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Objectives and questions: Severe Traumatic Brain Injury (TBI) is a leading cause of mortality and long-term disability among adults with Polytrauma (PT) worldwide. Despite advances in critical care, outcomes after TBI remain heterogeneous and difficult to predict. Current diagnostic and prognostic approaches – based on clinical assessment, imaging, and established blood-based biomarkers – have limited sensitivity for detecting TBI-specific signals in the complex polytrauma setting.This pilot study aimed to identify TBI-specific proteomic signatures in a heterogeneous cohort of severely injured polytrauma patients by analyzing the total plasma proteome and an extracellular vesicle (EV)-enriched plasma protein fraction.

Material and methods: 83 severely injured adult trauma patients (Injury Severity Score [ISS] ≥16) were included: isolated severe TBI (TBI; AIShead ≥4 and all other AIS ≤1, n =24), polytrauma with TBI (PT-TBI; AIShead ≥4 + other AIS ≥3, n =29), and polytrauma without TBI (PT; AIShead =0, n =30). 24 age- and sex-matched healthy volunteers served as controls. Proteomic profiling of plasma and plasma-derived EVs was performed using HPLC-MS/MS. Differentially expressed proteins (DEPs) underwent pathway and functional enrichment analyses. Associations with clinical parameters were assessed by Spearman’s rank correlation.

Results: The EV-enriched plasma fraction revealed a substantially higher number of DEPs compared with whole plasma (846 vs. 258), underscoring the increased sensitivity of EV-based proteomics. Across both proteomes, most dysregulated proteins were associated with PT, whereas fewer were linked to unspecified trauma, and only a subset was specific to isolated TBI or PT-TBI. In PT, upregulated plasma proteins were predominantly involved in glycolysis and carbon metabolism, whereas EV-associated proteins were selectively enriched in pathways related to cellular translation and protein catabolic processes, indicating distinct biological signatures captured by EVs. TBI-associated DEPs identified in plasma were mainly related to plasminogen activation, complement, and platelet activation, while EV-derived TBI-associated proteins preferentially mapped to anticoagulation and negative regulation of fibrinolysis. Several TBI-associated proteins correlated with trauma severity, with only AQR showing a moderate negative correlation with Glasgow Come Scale ([GCS], r = -0.472, p <0.0001). Notably, among identified candidate TBI biomarkers, only three proteins – all derived from EV-associated fraction – were CNS-specific.

Discussion and conclusions: EV-enriched plasma proteomics markedly increases analytical sensitivity compared with whole plasma analysis, uncovering distinct molecular pathways not captured by conventional analyses. Although most proteomic alterations reflect systemic effects of polytrauma, EVs reveal TBI-associated proteins that correlate with injury severity and neurological status (GCS), supporting their value as a complementary platform for TBI biomarker discovery and patient stratification.