Purification and Characterization of Multisquamase, the Prothrombin Activator Present in Echis Multisquamatus Venom

• Published in: Thrombosis research 1997-11, Vol.88 (3), p.309-316
• Main Authors: Petrovan, Ramona J, Govers-Riemslag, José W.P, Nowak, Götz, Hemker, H.Coenraad, Rosing, Jan, Tans, Guido
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Blood coagulation; carinatus; Echis; Echis multisquamatus; Enzymes - isolation & purification; Enzymes - metabolism; Investigative techniques, diagnostic techniques (general aspects); Medical sciences; Metalloendopeptidases - isolation & purification; Metalloendopeptidases - metabolism; Prothrombin - metabolism; Prothrombin activation; Snake venom; Thrombin - metabolism; Viper Venoms - isolation & purification; Viper Venoms - metabolism
• ISSN: 0049-3848; 1879-2472
• DOI: 10.1016/S0049-3848(97)00258-2

The venom of Echis multisquamatus (Central Asian sand viper) contains a single prothrombin activator, designated multisquamase, which is structurally and functionally different from ecarin, the prothrombin activator from the venom of Echis carinatus (saw-scaled viper). Multisquamase is comprised of a 58000 Mr and a 23000 Mr subunit that consists of two disulfide-linked chains of 12000 Mr and 10000 Mr, respectively. In contrast to ecarin, which activates prothrombin and prethrombin 1 at comparable rates, and whose activity is hardly affected by Ca 2+ or by changes in ionic strength, multisquamase hardly activates prethrombin 1; prothrombin activation requires Ca 2+ and is strongly inhibited at high ionic strength. The most favourable kinetic parameters are observed at 1 mM Ca 2+ and at low ionic strength (K m = 0.085 μM and k cat = 0.68 s −1 at I ≅ 0.04). An increase in ionic strength considerably reduces the rate of prothrombin activation, due to an increase of the K m (K m = 0.8 μM and k cat = 1.03 s −1 at I ≅ 0.2). Studies in plasmas from patients on oral anticoagulant therapy show that E. Multisquamatus venom only activates carboxylated prothrombin, whereas E. carinatus activates both prothrombin and descarboxyprothrombin. Thus, multisquamase-dependent prothrombin activation appears to require post-translational modification of the gla-domain. This venom prothrombin activator may, therefore, become a useful tool to quantitate prothrombin and descarboxyprothrombin in cases where vitamin K-dependent carboxylation of prothrombin is impaired.