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Mitochondria-induced platelet procoagulant activity
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Figure 1. Extracellular mitochondria were released from injured cells and bound platelets. (A, B) Extracellular mitochondria (exMT) from mice with traumatic brain injury (TBI) were detected by MitoTracker Green (# exMT/100 mL plasma) (A) and anti-cardiolipin antibody (n=12, paired t test) (B). (C) Platelet-exMT complexes were detected in the blood of TBI mice using CD41a and TOM22 antibodies (n=8, paired t test ). (D, E) After co-incubation for 30 min at 37°C, exMT also bound platelets in vitro, as shown by a representative histogram (D) in a dose-dependent manner (n=30, one-way analysis of variance) (E). (F, G) Transmission electron microscopy (see Online Supplementary Methods) showed exMT binding to the body (F) and filopodia (G) of platelets (bar = 500 nm; arrow: mitochondria; *: platelet; represen- tatives of 98 images reviewed).
(Figure 2B). Quantitatively, 13.7 ± 9.1% of platelets were exMT-bound after 30 min co-incubation (Figure 2C), which was consistent with the level of exMT-bound platelets found in TBI mice (Figure 1C). This exMT- platelet interaction was blocked by the phosphatidylser- ine-binding proteins annexin V and lactadherin (Figure 2C)28,31 and partially by a CD36 antibody (Figure 2D), which blocks the CD36-mediated binding of endothelial microvesicles to platelets,29 suggesting that exMT bind platelets through cardiolipin exposed on the mitochondria and CD36 expressed on platelets. Furthermore, ATP pro- duction was dose-dependently increased in the exMT- treated platelets at levels that were significantly higher than in platelets activated with ADP (Figure 2E). We were unable to determine whether the ATP increase was caused by platelet-bound exMT or by exMT-induced ATP pro- duction of platelets. Finally, when added to platelets that had been treated with exMT, FITC-conjugated annexin V bound 16.2 ± 6.3% of platelets (Figure 2F).
Extracellular mitochondria were metabolically viable and activated platelets
We found that exMT from TBI mice stimulated platelets to express CD62p and this activity was partially blocked by the antioxidant glutathione (GSH) (Figure 3A). One pit-
fall of this experiment was that exMT purified from plas- ma of TBI mice were derived from multiple cells. It was technically challenging to separate brain-derived exMT from those of other cells, without damaging the viability of exMT. To address this concern, we tested exMT from brains subjected to freeze-thaw injury in vitro.9 These exMT produced ATP (Figure 3B) and ROS (Figure 3C). The ROS production was blocked by 50 mM of the anti-oxi- dant NAC and enhanced by 200 mM of the oxidant TBHP. The two agents have previously been shown to block and enhance ROS production of mammalian cells, respective- ly.32;27 This metabolic viability was further validated by the lack of ROS production from paraformaldehyde-fixed exMT (Figure 3C). After 30 min of incubation with exMT, both mouse (Online Supplementary Figure S2) and human platelets (Figure 3D) expressed CD62p on their surfaces. The exMT-induced CD62p expression was reduced by the antioxidants GSH (20 mM), L-cysteine (0.5 mM), and NEM (2 mM) (Figure 3E). Testing the three thiol-modifying agents was necessary because GSH and L-cysteine reversibly interact with extracellular and intracellular oxi- dants, respectively, whereas NEM forms irreversible sulfur bonds with thiols. The exMT-treated platelets also increased calcium influx (Online Supplementary Figure S3), formed complexes with leukocytes (Figure 3F), and
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