Draculin, the anticoagulant factor in vampire bat saliva, is a tight-binding, noncompetitive inhibitor of activated factor X

Ana Z. Fernandez, Alfonso Tablante, Suzette Beguin, H. Coenraad Hemker, Rafael Apitz-Castro*

*Corresponding author for this work

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    Abstract

    The kinetic mechanism of action of Draculin on activated Factor X (FXa) is established. Draculin inhibits activated Factor X within seconds of incubation at near equimolar concentration (2-6 times on molar basis). Fitting the data to the equation for a tight-binding inhibitor gives a value for K-i(K-d) = 14.8 +/- 1.5 nM. The formation of the Draculin-FXa complex can be explained by a two-step mechanism, where for the first, reversible step, k(on) = 1.117 (+/- 0.169, S.E.M.) x 10(6) M-1 s(-1) and k(off) = 15.388 (+/- 1.672) x 10(-3) s(-1), while for the second, irreversible step, which is concentration-independent, k(2) = 0.072 s(-1). K-d obtained from k(off)/k(on) = 13.76 nM. Lineweaver-Burk plot shows a noncompetitive behavior. This noncompetitive mode of inhibition of Draculin is supported by the observation that Draculin, at concentrations giving complete inhibition, does not impair binding of p-aminobenzamidine to FXa. Moreover, under the same conditions, Draculin induces < 14% decrease of the fluorescence intensity of the p-aminobenzamidine-FXa complex. We conclude that Draculin is a noncompetitive, tight-binding inhibitor of FXa, a characteristic so far unique amongst natural FXa inhibitors. (C) 1999 Elsevier Science B.V. All rights reserved.
    Original languageEnglish
    Pages (from-to)135-142
    Number of pages8
    JournalBiochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
    Volume1434
    Issue number1
    DOIs
    Publication statusPublished - 14 Sept 1999

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