Objective To identify novel glycosaminoglycans from Linckia laevigata, elucidate their fine structures, and evaluate their anticoagulant potential as inhibitors of the intrinsic coagulation pathway. Methods Total polysaccharides were extracted from the starfish L. laevigata using a combination of enzymatic and alkaline hydrolysis, and then purified by strong anion-exchange chromatography. Oligosaccharides were prepared through deacetylation-deamination depolymerization. A “bottom-up” strategy integrating high-performance gel permeation chromatography (HPGPC), 1-phenyl-3-methyl-5-pyrazolone derivatization high-performance liquid chromatography (PMP-HPLC), infrared spectroscopy (IR), and one-dimensional/two-dimensional nuclear magnetic resonance (1D/2D NMR) was employed for structural characterization. In vitro anticoagulant activity was assessed by activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT) assays, while inhibition of intrinsic factor Xase (iFXase) was measured via a chromogenic substrate assay. Results A homogeneous sulfated glycosaminoglycan, designated LLGAG (molecular weight, M_w≈38 000), was obtained with a monosaccharide composition of iduronic acid (IdoA)∶N-acetylgalactosamine (GalNAc)∶galactose (Gal) ≈34.4∶59.7∶5.9. The polymer core comprised disaccharide units featuring the L-IdoA_2S3S-α-1,3-D-GalNAc_4S6S (2S3S4S6S) motif, together with 3S4S6S and 2S3S6S motifs, representing a rare and novel dermatan sulfate-like glycosaminoglycan. In vitro, LLGAG significantly prolonged APTT and TT, showing activity comparable to that of the clinical gold-standard enoxaparin sodium. Its half-maximal inhibitory concentration (IC₅₀) for iFXase inhibition was (17.75 ± 1.12) ng/mL, approximately 2.8-fold more potent than that of enoxaparin sodium. Conclusion This study identified a structurally unique glycosaminoglycan from L. laevigata, elucidated its precise structure, and demonstrated its potent iFXase inhibitory activity, thus providing both a theoretical foundation and a candidate molecule for the development of marine-derived heparin alternatives.

Linckia laevigata (Linnaeus)  /  polysaccharide  /  glycosaminoglycan  /  structural characterization  /  anticoagulant activity  /  intrinsic factor Xase