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.