Ixolaris binding to factor X reveals a precursor state of factor Xa heparin-binding exosite

Robson Q. Monteiro, Alireza R. Rezaie, Jong Sup Bae, Eric Calvo, John F. Andersen, Ivo M.B. Francischetti

Research output: Contribution to journalArticlepeer-review

Abstract

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 (ΔH) 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 (∼1 nM). Using a series of mutants on the HBPE, we identified the most important amino acids involved in zymogen/Ixolaris interaction - Arg-93 ⋙ Arg-165 ≥ Lys-169 > Lys-236 > Arg-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. Published by Cold Spring Harbor Laboratory Press.

Original languageEnglish (US)
Pages (from-to)146-153
Number of pages8
JournalProtein Science
Volume17
Issue number1
DOIs
StatePublished - Jan 2008
Externally publishedYes

Keywords

  • Heparin-binding exosite
  • Intrinsic tenase
  • Proexosite
  • Prothrombinase
  • Tissue factor

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Fingerprint

Dive into the research topics of 'Ixolaris binding to factor X reveals a precursor state of factor Xa heparin-binding exosite'. Together they form a unique fingerprint.

Cite this