Blood coagulation induced by the venom of Bothrops atrox. 2. Identification, purification, and properties of two factor X activators

Abstract

We have characterized and purified the two components of the venom of Bothrops atrox that activate the coagulation factor X. Activator 1 and activator 2 were separated by ion-exchange chromatography but otherwise presented similar characteristics. They consist of a heavy polypeptide of Mr 59,000 and either one or two light chains forming a doublet of Mr 14,000-15,000. They are inactive on synthetic substrates and on prothrombin or fibrinogen and thus appear to act specifically on factor X. They are not sensitive to inhibitors of serine proteases or thiol esterases. The activation of factor X is activated by Ca2+ ions with a Hill coefficient of 2.4 and is inhibited by Hg2+, Ba2+, and Cd2+. Its pH dependency suggests that the activity depends on the ionization of a group with an apparent pK of 6.9. We studied the cleavage of purified bovine factor X by B. atrox activators and compared it to that obtained with the factor X activator from Vipera russelli venom. Like the physiological activators, the venom's activators cleave the heavy chain of factor X, producing the activated factor Xa alpha. They produce however two other cleavages: one near the N-terminal end of the heavy chain of factor X, generating factor Xmu, and a second one located at one extremity of the heavy chain of factor Xa alpha, generating factor Xav.

Moojenactivase, a novel pro-coagulant PIIId metalloprotease isolated from Bothrops moojeni snake venom, activates coagulation factors II and X and induces tissue factor up-regulation in leukocytes

“…As for factor X, we observed that MooA induced the cleavage of the zymogen heavy chain at Arg 194 -Ile 195 , which led to the formation of enzymatically active factor Xa. A similar pattern of proteolytic activation has also been observed for endogenous factor VII/Tissue factor complex and other SVMPs (Hofmann and Bon 1987b;Takeya et al 1992;Siigur et al 2001;Samel et al 2003;Sun et al 2010).…”

Snake Venom Activators of Factor X: An Overview

“…With only a few exceptions, efficient activation of factor X critically depends upon the presence of CaCl 2 . Dose-response curves for CaCl 2 typically show a sigmoidal dependency with Hill coefficients ranging from 2.4 to 7.9 [11][12][13]. Since the mechanism of factor X activation by the activator present in Russell's viper venom (RVV-X) is quite well understood and can be regarded as an example for the other venom activators belonging to the group of metalloproteases, we will describe this activator in more detail.…”

“…Although in the original publications concerning the activators purified from B. atrox and from Cerastes cerastes venom, it was proposed that the purified activators showed heterogeneity because two light chains were observed on SDS-PAGE [11,32], it is very likely that these activators, like RVV-X, consist of a heavy chain containing the metalloprotease domain and two Ctype lectin light chains held together by disulfide bond(s). Hoffmann and Bon [11] isolated two factor X activators from B. atrox venom, but apart from differences in the purification both activators show similar functional characteristics. In addition, it was noted that these activators cleave the heavy chain of factor X at two additional positions, an activity not seen with RVV-X.…”

Proteases from <i>Vipera lebetina </i>Venom Affecting Coagulation and Fibrinolysis

“…The activation effect of factor X by VLFXA was measured by the amidolytic activity of the factor Xa that was formed according to the method described by Hofmann and Bon [29]. Bovine or human factor X was used as substrate for VLFXA.…”