Isolation of the Anticoagulant and Procoagulant Fractions of the Venom of Iranian Endemic Echis carinatus.

Abstract

Background: The venom of Echis carinatus contains both procoagulant and anticoagulant components that can either promote or block the blood coagulation cascade, and some of these components affect platelet function in different ways.

Objectives: The present study focuses on setting up a procedure for the purification of crude venom and designing appropriate clotting tests in order to characterize the procoagulant and anticoagulant fractions of E. carinatus venom.

Methods: Chromatographic methods, including gel filtration, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography (HPLC), were applied for purifying these fractions. Coagulant activity testing, prothrombin time (PT), and activated partial thromboplastin time (APTT) were used to determine procoagulant and anticoagulant properties. For measuring molecular weight, 15% SDS-PAGE electrophoresis with a molecular weight standard ranging from 6.5 to 200 kDa was used.

Results: We obtained five fractions named F₁, F₂, F₃, F₄, and F₅. The F₁ and F₂ fractions showed procoagulant activity, and the F₅ fraction had anticoagulant activity. The molecular weight of F_2.4.2 from fraction F₂ and F_5.1 from fraction F₅ were analyzed by SDS-PAGE electrophoresis under the reducing condition. These factors were identified as a single protein band at the end of purification. The molecular weights of these purified fractions were estimated to be 7.5 kDa and 38 kDa for F_5.1(b) and F_2.4.2(b), respectively.

Conclusions: Our findings suggest an efficient and suitable procedure for the identification and purification of the procoagulant and anticoagulant factors of the venom of Iranian E. carinatus using the PT and APTT assays.