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 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.
Fernandez
A Z
AZ
Lab. Trombosis Experimental, Centro de Biofísica y Bioquímica, IVIC, Ap. 21827, Caracas, Venezuela.
Tablante
A
A
Beguín
S
S
Hemker
H C
HC
Apitz-Castro
R
R
D000818:Animals; D000925:Anticoagulants / Q000737:chemistry*; D001550:Benzamidines / Q000737:chemistry; D002685:Chiroptera; D002851:Chromatography, High Pressure Liquid; D065427:Factor Xa Inhibitors; D006023:Glycoproteins / Q000737:chemistry*; D007700:Kinetics; D011485:Protein Binding; D012463:Saliva / Q000737:chemistry*; D012471:Salivary Proteins and Peptides / Q000737:chemistry*
D000925:Anticoagulants; D001550:Benzamidines; D065427:Factor Xa Inhibitors; D006023:Glycoproteins; D012471:Salivary Proteins and Peptides; C094183:draculin; C006500:4-aminobenzamidine