Endothelial PAR1 drives generation of neutrophil extracellular traps

Introduction Neutrophils release DNA extracellularly to form neutrophil extracellular traps (NETs) in a process called NETosis. NETs play key roles in homeostasis such as antimicrobial defence, immunothrombosis, and immune regulation, but dysregulated NETosis is linked to diseases like pre-eclampsia, autoimmune disorders, and sepsis. Endothelial cells (ECs) interact directly with neutrophils via selectin and integrin receptors to aid activation and transmigration, while EC-secreted factors also influence NETosis. The lung has a dense EC-lined capillary network and constant environmental exposure making NETs vital for pulmonary pathogen defence. Protease-activated receptor 1 (PAR1) is expressed by immune and endothelial cells and activated by thrombin, mediating signals during thrombosis and haemostasis. Thrombin increases circulating leukocytes in wildtype mice but not in global PAR1 knockout mice. In mesenteric ischemia reperfusion injury, EC-specific PAR1 knockout ( F2r ΔEC ) reduces NETs at injury sites despite unchanged leukocyte adhesion. This led us to explore endothelial PAR1’s role in neutrophil counts and NETosis. Method F2r ΔEC mice were generated using Cre-loxP technology with Cre recombinase under the VE-Cadherin promoter, specific to ECs. Blood was collected for cell count analyses or isolation of plasma. Lung and colon tissues were collected for protein lysates. Blood cell counts were assessed with a haematology analyzer, and bone marrow neutrophils were isolated using fractionated centrifugation with a density gradient.Isolated neutrophils were treated with protein lysates from lung and colon or plasma from F2r ΔEC mice or wildtype controls as well as lipopolysaccharide (LPS) and H₂O₂. NET quantity was measured microscopically with cell-impermeable SYTOX orange. Gene expression was analysed by RT-qPCR. Results F2r ΔEC mice exhibited mild neutropenia and monocytosis. Endothelial PAR1 knockout did not affect neutrophil NETosis capacity when stimulated with LPS or H₂O₂. However, wildtype neutrophils exposed to plasma from F2r ΔEC mice showed strongly reduced NET formation versus plasma from wildtype mice, correlating with decreased expression of NETosis-related enzymes myeloperoxidase (MPO), neutrophil elastase (NE) and peptidylarginine deiminase 4 (PAD4). Lung gene expression of von Willebrand factor (VWF) and granulocyte colony-stimulating factor (G-CSF) was also lower in F2r ΔEC mice. Interestingly, wildtype neutrophils treated with lung protein lysates from F2r ΔEC mice had increased NETosis, in contrast to treatment with colon lysates. Conclusion These results suggest endothelial PAR1 promotes NETosis primarily by influencing circulating factors rather than altering neutrophils’ intrinsic NETotic capacity. Additionally, endothelial PAR1 may facilitate neutrophil mobilization from the bone marrow through regulation of G-CSF expression. Notably, endothelial PAR1 deficiency differentially affects the ability of protein lysates from lung and colon tissues to induce NETosis. Publication History Article published online: 17 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany