Ixolaris binding to factor X reveals a precursor state of factor Xa heparin-binding exosite
| العنوان: | Ixolaris binding to factor X reveals a precursor state of factor Xa heparin-binding exosite |
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| المؤلفون: | Alireza R. Rezaie, Robson Q. Monteiro, Jong-Sup Bae, John F. Andersen, Ivo M.B. Francischetti, Eric Calvo |
| المصدر: | Protein Science. 17:146-153 |
| بيانات النشر: | Wiley, 2007. |
| سنة النشر: | 2007 |
| مصطلحات موضوعية: | Factor VIIa, Plasma protein binding, Calorimetry, Biochemistry, Article, Factor IXa, Tissue factor, chemistry.chemical_compound, Ticks, Prothrombinase, Zymogen, Animals, Humans, Salivary Proteins and Peptides, Binding site, Molecular Biology, Binding Sites, Heparin, Chemistry, Factor X, Isothermal titration calorimetry, Kinetics, Factor Xa, Chromatography, Gel, Biophysics, Protein Binding |
| الوصف: | Ixolaris is a two-Kunitz tick salivary gland tissue factor pathway inhibitor (TFPI). In contrast to human TFPI, Ixolaris specifically binds to factor Xa (FXa) heparin-binding exosite (HBE). In addition, Ixolaris interacts with zymogen FX. In the present work we characterized the interaction of Ixolaris with human FX quantitatively, and identified a precursor state of the heparin-binding exosite (proexosite, HBPE) as the Ixolaris-binding site on the zymogen. Gel-filtration chromatography demonstrated 1:1 complex formation between fluorescein-labeled Ixolaris and FX. Isothermal titration calorimetry confirmed that the binding of Ixolaris to FX occurs at stoichiometric concentrations in a reaction which is characteristically exothermic, with a favorable enthalpy (DeltaH) of -10.78 kcal/mol. ELISA and plasmon resonance experiments also indicate that Ixolaris binds to plasma FX and FXa, or to recombinant Gla domain-containing FX/FXa with comparable affinities ( approximately 1 nM). Using a series of mutants on the HBPE, we identified the most important amino acids involved in zymogen/Ixolaris interaction-Arg-93Arg-165or = Lys-169Lys-236Arg-125-which was identical to that observed for FXa/Ixolaris interaction. Remarkably, Ixolaris strongly inhibited FX activation by factor IXa in the presence but not in the absence of factor VIIIa, suggesting a specific interference in the cofactor activity. Further, solid phase assays demonstrated that Ixolaris inhibits FX interaction with immobilized FVIIIa. Altogether, Ixolaris is the first inhibitor characterized to date that specifically binds to FX HBPE. Ixolaris may be a useful tool to study the physiological role of the FX HBPE and to evaluate this domain as a target for anticoagulant drugs. |
| تدمد: | 1469-896X 0961-8368 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::0dc5d035eb62f71850e5d8facf756e4bTest https://doi.org/10.1110/ps.073016308Test |
| حقوق: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....0dc5d035eb62f71850e5d8facf756e4b |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 1469896X 09618368 |
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