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Z. Zhao et al.
We also found that 55.2±12.6% of annexin-V-binding microvesicles in peripheral blood of TBI mice were mor- phologically intact extracellular mitochondria (exMT).9 These exMT promoted coagulation through the surface- exposed anionic phospholipid cardiolipin. The findings raise an important question: are these exMT metabolical- ly active and, if so, do they affect platelet function through redox-dependent mechanisms? The question is raised because exMT can be viable, transferred between cells, and influence the function of target cells.10-13 The influence of mitochondria on target cells has mostly been reported as protective,10,12,13 likely due to increased energy supply. However, reactive oxygen species (ROS) are gen- erated during ATP production in mitochondria14 and are known to activate platelets.15-17 Several studies have shown that platelets in patients with TBI are activated but aggregate poorly.18 This platelet phenotype has also been reproduced in rat and swine models of TBI, in which platelets are activated,19,20 accumulate on the pia mater,21 and contribute to thrombosis in the lesion bound- ary zone.22 The underlying mechanism for this unique TBI-associated platelet phenotype remains poorly under- stood. Here we discuss results from a study that was designed to investigate the effect of exMT on platelet activation and procoagulant activity through in vitro experiments and in mouse models.
Methods
Mouse models
C57BL/6J male mice (12-20 weeks and 22-25 g), obtained from the Jackson Laboratory (Bar Harbor, ME, USA) were sub- jected to fluid percussion injury8 and blood samples were collect- ed to quantify plasma exMT (Online Supplementary Methods).9 This mouse protocol was approved by the Institutional Animal Care and Use Committee of the BloodWorks Research Institute.
Flow cytometry
Platelet activation
We used an LSR II flow cytometer (Beckon Dickinson, San Jose, CA, USA) to detect platelet activation through several measure- ments: CD62p expression, the binding of PAC-1 antibody (BD Biosciences), which recognizes the active conformation of the fib- rinogen receptor αIIbβ3, annexin V binding,9 platelet-bound fib- rinogen and coagulation factor V,25,26 and the formation of platelet- leukocyte complexes (Online Supplementary Methods).
Calcium influx
We used flow cytometry to measure the exMT-induced calci- um influx in platelets labeled with 5 mM eFluor 514 Calcium Sensor Dye (Bioscience, San Diego, CA, USA) for 10 min at 37°C.8
Reactive oxygen species production
ExMT were suspended in Ca2+-free HEPES buffered Tyrode solution (138 mM NaCl, 5.5 mM glucose, 10 mM HEPES, 12 mM NaHCO3, 2.9 mM KCl, 0.4 mM NaH2PO4, 0.4 mM MgCl2, and 0.1% BSA; pH 7.2) and incubated for 30 min at 37°C with 10 mM of DCFH-DA dye (ThermoFisher).24 As controls, labeled exMT were treated with either 50 mM N-acetylcysteine (NAC, Life Technologies, Grand Island, NY, USA), which is a precursor of the antioxidant glutathione, or 200 mM tert-butyl hydroperox- ide (TBHP, Life Technologies), which increases intracellular ROS production.27 ExMT fixed with 5% paraformaldehyde were also examined as a control.
Image flow cytometry for the extracellular mitochondria-platelet interaction
As we previously described,28 platelet-rich plasma was incubat- ed for 30 min at 37°C with exMT labeled with MitoTracker Green and an APC-conjugated CD41a antibody (BD Bioscience). The platelets were washed in phosphate-buffered saline and fixed in 1% paraformaldehyde. Images were acquired at 60 x magnifica- tion to collect ≥20,000 cells from each sample using the Amnis ImageStream® X Mk II system (Amnis, Seattle, WA, USA). To dis- tinguish exMT bound to the platelet surface from those internal- ized, platelets were digested with 1% trypsin for 10 min at 37°C after incubation with exMT. Mouse mitochondrial-specific DNA was then amplified from these exMT-treated and trypsinized platelets using a protocol modified from our previous study.9
CD36-extracellular mitochondria interaction
For in vitro experiments, platelet-rich plasma was incubated for 30 min at 37°C with a monoclonal CD36 antibody (Abcam, ab17044, Cambridge, MA, USA) or isotype IgG, followed by incu- bation with MitoTracker Green-labeled exMT and a PE-CD41a antibody for 30 min. For in vivo experiments, blood samples from non-injured mice infused with MitoTracker Green-labeled exMT were analyzed for exMT-bound platelets (MitoTracker Green+/CD41a+), platelet counts (CD41a+ platelet counts in 60 s), platelet CD62p expression, and platelet-leukocyte complexes (CD41a+/ CD45+). The CD36 antibody used in this study recog- nizes both mouse and human CD36.29,30
Statistical analysis
Categorical (frequency) variables were expressed as percentages and continuous variables were expressed as the mean ± standard error of mean. The quantitative data were analyzed using SigmaPlot V. 11.2 (SYSTAT Software Inc., San Jose, CA, USA) for paired t tests, one way or repeated measures analysis of variance (ANOVA) as specified in each analysis. A P value of ≤0.05 was considered statistically significant.
Results
Extracellular mitochondria bound platelets in vivo and in vitro
Structurally intact (MitoTracker Green+) (Figure 1A) and cardiolipin-exposed (Figure 1B) exMT were detected in the peripheral blood of mice with acute TBI. The exMT formed complexes with approximately 12.3 ± 5.8% of cir- culating platelets that remained detectable for 6 h after TBI (Figure 1C). Low levels of exMT-platelet complexes were also detected in sham-treated mice and likely resuit- ed from surgery-induced injuries. When co-incubated for 30 min at 37°C in vitro, purified exMT from brains subject- ed to freeze-thaw injury also formed complexes with platelets (Figure 1D) in a dose-dependent manner (Figure 1E). Transmission electron microscopy showed that exMT interacted with the body (Figure 1F) and filopodia (Figure 1G) of platelets.
The Amnis® imaging flow cytometer detected exMT on the surface of platelets (Figure 2A, top panel), being endo- cytosed by platelets (Figure 2A, middle panel) and fused with platelet membranes (Figure 2A, bottom panel). The internalization of exMT by platelets was further validated by co-incubation of human platelets with mouse exMT for 30 min at 37°C followed by the amplification of mouse mitochondria-derived DNA from exMT-treated platelets that were trypsinized to remove surface-bound exMT
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haematologica | 2020; 105(1)