目的 从蓝指海星Linckia laevigata中挖掘新型糖胺聚糖，解析其精细结构，并评估其作为内源性凝血途径抑制剂的抗凝潜力。方法 采用酶解-碱解协同提取蓝指海星总多糖，强阴离子交换色谱纯化获得糖胺聚糖；经脱酰-脱氨基解聚制备寡糖，采用“bottom up”策略联合高效凝胶渗透色谱（high-performance gel permeation chromatography，HPGPC）、1-苯基-3-甲基-5-吡唑酮衍生化高效液相色谱（1-phenyl-3-methyl-5-pyrazolone derivatization high-performance liquid chromatography，PMP-HPLC）、红外光谱（infrared spectroscopy，IR）及一维/二维核磁共振（one-dimensional/two-dimensional nuclear magnetic resonance，1D/2D NMR）进行结构表征；以活化部分凝血活酶时间（activated partial thromboplastin time，APTT）、凝血酶时间（thrombin time，TT）、凝血酶原时间（prothrombin time，PT）评价体外抗凝活性，生色底物法测定内源性因子X酶（intrinsic factor Xase，iFXase）抑制活性。结果 制备得到结构均一的硫酸化糖胺聚糖LLGAG（M_w≈38 000），其单糖组成为艾杜糖醛酸（iduronic acid，IdoA）∶乙酰氨基半乳糖（N-acetylgalactosamine，GalNAc）∶半乳糖（galactose，Gal）≈34.4∶59.7∶5.9。该聚糖核心由L-IdoA_2S3S-α-1,3-D-GalNAc_4S6S（2S3S4S6S）基序的二糖单元构成，兼含3S4S6S及2S3S6S等基序，为自然界中罕见的新型硫酸皮肤素样糖胺聚糖。体外实验中，LLGAG显著延长APTT与TT，活性与临床抗凝金标准依诺肝素钠相当；抑制iFXase的半数抑制浓度（median inhibition concentration，IC₅₀）为（17.75±1.12）ng/mL，较依诺肝素钠强约2.8倍。结论 发现了1种结构新颖独特的蓝指海星来源糖胺聚糖，推断出其确切结构，发现其具有强效iFXase抑制活性，为开发海洋生物来源的肝素替代物提供理论依据与候选分子。

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.

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国家自然科学基金资助项目（82404503）；国家自然科学基金资助项目（32160220）；国家自然科学基金资助项目（82373788）；广西杰出青年科学基金（2020GXNSFFA297005）；中药壮瑶药创新药物教育部工程研究中心（ZYZYY2025034）