In response to bacteria, neutrophils release extracellular vesicles capable of initiating thrombin generation through DNA-dependent and independent pathways

Abstract

Abstract Neutrophils release extracellular vesicles, and some subsets of neutrophil-derived extracellular vesicles are procoagulant. In response to Staphylococcus aureus, neutrophils produce extracellular vesicles that associate electrostatically with neutrophil extracellular traps. DNA in neutrophil extracellular traps is procoagulant, but whether neutrophil extracellular vesicles produced during bacterial challenge have similar activity is unknown. Given that extracellular vesicle activity is agonist and cell-type dependent and coagulation contributes to sepsis, we hypothesized that sepsis-causing bacteria increase production of neutrophil-derived extracellular vesicles, as well as extracellular vesicle–associated DNA, and intact extracellular vesicles and DNA cause coagulation. We recovered extracellular vesicles from neutrophils challenged with S. aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa and measured associated DNA and procoagulant activity. Extracellular vesicles from S. aureus–challenged neutrophils, which were previously characterized, displayed dose-dependent procoagulant activity as measured by thrombin generation in platelet-poor plasma. Extracellular vesicle lysis and DNase treatment reduced thrombin generation by 90% and 37%, respectively. S. epidermidis, E. coli, and P. aeruginosa also increased extracellular vesicle production and extracellular vesicle–associated extracellular DNA, and these extracellular vesicles were also procoagulant. Compared to spontaneously released extracellular vesicles, which demonstrated some ability to amplify factor XII–dependent coagulation in the presence of an activator, only extracellular vesicles produced in response to bacteria could initiate the pathway. S. aureus and S. epidermidis extracellular vesicles had more surface-associated DNA than E. coli and P. aeruginosa extracellular vesicles, and S. aureus and S. epidermidis extracellular vesicles contributed to initiation and amplification of thrombin generation in a DNA-dependent manner. However, DNA on E. coli or P. aeruginosa extracellular vesicles played no role, suggesting that neutrophils release procoagulant extracellular vesicles, which can activate the coagulation cascade through both DNA-dependent and independent mechanisms.