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 AffiliationsFunding 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.
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.
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.
4
Li W,
Johnson DJ,
Esmon CT,
Huntington JA.
Structure of the antithrombin-thrombin-heparin ternary complex reveals the antithrombotic mechanism of heparin. Nat Struct Mol Biol 2004; 11 (09) 857-862
5
Olson ST,
Richard B,
Izaguirre G,
Schedin-Weiss S,
Gettins PG.
Molecular mechanisms of antithrombin-heparin regulation of blood clotting proteinases. A paradigm for understanding proteinase regulation by serpin family protein proteinase inhibitors. Biochimie 2010; 92 (11) 1587-1596
6
Petitou M,
Casu B,
Lindahl U.
1976-1983, a critical period in the history of heparin: the discovery of the antithrombin binding site. Biochimie 2003; 85 (1-2): 83-89
12
Zhao L,
Wu M,
Xiao C.
, et al. Discovery of an intrinsic tenase complex inhibitor: pure nonasaccharide from fucosylated glycosaminoglycan. Proc Natl Acad Sci U S A 2015; 112 (27) 8284-8289
14
Nagase H,
Enjyoji K,
Minamiguchi K.
, et al. Depolymerized holothurian glycosaminoglycan with novel anticoagulant actions: antithrombin III- and heparin cofactor II-independent inhibition of factor X activation by factor IXa-factor VIIIa complex and heparin cofactor II-dependent inhibition of thrombin. Blood 1995; 85 (06) 1527-1534
15
Nagase H,
Enjyoji K,
Shima M.
, et al. Effect of depolymerized holothurian glycosaminoglycan (DHG) on the activation of factor VIII and factor V by thrombin. J Biochem 1996; 119 (01) 63-69
17
Wu M,
Wen D,
Gao N.
, et al. Anticoagulant and antithrombotic evaluation of native fucosylated chondroitin sulfates and their derivatives as selective inhibitors of intrinsic factor Xase. Eur J Med Chem 2015; 92: 257-269
18
Li JZ,
Lian EC.
Aggregation of human platelets by acidic mucopolysaccharide extracted from Stichopus japonicus Selenka. Thromb Haemost 1988; 59 (03) 435-439
19
Fonseca RJ,
Oliveira SN,
Pomin VH,
Mecawi AS,
Araujo IG,
Mourão PA.
Effects of oversulfated and fucosylated chondroitin sulfates on coagulation. Challenges for the study of anticoagulant polysaccharides. Thromb Haemost 2010; 103 (05) 994-1004
20
Kitazato K,
Kitazato KT,
Nagase H,
Minamiguchi K.
DHG, a new depolymerized holothurian glycosaminoglycan, exerts an antithrombotic effect with less bleeding than unfractionated or low molecular weight heparin, in rats. Thromb Res 1996; 84 (02) 111-120
21
Kitazato K,
Kitazato KT,
Sasaki E,
Minamiguchi K,
Nagase H.
Prolonged bleeding time induced by anticoagulant glycosaminoglycans in dogs is associated with the inhibition of thrombin-induced platelet aggregation. Thromb Res 2003; 112 (1-2): 83-91
22
Gao N,
Lu F,
Xiao C.
, et al. β-Eliminative depolymerization of the fucosylated chondroitin sulfate and anticoagulant activities of resulting fragments. Carbohydr Polym 2015; 127: 427-437
24
Sheehan JP,
Walke EN.
Depolymerized holothurian glycosaminoglycan and heparin inhibit the intrinsic tenase complex by a common antithrombin-independent mechanism. Blood 2006; 107 (10) 3876-3882
28
Wu M,
Xu S,
Zhao J,
Kang H,
Ding H.
Preparation and characterization of molecular weight fractions of glycosaminoglycan from sea cucumber Thelenata ananas using free radical depolymerization. Carbohydr Res 2010; 345 (05) 649-655
29
Zhao L,
Lai S,
Huang R.
, et al. Structure and anticoagulant activity of fucosylated glycosaminoglycan degraded by deaminative cleavage. Carbohydr Polym 2013; 98 (02) 1514-1523
30
Barrow RT,
Parker ET,
Krishnaswamy S,
Lollar P.
Inhibition by heparin of the human blood coagulation intrinsic pathway factor X activator. J Biol Chem 1994; 269 (43) 26796-26800
34
Fay PJ,
Beattie TL,
Regan LM,
O'Brien LM,
Kaufman RJ.
Model for the factor VIIIa-dependent decay of the intrinsic factor Xase. Role of subunit dissociation and factor IXa-catalyzed proteolysis. J Biol Chem 1996; 271 (11) 6027-6032
35
Li B,
Suwan J,
Martin JG.
, et al. Oversulfated chondroitin sulfate interaction with heparin-binding proteins: new insights into adverse reactions from contaminated heparins. Biochem Pharmacol 2009; 78 (03) 292-300
37
Xiao C,
Lian W,
Zhou L.
, et al. Interactions between depolymerized fucosylated glycosaminoglycan and coagulation proteases or inhibitors. Thromb Res 2016; 146: 59-68
39
Lian W,
Wu M,
Huang N.
, et al. Anti-HIV-1 activity and structure-activity-relationship study of a fucosylated glycosaminoglycan from an echinoderm by targeting the conserved CD4 induced epitope. Biochim Biophys Acta 2013; 1830 (10) 4681-4691
41
Santos GR,
Glauser BF,
Parreiras LA,
Vilanova E,
Mourão PA.
Distinct structures of the α-fucose branches in fucosylated chondroitin sulfates do not affect their anticoagulant activity. Glycobiology 2015; 25 (10) 1043-1052
43
Griffiths AE,
Rydkin I,
Fay PJ.
Factor VIIIa A2 subunit shows a high affinity interaction with factor IXa: contribution of A2 subunit residues 707-714 to the interaction with factor IXa. J Biol Chem 2013; 288 (21) 15057-15064
44
Mourão PA,
Pereira MS,
Pavão MS.
, et al. Structure and anticoagulant activity of a fucosylated chondroitin sulfate from echinoderm. Sulfated fucose branches on the polysaccharide account for its high anticoagulant action. J Biol Chem 1996; 271 (39) 23973-23984
45
Zhang X,
Liu H,
Lin L.
, et al. Synthesis of fucosylated chondroitin sulfate nonasaccharide as a novel anticoagulant targeting intrinsic factor Xase complex. Angew Chem Int Ed Engl 2018; 57 (39) 12880-12885
46
Petitou M,
van Boeckel CA.
A synthetic antithrombin III binding pentasaccharide is now a drug! What comes next?. Angew Chem Int Ed Engl 2004; 43 (24) 3118-3133
