Page 261 - Haematologica April 2020

Programmed necrosis of platelets in WAS
result of the limited number of samples (Online Supplementary Figure S7B, C), it is interesting that patient #18 (indicated with a red arrow), who had normal-sized platelets and a mild phenotype, also had the least PS expo- sure upon immobilization and incidentally the highest platelet count.
Discussion
In this study, we show that the death of WAS platelets upon minor stimulation, such as fibrinogen attachment or
low-dose thapsigargin treatment, follows the pathway of mitochondrial necrosis. It is a rapid process associated with opening of mitochondrial permeability transition pores, which actually precedes PS exposure at the single platelet level, extracellular calcium-dependent, and it is downregulated by cyclophilin D and inositol trispohos- phate receptor antagonists, but not by apoptosis or necroptosis inhibitors. It is associated with decreased platelet ATP levels, and downregulation of the energy metabolism with CCCP, rotenone and oligomycin-pro- moted necrosis, but did not cause it by itself. This cell death phenomenon predominantly occurred in the
AB
CD
E
Figure 5. Dependence of phosphatidylserine exposure on mitochondria count.
Platelets that exposed phosphatidylserine (PS) during incubation on fibrinogen con- tained significantly fewer mitochondria than PS- cells. (A) Mean mitochondria num- ber in platelet subpopulations per patient with Wiskott-Aldrich syndrome (WAS) or per healthy donor (HD) for non-activated (N/A) fibrinogen-bound platelets. Each dot rep- resents one WAS patient (7 patients, 381 platelets) or HD (n=4, 567 platelets). (B) Averaged PS+ fraction ± standard deviation of the same WAS and HD platelets with different mitochondrial counts. (C) Averaged distribution of mitochondria per platelet (both subpopulations) for WAS patients (7 patients, 381 platelets) and HD (11 HD, 1,179 platelets). (D, E) Healthy activated platelets, overall 613 cells from seven HD activated with TRAP-6 (n=5, 306 cells) or thrombin (n=4, 307 cells). Platelets most likely to expose PS had fewer mitochondria. Mitochondria were counted by TMRM flu- orescence using a microscope after spreading for 20 min (before activation in exper- iments with activated platelets from HD); subpopulation were determined after an additional 30 min incubation. Each dot represents the mean of the mitochondria count in a patient or HD (A,C). P: Mann–Whitney U-test. TRAP-6: thrombin receptor agonist peptide-6: TMRM: tetramethylrhodamine methyl ester.
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