Anticoagulant activity of sulfated polysaccharides from the green seaweed Cladophora falklandica

PAULA X. ARATA; MARINA CIANCIA; LUCÍA KORDICH; IRENE QUINTANA

Amsterdam

Congreso; XXIV Congress of the International Society on Thrombosis and Haemostasis, ISTH; 2013

International Society on Thrombosis and Haemostasis, ISTH

Background: Some sulfated polysaccharides were found to have anticoagulant properties. Seaweeds from the Cladophorales synthesize sulfated xyloarabinogalactans. Aqueous extracts from C. falklandica showed as major constituents galactose and arabinose, and high percentage of sulfate. Taking into account the increasing interest in the development of new antithrombotic agents, these polysaccharides deserve to be investigated as potential anticoagulants. Aims: To evaluate the anticoagulant activity of the water extracts of C. falklandica. Methods: The room-temperature water extracts (CX1-CX3) were evaluated. Determinations of prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT) were assayed according to established methods. Normal platelet depleted citrated plasma (900 μL) was mixed with 100 μL of each polysaccharide extracts, in different concentrations, and incubated for 1 min at 37°C. Saline solution was used as control. TT-like assays were also performed with purified fibrinogen (3 mg/mL) instead of plasma. Results were expressed as ratios obtained by dividing the clotting time achieved with the extract by the time achieved with the control. In order to performed fibrinformation studies, clots were generated by adding thrombin (0.05 IU/mL) and calcium chloride (20 mmol/L) to the preincubated plasma. The kinetics was evaluated measuring optical density (OD) at 405 nm every one minute up to constant values. Assays were carried out in polystyrene strips, in the presence of different concentrations of CX2 (10 to 100 µg/mL) or saline solution as control. The sigmoid curves obtained (OD versus time) were characterized by three parameters: lag phase, maximum velocity achieved and final network OD. All assays were performed in quadruplicate. Results: The second room temperature water extract (CX2), comprises arabinose as the major component (60.6 %), galactose (23.5 %) and xylose (10.5 %), with a high percentage of sulfate, with a molar ratio carbohydrate: SO3 of 1:0.7. CX2 was the most active one in coagulation tests. This fraction significantly prolonged PT, APTT and TT, in concentration dependent manner; for CX2 solution (100 µg/mL) the ratios were 1.8, 5.2, and 4.1, respectively. For the TT-like assays the ratio was 2.7, for the same concentration of CX2. The kinetics analysis of fibrinformation with CX2 showed statistically significant differences versus control in lag phase (0.5 ± 0.0 min vs. 6.0 ± 0.0 min, p<0.001), Vmax (408.5 ± 30.4 min-1 vs. 212 ± 23.8 min-1, p<0.001) and in the final network OD (0.709 ± 0.002 vs 0.801± 0.011, p<0.001). Conclusions: The second water extract of C.falklandika showed anticoagulant activity by global coagulation tests with normal plasma; therefore CX2 could potentiate thrombin inhibition by antithrombin and/or heparin cofactor II. Besides, TT-like assays with purified fibrinogen suggest that, at least one of the mechanisms involved would be direct thrombin inhibition. By other hand, a probable procoagulant effect was shown by fibrinformation kinetics. In other to understand these findings further studies are currently undertaken.