DISSCUSSION
Assay for potencies and selectivity of anti-iXase activities of dHG-5 and its containing oliogosaccharides showed that dHG-5 showed potent anti-iXase activity and f.IXa-binding activity without obvious effects on the other coagulation factor targets, which indicated its high selectivity in anti-iXase. Silmilar to our previous report (Xiao, Zhaoet al. , 2019), it was notable that dHG-5 and its containing oligosaccharides seemed to enhance the activity of f.IXa in hydrolyzing its chromogenic substrate in the absence of f.VIIIa. This phenomena might be attributed to the conformational change of f.IXa induced by oligosaccharides. In fact, under physiological conditions, f.IXa combines its cofactor f.VIIIa, phospholipid and Ca2+to form iXase complex, which enhances the catalytic efficiency of f.IXa with million times (Duffy & Lollar, 1992; Mertens, Wijngaarden et al. , 1985). Thus, the direct effects of dHG-5 and its containing oligosaccharides on f.IXa activity was very faint when compared with their inhibition on f.IXa-f.VIIIa complex assembling, i.e., anti-iXase activities.
Assay for anticoagulant activities of dHG-5 and its containing oliogosaccharides showed that the anticoagulant mechanisms of dHG-5 and its containing oligosaccharides were AT-III- and HCII-independent, which were different from that of LMWH and DS. At the equivalent anticoagulant potency’s doses, these oligosaccharides showed approximate thrombosis inhibition, suggesting their antithrombotic activities were closely related to their anticoagulant activities. The results of pharmacodynamics of dHG-5 showed the linear kinetics, suggesting the predictable pharmacodynamic characteristics.
Through a series of studies, the pharmacological properties of dHG-5 and its containing oligosaccharides in anti-iXase, f.IXa-binding, anticoagulant and antithrombotic activities were clearly illuminated. dHG-5 showed strong anti-iXase activity, f.IXa-binding, anticoagulant and antithrombotic activities without activating f.XII and platelets. Within bounds, the anti-iXase, f.IXa-binding and anticoagulant activities of these oligosaccharides increased with the increase of dp, oHG-17 to -29 closed to the full activity. It indicated that the increase of dp obviously increased these activities of these oligosaccharides to a certain extent, even octasaccharide was the minimum structural unit required for the potent anti-iXase activity (Yin, Zhou et al. , 2018). Moreover, the activity potencies of dHG-5 in anti-iXase, f.IXa-binding, anticoagulation and anti-thrombosis were close to the weighted average sum of that of its containing oligosaccharides (Table 1). It indicated that there was no synergy or antagonism among these oligosaccharides, the similarity of dHG-5 and oHG-17 in Mw and activities also confirmed this. Thus pharmacological activities of dHG-5 could be explained well by the contribution of its containing oligosaccharides both qualitatively and quantitatively. Besides, the relationships between Mw and f.IXa-binding, anti-iXase, anticoagulant and antithrombotic potencies of dHG-5 containing oligosaccharides all fitted well with similar power function, which suggested the high dependency among these activities.
dHG-5 is an active ingredient consisting of a series of oligosaccharides homologs. As a multi-component drug, it is a great challenge to clarify the correlation of pharmacological activity between drug and its containing components. For instance, heparins and LMWHs have been widely used in clinic, but due to complexity of the composition, the types and composition ratios of their containing oligosaccharides could not be clarified (Loganathan, Wang et al. , 1990). Consequently, the correlation of pharmacological activity between these preparations and their containing oligosaccharides has not been reported. Our study showed that the chemical composition of dHG-5 was relatively clear. And the nine purified oligosaccharide components (oHG-5, -8, -11, -14, -17, -20, -23, -26 and -29) accounted for about 95% of dHG-5. And spectral analysis (1D/2D NMR and MS) confirmed that these oligosaccharide components had the regular structures and shared the common formula. Compared with heparins that have complex composition and diverse substituents, the chemical composition of dHG-5 was clear, which enabled the further study in structure-activity relationship and the correlation of pharmacological activities between dHG-5 and its containing oligosaccharides.
According to pharmacoeconomics, dHG-5 is suitable for developing a noval anticoagulant. Though the activities of oHG-17 are similar to that of dHG-5, the preparation process of pure oligosaccharide is complex, and not feasible for large-scale industrial production. While it is more feasible for the scale preparation of dHG-5. What’s more, the composition and proportion of dHG-5 containing oligosaccharides could be controlled well in the preparation process. Therefore, dHG-5 may be more suitable as an iXase inhibitor to be a novel anticoagulant applied in clinic.
dHG-5 may be more effective than other intrinsic coagulation inhibitors in antithrombosis. The effects of common coagulation pathway inhibitors and intrinsic coagulation pathway inhibitors on hemostatic function are different (Colman, 2006; Qiufang, Tucker et al. , 2010; Wheeler & Gailani, 2016; Woodruff, Xu et al. , 2013). Compared with available clinical drugs, selective intrinsic coagulation pathway inhibitors may have the characteristics of antithrombosis with low bleeding tendency (Lin, Zhao et al. , 2020). According to the cell-based coagulation model, f.XIIa and f.XIa inhibitors may have limited effects on coagulation amplification and propagation during thrombosis, while iXase inhibitors should be able to exhibitie more effective antithrombotic activity (Hoffman, 2003; Lin, Zhao et al. , 2020).
In conclusion, our data demonstrate the anti-iXase, f.IXa-binding, anticoagulant and antithrombotic activities of dHG-5 are contributed by that of its containing oligosaccharides in terms of a weighted average sum. These activities of dHG-5 containing oligosaccharides were positively correlated with their chain length to a certain degree, among which, the molecular weight of oHG-17 may be necessary to achieve full activity. Not only dHG-5 has the characteristic of antithrombosis with low bleeding tendency, but also clear chemical composition. This paper makes an important supplement to the preclinical study of dHG-5, which makes a good preparation for the entry into the clinical study.
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