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Mitochondria-induced platelet procoagulant activity
pholipids as indicated by annexin V binding (Figure 2F) and had elevated levels of fibrinogen (Figure 5A) and coagulation factor Va on their surfaces (Figure 5B), sug- gesting that exMT-treated platelets could promote coag- ulation.33,34 Consistent with this notion, exMT-treated platelets accelerated clot formation at a level comparable to that of collagen-activated platelets but lower than that of purified phosphatidylserine micelles (Figure 5C). When added to platelet-rich plasma, exMT-treated platelets significantly accelerated and enhanced throm- bin-induced clot retraction (Figure 5D), which measures the integrin αIIbβ3-dependent retraction of platelet- bound fibrin fibers.35,36 The effect of exMT was compara- ble to that of collagen-stimulated platelets. This procoag- ulant activity was independent of tissue factor, which was not detected on the surface of exMT-treated platelets (Online Supplementary Figure S6).
Anti-CD36 antibody blocked extracellular mitochondria-induced platelet activation
To measure exMT-induced platelet activation in vivo, non-injured mice were infused with purified exMT. Thirty minutes after infusion, platelets became exMT-bound
(Figure 6A), expressed CD62p (Figure 6B), and formed complexes with leukocytes (Figure 6C). Mice infused with exMT also developed thrombocytopenia (Figure 6D). MitoTracker-Green-labeled exMT were detected in approximately 50% of platelet-leukocyte aggregates (Figure 6E). These phenotypic changes of platelets were prevented by infusing mice with exMT, together with a CD36 antibody but not isotype IgG.
Discussion
We investigated whether morphologically intact but cardiolipin-exposed exMT are metabolically active and, if so, whether they activate platelets through ROS. We have made several novel observations that define an exMT- induced and redox-dependent intermediate phenotype of platelets.
First, despite the cardiolipin translocation,9 exMT found in TBI mice or released from brains subjected to freeze- thaw injury produced ATP and ROS (Figure 3B, C). Because metabolic activity of mitochondria requires a functional membrane, exMT that interact with platelets
ABCD
EFG
Figure 3. Extracellular mitochondria induced α-granule secretion of platelets through reactive oxygen species. (A) Extracellular mitochondria (exMT) purified from mice with traumatic brain injury stimulated platelets to express CD62 and this effect was blocked by 20 mM of glutathione (GSH) (n=45, one-way analysis of variance, ANOVA). (B, C) ExMT released from brains subjected to freeze-thaw injury produced ATP (n=4, one-way ANOVA) (B) and reactive oxygen species (ROS) (2.5x108/mouse of exMT; mean fluorescence intensity) at 37°C in vitro. The ROS production was blocked by the antioxidant N-acetylcysteine, enhanced by the oxidant tert-butyl hydroperoxide, and not detected in paraformaldehyde-fixed exMT (n=18, one-way ANOVA) (C). (D) Platelets incubated with exMT at a ratio of 1:1 or 1:5 for 30 min at 37°C expressed CD62p (n=24, one-way ANOVA). (E) The CD62p expression was reduced by 20 mM of GSH, 200 mM of L-cysteine, or 2 mM of NEM (n=30, one-way ANOVA). (F, G) ExMT-treated platelets formed complexes with leukocytes (n=24, one-way ANOVA) (F) and released von Willebrand factor (n=24, one-way ANOVA, Online Supplementary Methods) (G). For platelet activation, platelets stimulated with 5 mg/mL of collagen or 50 nM of thrombin were used as controls to mimic “coated platelets”.33 NAC: N-acetylcysteine; TBHP: tert-butyl hydroperoxide; MFI: mean fluorescence intensity; NEM: N-ethylmaleimide; vWF: von Willebrand factor.
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