Page 254 - Haematologica April 2020

S.I. Obydennyi et al.
ton.2 The severity of immunodeficiency varies between WAS patients, whereas the platelet defect (reduced num- ber and size) is the universal feature of the disease, and thrombocytopenia-related bleeding contributes greatly to mortality in untransplanted patients.3 Although major platelet functions are retained in WAS platelets, there is evidence of defects in these platelets that could potentially contribute additionally to bleeding.4
The specific mechanisms of thrombocytopenia in WAS remain elusive. Studies of megakaryocytes from patients produced evidence both in favor of defects in platelet pro- duction5 and against them.6 On the other hand, platelets from WAS patients and murine WAS protein knockouts had shortened lifespan and were subject to increased phagocytosis.7-9 In particular, it was shown previously that WAS platelets have increased phosphatidylserine (PS) exposure upon storage and activation,10,11 which could be one of the mechanisms involved in their accelerated clear- ance by splenic macrophages and possibly contribute to thrombocytopenia. Indeed, recent evidence from diverse eukaryotic systems suggests that the actin cytoskeleton has a role in regulating apoptosis via interactions with mitochondria.12 Changes to the dynamics of the actin cytoskeleton were implicated in the release of reactive oxygen species (ROS) from mitochondria and subsequent cell death.13 Interestingly, recent studies discovered that platelets from patients with deficiency of actin filament branching regulator Arp2/3 have major phenotype fea- tures similar to those observed in WAS:14,15 microthrombo- cytopenia, deficiency of dense granules and spreading.
Here we investigated the mechanism underlying cell death in platelet samples from a cohort of 35 WAS patients. The main conclusion is that the platelets are prone to PS exposure upon minor stimulation, which occurs as a result of mitochondrial permeability transition pore opening. We provide evidence for the two major mechanisms responsible for this: (i) an increased surface- to-volume ratio of these micro-platelets leading to dysreg- ulation of calcium homeostasis; (ii) a decreased number of mitochondria per platelet, which results in a dramatic increase of cytosolic calcium upon opening of the mito- chondrial permeability transition pores in a single mito- chondrion. Platelet size correlated with platelet count in the untreated WAS patients.
Methods
A full description of the methods and reagents is available in the Online Supplementary Material.
Patients and healthy donors
A total of 35 patients with WAS were included in the study (Table 1). The diagnosis was made according to the diagnostic cri- teria of the European Society for Immunodeficiencies and geneti- cally confirmed by identification of WAS mutations. Romiplostim was administered off-label at a dose of 9 μg/kg weekly, according to an institutional protocol. Twelve of 35 patients had a Zhu score of 1 or 2. Control samples included blood from children and healthy adults, as indicated in the experimental descriptions.
Blood collection and platelet isolation
Investigations were performed in accordance with the Declaration of Helsinki under approval of the Children's Center for Hematology Ethical Committee, and written informed consent was
obtained from all patients (or their parents) and donors. Washed platelets were prepared essentially as described previously.17
Confocal microscopy experiments: general design
Glass coverslips were cleaned and coated with 1 mg/mL fibrino- gen or monafram in phosphate-buffered saline. Washed platelets were attached to the protein-coated surface by incubating them at
Table 1. Characteristics of the patients with Wiskott-Aldrich syndrome.
Patient # Age (years)
1 12
2 13
3 0.9
4 2
5 11
6 0.8 7 4
8 12 9 0.6 10 10 11 17 12 12 13 14 14 10 15 0.5 16 1 17 0.6
18 5 19 0.9 20 7 21 2 22 1 23 1 24 2 25 2 26 2
27 7 28 11 29 7
30 0.5
31 9
32 8
33 3
34 17
35 2
WAS gene Disease mutation severity score*
c.777+1G>A 5
Romiplostim treatment
Platelet count 109/L
c .1453G>A, p.D485N
4
After 114
Before 52
After 337
Before 30 After 24
Before 38
After 82 After 34
After 27
Before 38 After 145
After 53
After 12 Before 7 After 34
Before 20 Before 31 Before 32 Before 77
After 170 After 42
Before 54 Before 19 Before 11 Before 55
After 192
Before 20
After 81
After 73
Before 16
After 60
After 20 Before 17 After 16
Before 36 After 72 After 37 After 147
Splenectomy 170
After 30
c.929_931+9del (c.929_931+ 5
9delAGGgtgagaccc)
c.223G>A, p.V75M 3
c.1201_1205dupCCACC, 5 p.P403HfsTer44
c.777+1G>A 3 c.4A>T, p.S2C 1
c.560-1G>A 5
c.631C>T, p.R211Ter 4
c.116T>C, p.L39P 2
c.223G>A, .V75M 2
c.134C> , p.T45M 2
c.4A>T, p.S2C 2
c.4A>T, p.S2C 2
c.4A>T, p.S2C 2
c.961 >T, p.R321Ter 3
c.107_108delTT, 5 p.F36Ter
c.413G>A, p.R138Q 1 c.37C>T, p.R13Ter 3 c.961C>T, p.R321Ter 3 c.559+5G>A 5 c.631C>T, p.R211Ter 5 c.273+2T>C 5 c.281G>C, p.R94P 1 c.777+2del4(GAGT) 5 c.2T>C, p.M1R 4
c.961 C T, p.R321Ter 3
c.559+5G>A 4
c.107_108delTT, 5 p. F36Ter
c.314T>C, p.L105P 1 c.143 > , p.47I>T 3 c.223 G>A, .V75M 3
c.1430G>A, p.R477K 2
c.559+5G>A 5
c.267 G>A, .R86H 2
* Scoring as suggested by Ochs et al.40
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