Pathway-level proteomics identifies subcellular trafficking dysregulation as a molecular signature in severe traumatic brain injury

BACKGROUND: Traumatic brain injury (TBI) remains a leading cause of death and disability worldwide in intensive care units, yet no targeted neuroprotective therapies exist despite over 300 clinical trials. This therapeutic failure stems partly from biological heterogeneity and the limitations of single-biomarker approaches that cannot capture the multifaceted pathophysiology of secondary brain injury. Systems biology approaches examining dysregulated pathways rather than isolated proteins may reveal candidate endotypes suitable for patient stratification and identify potential therapeutic targets. METHODS: We conducted an exploratory, case-control study, in adult critically-ill patients comparing 10 severe TBI (sTBI) patients (median GCS 5.5) with 10 age-and sex-matched healthy controls. Plasma samples were collected upon admission to the ICU, within 24 h of injury. High-throughput proximity extension assays quantified 1196 plasma proteins, identifying multiple differentially expressed proteins. Gene set enrichment analysis interrogated Reactome pathways and Gene Ontology terms. Protein-protein interaction networks were constructed using the STRING database, and associations between enriched pathways and clinical variables were examined. RESULTS: We identified 348 significantly differentially abundant proteins between groups. Gene set enrichment analysis revealed 19 enriched Reactome pathways and 12 Gene Ontology terms, predominantly reflecting immune activation, inflammation, cellular stress responses, and intracellular trafficking. Strikingly, membrane trafficking pathways, including clathrin-mediated endocytosis and Golgi transport, emerged as significantly enriched, representing an underrecognized mechanism in sTBI pathophysiology. Protein-protein interaction analysis identified four functional clusters, with IL6 and IL10 as densely connected hub proteins coordinating inflammatory responses. Membrane trafficking pathways were associated with clinical outcomes including both length of hospital and intensive care unit stay, as well as need for neurosurgical intervention, in sTBI patients. CONCLUSIONS: By profiling the expression of 1196 plasma proteins in parallel, this study provides one of the most comprehensive proteomic characterizations of critically-ill adults with severe traumatic brain injury to date. Systems biology analyses suggest that sTBI may trigger coordinated pathway-level dysregulation extending beyond inflammation to include subcellular trafficking machinery. The association between membrane trafficking pathways and clinical outcomes suggests these may represent a candidate molecular endotype with prognostic relevance. These hypothesis-generating findings support pathway-based approaches for patient stratification and therapeutic targeting in sTBI, though validation in larger cohorts is required.