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Z. Zhao et al.
released von Willebrand factor (Figure 3G). The exMT- induced CD62 expression and platelet-leukocyte aggrega- tion were detected in 10-23% of platelets, which was con- sistent with the percentages of exMT-bound platelets found in TBI mice (Figure 1). We used platelets stimulated with collagen and thrombin as the control because this exMT-induced platelet phenotype resembled that of “coated platelets”.33,34 Together, these data suggested that exMT from TBI mice and those released from brains sub- jected to freeze-thaw injury in vitro were metabolically viable and activated platelets in an oxidant-dependent manner.
Using hopping probe ion conductance microscopy, we continuously monitored morphological changes of platelets adherent to fibrinogen in real-time (Figure 4A, top panel). After stimulation with exMT, adherent platelets underwent drastic membrane disintegration as exemplified in the middle panel of Figure 4A. This exMT- induced membrane disintegration was prevented by 20 μM GSH (Figure 4A, bottom row). Consistent with the exMT-induced platelet disruption, CD41a+ platelet- derived membrane microvesicles were detected in the supernatant of exMT-treated platelets (Figure 4B) and
platelet counts were reduced after treatment with exMT (Figure 4C). The production of platelet microvesicles and the reduction of platelet counts were partially blocked by the anti-oxidant GSH.
In contrast to their induction of α-granule secretion, exMT at comparable doses failed to induce platelet aggre- gation (Figure 4D) and did not enhance the formation of platelet thrombosis on the collagen matrix under arterial shear stress (Online Supplementary Figure S4A-C). Furthermore, the exMT-treated platelets aggregated nor- mally in response to collagen (Figure 4E) and ADP (Figure 4F), and were moderately primed for activation by sub- threshold concentrations of ADP and collagen (Online Supplementary Figure S4D). Neither the binding of PAC-1 antibody (Online Supplementary Figure S5), which recog- nizes the active conformation of integrin αIIbβ3, nor the surface density of the integrin αIIbβ3 (Figure 4G) was changed after exMT treatment, as compared to platelets stimulated with collagen.
Extracellular mitochondria-treated platelets were procoagulant
ExMT-treated platelets also expressed anionic phos-
AB
C
DEF
Figure 2. Anionic phospholipid and CD36 mediated the extracellular mitochondria-platelet interaction. (A) Amnis® flow cytometric images show extracellular mito- chondria (exMT) binding a platelet (top panel), endocytosed by a platelet (middle panel), and fused with platelet membrane (bottom panel) after 30 min co-incubation at 37°C (representative images from 20,000 images randomly selected). (B) Platelets internalized exMT in a dose dependent manner, as determined by the pres- ence of mouse mitochondrial DNA in mouse exMT-treated human platelets that were trypsinized to remove surface-bound exMT (n=20, one-way analysis of variance, ANOVA). (C, D) The formation of complexes between platelets and MitoTracker-Green+ exMT (1:1 ratio) was blocked by 20 mg/mL of annexin V or 200 mg/mL of lac- tadherin (n=56, one-way ANOVA) (C) and by an anti-CD36 antibody (n=20, one-way ANOVA) (D). (E) ATP production was increased in exMT-treated platelets (n=24, one-way ANOVA). (F) FITC-conjugated annexin V bound platelets that were pretreated with exMT (left: a representative flow cytometric histogram; right: a summary from 12 experiments; paired Student t test).
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haematologica | 2020; 105(1)