RESUMO
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.
Assuntos
Anticoagulantes/química , Inibidores do Fator Xa , Glicoproteínas/química , Saliva/química , Proteínas e Peptídeos Salivares/química , Animais , Benzamidinas/química , Quirópteros , Cromatografia Líquida de Alta Pressão , Cinética , Ligação ProteicaRESUMO
Draculin, a glycoprotein isolated from vampire bat (Desmodus rotundus) saliva, is a natural anticoagulant which inhibits activated coagulation factors IX (IXa) and X (Xa). The observation that under captivity conditions, the anticoagulant activity present in vampire bat saliva is dependent upon the salivation protocol, led us to investigate the possible relationship between the expression of biological activity of native draculin and the post-translational glycosylation of the protein backbone. Daily salivation of vampire bats yields a saliva that progressively decreases in anticoagulant activity, without any significant change in overall protein content, or in the amount of protein specifically recognized by a polyclonal anti-draculin antibody. Anticoagulant activity of the saliva is restored after a 4-day period of rest. Besides the marked difference in anticoagulant activity, purified native draculin, obtained from high- and low-activity saliva, shows significant differences in: (a) composition of the carbohydrate moiety, and (b) Glycosylation pattern. Furthermore, controlled chemical deglycosylation of native draculin, under conditions that do not affect the polypeptide backbone, progressively leads to complete loss of the biological activity. Our present results implicate that correct glycosylation of draculin is a seminal event for the expression of the biological activity of this glycoprotein.