Platelets and PEVs contain proteasome. (A) Proteasome 20S α subunit, CD41, TOM20, TSG101 and actin in human PEVs (18 000g fraction) and platelet (PLTs) preparations (20 μg protein per lane) were assessed by immunoblot analysis. Results are representative of 5 distinct preparations. (B-E) Protein quantifications were assessed by densitometry with laboratory imaging software (BioRad). Results were normalized to actin and expressed as arbitrary units (AU). Mean ± SEM (n = 5). (D) *P < .05 (paired Student t test). (F) Proteasome function was assessed by measuring trypsin-like, caspase-like, or chymotrypsin-like activity of PEVs and platelets treated or not with epoxomicin using the Proteasome-Glo chymotrypsin-like, trypsin-like, and caspase-like cell-based assays. Twenty and 10 μg of proteins was used for platelets and PEVs, respectively. Mean ± SEM (n = 6). *P < .05, **P < .01, ***P < .001 (Mann-Whitney U test). (G) TEM visualization of immunogold labeling of proteasome 20S α subunit in PEVs released from thrombin (0.5 U/mL)-activated platelets (arrowheads). Data are representative of 3 independent experiments. (H) hs-FCM analysis of resting platelets and thrombin (0.5 U/mL)–activated platelets. Two distinct populations of PEVs, (larger PEVs [∼17% of these PEVs contain active proteasome] and smaller PEVs) that do not contain active proteasome (n = 20). Data are the mean ± SEM. **P < .01; ***P < .001; ****P < .0001 (Kruskal–Wallis). (I-J) Controls were performed to assess the specificity of PEV detection using hs-FCM. Sensitivity of CD41⁺proteasome+ PEVs to competition by epoxomicin, ultracentrifugation (Ultracentri), or 0.05% Triton X-100 and unlabeled samples are presented as the percentage of untreated (Control). Data are the mean ± SEM of 5 independent experiments. ****P < .0001 vs control (paired Student t test). (K) Confocal microscopy visualization of proteasome content associated with platelets (left) and PEVs (right). Visualization of CD41, wheat germ agglutinin (WGA) to determine plasma membrane surface and proteasome (LWA300), and merger of the stains is displayed in the region of interest (ROI). Populations originating from dashed-line squares and represented in ROI are triple positives (arrowheads) or CD41⁺ and WGA⁺ but proteasome− (arrows). SEM, standard error of the mean; TEM, transmission electron microscopy.