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 hemorrhagic 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, tumor necrosis factor α, interleukin 6, high mobility group box 1, hydrogen peroxide, and hypoxia. Endothelial injury markers (syndecan-1 and thrombomodulin), hemostatic protein expression, coagulation, and fibrinolysis were analyzed using enzyme-linked immunosorbent assay, immunofluorescence, global hemostasis assays, and RNA sequencing. Plasma from healthy donors and trauma patients was used to assess clinical relevance. Traumatized 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, whereas procoagulant tissue factor increased, heightening thrombogenic potential. Initially, traumatized ECFCs promoted fibrinolysis via thrombomodulin shedding but later secreted antifibrinolytic plasminogen activator inhibitor 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 hemostatic 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.