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 ≥ 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.
Original language | English (US) |
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Pages (from-to) | 871-877 |
Number of pages | 7 |
Journal | Biochemical Journal |
Volume | 387 |
Issue number | 3 |
DOIs | |
State | Published - May 1 2005 |
Externally published | Yes |
Keywords
- Blood feeding
- Factor Xa
- Heparin-binding exosite
- Prothrombin
- Tick saliva
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology