# Bioinformatics-Guided Identification and Quantification of Biomarkers of Crotalus atrox Envenoming and Its Neutralization by Antivenom

## TL;DR

This study identifies biomarkers in mice plasma extracellular vesicles that change after snake venom exposure and antivenom treatment, which could improve snakebite diagnosis and treatment.

## Contribution

The study introduces new biomarkers from extracellular vesicles that reflect physiological changes due to snake venom and antivenom effects.

## Key findings

- Extracellular vesicle proteins like Slc25a4, Rps8, and others were significantly altered by venom and antivenom.
- Biomarkers indicate changes in mitochondrial function, lipid metabolism, immunity, and cytolysis.
- These findings could enhance diagnostic tools for snakebite envenoming.

## Abstract

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 min, the mice received saline or antivenom (Antivipmyn) by intravenous injection. After 24 h, 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 toward 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.

•Mass spectrometry-based proteomics of mice plasma extracellular vesicles.•Murine plasma exosomes contain proteins sensitive to envenoming and antivenom.•Protein abundance changes in plasma extracellular vesicles post venom exposure.•Snake venom causes changes in lipid metabolism, hemostasis, and immune response.•Potential diagnostic and prognostic protein biomarkers of snakebite envenoming.

Mass spectrometry-based proteomics of mice plasma extracellular vesicles.

Murine plasma exosomes contain proteins sensitive to envenoming and antivenom.

Protein abundance changes in plasma extracellular vesicles post venom exposure.

Snake venom causes changes in lipid metabolism, hemostasis, and immune response.

Potential diagnostic and prognostic protein biomarkers of snakebite envenoming.

This study focus on mice plasma extracellular vesicles (EV) released after mice exposure to snake venom. We exposed mice to Crotalus atrox whole venom and antivenom intravenously and then checked the plasma EV response through their protein cargo. Here is an overview of murine plasma EV proteome with significant changes in protein abundance for potential biomarkers associated with venom and antivenom exposure. These have the potential to enhance current diagnostic tools for snakebite management, thereby contributing to the diagnosis and prognosis of snakebite envenoming.