Quantitative mass spectrometry-based proteomics of extracellular vesicles (EVs) provides systems-level exploration for the analysis of snakebite envenoming (SBE) as the venom progresses, causing injuries such as hemorrhage, trauma, and death. Predicting EV biomarkers has become an essential aspect of this process, offering an avenue to explore the specific pathophysiological changes that occur after envenoming. As new omics approaches emerge to advance our understanding of SBE, further bioinformatics analyses are warranted to incorporate the use of antivenom or other therapeutics to observe their global impact on various biological processes. Herein, we used an in vivo BALB/c mouse model and proteomics approach to analyze the physiological impacts of SBE and antivenom neutralization in intact animals; this was followed by bioinformatics methods to predict potential EV biomarkers. Groups of mice (n=5) were intramuscularly injected with Saline or Crotalus atrox venom. After 30 minutes, the mice received saline or antivenom (ANTIVIPMYN®) by intravenous injection. After 24 hours, blood was collected to extract the plasma to analyze the EV content and determine the exposome of C. atrox venom as well as the neutralizing capabilities of the antivenom. The predicted biomarkers consistently and significantly sensitive to antivenom treatment are Slc25a4, Rps8, Akr1c6, Naa10, Sult1d1, Hadha, Mbl2, Zc3hav, Tgfb1, Prxl2a, Coro1c, Tnni1, Ryr3, C8b, Mycbp, and Cfhr4. These biomarkers pointed towards specific physiological alterations, causing significant metabolic changes in mitochondrial homeostasis, lipid metabolism, immunity, and cytolysis, indicating hallmarks of traumatic injury. Here, we present a more comprehensive view of murine plasma EV proteome and further identify significant changes in abundance for potential biomarkers associated with antivenom treatment. The predicted biomarkers have the potential to enhance current diagnostic tools for snakebite management, thereby contributing significantly to the evolution of treatment strategies in the diagnosis and prognosis of SBE.

Keywords: Antivenom; Extracellular Vesicles; Proteomics; Snakebite; Systems Biology.

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