A novel in vitro model of trauma-induced endotheliopathy provides a platform for studying mechanisms of coagulopathy

Trauma-induced coagulopathy (TIC) significantly contributes to trauma-related mortality, driven by dysregulated coagulation and fibrinolysis. Endotheliopathy of trauma (EoT) is central to TIC, yet its underlying mechanisms remain unclear. Current in vitro models fail to replicate the complex trauma environment, including haemorrhagic shock, tissue injury, and inflammation. This study aimed to develop a novel in vitro model of EoT that mimics key TIC features, enabling the investigation of endothelial contributions to TIC. Endothelial colony-forming cells (ECFCs) were exposed to trauma-relevant factors, including epinephrine, TNF-α, IL-6, HMGB1, hydrogen peroxide, and hypoxia. Endothelial injury markers (syndecan-1, thrombomodulin), haemostatic protein expression, coagulation, and fibrinolysis were analysed using ELISA, immunofluorescence, global haemostasis assays, and RNA sequencing. Plasma from healthy donors and trauma patients was used to assess clinical relevance. Traumatised ECFCs exhibited progressive dysfunction, with early surface damage and sustained fibrinolytic dysregulation. Transcriptomic analysis showed activation of inflammatory pathways, metabolic shifts, and epigenetic changes. Surface expression of anticoagulant proteins decreased, while procoagulant tissue factor increased, heightening thrombogenic potential. Initially, traumatised ECFCs promoted fibrinolysis via thrombomodulin shedding but later secreted antifibrinolytic PAI-1, mimicking the biphasic TIC phenotype. Plasma assays revealed thrombin generation and clot lysis changes similar to trauma patients. This in vitro model successfully replicates EoT and TIC-associated haemostatic imbalances, capturing the time-dependent evolution of endothelial dysfunction. It provides mechanistic insights into TIC and serves as a platform for testing targeted interventions to mitigate endothelial-driven coagulopathy in trauma. RNAseq profiling of endothelial colony-forming cells (ECFCs) from 6 healthy donors treated with a cocktail simulating systemic traumatic injury conditions to induce endotheliopathy.