title: Identification of AcAP5 as a novel factor Xa inhibitor with both direct and allosteric inhibition.
abstract: Ancylostoma caninum anticoagulant peptide 5 (AcAP5) is a potent inhibitor for coagulation factor Xa (FXa). Previous studies show that AcAP5 binds to FXa at the active site, and/or the exosite. The active site-binding contributes to direct blocking of FXa catalytic activity, but the effect of exosite-binding and the underlying mechanism remain unknown. To investigate whether and how the exosite-binding affects FXa function, we prepared several AcAP5 mutants with modifications to the active site-binding or exosite-binding region. Their FXa-inhibiting and anticoagulant activities were examined both in vitro and in rabbit plasma, and the interactions with FXa were analyzed using in silico molecular modeling, docking, and molecular dynamics simulation. Mutants abolishing either active site- or exosite-binding resulted in a dramatic decrease in their anti-FXa and anticoagulant activities. Elongation of AcAP5 exosite-binding region also impaired the FXa-inhibiting activity. Computational analysis demonstrated that the conformation of FXa becomes more rigid due to exosite-binding with AcAP5, which consequently affects its catalytic activity. Our results suggest that both active site- and exosite-binding contribute to the FXa inhibitory activity of AcAP5.
authors.affiliation: Department of Molecular and Cellular Pharmacology, Peking University School of Pharmaceutical Sciences, Beijing, China.
authors.forename: Yuanjun
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: State Key Laboratory of Bioactive Substances and Function of Natural Medicine, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Department of Molecular and Cellular Pharmacology, Peking University School of Pharmaceutical Sciences, Beijing, China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Neuroscience, Temple University Lewis Katz School of Medicine, Philadelphia, PA, USA. Electronic address: whu@temple.edu.
authors.forename: Wenhui
authors.initials: W
authors.lastname: Hu
authors.affiliation: Department of Molecular and Cellular Pharmacology, Peking University School of Pharmaceutical Sciences, Beijing, China. Electronic address: wangyinye@bjmu.edu.cn.
authors.forename: Yinye
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000494:pharmacology*; D001665:Binding Sites; D020134:Catalytic Domain; D004353:Drug Evaluation, Preclinical / Q000379:methods; D015951:Factor Xa / Q000737:chemistry / Q000378:metabolism; D065427:Factor Xa Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D015801:Helminth Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D008297:Male; D056004:Molecular Dynamics Simulation; D009154:Mutation; D011817:Rabbits
chemical_list: D000925:Anticoagulants; D065427:Factor Xa Inhibitors; D015801:Helminth Proteins; D015951:Factor Xa
keywords: AcAP5; Active site; Computational simulation; Exosite; Factor Xa; Inhibition