Changes in the pattern of plasma extracellular vesicles after severe trauma.

Sahithi J Kuravi,Antonio Belli,Gerard B Nash,Clara M Yates,Jon Hazeldine,Paul Harrison,Mark Midwinter,Mark Foster,Chris Watson,Peter Hampson

doi:10.1371/journal.pone.0183640

Abstract

BackgroundExtracellular 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 findings37 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.ConclusionsEV 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.

Highlights

Systemic complications following severe traumatic injury can lead to late morbidity and mortality [1, 2]
We found that Extracellular vesicles (EV) concentrations measured by nanoparticle tracking analysis (NTA) were significantly increased in trauma patients compared to healthy controls, and remained elevated over days
EV counted by flow cytometry (FC) represent a small sub-population of the total load detected by NTA

Summary

Introduction

Systemic complications following severe traumatic injury can lead to late morbidity and mortality [1, 2]. Along with increased release of soluble mediators such as cytokines, elevated numbers of extracellular vesicles have been reported in circulating blood after severe trauma [6, 7]. Whether the levels or bioactivity of these vesicles influence the inflammatory complications of traumatic injury remains uncertain. EV contain membrane proteins, mRNA, miRNA, cytokines and chemokines [9,10]. They may be vectors of intercellular exchange with significant roles in coagulation, inflammation, immune responses, angiogenesis and cancer [11,12,13]. Extracellular vesicles (EV) released into the circulation after traumatic injury may influence complications. We evaluated the numbers of EV in plasma over 28 days after trauma and evaluated their pro-coagulant and inflammatory effects.

Methods

Results

Conclusion