Mechanisms of increased mitochondria-dependent necrosis in Wiskott-Aldrich syndrome platelets

Abstract

Wiskott-Aldrich syndrome (WAS) is associated with thrombocytopenia of unclear origin. We investigated real-time cytosolic calcium dynamics, mitochondrial membrane potential and phoszphatidylserine (PS) exposure in single fibrinogen-bound platelets using confocal microscopy. The WAS platelets had higher resting calcium levels, more frequent spikes, and their mitochondria more frequently lost membrane potential followed by PS exposure (in 22.9% of platelets vs 3.9% in controls; P<0.001) after the collapse of the last mitochondria. This phenomenon was inhibited by the mitochondrial permeability transition pore inhibitor cyclosporine A, as well by xestospongin C and lack of extracellular calcium. Thapsigargin by itself caused accelerated cell death in the WAS platelets. The number of mitochondria was predictive of PS exposure: 33% of platelets from WAS patients with fewer than five mitochondria exposed PS, while only 12% did among those that had five or more mitochondria. Interestingly, healthy donor platelets with fewer mitochondria also more readily became procoagulant upon PAR1/PAR4 stimulation. Collapse of single mitochondria led to greater cytosolic calcium increase in WAS platelets if they had one to three mitochondria compared with platelets containing higher numbers. A computer systems biology model of platelet calcium homeostasis showed that smaller platelets with fewer mitochondria could have impaired calcium homeostasis because of higher surface-to-volume ratio and greater metabolic load, respectively. There was a correlation (C=0.81, P<0.02) between the mean platelet size and platelet count in the WAS patients. We conclude that WAS platelets readily expose PS via a mitochondria-dependent necrotic mechanism caused by their smaller size, which could contribute to the development of thrombocytopenia.

Figure 1.

Exposure of phosphatidylserine by Wiskott-Aldrich syndrome platelets upon fibrinogen binding. (A) Confocal microscopy images of healthy (left) and Wiskott-Aldrich syndrome (WAS) (right) platelets after spreading for 30 min on a fibrinogen surface in the presence of 1.5 μM Ca2⁺. The platelets are labeled with CD61 (green) and annexin V (red); scale bar: 10 μm. (B) Phosphatidylserine-positive (PS⁺) fraction of platelets from the WAS patients (27 patients, >7,500 cells), adult healthy (18 donors, >6,500 cells) and 0- to 7-year old children without WAS (6 children, age: 0, 0, 2, 3, 4, 7 years, 2,300 platelets) on a fibrinogen surface. (C) PS⁺ fraction of WAS platelets on the fibrinogen surface, showing a comparison of romiplostim-treated and untreated WAS patients, P=0.94. (D) Monafram-coated coverslips did not change the PS⁺ fraction, P=0.86, n=4, 2,500 platelets. w/o: without; FG: fibrinogen.

Figure 2.

Functional response of the Wiskott-Aldrich syndrome and healthy platelets. (A-G) Whole blood platelets were stimulated (designated by A) or not (designated N/A) with thrombin receptor agonist peptide-6 plus collagen-related peptide and analyzed by flow cytometry. Parameters shown for healthy children (n=21, age 0-13 years, median 5.0) and Wiskott-Aldrich patients (17 treated with romiplostim, 11 non-treated) are: platelet size, determined from the forward scatter measured by mean fluorescence intensity (MFI) (A); CD42b level, MFI (B); CD61 level, MFI (C); PAC1-positive platelets, % (D); CD62p-positive platelets, % (E); dense granule release determined by mepacrine level, MFI (F); and phosphatidylserine-positive platelet fraction, % (G). P: Mann–Whitney U-test, P*:Wilcoxon signed-rank test. FSC: forward scatter, WAS: Wiskott-Aldrich syndrome; PS⁺: phosphatidylserine-positive.

Figure 3.

