Patrick Durand

Anticoagulant properties of a fucoidan fraction

Fucoïdans are a family of high molecular weight sulphated polysaccharides in the Mr range 8 x 10(5) -10(6), widely dispersed in brown seaweed cell wall. When extracted from several brown algae, they exhibit anticoagulant properties. The chemical degradation of a crude extract, from Pelvetia canaliculata, was undertaken to obtain a low molecular weight polysaccharide (Mr 20,000 +/- 5,000) with the purpose of a possible clinical use. Its anticoagulant potency was investigated through the inhibition of factor IIa and factor Xa in the presence of antithrombin III or heparin cofactor II. The degraded fucoïdan revealed a potent antithrombin activity: studied in an antithrombin III depleted plasma or in the presence of purified heparin cofactor II, the fucoïdan was as efficient as heparin and dermatan sulphate on heparin cofactor II potentiation, at the same concentration by weight. In whole plasma or in the presence of the purified inhibitor, an anti-factor IIa activity mediated by antithrombin III was detected (30 times less potent than for heparin, on a weight to weight basis). In contrast, no anti-factor Xa activity was detected in the presence of the degraded fucoïdan, under the same experimental conditions. These fucoïdans, by-products of alginates preparation in the food and cosmetologic industries, are obtained easily. Thus, they may represent a cheap and easy source of a new type of anticoagulants.

Structural aspects of fish skin collagen which forms ordered arrays via liquid crystalline states

The ability of acid-soluble type I collagen extracts from Soleidae flat fish to form ordered arrays in condensed phases has been compared with data for calf skin collagen. Liquid crystalline assemblies in vitro are optimized by preliminary treatment of the molecular population with ultrasounds. This treatment requires the stability of the fish collagen triple helicity to be controlled by X-ray diffraction and differential scanning calorimetry and the effect of sonication to be evaluated by viscosity measurements and gel electrophoresis. The collagen solution in concentrations of at least 40 mg ml(-1) showed in polarized light microscopy birefringent patterns typical of precholesteric phases indicating long-range order within the fluid collagen phase. Ultrastructural data, obtained after stabilization of the liquid crystalline collagen into a gelated matrix, showed that neutralized acid-soluble fish collagen forms cross-striated fibrils, typical of type I collagen, following sine wave-like undulations in precholesteric domains. These ordered geometries, approximating in vivo situations, give interesting mechanical properties to the material.

New natural polysaccharides with potent antithrombic activity fucans from brown algae

Fucans extracted from brown algae exhibit anticoagulant properties, as demonstrated by coagulation assays and kinetic study of the inactivation of thrombin in the presence of antithrombin III. These derivatives exert a direct antithrombic activity by an antithrombin III-independent pathway. Besides, their anticoagulant activity is not mediated by a potency to inhibit factor Xa. Relatively low-molecular-mass fractions (50,000 Daltons) prepared by chromatographic fractionation of the crude fucans also exhibit anticoagulant activity. They may prove useful as anticoagulant drugs.

Comparison of artificial light photobioreactors and other production systems using Porphyridium cruentum

A new type of preparative photobioreactor for high quality production of microalgae is developed for hatchery-nursery of marine animals, as well as for fine chemicals extraction. Of modular conception, two artificial light photobioreactors in plastic and stainless steel are designed so as to provide strictly controlled conditions in an attempt to increase quality and diminish cost prices. They are assessed for production of Porphyridum cruentum and compared to conventional transparent tanks and solar photobioreactors. The concentration and productivity obtained are ten-fold higher than with hatchery tanks, which leads to a significant drop in cost price of biomass. Comparison is also made with a 10 m2 solar photobioreactor operated in the south of France, for which biomass cost price is half that of 1.5 m2 artificial light photobioreactor. Extrapolations erasing size discrepancy show that the cost price of the two technologies are not very different.

Purification, kinetical and molecular characterizations of a serine collagenolytic protease from greenshore crab (Carcinus maenas) digestive gland☆

A serine collagenolytic protease was purified from a water soluble fraction of greenshore crab digestive gland by acidic precipitation, gel filtration on a Sephadex G-50 column, ion-exchange chromatography on a Fractogel TSK DEAE column, immobilized metal ion affinity chromatography (IMAC) on IDA (Cu2+) Sepharose 6B and ion-exchange chromatography on Hyper D column. The molecular mass of the monomeric Carcinus serine collagenase (CSC) was estimated to be 23,000 by SDS PAGE and its isoelectric point was found to be 4.0. The CSC is optimally active at pH 7 and 30 degrees C and is stable over a month at room temperature. The CSC activity is strongly inhibited by PMSF, 3,4-DCI, soybean trypsin inhibitor, alpha 1 proteinase inhibitor and elastatinal. The CSC hydrolyzes native collagen (Type I and III). CSC N-terminal sequence is similar to shrimp chymotrypsin-like protease and crab collagenolytic protease sequences. Kinetic parameters of the CSC were determined using some peptidyl-p-nitroanilides. The catalytic efficiency (kcat/Km) is Leu > Phe > Ala.

Relationships between chemical characteristics and anticoagulant activity of low molecular weight fucans from marine algae

Anticoagulant activity of fucans is closely linked to their chemical composition. The anticoagulant properties of various fractions extracted from different algae species were studied to assess the relationship between fucan structure and activity. For this purpose, cell walls were isolated from the brown algae and then submitted to sequential chemical extraction leading to high molecular weight fucans (HMWF). Different low molecular weight fucans (LMWF) were obtained by acidic degradation or radical process depolymerization of HMWF. Part of them were fractionated according to their molecular weight and charge density. We have demonstrated that the anticoagulant activity mainly correlated to the sulfate groups content and to the molecular weight distribution. The best results were obtained with fractions extracted from Ascophyllum nodosum. The anticoagulant and antithrombotic activities of each fraction were studied in vitro, ex vivo and in vivo. Affinity chromatography on stationary phase coupled with heparin binding plasma proteinase inhibitors was paerformed on the most active fractions. The high affinity fragments were purified and identified mostly sas α (1,2) and α (1,3 )linked units of 4-sulphuryl-fucose with sulfate or some branching at position 2 or 3.