title: Ixolaris: a factor Xa heparin-binding exosite inhibitor.
abstract: Ixolaris is a two-Kunitz TFPI (tissue factor pathway inhibitor) from the tick salivary gland. In contrast with human TFPI, Ixolaris binds tightly to the zymogen FX (Factor X) and to dansyl-Glu-Gly-Arg-chloromethyl ketone-treated FXa (DEGR-FXa; active-site-blocked FXa), indicating that exosites are involved in the FX(a)-Ixolaris interaction. Here we provide evidence that Ixolaris binds specifically to the FXa HBE (heparin-binding exosite), since (i) it markedly decreases the inhibition of FXa by the antithrombin-heparin but not the antithrombin-pentasaccharide complex, (ii) it impairs FXa binding to Sepharose-immobilized heparin, and (iii) it allosterically modulates the catalytic activity of FXa for small chromogenic substrates (S-2765). By using a series of recombinant FXa mutants in which the HBE is mutated, we have identified the importance of amino acids involved in the enzyme-inhibitor interaction as being in the following order: Arg-93>>Arg-165> or =Lys-169>Lys-236>Lys-96>Arg-240>Arg-125. Ixolaris at appropriate concentrations also inhibits thrombin formation in vitro by the assembled prothrombinase complex, a process that is critically dependent on the FXa HBE. Ixolaris is the first inhibitor characterized to date that binds specifically to the FXa HBE.
authors.affiliation: Instituto de Bioquímica Médica, Programa de Biologia Estrutural, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-590, Brazil.
authors.forename: Robson Q
authors.initials: RQ
authors.lastname: Monteiro
authors.forename: Alireza R
authors.initials: AR
authors.lastname: Rezaie
authors.forename: José M C
authors.initials: JM
authors.lastname: Ribeiro
authors.forename: Ivo M B
authors.initials: IM
authors.lastname: Francischetti
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D015951:Factor Xa / Q000737:chemistry*; D065427:Factor Xa Inhibitors; D006493:Heparin / Q000737:chemistry*; D011485:Protein Binding; D011516:Prothrombin / Q000737:chemistry; D012471:Salivary Proteins and Peptides / Q000737:chemistry*
chemical_list: D065427:Factor Xa Inhibitors; C455900:Ixolaris protein, Ixodes scapularis; D012471:Salivary Proteins and Peptides; D011516:Prothrombin; D006493:Heparin; D015951:Factor Xa