Page 255 - Haematologica April 2020

Programmed necrosis of platelets in WAS
1.3x105/μL (or the maximal concentration attainable for WAS under conditions of thrombocytopenia) for 20 min and rinsing with buffer A with 1.5 mM CaCl2. The PS+ fraction was counted after incubation for a further 30 min.
Cytosolic calcium signaling and mitochondrial membrane potential change
The methodology for these investigations was essentially as described elsewhere.18 Calibration for ratiometric measurements was performed separately for healthy and WAS platelets. Calcium concentrations were calculated using an equation for ratiometric indicators.19 Tetramethylrhodamine methyl ester (TMRM) was used to detect mitochondrial potential dynamics.
Characterization of platelet response to TRAP-6 in platelet-rich plasma
Samples of platelet-rich plasma were diluted with platelet-poor plasma to a final concentration of 20,000/μL and buffered with HEPES at pH 7.4 (100 mM final concentration). The irreversible thrombin inhibitor Phe-Pro-Arg chloromethyl ketone (PPACK) was added to the final concentration of 100 μM in order to block spontaneous thrombin generation upon recalcification. Platelets were recalcified by addition of calcium chloride (final concentra- tion 20 mM, which corresponds to 2 mM of free calcium20) and activated by 25 μM TRAP-6 (thrombin receptor agonist peptide-6) for 5 min at room temperature.
ATP measurement in platelets
In brief, platelets of the samples were lyzed (by addition of 90 μL dimethylsulfoxide to 10 μL platelet suspension) and analyzed by luciferase-luciferin assay as described previously21,22 while the other part was stained with CD61-FITC and annexin V-Alexa Fluor 647 and analyzed by flow cytometry.
Flow cytometry characterization of platelet functional activity
The experiments were performed essentially as described previ- ously23,24 with minor modifications.
Transmission electron microscopy
The protocol was essentially as described elsewhere.25
Statistics
Data are presented as means ± standard deviations. The statis- tical significance of the differences between groups was deter- mined with the non-parametric Mann–Whitney U-test (P) or Wilcoxon signed-rank test (P*) for paired samples. Differences were considered to be statistically significant when the P-value was <0.05.
Computational modeling of platelet calcium homeostasis
A systems biology model of platelet calcium signaling was based on one developed previously.18 In contrast to its predecessor pure calcium signaling/homeostasis models,26,27 it had several mito- chondrial compartments and included equations describing dependence of ATP production and calcium pump activity on mitochondrial inner membrane potential.
Results
Phosphatidylserine exposure by fibrinogen-immobilized Wiskott-Aldrich syndrome platelets
In order to gain insight into the mechanisms of increased/accelerated PS exposure of WAS platelets, we
investigated dynamics of status change by the single fib- rinogen-bound cells (Figure 1A). Unexpectedly, this rela- tively mild method of platelet immobilization produced massive spontaneous PS exposure in the WAS platelets during 30 min without any additional stimulation com- pared with PS exposure on platelets from either healthy adults (n=18) or children without WAS (n=6, aged 0-7 years, median 2.5 years) (Figure 1B). The smaller num- ber of adherent platelets in some of the WAS patients could lead to some underestimation of their PS+ platelets, so that the effect could be even greater. Taking into account that some of the patients received romi- plostim, previously shown to potentially affect platelet function.28,29 it was reasonable to evaluate its effect on platelets separately.30,31 However, there was no statisti- cally significant difference between PS externalization by platelets from untreated WAS patients and from those on romiplostim (Figure 1C): in both groups, approximately 20% of platelets, on average, exposed PS. The phenomenon was not fibrinogen-specific, as platelets attached to the αIIbβ3 antagonist monafram pro- duced the same results (Figure 1D).
Functional activity of the Wiskott-Aldrich syndrome platelets
To thoroughly characterize the status of the WAS platelets involved in the study, we analyzed them by diluted whole blood flow cytometry using a comprehen- sive set of functional markers (Figure 2 and Online Supplementary Figure S1). Platelets were either left in the resting state or subjected to potent dual stimulation with TRAP-6 and collagen-related peptide (CRP). As a control, we used a group of 20 healthy children (9 boys and 11 girls, aged 0-13 years, median 5 years). The WAS platelets had significantly decreased forward scatter and levels of major surface glycoproteins (Figure 2A-C) reflecting their decreased size. There were two interesting exceptions: patients 5 and 18 had normal forward scatter. Patient 5 was splenectomized, which could be a plausible explana- tion of his larger platelets, while patient 18 had an excep- tionally mild WAS phenotype. The size-independent parameter of shape change evaluated as the light scatter- ing ratios for the resting/stimulated platelets (Online Supplementary Figure S1A, B) was significantly decreased in WAS. In order to take into account the difference in platelet size and surface area, we evaluated integrin αIIbβ3 activation and α-granule release by either the percentage of PAC1+ (Figure 2D) or CD62P+ (Figure 2E) platelets, or by normalizing the data on CD61 fluorescence intensity, as described elsewhere31 (Online Supplementary Figure S1). In both cases, the response to activation was somewhat decreased in WAS platelets compared with healthy platelets. However, there was a relative increase in base- line platelet activation in the WAS patients compared with the controls, as judged by integrin (Online Supplementary Figure S1D) and P-selectin (Online Supplementary Figure S1F) surface expression of resting platelets. Dense granule release in WAS was essentially lower than in healthy chil- dren (Figure 2F). Interestingly, the completely size-inde- pendent response of procoagulant platelet formation eval- uated as a percentage of annexin V+ cells was also clearly decreased (Figure 2G). Neither of these phenomena changed in the romiplostim-treated patients compared with untreated ones. Analysis with TRAP stimulation in diluted platelet-rich plasma revealed a minor increase in
haematologica | 2020; 105(4)
1097