Antithrombotic activities of fucosylated chondroitin sulfates and their depolymerized fragments from two sea cucumbers
Introduction
Thrombotic diseases are reported to contribute to 30% of early deaths globally (Mackman, 2008, Wang et al., 2014, Zhao et al., 2012). Arterial or venous blood clots can cause acute coronary syndrome (ACS) or venous thromboembolisms which are the 3rd leading cause of cardiovascular-event associated deaths (Mega & Simon, 2015). Many antithrombotic drugs including antiplatelets and anticoagulants are available for treating coagulopathies, and heparin (HP) is one of the most commonly prescribed drugs effective for reducing arterial and venous thrombosis in patients with cardiovascular disease. However, these compounds can cause unwanted bleeding that may limit their use and a contaminated batch of HP caused more than 100 deaths and many adverse reactions in the US and Germany (Pan et al., 2010). Low-molecular-weight heparins (LMWH) are also available and have less associated unwanted bleeding (Quinlan, McQuillan, & Eikelboom, 2004) and these drugs complement the diverse additional anticoagulants on the US market, but better drugs are always desired.
Fucosylated chondroitin sulfate (FCS) isolated from sea cucumbers is an important glycosaminoglycan (GAG), which had a chondroitin sulfate-like core backbone consisted of repeating units of β-1,4-glucuronic acid (→4GlcAβ1 → ) and β-1,3-N-acetyl-galactosamine (→3GalNAcβ1→) with α-fucose branches linked to the O-3 position of GlcA residues (Fig. 1). The extent of GalNAc sulfation varied among sea cucumber species (Kariya, Sakai, Kaneko, Suzuki, & Kyogashima, 2002; Vieira, Mulloy, & Mourão, 1991; Wu et al., 2015; Yoshida, Minami, Nemoto, Numata, & Yamanaka, 1992). FCS isolated from Ludwigothure agrisea was 53% 6-sulfated GalNAc units (Vieira & Mourão, 1988), and the polysaccharide from Stichopus japonicas contained all 4,6-disulfated GalNAc residues (Nagase et al., 1995). Sulfation patterns of fucose branches differed between species and/or among preparations (Chen et al., 2011; Fonseca, Santos, & Mourão, 2009; Luo et al., 2013; Matsuhiro, Osorio-Román, & Torres, 2012; Mourão et al., 1996; Wu et al., 2012; Wu, Xu, Zhao, Kang, & Ding, 2010; Ye, Xu, & Li, 2012) and fucose side chains were non-, 3-, 4-, 2,4- or 3,4-disulfation patterns.
Sulfation patterns and structures of FCS accounted for anticoagulant and antithrombotic activity (Buyue and Sheehan, 2009, Chen et al., 2013, Fonseca et al., 2010, Fonseca and Mourão, 2006, Mourão et al., 2001; Mourão, Giumaraes, Mulloy, Thomas, & Gray, 1998; Pacheco, Vicente, Zancan, & Mourão, 2000; Zancan and Mourão, 2004, Zhao et al., 2013). Mammalian chondroitin sulfate without fucosylated side chains had no anticoagulant or antithrombotic activity, and modifications such as desulfation or defucosylation eliminated this activity (Mulloy, Mourão, & Gray, 2000; Wu et al., 2012).
Chen’s group compared anticoagulant activity of FCSs from four sea cucumber species with different sulfation patterns, and polysaccharides containing 95.9% 2,4-disulfated fucose branches had more anticoagulant effects (Chen et al., 2011). Additionally, FCS could reduce venous and arterial thrombosis in a rat model but unwanted bleeding was a problem (Fonseca et al., 2009). Depolymerization was considered effective for reducing bleeding side effect. Low-molecular-weight FCSs had antithrombotic activity with less bleeding than both UFH and LMWH (Kitazato, Kitazato, Nagase, & Minamiguchi, 1996; Wu et al., 2010; Yang, Wang, Jiang, Lv, &, Zhang et al., 2015; Yang, Wang, Jiang, & Lv, 2015; Zhao et al., 2015).
In this study, FCSc was isolated from Cucumaria frondosa, and its low molecular weight fragments were prepared with Cu2+ catalytic free-radical depolymerization. Physicochemical properties were analyzed by comparing this to FCSt from Thelenota ananas. Detailed structural information was characterized by disaccharide composition and nuclear magnetic resonance (NMR) analysis. Furthermore, anticoagulant and antithrombotic activity were evaluated in vitro and in vivo to understand the relationship between molecular weight, sulfation patterns, and biological activity of FCS.
Section snippets
Reagents and animals
LMWH and HP from porcine intestinal mucosa were purchased from Sigma (St. Louis, MO). Q-Sepharose Fast Flow resin was purchased from GE Healthcare (Uppsala, Sweden). Antithrombin (AT), bovine FXa, human thrombin (FIIa), heparin cofactor II (HCII), chromogenic substrate S-2765 and S-2238 were purchased from Adhoc International Technologies Co., Ltd, (Beijing, China). All other chemicals and reagents were of analytical grade.
Male Wistar rats (250–270 g) and male Kunming mice (18–22 g) were
Preparation of native FCSs and dFCSs
The yields of FCSc and FCSt isolated from C. frondosa and T. ananas were 0.60% and 0.65%, respectively. Separation of FCSc on a Q Sepharose Fast Flow column displayed one single peak during the elution with 1.5 M NaCl (Fig. 2a). FCSc purification on a Sephacryl S-300 column revealed a single and symmetrical sample peak as well (Fig. 2b). Depolymerization of native FCS with H2O2 in the presence of Cu2+ was monitored by GPC at different time points. To compare the antithrombotic activity of native
Conclusion
In this study, antithrombotic and anticoagulant activities of four FCS fractions with different molecular weights and sulfation patterns from two sea cucumbers were compared respectively. Low molecular weight fragments including dFCSc and dFCSt had better antithrombotic-hemorrhagic ratios than their native forms, and even the positive drugs HP and LMWH in a rat arterial thrombosis model. dFCSc and dFCSt containing diverse sulfated fucose branches had similar antithrombotic effects and bleeding
Acknowledgements
This work was supported in part by the Natural Science Foundation of China Shandong Joint Fund for Marine Science Research Centers (U1406402), the National Science & Technology Support Program of China (2013BAB01B02), Taishan scholar project special funds and Ocean University of China (OUC 201562031). ‘The authors thank Xiuli Zhang and Cong Wang, Ocean University of China, for providing technical support of NMR operation.
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