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.