Enzymatic and chemical depolymerization of Fucus evanescens fucoidan: Impact on structure and hemostatic activity

Fucoidans are fucose-rich, sulfated polysaccharides with potent anticoagulant properties. However, their application is limited by the structural heterogeneity and the platelet-activating effects associated with their high molecular weight (Mw). Low-Mw derivatives alleviate these issues but often show diminished anticoagulant potency. In the present study, we systematically compared enzymatic and chemical depolymerization methods to generate low-Mw Fucus evanescens fucoidan derivatives and evaluated how structural modifications influence their anticoagulant and platelet-modulating activities. Fucoidan derivatives were produced using four GH107 endo-fucanases with different sulfation specificities, as well as mild acid hydrolysis and hydrogen peroxide treatment. The structural characterization of the obtained derivatives was carried out using size-exclusion chromatography (SEC), nuclear magnetic resonance (NMR) spectroscopy, and a novel enzyme-based sulfation pattern mapping assay. Hemostatic properties were assessed via coagulation assays, coagulation factors inhibition, platelet aggregation, and measurements of whole-blood clot stability. Short-duration acid hydrolysis (1 h) and controlled peroxide treatment (12 h) yielded low-Mw derivatives with only slight anticoagulant activity reductions, whereas prolonged chemical treatment substantially decreased potency. Enzymatic depolymerization produced more defined low- and medium-Mw derivatives with higher yields and structure-dependent activity. Structure-activity analysis revealed that anticoagulant efficacy primarily depends on sulfation pattern (notably 2,4-di-sulfation), and molecular weight thresholds (6-10 kDa), rather than total sulfate content. Similarly, depolymerization reduced platelet aggregation by >45 %, with activation absent below 10 kDa. However, elevated 2,4-di-sulfation correlated positively with platelet aggergation. These findings highlight the critical balance between Mw and 2,4-di-sulfation in regulating hemostasis, providing a framework for designing safer, more effective fucoidan-based therapeutics.