Changes in the pattern of plasma extracellular vesicles after severe trauma

Background Extracellular vesicles (EV) released into the circulation after traumatic injury may influence complications. We thus evaluated the numbers of EV in plasma over 28 days after trauma and evaluated their pro-coagulant and inflammatory effects. Methods and findings 37 patients suffering trauma with an injury severity score >15 were studied along with 24 healthy controls. Plasma samples were isolated by double centrifugation (2000g 20min; 13000g 2min) from blood collected from within an hour up to 28 days after injury. Plasma EV were counted and sized using nanoparticle tracking analysis (NTA); counts and cellular origins were also determined by flow cytometry (FC) using cell-specific markers. Functional effects were tested in a procoagulant phospholipid assay and in flow-based, leukocyte adhesion assay after endothelial cells (EC) were treated with EV. We found that EV concentrations measured by NTA were significantly increased in trauma patients compared to healthy controls, and remained elevated over days. In addition, or FC showed that patients with trauma had higher numbers of EV derived from platelets (CD41+), leukocytes (CD45+) and endothelial EC (CD144+). The increases were evident throughout the 28-day follow-up. However, the FC count represented <1% of the count detected by NTA, and only 1–2% of EV identified using NTA had a diameter >400nm. The procoagulant phospholipid activity assay showed that patient plasma accelerated coagulation on day 1 and day 3 after trauma, with coagulation times correlated with EV counts. Furthermore, treatment of EC for 24 hours with plasma containing EV tended to increase the recruitment of peripheral flowing blood mononuclear cells. Conclusions EV counted by FC represent a small sub-population of the total load detected by NTA. Both methods however indicate a significant increase in plasma EV after severe traumatic injury that have pro-coagulant and pro-inflammatory effects that may influence outcomes.## 研究背景 创伤后释放进入循环系统的细胞外囊泡（Extracellular vesicles, EV）可能会影响并发症的发生。本研究旨在评估创伤后28天内血浆中细胞外囊泡的数量，并探究其促凝血与促炎效应。 ## 研究方法与结果 本研究共纳入37名损伤严重度评分>15的创伤患者与24名健康对照者。血浆样本采用双重离心法（2000g离心20分钟；13000g离心2分钟）分离，采血时间覆盖创伤后1小时至28天。采用纳米颗粒追踪分析（Nanoparticle Tracking Analysis, NTA）对血浆中的细胞外囊泡进行计数与粒径表征；同时借助流式细胞术（Flow Cytometry, FC）结合细胞特异性标志物，确定细胞外囊泡的计数结果及其细胞来源。功能效应检测通过两项实验完成：其一为促凝血磷脂活性实验，其二为经细胞外囊泡处理后的内皮细胞（Endothelial Cells, EC）流动式白细胞黏附实验。 研究结果显示：与健康对照者相比，创伤患者经纳米颗粒追踪分析检测得到的细胞外囊泡浓度显著升高，且该升高状态可持续多日。流式细胞术检测结果表明，创伤患者体内源自血小板（CD41+）、白细胞（CD45+）及内皮细胞（CD144+）的细胞外囊泡数量均显著增加，且该升高趋势在28天的随访周期内持续存在。不过，流式细胞术检测到的细胞外囊泡数量仅占纳米颗粒追踪分析检测总量的<1%，且通过纳米颗粒追踪分析鉴定的细胞外囊泡中，仅1%~2%的粒径大于400nm。促凝血磷脂活性实验显示，创伤后第1天与第3天，患者血浆可加速凝血进程，凝血时间与细胞外囊泡计数呈显著相关性。进一步实验发现，用含细胞外囊泡的血浆处理内皮细胞24小时后，外周循环血单核细胞的招募水平呈现升高趋势。 ## 研究结论 流式细胞术检测到的细胞外囊泡仅占纳米颗粒追踪分析检测总细胞外囊泡负荷的极小一部分。但两种检测方法均证实，严重创伤后患者血浆中的细胞外囊泡数量显著升高，且这类细胞外囊泡兼具促凝血与促炎效应，可能对创伤患者的临床预后产生影响。