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Z. Zhao et al.
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Figure 4. Extracellular mitochondria induced platelet disintegration, not aggregation. Platelets adherent to immobilized fibrinogen were treated with phosphate- buffered saline (PBS), extracellular mitochondria (exMT), or exMT + 20 mM of glutathione (GSH) for 30 min at 37°C and repeatedly scanned for up to 80 min by hop- ping probe ion conductance microscopy. (A) The images show adherent platelets treated with PBS (top panel), exMT at a 1:1 ratio with platelets (middle panel; arrow: membrane disintegration of an adherent platelet), and GSH-treated exMT (bottom panel). The images are representative of three to six independent experiments. (B) The supernatants were collected and stained for CD41a+ microvesicles by flow cytometry (n=18, one-way analysis of variance, ANOVA). (C) Platelet counts before and after exMT treatment either alone or with GSH (n=15, one-way ANOVA). (D-F) ExMT induced minimal platelet aggregation (D) (n=24, one-way ANOVA), but exMT- treated platelets aggregated normally when stimulated with collagen (E) or ADP (F) (n=54, repeated measures ANOVA). (G) CD61 expression on exMT-treated platelets was comparable to that on untreated platelets but increased upon stimulation with 5 mg/mL of collagen (n=24, one-way ANOVA). MV: microvesicles; MFI: mean fluorescence intensity.
Platelet-bound exMT may have interfered with the fib- rinogen coupling of platelets through steric hindrance. This is unlikely because exMT-bound platelets aggregat- ed after stimulation with collagen or ADP at levels com- parable to those of platelets that had not been treated with exMT (Figure 4E, F). (ii) ExMT only activated 10- 25% of platelets, insufficient to induce platelet aggrega- tion. A similar effect was observed with platelets simulta- neously treated with two antibodies against the integrins αIIb and β3,41 suggesting that α-granule secretion alone is insufficient to induce platelet aggregation, but may prime platelets for activation by other agonists. (iii) Some or all exMT-stimulated platelets underwent drastic membrane disintegration to produce microvesicles (Figure 4A, B), thereby becoming unavailable for aggregation. The third possibility is supported by the reduction of platelet counts after exMT treatment (Figure 4C). This exMT- induced intermediate platelet phenotype resembles “coat-
are likely those with an intact membrane. The metaboli- cally active exMT formed complexes with platelets (Figures 1 and 2) which remained detectable in the circu- lation for at least 6 h after TBI (Figure 1C).9 Cardiolipin on exMT and CD36 on platelets mediated the exMT-platelet interaction because: (i) cardiolipin is the dominant anion- ic phospholipid expressed on exMT9 and (ii) the exMT- platelet interaction was blocked by the phospholipid binding proteins annexin V and lactadherin (Figure 2C) and by an antibody against CD36 (Figure 2D), which is a phospholipid receptor37 that is expressed on platelets38,39 and promotes phospholipid-mediated endocytosis.40
Second, exMT induced platelets to secrete their α-gran- ule proteins (Figures 3 and 5) but failed to induce them to aggregate (Figure 4D) or promote platelet thrombus for- mation on the collagen matrix under arterial shear stress (Online Supplementary Figure S4A-C). There are several possible explanations for this apparent discrepancy. (i)
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haematologica | 2020; 105(1)