Potencies and Selectivity of Anti-iXase Activities of dHG-5 and its Containing Oliogosaccharides
Previous research showed that some depolymerized FG and oligosaccharides retained the potent activity for inhibiting iXase without the adverse effects of native FG (Zhao, Wu et al. , 2015). Herein, the anti-iXase activities and selectivity of dHG-5 and its containing oligosaccharides were investigated.
Anti-iXase activity. The activities of dHG-5 and its containing oligosaccharides in inhibiting iXase were evaluated based on their IC50 (Figure 2). dHG-5 had small IC50 of 14.0 nM of anti-iXase. For dHG-5 containing oligosaccharides, oHG-5 had the largest IC50 (> 3000 nM) of anti-iXase, the IC50 of anti-iXase of oligosaccharides with dp 8~14 decreased significantly (156 nM ~ 27.1 nM) with the extending of their chain length, while the IC50 of anti-iXase of oligosaccharides with dp 17~29 had small changes (17.2 nM ~ 2.93 nM), and the IC50 of anti-iXase of oHG-17 approximated to that of dHG-5 (Figure 2). The functional relationship between the Mw (x ) and IC50 of anti-iXase of dHG-5 containing oligosaccharides (y ) was fitted well with the power function (classical Freundlich model y = a× xb ): y = 1.66 × 1013 × x -3.25(R2 = 0.997) (Figure 2B). Further calculation showed that the anti-iXase potency of dHG-5 (100 U, used as standard) was close to the weighted average sum of that of its containing oligosaccharides (Table 1)
f.IXa-binding affinity. Previous studies found that FG oligosaccharides inhibited iXase activity by binding to f.IXa and disrupting the formation of iXase complex (John P. Sheehan & Walke, 2006; Xiao, Lian et al. , 2016; Xiao, Zhao et al. , 2019). Herein, the f.IXa-binding affinity of dHG-5 and its containing oligosaccharides were assessed by the competition study using bio-layer interferometry (BLI). The kinetics parameters of f.IXa-oHG-11 binding were determined (Figure 3A). The interaction of f.IXa and oHG-11 fitted well with the 2:1 heterogeneous ligand binding model, with high affinity (Table S1).
The abilities of dHG-5 containing oligosaccharides competitively binding to f.IXa (100 nM) were evaluated by determining their IC50 values for inhibiting f.IXa to bind to immobilized oHG-11 (Figure 3B). dHG-5 had small IC50 of 0.430 µM of f.IXa-binding. For dHG-5 containing oligosaccharides, oHG-5 had the largest IC50 (~ 12.9 µM) of f.IXa-binding, the IC50 of f.IXa-binding of oligosaccharides with dp 8~14 decreased significantly (3.29 µM ~ 1.20 µM) with the extending of their chain length, while the IC50 of f.IXa-binding of oligosaccharides with dp 17~29 were similar (0.416 µM ~ 0.284 µM), and the IC50 of f.IXa-binding of oHG-17 approximated to that of dHG-5. The functional relationship between the Mw (x ) and IC50of f.IXa-binding of dHG-5 containing oligosaccharides (y ) was fitted well with the power function: y = 3.36 × 1010 × x-2.06 (R2 = 0.970) (Figure 3C). Further calculation showed that the f.IXa-binding potency of dHG-5 (100 U, used as standard) was close to the weighted average sum of that of its containing oligosaccharides (Table 1)
Effects of dHG-5 and its oligosaccharides on other coagulation factor targets. To investigate the anti-iXase selectivity of dHG-5 and its containing oligosaccharides, their effects on f.IIa, f.VIIa, TF-f.VIIa, f.Xa, f.XIa and f.XIIa in the presence or absence of AT-III were assayed. Concentrations of dHG-5 and its containing oligosaccharides that resulted in 50% or 90% iXase inhibition were used in assays.
At the concentrations of inhibiting 50% iXase, dHG-5 and its containing oligosaccharides showed no obvious effects on the activities of f.IIa, f.VIIa, f.Xa, f.XIa and f.XIIa in the presence or absence of AT-III (Figure 4A), except that oHG-5 (had weakest anti-iXase activity) obviously inhibited f.VIIa in the absence of AT-III (Figure 4B). At much higher concentrations of inhibiting 90% iXase, in the presence of AT-III, dHG-5 and its containing oligosaccharides showed little or very weak effects on f.IIa, f.XIa and f.XIIa (Figure 4C-D). Although oHG-5, -8, -11, and -14 showed slight to moderate inhibition (14.2% ~ 41.8%) of f.Xa (Figure 4C-D), dHG-5 and oligosaccharides with dp > 14 had no obvious effects on f.Xa. While dHG-5 and its containing oligosaccharides enhanced the activity of f.IXa in hydrolyzing its chromogenic substrate in the absence of f.VIIIa (Figure 4E-F).