Activated platelets release extracellular mitochondria. (A) Platelet-free supernatants resulting from the isolation of thrombin-activated platelets consume O₂ via the electron transport chain following cell permeabilization with saponin detergent (50 μg/mL). No O₂ consumption is detected in supernatants obtained from resting platelets (n = 4; data are mean ± SEM). (B) Three predicted types of extracellular microparticles (MPs) produced on platelet activation: mitochondria (freeMitos), mitochondria-containing MPs (mitoMPs), and MPs lacking mitochondria (MPs). (C) Isolation of freeMitos using anti-TOM22 microbeads (or IgG control) in thrombin-stimulated platelets and mtDNA quantification (n = 4; data are mean ± SEM, **P < .005, Student t test). (D) TEM visualization of freeMitos (white arrows), mitoMPs (black arrows), and MPs (black arrowheads) released from thrombin-activated platelets. (E) Three-dimensional CSLM reconstruction of the supernatant of thrombin-activated platelets. Populations represented in image are platelets (black arrow), MPs (white arrows), mitoMPs (white arrowheads), and freeMitos (black arrowheads). (F) High-sensitivity flow cytometry (hs-FCM) analysis of resting platelets (upper panel, top right quadrant) and thrombin-activated platelets, which show 3 additional, distinct populations of particles, ie, freeMitos (bottom panel, top left quadrant, blue), mitoMPs (bottom panel, top right quadrant, pink), and mitochondria-free MPs (bottom panel, bottom right quadrant, red). Bottom left quadrant of both upper and lower panels represents background noise (gray). FSC-PMT and SSC dot plots of platelets (first right panel) and 3 populations of microparticles: freeMitos (second right panel), mitoMPs (third right panel), and MPs (fourth right panel). The relative diameters are presented according to size-defined microsphere calibrations. (G) Release of (left) freeMito, (center) mitoMPs, and (right) MPs from thrombin-activated platelets require intact actin microfilament dynamics. Mitochondrial release is significantly reduced on addition of actin inhibitors (cytochalasin [B,D-E] and latrunculin [A]), but not tubulin polymerization inhibitor (nocodazole) (n = 4; data are mean ± SEM, *P < .05, **P < .005, and ***P < .001, Student t test). (H) Heat-aggregated IgG (HA-IgG), thrombin, collagen, cross-linked collagen related peptide (CRP-XL), and phorbol 12-myristate 13-acetate (PMA) trigger the release of (left) extracellular freeMitos, (center) mitoMPs, and (right) MPs quantified by hs-FCM (n = 4; data are mean ± SEM. *P < .05, **P < .005, and ***P < .001 vs supernatant from resting platelets, Student t test).