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

doi:10.1110/ps.073016308

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

Robson Q. Monteiro¹, Alireza R. Rezaie², Jong-Sup Bae², Eric Calvo³, John F. Andersen³, and Ivo M.B. Francischetti³

¹ Instituto de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil
² Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA
³ Vector Biology Section, Laboratory of Malaria and Vector Research, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-8132, USA

(RECEIVED May 22, 2007; FINAL REVISION September 28, 2007; ACCEPTED October 3, 2007)

Ixolaris is a two-Kunitz tick salivary gland tissue factor pathwayinhibitor (TFPI). In contrast to human TFPI, Ixolaris specificallybinds to factor Xa (FXa) heparin-binding exosite (HBE). In addition,Ixolaris interacts with zymogen FX. In the present work we characterizedthe interaction of Ixolaris with human FX quantitatively, andidentified 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 formationbetween fluorescein-labeled Ixolaris and FX. Isothermal titrationcalorimetry confirmed that the binding of Ixolaris to FX occursat stoichiometric concentrations in a reaction which is characteristicallyexothermic, with a favorable enthalpy ( ${Delta}$ H) of –10.78 kcal/mol.ELISA and plasmon resonance experiments also indicate that Ixolarisbinds to plasma FX and FXa, or to recombinant Gla domain-containingFX/FXa with comparable affinities ( $~$ 1 nM). Using a series ofmutants on the HBPE, we identified the most important aminoacids involved in zymogen/Ixolaris interaction—Arg-93>>> Arg-165 $≥$ Lys-169 > Lys-236 > Arg-125—whichwas identical to that observed for FXa/Ixolaris interaction.Remarkably, Ixolaris strongly inhibited FX activation by factorIXa 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 inhibitsFX interaction with immobilized FVIIIa. Altogether, Ixolarisis the first inhibitor characterized to date that specificallybinds to FX HBPE. Ixolaris may be a useful tool to study thephysiological role of the FX HBPE and to evaluate this domainas a target for anticoagulant drugs.

Keywords: tissue factor; heparin-binding exosite; proexosite; intrinsic tenase; prothrombinase

Reprint requests to: Ivo M.B. Francischetti, Laboratory of Malariaand Vector Research, National Institutes of Allergy and InfectiousDiseases, National Institutes of Health, 12735 Twinbrook Parkway,Twinbrook III Building, Room 2E-28, Bethesda, MD 20892-8132,USA; e-mail: ifrancischetti{at}niaid.nih.gov; fax: (301) 480-2571.

Abbreviations: [F]-Ixolaris, Ixolaris labeled at the N-terminalby fluorescein; FX, factor X; FXa, factor Xa; HBE, heparin-bindingexosite; ITC, isothermal titration calorimetry; PC, L- ${alpha}$ -phosphatidylcholine;PEG, polyethylene glycol; PS, L- ${alpha}$ -phosphatidylserine; S-2222,Bz-Ile-Glu( ${gamma}$ -OR)-Gly-Arg-pNA·HCl; TABE, thrombin-anionbinding exosite; TFPI, tissue factor pathway inhibitor; SPR,surface plasmon resonance.

Article published online ahead of print. Article and publicationdate are at http://www.proteinscience.org/cgi/doi/10.1110/ps.073016308.

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