Characterization, chemical modifications and in vitro anticoagulant properties of an exopolysaccharide produced by Alteromonas infernus

Abstract

A new low-molecular-weight`heparin-like' component was obtained from an exopolysaccharide produced by a mesophilic strain found in deep-sea hydrothermal vents. Data concerning the structure of the native high-molecular-weight exopolysaccharide (10 6 g/mol, 10% sulfate content) are reported for the first time. Two depolymerization processes were used to obtain low-molecular-weight (24^35U10 3 g/mol) oversulfated fractions (sulfate content 20 or 40). Nuclear magnetic resonance studies indicated that after sulfation (40%), the low-molecularweight fraction obtained by free radical depolymerization was less sulfated in the 6-O-position than the fraction depolymerized by acid hydrolysis. The free radical depolymerized product also had sulfated residues in the 4-O-position and disulfated ones in the 2,3-O-positions. Moreover, the compounds generated by the free radical process were more homogeneous with respect to molecular mass. Also for the first time, the anticoagulant activity of the low-molecular-weight exopolysaccharide fractions is reported. When the fractions obtained after sulfation and depolymerization were compared with heparins, anticoagulant activity was detected in oversulfated fractions, but not in native exopolysaccharide. The free radical depolymerized fraction inhibited thrombin generation in both contact-activated and thromboplastinactivated plasma, showing a prolonged lag phase only in the contact-activated assay. Affinity co-electrophoresis studies suggested that a single population of polysaccharide chains binds to antithrombin and that only a subpopulation strongly interacts with heparin cofactor II.

Prospecting exopolysaccharides produced by selected bacteria associated with marine organisms for biotechnological applications

“…This negative charge helps buffering the cells against fluctuating salinity experienced in the marine environments [36] . Sulfated EPSs isolated from bacteria have shown to possess anticoagulant, antioxidant and bone healing properties [8,37] , similarly acetyl and phosphate groups in the EPS also render biologically important properties [38,39] . Ability of the EPS to scavenge free radicals is another property of biological importance.…”

Fermentation Technologies for the Optimization of Marine Microbial Exopolysaccharide Production

“…Vincent et al [32] described the isolation of Alteromonas sp. from the epidermis of a polychaete annelid, Alvinella pompejana , living in the deep sea hydrothermal vents of the East Pacific Rise, able to produce a heteropolysaccharide with high levels of uronic acids and pyruvate, and showing anticoagulant and bone healing properties [33,34,35]. Bozzi et al .…”

Structural studies of the main exopolysaccharide produced by the deep-sea bacterium Alteromonas infernus

“…The molecular weight of this LMW-EPS was determined as previously described by high-performance size-exclusion chromatography (HPSEC) using pullulan as standard. 7 Pullulan is a neutral glucan, whereas LMW-EPS is highly negatively charged polysaccharide. Consequently, such a calibration, which does not allow an exact measure of molecular mass, was used only for having a rough estimation of MW and homogeneity.…”

“…8 Affinity co-electrophoresis experiments performed in the presence of antithrombin or heparin co-factor II showed that all chains of sulfated LMW-EPS bound antithrombin strongly while only a subpopulation did the same with heparin co-factor II. 7 This latter result strongly suggests that the anticoagulant activity is modulated not only by the sulfate content, but also by the position of sulfate groups along the polysaccharide chain.…”

“…Nevertheless, several oversulfated low-molecular-weight EPS fractions (sulfated LMW-EPS), with uronic acid and sulfate contents comparable to those of heparin, have been prepared by partial hydrolysis and chemical sulfation; 6 some of these fractions displayed interesting anticoagulant activity. 7 They were less potent than low-molecular-weight heparin and unfractionated heparin in activated partial thromboplastin time (APTT) assays (2.5 and 6.5 times, respectively), but more potent than LMW dermatan sulfate. 8 Affinity co-electrophoresis experiments performed in the presence of antithrombin or heparin co-factor II showed that all chains of sulfated LMW-EPS bound antithrombin strongly while only a subpopulation did the same with heparin co-factor II.…”