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

The kinetic mechanism of action of Draculin on activated Factor X (FXa) is established. Draculin inhibits activated Factor X within seconds of incubat ion 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 exp lained 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 (+/- 1.6 72) x 10(-3) s(-1), while for the second, irreversible step, which is conce ntration-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 noncom petitive mode of inhibition of Draculin is supported by the observation tha t Draculin, at concentrations giving complete inhibition, does not impair b inding of p-aminobenzamidine to FXa. Moreover, under the same conditions, D raculin induces < 14% decrease of the fluorescence intensity of the p-amino benzamidine-FXa complex. We conclude that Draculin is a noncompetitive, tig ht-binding inhibitor of FXa, a characteristic so far unique amongst natural FXa inhibitors. (C) 1999 Elsevier Science B.V. All rights reserved.