![Figure 3. Extracellular mitochondria are present in various situations where platelets are known to be activated. (A) Platelet mitoMPs (CD41+MitoTracker+) are found in higher concentrations in the synovial fluid of RA patients (n = 20) than in the synovial fluid of osteoarthritis patients (OA, n = 14; data are mean ± SEM, *P < .05, Mann-Whitney test). (B) FreeMitos are detected in fresh SF of RA patients. Isolation of freeMitos in RA SF (from 3 different patients) with anti-TOM22 microbeads (or control IgG; supplemental Figure 3) and mtDNA quantification. (C) TEM imaging of (left) a freeMito and (right) a mitoMP from fresh RA SF. (D) O2 consumption is observed in PFP obtained at the indicated time intervals from platelet storage bags. (E) Isolation of freeMitos (supplemental Figure 3) in PFP along with mtDNA quantification reveals an abundance of freeMito at day 5 (n = 6; data are mean ± SEM, *P < .05 vs day 0, paired Student t test). (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) TEM imaging of PFP collected on day 5 confirming the presence of (left) freeMitos and (right) mitoMPs. (H) Mitochondrial membrane potential is detected in freeMitos and mitoMPs collected from PFP, as measured by a JC-1 assay using hs-FCM (red to green ratio) (n = 5; data are mean ± SEM). (I) Extracellular mitochondria (as detected by mtDNA quantification) are found at higher concentration in PFP of platelet storage bags that have cause adverse transfusion reaction to the recipient (no adverse reaction group [n = 61] vs adverse reaction group [n = 74] matched in term of storage duration; data are mean ± SEM, ***P < .001, Student t test). Adverse reaction measured include mainly febrile nonhemolytic reactions, skin manifestations such as itching or skin rash, and cardiovascular events such as hypotension or tachycardia.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/124/14/10.1182_blood-2014-05-573543/4/2173f3.jpeg?Expires=1723198844&Signature=cIMggq2HsoCH1EEdQj9xkk5VYtLAEfNEjz17SoP2xSiSFsK1uZEPQr4Jay7Aq4TrDBccQ6Zc4-mnLCXEnftOOB9m9urt6F2-bEZxZncvbe~bFvuyda2~N0jekm490RHWMogmJaLO2j2pl3dNN8ni-gSfzrYp7IPH8iNUGUqUiXzcf4z5r1mdNFu72QNBn1BrjgGC0oOY8kNHSXx5OAbLXv7tt6pcTrtqcsgBYctooAMnvKpzkc6EwcFp0SVVT4FfeCwCokatXmdMaFIArQn5V27W39s1g2S5OKWQgfkGdsSkP77IJL4hvDsp9FOHQw0vCVc7KXpBS0RgxYbiCOAE2Q__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Extracellular mitochondria are present in various situations where platelets are known to be activated. (A) Platelet mitoMPs (CD41+MitoTracker+) are found in higher concentrations in the synovial fluid of RA patients (n = 20) than in the synovial fluid of osteoarthritis patients (OA, n = 14; data are mean ± SEM, *P < .05, Mann-Whitney test). (B) FreeMitos are detected in fresh SF of RA patients. Isolation of freeMitos in RA SF (from 3 different patients) with anti-TOM22 microbeads (or control IgG; supplemental Figure 3) and mtDNA quantification. (C) TEM imaging of (left) a freeMito and (right) a mitoMP from fresh RA SF. (D) O2 consumption is observed in PFP obtained at the indicated time intervals from platelet storage bags. (E) Isolation of freeMitos (supplemental Figure 3) in PFP along with mtDNA quantification reveals an abundance of freeMito at day 5 (n = 6; data are mean ± SEM, *P < .05 vs day 0, paired Student t test). (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) TEM imaging of PFP collected on day 5 confirming the presence of (left) freeMitos and (right) mitoMPs. (H) Mitochondrial membrane potential is detected in freeMitos and mitoMPs collected from PFP, as measured by a JC-1 assay using hs-FCM (red to green ratio) (n = 5; data are mean ± SEM). (I) Extracellular mitochondria (as detected by mtDNA quantification) are found at higher concentration in PFP of platelet storage bags that have cause adverse transfusion reaction to the recipient (no adverse reaction group [n = 61] vs adverse reaction group [n = 74] matched in term of storage duration; data are mean ± SEM, ***P < .001, Student t test). Adverse reaction measured include mainly febrile nonhemolytic reactions, skin manifestations such as itching or skin rash, and cardiovascular events such as hypotension or tachycardia.
