Effects of Native Fucosylated Glycosaminoglycan, Its Depolymerized Derivatives on Intrinsic Factor Xase, Coagulation, Thrombosis, and Hemorrhagic Risk

Abstract

A native fucosylated glycosaminoglycan from sea cucumber Holothuria fuscopunctata (nHG), mainly branched with Fuc3S4S, exhibited potent anticoagulant activity by intrinsic tenase iXase (FIXa-FVIIIa complex) and antithrombin-dependent factor IIa (FIIa) inhibition, but also had the effects of FXII activation and platelet aggregation. For screening a selective iXase inhibitor, depolymerized nHG (dHG-1 ∼ –6) and a pure octasaccharide (oHG-8) were prepared. Like nHG, dHG-1 ∼ –6 and oHG-8 could potently inhibit iXase, and competitive binding assay indicated that dHG-5 and oHG-8 could bind to FIXa. Nevertheless, dHG-5 and oHG-8 had no effects on FXII and platelet activation. nHG, dHG-5, and oHG-8 could significantly prolong the activated partial thromboplastin time of human, rat, and rabbit plasma. In the rat deep venous thrombosis model, dHG-5 and oHG-8 showed potent antithrombotic effects in a dose-dependent manner, while the thrombus inhibition rate of nHG at high dose was markedly reduced. Additionally, dHG-5 and oHG-8 did not increase bleeding at the doses up to 10-fold of the effectively antithrombotic doses compared with nHG and low molecular weight heparin in the mice tail-cut model. Considering that dHG-5 possesses strong anti-iXase and antithrombotic activities, and its preparation process is simpler and its yield is higher compared with oHG-8, it might be a promising antithrombotic candidate.

The components and activities analysis of noval anticoagulant candidate dHG-5

“…The main type of FucS branches in the native FG wasL -fucose-3,4-disulfates (Fuc 3S4S ), and the minor wereL -fucose-2,4-disulfates (Fuc 2S4S ) andL -fucose-4-sulfates (Fuc 4S ). The depolymerized fraction, dHG-5, was prepared from native FG by β-eliminative depolymerization as previously described (Zhou, Gao et al , 2020) and the high performance gel permeation chromatography (HPGPC) profiles of dHG-5 ( Figure 1A and Figure S2) showed that dHG-5 was the mixture of oligosaccharides with different degree of polymerization (dp). The molar percentages of the main oligosaccharides and those with dp > 29) in dHG-5 were estimated as 4.…”

“…Depolymerized FG (dFG) with average molecular weight (Mw) less than 12 kDa showed significantly improved selectivity of anti-iXase activity and had no obvious effects on platelets and f.XII (Kitazato, Kitazato et al , 1996;Wu, Wen et al , 2015). Among them, a 5.2 kDa-dFG fromHolothuria fuscopunctata (dHG-5) was developed in our group as a promising anticoagulant candidate, due to its advantages in mature preparation process, anti-iXase inhibitor selectivity, antithrombotic activity (Zhou, Gao et al , 2020).…”

Characterization of the Hydrolysis Kinetics of Fucosylated Glycosaminoglycan in Mild Acid and Structures of the Resulting Oligosaccharides

“…In the 1 H NMR spectrum (Figure 5A), five signals between 5.2 and 5.6 ppm could be assigned to five α anomeric protons of fucose with distinct sulfation patterns [3,24], since their 3 J H1-H2 values (3.6-4.6 Hz) were in agreement with that of α-anomers. They could be identified as α anomers of 2,4-O-disulfated fucose (Fuc2S4S, type I), 2-O-monosulfated fucose (Fuc2S, type II), 3,4-O-disulfated fucose (Fuc3S4S, type III), 3-O-monosulfated fucose (Fuc3S, type IV), and 4-O-monosulfated fucose (Fuc4S, type V), respectively, according to previous reports [3,4,13,15].…”

Structural Characterization and Heparanase Inhibitory Activity of Fucosylated Glycosaminoglycan from Holothuria floridana

“…The elution solvent was 0.1 M NaCl. The flow rate was 0.5 mL/min, and the temperature of the column was 40 • C. For Mw calculation, the Shodex OH-pak SB-804 HQ column (8 mm × 300 mm) was calibrated by a series of fucosylated glycosaminoglycan fractions from H. fuscopunctata with Mw of 39.9, 27.8, 14.9, 8.24, 5.30 and 3.12 kDa, determined by a size-exclusion chromatography with multi-angle laser light scattering [36].…”