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Exploration of the biological effects of a basic Phospholipase A2 from Agkistrodon piscivorus piscivorus venom
* , , , , *
1  National Natural Toxins Research Center
2  Texas A&M University- Kingsville

Published: 13 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Background: Phospholipases A2 (PLA2s) are found in abundance in many North American snake species. They are responsible for a wide array of pharmacological effects on tissues both locally and systemically. In A.p.piscivorus (A.p.p.), these toxins make up a significant portion of venom constituents. A basic PLA2 was recently isolated through reverse phase HPLC and identified as a D-49 PLA2 (A.p.p PLA2). After testing activities on an in vivo model, the release of pro-inflammatory mediators, systemic myotoxicity, and hemolytic effects were observed. This study aims to explore the hematological, myotoxic and pro-inflammatory activities of this toxin using in vitro models.

Methods: Whole blood was used to test hemolytic activity of A.p.p PLA2 in vitro and, through the SONOCLOT analyzer, the effects on the hemostatic system. Moreover, we tested cell viability, expression of cell activation molecules, and cell damage markers on human umbilical vein endothelial cells (HUVEC) representing the vascular system and a myoblast cell line (C2C12) as a muscle model. Cells were incubated with A.p.p PLA2 over different times. Cell viability was tested using MTT assay, the expression and release of pro-inflammatory and hemostatic mediators were done using flow cytometry and ELISA. Muscle damage was detected evaluating creatine kinase (CK) release.

Discussion/ Conclusion: It was observed that A.p.p PLA2 caused significant hemolytic activity and substantial changes in the coagulation system and mild changes in platelet function in whole human blood. Likewise, this toxin altered cell viability in C2C12 but not in HUVEC. Endothelial cells were also activated when incubated both at 3 h and 24 h. Additionally, C2C12 released IL-6 and CK, which are markers of cell damage. This data can be used for further experimentation to characterize enzymes belonging to this family to produce specialized antivenoms that target PLA2s and their biological activities.

Keywords: Phospholipases A2; Agkistrodon piscivorus piscivorus; myotoxicity; hemostatic system; inflammation; cell activation