Sulfation of fucogalactan from Agaricus bisporus: Different patterns in the chemical structure and their effects on anticoagulant activity.

A fucogalactan from Agaricus bisporus was sulfated by two methodologies based on an optimized sulfation method. The direct action of chlorosulfonic acid and SO3-pyridine complex over the sulfation reaction and its effects on anticoagulant activity were evaluated. The products of chemical sulfations were two sulfated fucogalactans named E100 and ESL respectively. Clotting assays (APTT, PT and TT) showed that both sulfated polysaccharides have anticoagulant activity, and that ESL was more potent compared to E100. The FXa, T and FXIIa activities in the presence of the sulfated polysaccharides were determined. The better anticoagulant activity of ESL could be related to anti-FXIIa activity and also probably to its higher bioavailability. The HPSEC analysis showed similar Mw of 1.08×104gmol-1 and 1.00×104gmol-1 for E100 and ESL respectively. NMR and methylation analyses indicated a heterogeneous sulfation pattern for E100, whereas ESL showed conserved unsulfated (1→6)-linked α-d-Galp residues in the main-chain and a more homogeneous sulfation pattern. The DS values of ESL and E100 were 1.0 and 2.8 respectively, indicating that the sulfation pattern is more important for the anticoagulant activity than the amount of sulfate.

Slight differences in sulfation of algal galactans account for differences in their anticoagulant and venous antithrombotic activities.

We compared sulfated galactans (SGs) from two species of red algae using specific coagulation assays and experimental models of thrombosis. These polysaccharides have an identical saccharide structure and the same size chain, but with slight differences in their sulfation patterns. As a consequence of these differences, the two SGs differ in their anticoagulant and venous antithrombotic activities. SG from G. crinale exhibits procoagulant and prothrombotic effects in low doses (up to 1.0 mg/kg body weight), but in high doses (>1.0 mg/kg) this polysaccharide inhibits both venous and arterial thrombosis in rats and prolongs ex-vivo recalcification time. In contrast, SG from B. occidentalis is a very potent anticoagulant and antithrombotic compound in low doses (up to 0.5 mg/kg body weight), inhibiting venous experimental thrombosis and prolonging ex-vivo recalcification time, but these effects are reverted in high doses. Only at high doses (>1.0 mg/kg) the SG from B. occidentalis inhibits arterial thrombosis. As with heparin, SG from G. crinale does not activate factor XII, while the polysaccharide from B. occidentalis activates factor XII in high concentrations, which could account for its procoagulant effect at high doses on rats. Both SGs do not modify bleeding time in rats. These results indicate that slight differences in the proportions and/or distribution of sulfated residues along the galactan chain may be critical for the interaction between proteases, inhibitors and activators of the coagulation system, resulting in a distinct pattern in anti- and procoagulant activities and in the antithrombotic action.

An algal sulfated galactan has an unusual dual effect on venous thrombosis due to activation of factor XII and inhibition of the coagulation proteases.

Sulfated galactan from the red alga Botryocladia occidentalis has a potent anticoagulant activity, due to its ability to enhance thrombin and factor Xa inhibition by antithrombin and/or heparin cofactor II. It is less active than unfractionated heparin in arterial thrombosis, but in a venous thrombosis presents a dual effect, inhibiting thrombosis in low but not in high doses. This dual effect on venous thrombosis is a consequence of two actions, one that inhibits thrombin and factor Xa and one that induces factor XII activation. In order to dissociate these effects, we prepared derivatives of the sulfated galactan with low molecular weights. Two fractions that were similar in size to unfractionated heparin and low-molecular-weight heparin were obtained. As the molecular weight decreased, the ability to activate factor XII and to promote inhibition of coagulation proteases in the presence of antithrombin and heparin cofactor II diminished. At approximately 5 kDa, the sulfated galactan fragment had no effect on factor XII activation, and showed the same effect as unfractionated heparin in a venous thrombosis model. The approximately 5-kDa fragment is an antithrombotic with several advantages: i) It is as active as unfractionated heparin in venous thrombosis, but it has little activity in arterial thrombosis; ii) It inhibits venous thrombosis with very little anticoagulant effect; iii) It does not cause bleeding; and iv) It is not obtained from mammals.