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

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.

The “Vampirome”: Transcriptome and proteome analysis of the principal and accessory submaxillary glands of the vampire bat Desmodus rotundus, a vector of human rabies

“…The glands secrete factors that inhibit clot formation and dissolve already formed clots, thus keeping blood flowing freely in a bite wound and enabling bats to drink their meal [8, 9]. Accordingly, saliva of vampire bats has been described to contain an uncharacterized platelet aggregation inhibitor [8, 10] and one anticoagulant (draculin) targeting FXa [11, 12]. Surprisingly, the only antihemostatic agent characterized thus far at the molecular level is a family of plasminogen activators: Desmodus salivary plasminogen activator (DSPA) α1 (DSPAα1, Desmoteplase), DSPAα2, DSPAβ, and DSPAγ [13–16].…”

Snake Venom Activators of Factor X: An Overview

“…One of the targets to initiate or interfere with blood coagulation appears to be at the level of factor X or factor Xa since many proteins have been identified in snake venoms that are capable of activating factor X or inhibiting factor Xa. Some of the inhibitors show a remarkable specificity and have been found in blood-sucking animals such as vampire bats [3], ticks [4] or leeches [5,6].…”

Desmoteplase: discovery, insights and opportunities for ischaemic stroke

“…Due to the relevant abundance of D. rotundus compared with the other two species, the saliva of this particular species has been studied in greater detail. Over the years, the saliva of D. rotundus has been shown to contain a highly targeted fibrinolytic system enzyme, a potent platelet aggregation inhibitor (Hawkey, 1967) and a novel anti‐coagulant (Apitz‐Castro et al ., 1995; Fernandez et al ., 1999). A vasoactive compound has been detected but not characterized in any detail (Hawkey, 1966).…”