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.