Flowchart of methods used for EV isolation and obtained results. From top left, EVs were isolated from 3.2% citrate plasma by differential ultracentrifugation technique. (A) EVs were analyzed using nanoparticle tracking analysis with Nanosight LM10 (Malvern Instruments Ltd). In the example shown, particle size was between 100 and 200 nm, consistent with the expected size of EVs. (B) Three sets of pooled samples with controls (n = 5) were analyzed in 3 proteomic experiments by TMT and MS/MS analysis. Additionally, 4 and 2 individual samples from the first and second sample pools, respectively, were also analyzed separately in the TMT experiments. (C) Gene Ontology analysis using Fun Rich software v3 on the 3 data sets regarding their cellular components. The majority of the identified proteins were components of EVs: 81.78%, 82.49%, and 80.29% from the first, second, and third data set, respectively. The total number of EV proteins identified was 645 EV proteins with 212 (32.9%) EV proteins common across the 3 separate experiments.