Investigation of coagulation activity of obtained fraction from the venom of Macrovipera lebetina

Introduction: Every year, many people are traumatized and severely injured by accidents, and in most cases, failure to control the bleeding resulting from these accidents results in deaths. Some researches focused on the coagulation protein molecules in the venom of animal, including snakes. Vipers venom usually causes coagulation and hemorrhagic disorders, and these compounds provide a rich source of pharmacological proteins for the study of coagulation cascades.Materials & Methods: In this study, the venom of Macrovipera lebetina was preparation and Prothrombin time (PT) test was performed on venom. Chromatography was done by Gel filtration and the fractions were determined by the PT test. Chromatography was performed by reverse phase HPLC to obtain purer fractions and again the fractions were tested for coagulation properties. The molecular weight of the active fraction was calculated by SDS-PAGE. To investigate the fractional toxicity, hemolytic activity assay was performed (in vitro). In the animal study, internal and external bleeding injuries were assessed.Results: Based on the results Six fractions of Gel filtration chromatography were observed. Fraction N.6 had coagulation activity and was selected for further purification by reverse phase HPLC chromatography. A total of 13 peaks were observed in the chromatogram and collected. Fraction N.10 had more coagulation activity than the other fractions. Fraction N.10 caused to coagulation of human plasma in 1 second. The peptide weight of fraction N.10 was estimated to be approximately 6 kDa. The fraction N.10 had no hemolytic activity and it killed 50% of mice at the dose of 5.7μg/μl. In the external injury model, the bleeding time was reduced from 88 seconds to 11 seconds, and in the internal injury model, the bleeding time was reduced from 36 seconds to 17 seconds.Discussion: This study showed that the isolated fraction from the venom of Macrovipera lebetina had coagulation activity in low-dose. Natural compounds, such as peptides extracted from animal toxins, can be used to counter the side effects of chemical drugs. The results of this study indicate that the purified peptide can have therapeutic potential in the future and prevent trauma-related bleeding.

Keywords:

Macrovipera lebetina , Natural coagulants , Snake venom , Prothrombin time