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C.C.F.M.J. Baaten et al.
ed a depolarization of the platelet mitochondria, which was independent of diagnosis or treatment class (Kruskal- Wallis H test, P=0.656 and P=0.126, respectively). The low TMRE fluorescence correlated well with the reduced platelet responsiveness (Spearman’s rho=0.569, P=0.001). However, cyclosporin A-induced inhibition of mitochon- drial permeability pore formation did not affect PS expo- sure (data not shown).
We subsequently assessed platelet mitochondrial activity
by measuring mitochondrial respiration via high-resolution respirometry.27 With saturating amounts of complex I-II substrates of the oxidative phosphorylation (OXPHOS) chain, i.e., pyruvate, malate, ADP, glutamate and succinate, the maximal ADP-supported respiration of mitochondria was significantly lower in platelets from patients than from controls (Figure 5B). To exclude that the mitochondrial content was altered, we measured the citrate synthase activity.28 However, this was unchanged in the patients’
A
B
C
Figure 3. Impaired platelet spreading and Ca2+ signaling of platelets from patients. (A) Platelets from patients or healthy controls were allowed to spread on a fib- rinogen surface for 10 min, after which microscopic images were captured. Spreading state per platelet was classified in three stages based on morphology: (i) filopo- dia, (ii) lamellipodia, or (iii) fully spread. Percentages of platelets per category are shown. Medians (with IQR) for nine patients, seven control subjects. (B, C) Fluo-4- loaded platelets from patients (n=7) and controls (n=5) were stimulated with thrombin (4 nM), CRP-XL (10 μg/mL) or thapsigargin (0.5 μM) in the presence of 2 mM CaCl2. Changes in Fluo-4 fluorescence were measured in time by flow cytometry. (B) Representative Fluo-4 traces in time. (C) Relative increases in cytosolic Ca2+. Medians with IQR, **P<0.01. Overall platelet responsiveness of the patients was 31.5 – 57.9% (IQR). CRP: collagen-related peptide.
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haematologica | 2018; 103(9)