Blood coagulation induced by the venom of Bothrops atrox. 1. Identification, purification, and properties of a prothrombin activator

Biochemistry. 1987 Feb 10;26(3):772-80. doi: 10.1021/bi00377a018.

Abstract

In this paper, we show that the procoagulant action of Bothrops atrox venom is due in part to a protein component that activates prothrombin. The venom prothrombin activator was purified by ion-exchange chromatography and gel filtration. It was separated from a protease by affinity chromatography in a p-aminobenzamidine-CH-Sepharose column. It is a protein of about Mr 70,000, consisting of a single polypeptide chain. We have studied the kinetics of activation of prothrombin under different experimental conditions. The prothrombin activator from B. atrox venom is insensitive to reagents of serine and thiol proteases but is inactivated by ion chelators and by various divalent ions. These results suggest that it is a metalloenzyme. The prothrombin activator from B. atrox venom is inactive on the chromogenic substrates S-2337 and S-2238, and it is selective for prothrombin since it does not act on other blood coagulation factors such as fibrinogen and factor X. We have also studied the pattern of peptide cleavages produced in the human prothrombin molecule during the activation by the activator from B. atrox venom and compared it to that obtained with ecarin, a prothrombin activator from Echis carinatus venom. In the presence of thrombin inhibitors, e.g., hirudin, we found that the activators from B. atrox venom and ecarin act in a similar, or identical, manner by producing a thrombin intermediate, meizothrombin. In the absence of thrombin inhibitors, several peptides are generated, and alpha-thrombin is produced as a consequence of meizothrombin action.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blood Coagulation / drug effects*
  • Cattle
  • Chromatography, Affinity
  • Crotalid Venoms / pharmacology*
  • Electrophoresis, Polyacrylamide Gel
  • Endopeptidases / isolation & purification
  • Endopeptidases / pharmacology*
  • Enzyme Activation
  • Kinetics
  • Metalloendopeptidases*
  • Prothrombin / drug effects
  • Prothrombin / isolation & purification
  • Prothrombin / metabolism*

Substances

  • Crotalid Venoms
  • Prothrombin
  • Endopeptidases
  • Metalloendopeptidases
  • prothrombin activator protein, Bothrops atrox venom