Deparment of Biology, Central Michigan University, 1200 S Franklin St., Mt. Pleasant, MI 48859, United States.
Biological Sciences Department, California Polytechnic State University, San Luis Obispo, 1 Grand Avenue, San Luis Obispo, CA 93407, United States.
J Leukoc Biol. 2024 Nov 27;116(6):1223-1236. doi: 10.1093/jleuko/qiae125.
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.
中性粒细胞释放细胞外囊泡，其中一些中性粒细胞衍生的细胞外囊泡具有促凝作用。在金黄色葡萄球菌的刺激下，中性粒细胞产生的细胞外囊泡通过静电作用与中性粒细胞细胞外陷阱结合。中性粒细胞细胞外陷阱中的 DNA 具有促凝作用，但在细菌刺激期间产生的中性粒细胞细胞外囊泡是否具有类似的活性尚不清楚。鉴于细胞外囊泡的活性取决于激动剂和细胞类型，并且凝血参与了败血症，我们假设引起败血症的细菌会增加中性粒细胞衍生的细胞外囊泡以及与细胞外囊泡相关的 DNA 的产生，完整的细胞外囊泡和 DNA 会导致凝血。我们从金黄色葡萄球菌、表皮葡萄球菌、大肠杆菌和铜绿假单胞菌刺激的中性粒细胞中回收细胞外囊泡，并测量相关的 DNA 和促凝活性。以前已被表征的金黄色葡萄球菌刺激的中性粒细胞来源的细胞外囊泡，在血小板贫血浆中测量的凝血酶生成中显示出剂量依赖性的促凝活性。细胞外囊泡裂解和 DNA 酶处理分别使凝血酶生成减少了 90%和 37%。表皮葡萄球菌、大肠杆菌和铜绿假单胞菌也增加了细胞外囊泡的产生和与细胞外囊泡相关的细胞外 DNA，并且这些细胞外囊泡也是促凝的。与在激活剂存在下具有一些能力来放大因子 XII 依赖性凝血的自发释放的细胞外囊泡相比，只有响应细菌产生的细胞外囊泡才能启动该途径。金黄色葡萄球菌和表皮葡萄球菌的细胞外囊泡比大肠杆菌和铜绿假单胞菌的细胞外囊泡具有更多的表面相关 DNA，并且金黄色葡萄球菌和表皮葡萄球菌的细胞外囊泡通过 DNA 依赖性方式有助于凝血酶生成的起始和放大。然而，大肠杆菌或铜绿假单胞菌细胞外囊泡上的 DNA 不起作用，这表明中性粒细胞释放促凝细胞外囊泡，这些细胞外囊泡可以通过 DNA 依赖和非依赖机制激活凝血级联。
