Ixolaris binding to factor X reveals a precursor state of factor Xa heparin-binding exosite.
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-93 >>> Arg-165 > or = 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.
Monteiro
Robson Q
RQ
Instituto de Bioquimica Medica, Centro de Ciencias de Saude, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Rezaie
Alireza R
AR
Bae
Jong-Sup
JS
Calvo
Eric
E
Andersen
John F
JF
Francischetti
Ivo M B
IM
D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D002850:Chromatography, Gel; D015942:Factor VIIa / Q000378:metabolism; D005170:Factor X / Q000737:chemistry* / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism*; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D012471:Salivary Proteins and Peptides / Q000737:chemistry* / Q000378:metabolism*; D013987:Ticks
C455900:Ixolaris protein, Ixodes scapularis; D012471:Salivary Proteins and Peptides; D005170:Factor X; D006493:Heparin; D015942:Factor VIIa; D015951:Factor Xa