Dynamics of cytoplasmic calcium, mitochondrial potentials and phosphatidylserine exposure of single platelets. Plots show dynamics of intracellular calcium concentration and annex-in V binding to single platelets during incubation on fibrinogen in the presence of 1.5 mM of extracellular calcium. (A) Averaged calcium dynamics (± standard deviation) for non-activated (N/A) platelets from patients with Wiskott-Aldrich syndrome (WAS) (4 patients, 34 platelets) and for platelets from healthy donors (HD) which were either activated with 10 μM TRAP (3 HD, 30 platelets) or not activated (3 HD, 26 platelets). (B) Dynamics for a single healthy phosphatidylserine-negative (PS⁻) platelet; (C) the same for a WAS PS⁻ platelet; (D) the same for a WAS PS⁺ platelet. The TMRM signal is represented as a number of TMRM-positive mitochondria in the platelet (B-D). Intracellular events leading to PS exposure induced with mitochondria collapse with following cytoplasmic calcium increase and PS exposure. All three processes were almost simultaneous, lasting decades of seconds. Both WAS (E) and normal (not shown) PS⁺ platelets lost their mitochondrial potentials. Scale bar: 1 μm for all microscopic images. TRAP: thrombin receptor agonist peptide; TMRM: tetramethylrhodamine methyl ester.

Figure 4.

Prevention of mitochondrial permeability transition pore opening affects spontaneous phosphatidylserine exposure of Wiskott-Aldrich syndrome platelets. (A) Phosphatidylserine (PS) exposure of fibrinogen-spread platelets incubated in the absence or presence of 5 μM cyclosporine A (3,900 platelets from 11 patients and 4,000 platelets from 6 healthy donors). (B) PS exposure of platelets incubated with DMSO or programmed cell death inhibitors calpeptin (200 μM, 20 min incubation), Nec-1 (50 μM, 50 min incubation) and Z-VAD-FMK (50 μM, 50 min incubation) (100-300 platelets were observed for each dot). (C) Modulation of intracellular calcium signaling in spread platelets by xestospongin C (3 μM, 50 min); thapsigargin (TG, 1 μM, 30 min); with lactadherin and without addition of 1.5 mM CaCl₂ (n=5, 6,800 platelets). (D) Flow cytometry analysis of Wiskott-Aldrich syndrome (WAS) platelet PS exposure. Incubation of platelets in suspension with 1 mM TG in the presence of 1.5 mM CaCl₂ for 10 min induced a PS⁺ platelet fraction comparable to that with fibrinogen-spreading for both WAS patients (n=7, mean ± standard deviation: 19.7%±11.8%) and healthy donors (n=11, 6.6%±8.0%). (E) Flow cytometry analysis of WAS platelet PS exposure in suspension without addition of CaCl₂(n=3); (F) Analysis of the mitochondrial inhibitors in suspension at TG treatment (n=3); (G, H) ATP levels (G) versus the number of PS⁺ platelets (H): in healthy donors and WAS patients at TG and CCCP treatment (n=3). CsA: cyclosporine A, DMSO: dimethylsulfoxide; HD: healthy donor.

Figure 5.

Dependence of phosphatidylserine exposure on mitochondria count. Platelets that exposed phosphatidylserine (PS) during incubation on fibrinogen contained significantly fewer mitochondria than PS- cells. (A) Mean mitochondria number 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 represents 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 fluorescence using a microscope after spreading for 20 min (before activation in experiments 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.

Figure 6.

Increased cytosolic calcium as a result of downsizing: computer systems biology simulation of calcium signaling in normal and Wiskott-Aldrich syndrome platelets. Wiskott-Aldrich syndrome (WAS) platelets were assumed to have the same content of signaling proteins, scaled to the respective volume of compartments. (A, B) Stochastic simulation of the activation of normal platelets containing two (A) or four (B) mitochondria with 10 nM thrombin. With the collapse of one mitochondrion the average cytosolic calcium increases 1.5-fold (A) in the case of two mitochondria or does not change (B) in the case of four mitochondria. (C-E) Stochastic and deterministic simulations of normal and WAS platelets stimulated with 1 nM thrombin.