Analysis of mouse plasma proteome reveals systemic effects of Bothrops jararaca snake envenomation

INTRODUCTION: Envenoming by viperid snakes is a critical public health problem in Brazil and it is recognized by the World Health Organization as an important Neglected Tropical Disease, causing a high percentage of permanent disability and death. In Brazil, Bothrops snakes are responsible for approximately 20,000 snakebites annually. Although it is well established that systemic effects of Bothrops envenomation comprise coagulopathy, hemorrhage and renal failure, the molecular mechanisms involved in the disturbance of plasma proteins are poorly understood. Proteomic analysis based on mass spectrometry has been used as a powerful tool in order to better understand systemic effects related to snakebite envenomation. OBJECTIVES: i) to characterize changes in plasma protein abundance in animals injected with venom in the thigh muscle, using mass spectrometry; ii) to characterize the impact of bothropic antivenom upon the plasma proteome. METHODS: In vivo experiments were carried out after approval by the animal research ethics committee of Butantan Institute under no. CEUA 9991131219. Swiss mice were injected with B. jararacavenom (1.6 mg/kg) in the gastrocnemius muscle, mimicking a snakebite, and 1 h later with antivenom (1.6 mg/kg; i.v. tail). After 3 h, 6 h, and 24 h, citrated blood samples were collected and centrifuged to obtain plasma, which was further processed to deplete three abundant proteins (albumin, IgG and transferrin) using a multiple affinity removal column. Samples of protein-depleted plasma were submitted to the single-pot, solid-phase-enhanced sample preparation (sp3) protocol followed by trypsin digestion and analysis by LC-MS/MS using a QExactive mass spectrometer, and data-independent acquisition. Data were searched in Spectronaut 18, and MSstats R package was used for statistical analysis. RESULTS: An average of 7,000 peptides and 600 unique proteins were identified. The number of significantly differentially abundant proteins increased over time in response to envenomation. After 6 h of venom injection, an increase of acute phase proteins was detected, including lipocalin 2, apolipoproteins and serpins, whereas fibrinogen, vWF and factor XIII were decreased. After 24 h, several inflammatory markers, such as complement C3, alpha-2-macroglobulin, serum amyloid A1 and A2, serum amyloid P component (Apcs), creatine kinase and lactate dehydrogenase were detected as increased. CONCLUSIONS: Murine envenomation by B. jararaca resulted in changes in the plasma protein profile compatible with inflammation in addition to alterations in hemostasis, and indicated leakage of proteins due to muscle damage, and possibly from kidney tissue. Under the experimental conditions of this study, antivenom injection did not reduce the number of differentially abundant proteins.

Keywords: Bothrops jararaca, snakebite, envenomation, anti-bothropic antivenom, proteomics, mass spectrometry.