Single vesicle analysis reveals the release of tetraspanin positive extracellular vesicles into circulation with high intensity intermittent exercise.

Small extracellular vesicles (sEVs) are released from all cell types and participate in the intercellular exchange of proteins, lipids, metabolites and nucleic acids. Proteomic, flow cytometry and nanoparticle tracking analyses suggest sEVs are released into circulation with exercise. However, interpretation of these data may be influenced by sources of bias introduced by different analytical approaches. Seven healthy participants carried out a high intensity intermittent (HIIT) cycle protocol consisting of 4×30s at a work-rate corresponding to 200% of individual watt max interspersed by 4.5 minutes of active recovery. EDTA treated blood was collected pre and immediately post the final effort. Platelet poor (PPP) and platelet free plasma (PFP) was derived by one or two centrifugal spins at 2500g respectively (15 min, room temperature). Platelets were counted on an automated haemocytometer. Plasma samples were assessed via the Exoview R100 platform which immobilises sEVs expressing common tetraspanin markers CD9, CD63, CD81 and CD41a on microfluidic chips and with the aid of fluorescence imaging, counts their abundance at a single sEV resolution, importantly, without a pre-isolation step. There was a lower number of platelets in the PFP than PPP, which was associated with a lower number of CD9, CD63 and CD41a+ sEVs. HIIT induced an increase in fluorescent counts in CD9, CD63 and CD81 positive sEVs in both PPP and PFP. These data support the concept that sEVs are released into circulation with exercise. Furthermore, platelet free plasma is the preferred, representative analyte to study sEV dynamics and phenotype during exercise. KEY POINTS: Small extracellular vesicles (sEV) are nano-sized particles containing protein, metabolites, lipid and RNA and can be transferred from cell to cell. Previous findings implicate sEVs are released into circulation with exhaustive, aerobic exercise, but since there is no gold standard method to isolate sEVs, these findings may be subject to bias introduced by different approaches. Here, we use a novel method to immobilise and image sEVs, at a single vesicle resolution, to show sEVs are released into circulation with high intensity, intermittent exercise. Since platelet depletion of plasma results in a reduction in sEVs, platelet free plasma is the preferred analyte to examine sEV dynamics and phenotype in the context of exercise. Abstract figure legend Platelet free plasma was derived from 7 healthy participants pre and post a high intensity, intermittent exercise protocol (HIIT). Samples were directly analysed via a microfluidic chip array, which immobilises small extracellular vesicles (sEV) expressing the tetraspanin protein markers CD9, CD63, CD81 and CD41a and determines sEV count and protein expression via fluorescence intensity on a single sEV basis. HIIT resulted in an increase in the number of CD9, CD63, CD81 and CD41a positive sEV in circulation, with an associated increase in CD9, CD63 and CD81 protein expression. Since platelets are known to release sEV, also analysed were sEV counts in platelet free versus platelet poor plasma. Since there was a significant reduction in CD9, CD63 and CD41a positive sEV associated with a decrease in platelets, platelet free plasma is likely the most representative analyte when examining sEV dynamics during exercise. This article is protected by copyright. All rights reserved.