: carboxymethylation of hydroxyl groups on random D-glucose units (CM), conversion of carboxylate groups to benzylamide structures (B), and sulfonation of the phenyl ring (S). These polymers exhibit a heparin-like anticoagulant capacity which is closely related to their CMS ratio. Evidence is presented to show that the distribution of S-containing groups along the polymer backbone can be optimized to get the best anticoagulant activity. Indeed, the anticoagulant activity reached a maximum after a sulfonation time of 1 h.

Anticoagulant activity of dextran derivatives.

Carboxymethyl dextran benzylamide sulfonate/sulfates (CMDBS) are synthetic polysaccharides with anticoagulant activity. We synthesized eight different highly substituted CMDBS and one CMDSu. We studied both their anticoagulant activity and the catalysis of thrombin (T) inhibition by heparin cofactor II (HCII) and antithrombin (AT) in the presence of these dextran derivatives relative to heparin and dextran sulfate (DXSu). The anticoagulant activity of CMDBS was due both to direct thrombin inhibition and to catalysis of thrombin inhibition by HCII. The anticoagulant and catalytic activities of CMDBS were related mainly to their molecular weight and sulfate content. The interaction of the dextran derivatives with thrombin does not involve the active site of the enzyme. A kinetic study showed that all the CMDBS exhibited higher affinity for thrombin than heparin did but lower affinity than DXSu did, suggesting that the benzylamide and sulfate groups potentiate the interaction between the dextran derivatives and thrombin. This study shows that the mechanism by which the dextran derivatives inhibit thrombin is original and is related to preferential interaction with thrombin; this both inhibits the clotting activity of the enzyme and increases the reaction rate of thrombin inhibition by HCII.

Carboxymethylation of dextran in aqueous alcohol as the first step of the preparation of derivatized dextrans

Carboxymethyldextrans (CMD) with a degree of substitution of carboxymethyl groups DS > 0.9 are precursors for the synthesis of derivatized dextrans termed CMDBS, which present heparin-like biological properties. Syntheses of CMD in water/organic solvent mixtures using monochloroacetic acid (MCA) and strong alkaline conditions allowed to obtain highly substituted CMD with good reproducibility and high yields. The influences of, respectively, individual concentrations of reactants, composition and temperature of reaction medium, and allowed reaction time have been examined. The most favorable conditions for dextran carboxymethylation were obtained with 3.8 M NaOH and a [MCA]/[dextran] ratio of 2.5, allowed to react at 60°C for 90 min, in a reaction medium consisting of a tert-butanol/water (alternatively isopropanol/water) 85 : 15 (v/v) mixture. A DS of about 1.0 was obtained in one step, as compared to a DS less than 0.6 when using aqueous conditions. Improvements on overall CMD substitution levels (up to DS of 1.47) were achieved by merely repeating the reaction i. e. using the CMD products of the first reaction as substrate in the second. Carboxymethyldextrane (CMD) mit einem Substitutionsgrad der Carboxymethylgruppen DS > 0.9 sind Vorstufen fur die Synthese von derivatisierten Dextranen (CMDBS) mit heparinahnlichen biologischen Eigenschaften. Die Herstellung von CMD in wasrigen organischen Losungsmitteln und in Gegenwart von Monochloressigsaure (MCA) unter stark alkalischen Bedingungen fuhrt mit guter Reproduzierbarkeit und hohen Ausbeuten zu hochsubstituierten CMD. Der Einflus der Konzentration der Reaktanden, der Zusammensetzung und Temperatur des Reaktionsmediums sowie der Reaktionszeit wurden untersucht. Die gunstigsten Bedingungen fur die Carboxymethylierung von Dextran zeigten sich mit 3.8 M NaOH, einem [MCA]/[Dextran]-Verhaltnis von 2.5, einer Reaktionstemperatur von 60°C und einer Reaktionsdauer von 90 min in einer 85 : 15 (v/v) tert-Butanol/Wasser (alternativ Isopropanol/Wasser)-Mischung als Reaktionsmedium. Ein Substitutionsgrad von etwa 1.0 wurde mit einem Reaktionsschritt erhalten, wahrend unter wasrigen Bedingungen ein DS von weniger als 0.6 resultierte. Verbesserungen des CMD-Substitutionsgrades (bis zu DS 1.47) wurden durch wiederholte Reaktion erzielt.

Polysaccharides from the skin of the ray Raja radula. Partial characterization and anticoagulant activity

INTRODUCTION The polysaccharide fraction from the skin of the ray Raja radula was extracted, characterized and assayed for anticoagulant activity. MATERIALS AND METHODS A whole polysaccharidic fraction was extracted from the skin of the ray Raja radula by papain digestion followed by cetylpyridinium chloride and ethanol precipitation and was subjected to gel chromatography and anion exchange chromatography, acetate cellulose electrophoresis and characterized by physicochemical procedures. APTT and anti Xa assays were performed to assess the anticoagulant activity of the polysaccharidic fractions in comparison with unfractionated heparin. RESULTS Gel and anion-exchange chromatography revealed two negatively charged polysaccharidic populations different in both molecular weight and charge. Infrared spectra suggested the occurrence of uronic acids and acetylated hexosamines. The second polysaccharide was highly sulfated, with a sulfate content of approximately 29%. These data suggested that dermatan sulfate (DS) is the sulfate rich polysaccharide whereas hyaluronic acid (HA) is the polysaccharide devoid of sulfate groups. Molecular mass characterization indicated that their average molecular masses were 22 kDa and 85 kDa, respectively. The sulfated polysaccharide, i.e. presumably DS, accounted alone for the observed concentration-dependent anticoagulant activity which was, as measured by APTT, 2 to 3-fold lower than that of heparin. In addition, it had a significant anti-Xa activity. CONCLUSION A major-sulfated polysaccharide, likely a dermatan sulfate, was extracted from the ray Raja radula skin. The results indicated that it exhibited a high anticoagulant activity and suggested that it was mediated by both heparin cofactor II and antithrombin.