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).