Introduction
Cardiovascular disease has been causing high morbidity and mortality in worldwide (Benjamin, Virani et al. , 2018; Timmis, Townsendet al. , 2017). Among which, venous thromboembolism, such as deep venous thrombosis and pulmonary embolism, is one of the main complications of major surgery and cancer, et al , which remains to be a global disease burden (Becattini, Cohen et al. , 2016; Carpenter, Richardson et al. , 2018; Jiménez, Bikdeli et al. , 2019).
According to the classic coagulation cascade model, intrinsic coagulation pathway is closely involved in pathological thrombosis while dispensable in physiological hemostasis (Colman, 2006; Qiufang, Tuckeret al. , 2010; Wheeler & Gailani, 2016; Woodruff, Xu et al. , 2013). It is initiated by polyanions such as polyphosphate and endotoxin produced in infection processes (Campello, Henderson et al. , 2018; Long, Kenne et al. , 2016). In the cell-based coagulation model, pathological thrombosis might also be initiated by blood-born tissue factor (TF) (e.g., TF derived from monocytes) under pathological conditions such as inflammation (Hoffman, 2003). After the initiation of coagulation by TF, f.VIII (factor VIII) and platelets are activated by trace amount of thrombin produced in the extrinsic coagulation pathway. Then f.VIIIa (activated factor VIII) forms complex with f.IXa (activated factor IX) in the surfaces of activated platelets, which is essential in the amplification and propogation phases of coagulation, and thus thrombosis (Smith, 2010). The f.VIIIa-f.IXa complex is also known as intrinsic Xase (iXase), the last and rate-limiting enzyme in the intrinsic coagulation pathway (Ahmad, Rawala-Sheikh et al. , 1992).
Based on the above theries, selectively inhibiting the intrinsic coagulation pathway is recognized to be a promising strategy for safe antithrombotic therapy (Wheeler & Gailani, 2016). In recent years, inhibitors targeting activated factor XII (f.XIIa), XI (f.XIa) or IX (f.IXa) are being developed, and showed promising preclinical results (Pinto, Orwat et al. , 2017; Quan, Pinto et al. , 2018; Robert, Bertolla et al. , 2008; Wang, Beck et al. , 2010). While anti-iXase drug candidate has not been reported, according to both coagulation model, it should be a good target for effective and safe antithrombosis (Lin, Zhao et al. , 2020).
Fucosylated glycosaminoglycan (FG) from sea cucumbers, consisting of the chondroitin sulfate-like backbone and the unique branches of sulfated fucoses, is a potent iXase inhibitor considered as a lead compound for novel anticoagulant (V. H. Pomin, 2014). Native FG exhibited potent anticoagulant and antithrombotic activities, but also had the undesired effects of platelets aggregation and factor XII (f.XII) activation (Fonseca, Santos et al. , 2009; J. Z. Li, Bao et al. , 1985). 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).
Like the heterogenous polysaccharide heparin, dHG-5 is a multi-component compound and composed of a series of oligosaccharides. However, it’s unclear that these oligosaccharides’ structures and contents contained in dHG-5, and how these oligosaccharides contribute to the pharmacological activities of dHG-5. We now aimed 1) to purify and identify these oligosaccharide components, 2) to characterize the pharmacological activities of these oligosaccharide components, 3) to reveal the contribution of these oligosaccharide components to dHG-5 in pharmacological behaviors. Besides, we have systematically evaluated the pharmacological activities of dHG-5 in target selectivity, anticoagulation, antithrombosis, bleeding risk and pharmacodynamics.