Caroline Coelho Ferreira¹, Lisele Maria Brasileiro Martins^2,3, Sofia Angiole Cavalcante¹, Thaís Pinto Nascimento³, Marco Aurelio Sartim^3,4, Paulo Costa Carvalho⁵, Juliana de Saldanha da Gama Fischer Carvalho⁵, Marlon Mariano Dias dos Santos⁵, Amanda Camillo Andrade⁵, Jacqueline de Almeida Gonçalves Sachett^2,3, Wuelton Marcelo Monteiro^2,3, Priscila Ferreira de Aquino¹

Introduction: The Amazon region of Brazil reports one of the highest rates of snakebite envenoming, with Bothrops atrox responsible for the majority of cases. Despite ongoing research on its clinical manifestations, additional proteomic studies can help clarify how systemic responses unfold over time. Objective: Characterize the protein profile of B. atrox envenoming in victims’ urine identifying time-dependent dynamics post-envenoming. Methodology: Urine samples were collected from three groups: early-treated patients (1–4 h, n = 5), late-treated patients (8–13 h, n = 5), and healthy controls (n = 5). Protein extraction was made with methanol precipitation followed by Urea/Thiourea, reduction with dithiothreitol (DTT), alkylation with iodoacetamide (IAA), incubation with DTT and then digestion with trypsin. The peptide mixture was desalted, concentrated, resuspended in acid formic, and analyzed using an Ultimate 3000 Basic online with a Fusion Lumos Orbitrap mass spectrometer set in data-dependent acquisition mode (DDA). Data analysis was performed using the software Patternlab for Proteomics V (PLV) applying a peptide spectrum match for identification, and XIC for protein relative quantification. The biological processes associated with the proteins found were investigated using UNIPROT, Human Protein Atlas databases, Gene Ontology (GO) and Panther. Results: A total of 13,721 peptides and 1,680 proteins were identified, with 1,030 quantified using at least two unique peptides. Among them, 73 were exclusive in the early-treated and 83 in the late-treated group. In the early-treated group, exclusive proteins were primarily related to immediate responses such as cellular stress, tissue injury, and immune recognition. In the late-treated group, proteins were associated with recovery mechanisms, such as Angiopoietin-related protein 3 (ANGPTL3), reflecting metabolic adjustments. However, in the early-treated group, differentially abundant proteins included components of the complement system (C3 and factor D), inflammatory signaling (phosphatidylethanolamine-binding protein 4 - PEBP4), and innate immune recognition (N-acetylmuramoyl-L-alanine amidase - NAMLA), indicating early activation of immune pathways. STRING analysis revealed a coordinated innate immune response through functionally coupled recruitment mechanisms. In the late-treated group, the proteomic profile was marked by proteins involved in coagulation regulation, plasma transport, adaptive immunity, and initiation of tissue repair. Proteins such as Retinol binding protein 4 (RBP4), Thyroid hormone-binding protein (TTR), albumin, serotransferrin, and Beta-2-microglobulin (B2M) were more abundant, potentially indicating increased vascular permeability, tubular dysfunction, or systemic protein overflow during recovery. Together, these findings illustrate a possible protein signature between the two phases post-envenoming, reflecting a transition from an acute immune-inflammatory state to a recovery-oriented response. Conclusion: This study provides an overview of the systemic effects of B. atrox envenoming based on urinary protein signatures. The identification of distinct protein patterns contributes to a broader understanding of the pathophysiological processes triggered by envenomation and can support future efforts in biomarker discovery.

Agradecimentos: CAPES – Finance Code 001; FAPEAM (Kunhã Program – Edital Nº 008/2022); POSGRAD Program.