Thrombin concentration shapes endothelial extracellular vesicle profiles with divergent inflammatory functions

ABSTRACT: Thrombin, a central enzyme in the coagulation cascade, also regulates diverse cellular processes, including inflammation and vascular barrier function, primarily by activating protease-activated receptor 1. Previous studies demonstrated that thrombin elicits concentration-dependent, opposing effects; low concentrations confer anti-inflammatory and barrier-protective responses, whereas high concentrations promote inflammation and barrier disruption. The underlying mechanisms, however, remain incompletely understood. Here, we showed that thrombin stimulates extracellular vesicle (EV) release from endothelial cells across a broad concentration range and that EVs generated at low vs high thrombin concentrations carry distinct microRNA (miR) cargo. Low-thrombin EVs mediate cytoprotective responses via the transfer of miR-409-5p, which targets ubiquitin-specific protease 7 that promotes inflammation via the NF-kB signaling pathway in recipient cells, whereas high-thrombin EVs disrupt barrier integrity and promote inflammation through delivery of miR-155-5p, a regulator of suppressor of cytokine signaling 1 that acts as a crucial negative regulator of the cytokine signaling pathway. Functional manipulation of these EVs confirmed the causal roles. Incorporation of anti-miR-409-5p abrogated the protective effects of low-thrombin EVs, whereas anti-miR-155-5p suppressed the cytopathic effects of high-thrombin EVs. Moreover, control EVs engineered to carry a miR-409-5p mimic reproduced the anti-inflammatory and barrier-protective phenotype of low-thrombin EVs. Collectively, these findings identified EV-associated miRs as key mediators of the concentration-dependent dual actions of thrombin, which may open the therapeutic potential of EVs engineered to deliver selective miRs or anti-miRs for the treatment of inflammatory vascular diseases.