Abstract
A nonasaccharide (FG9) derived from natural fucosylated glycosaminoglycan (FG) is
identified as a selective intrinsic factor Xase complex (FIXa-FVIIIa-Ca2+-phospholipid, FXase) inhibitor that possesses potential inhibition of venous thrombus
in rats and shows negligible bleeding risk. The mechanism and molecular target of
the nonasaccharide for intrinsic FXase inhibition were systematically investigated
and compared with low molecular weight heparin (LMWH). Our results showed that FG9
dose-dependently inhibited FX activation by intrinsic FXase complex in a noncompetitive
inhibition pattern, where the apparent affinity for FG9 was approximately 1.8-fold
higher than that for LMWH. FG9 displayed no inhibitory effect on the activity of FIXa/phospholipid,
and did not affect the decay rate of FVIIIa activity. FG9 reduced the apparent affinity
of FIXa for FVIIIa in a dose-dependent manner, and accelerated the decay of intrinsic
FXase complex activity. FG9 bound to FIXa with high affinity and the FIXa binding
sites of FG9 were overlapped with that of LMWH, and the ability of FG-derived oligosaccharides
to bind FIXa required the minimum 9 degrees of polymerization. FG9 derivatives were
prepared and their structures were confirmed by one-dimensional/two-dimensional nuclear
magnetic resonance. Structure–activity relationship studies showed that carboxy reduction
significantly weakened its anti-FXase activity and binding affinity to FIXa, while
the effects of carboxyl ethyl esterification and deacetylation were relatively weaker.
Overall, our results suggest that the nonasaccharide FG9 strongly inhibits intrinsic
FXase complex activity via binding to FIXa and disrupting FIXa–FVIIIa interactions,
and the free carboxyl groups of FG9 are required for its potent anti-FXase activity.
Keywords
nonasaccharide from fucosylated glycosaminoglycan - intrinsic factor Xase complex
- factor IXa - anticoagulant
