Thromb Haemost 2019; 119(05): 705-715
DOI: 10.1055/s-0039-1681047
Coagulation and Fibrinolysis
Georg Thieme Verlag KG Stuttgart · New York

Nonasaccharide Inhibits Intrinsic Factor Xase Complex by Binding to Factor IXa and Disrupting Factor IXa–Factor VIIIa Interactions

Chuang Xiao
1  State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
2  College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China
3  School of Pharmaceutical Science and Yunnan Key Laboratory of Pharmacology for Natural Products, Kunming Medical University, Kunming, China
,
Longyan Zhao
1  State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
2  College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China
4  School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan, China
,
Na Gao
1  State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
2  College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China
4  School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan, China
,
Mingyi Wu
1  State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
,
Jinhua Zhao
1  State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
› Author Affiliations
Funding This work was funded in part by the National Natural Science Foundation of China (31600649, 81773737, 81673330 and 81872774), Yunnan Provincial Science and Technology Department in China (2016FA050), and a grant from Youth Innovation Promotion Association (2017435), Chinese Academy of Sciences.
Further Information

Publication History

20 September 2018

17 January 2019

Publication Date:
21 March 2019 (eFirst)

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.

Authors' Contributions

C.X. performed research, analysed data and wrote the manuscript; L.Z. and N.G. performed research; M.W. revised the manuscript; and J.Z. designed the research, analysed data and revised the manuscript.


Supplementary Material