Page 181 - Haematologica3

concentration is lower, it assembles with the ORAI1 Ca2+ (ICRAC) channels in the plasma membrane to mediate store-operated Ca2+ entry (SOCE).1-4 Therefore, the con- ventional test for SOCE (i.e. for STIM1 and ORAI1 activ- ity) is to provoke depletion of the STIM1-linked Ca2+ store with the endoplasmic Ca2+ -ATPase inhibitor thapsi- gargin, and then measure Ca2+ entry through ORAI1 channels upon addition of extracellular CaCl2.5,6
Both ORAI1 and STIM1 have non-redundant roles in patroling and defense functions of white blood cells. In platelets, ORAI1 as well as STIM1 is considered to enhance the Ca2+ signal generation, induced in particular by protein tyrosine kinase-linked receptors, such as gly- coprotein VI (GPVI). In mouse, both ORAI1 and STIM1 are implicated in hemostasis and arterial thrombus for- mation.7-10 Murine knockout studies have indicated that the ORAI1-STIM1 Ca2+ signaling contributes to multiple platelet activation processes, such as adhesiveness via integrin activation, granule release, aggregation of platelets, and procoagulant activity.8,11,12 However, in humans, the consequences of defective ORAI1 or STIM1 activity in platelets have not been thoroughly investigat- ed.
In humans, dysfunctional mutations in the ORAI1 or STIM1 genes are very rare.13 The patients described with such mutations usually suffer from severe immunodefi- ciency, congenital myopathy, ectodermal dysplasia or other Ca2+ -linked abnormalities.14-16 The immune deficien- cy can be attributed to the loss-of-function of leukocyte and lymphocyte subsets. Given the severity of the symp- toms, often already evident at a young age, these patients are commonly treated by allogeneic hematopoietic stem cell transplantation. The few patients described do not have an overt bleeding history, although they may peri- odically show autoimmune thrombocytopenia.16
Leukocyte adhesion deficiency type III (LAD-III) forms another severe immune disease, in this case accompanied by epistaxis or petechiae. It is associated with dysfunc- tional mutations in the FERMT3 gene of the integrin-reg- ulation protein, kindlin-3.13 LAD-III patients present with normal platelet count, but impaired platelet adhesion, which may explain the bleeding symptoms.17,18 These patients may require hematopoietic stem cell transplanta- tion during childhood.19
Recently, we developed a microspot-based assay for multiparameter assessment of whole blood thrombus for- mation under flow conditions.20 This assay proved to be valuable in characterizing platelet count and function abnormalities in patients with a variety of genetic bleed- ing disorders. Here, we used this integrative method to investigate overall platelet functions in 9 patients with severe immunodeficiencies, including parents, with a confirmed dysfunctional mutation in ORAI1, STIM1 or FERMT3. For 2 patients, we also investigated the effects of bone marrow transplantation. The results suggest a variable phenotypic penetrance on platelet properties in patients and relatives carrying these mutations.
Methods
Patients and controls
Blood was drawn from patients and healthy controls after full informed consent in compliance with the Declaration of Helsinki. The studies were approved by the local medical ethics
committees. Healthy controls had normal blood cell counts, had not received anti-platelet medication for at least two weeks, and did not have a known history of bleeding or immunodefi- ciency.
Blood samples from patients with immunodeficiency and their parents (all genotyped) were collected at the Department of Pediatrics and Adolescent Medicine, University Medical Center in Freiburg, and at the Children’s Hospital, University of Ulm, Germany (Table 1). Simultaneously, blood samples were taken from healthy donors serving as daily travel controls. Relevant patients' characteristics, including identified mutations and clinical history, are summarized in Table 1. Patient P1 showed homozygosity in the R91W mutation in the ORAI1, known to be linked to severe combined immunodeficiency and defective T-cell Ca2+ signaling.1 Heterozygosity in this mutation was confirmed for both parents (P2 and P3). Patient P4 carried a heterozygous G98S mutation in ORAI1, which was confirmed in the mother (P5). This mutation is reported to be linked to tubular aggregated myopathy-2.21 Patient P6 carried a heterozy- gous R429C mutation of STIM1, which is also linked to T-cell immunity.22 Patient P7 suffered from severe immunodeficiency, linked to a homozygous R573X mutation in kindlin-3 encoded by the FERMT3 gene. The 2 parents (P8 and P9) were heterozy- gous for this mutation. Two of the patients with severe immun- odeficiency, P1 and P7, were eligible for bone marrow transplan- tation. Blood samples in these cases were obtained before and at two or three months after transplantation, respectively. Only limited volumes of blood were available for investigation due to the young age of the patients.
Platelet Ca2+ responses
haematologica | 2018; 103(3)
Platelet function in genetic immune deficiencies
Rises in cytosolic [Ca2+] were measured in platelets after load- i
ing with Fura-2 acetoxymethyl ester (2.5 μM) by calibrated ratio fluorometry.23 Data are presented as nM increases in cytosolic [Ca2+].24
Multiparameter thrombus formation on microspots under flow
Whole blood thrombus formation was assessed under flow conditions, basically as described before.20 In brief, blood sam- ples (0.5 mL) were re-calcified in the presence of thrombin inhibitors, and perfused over a coverslip coated with three microspots (spot 1: type I collagen; spot 2: von Willebrand Factor (VWF)/rhodocytin; spot 3: VWF/fibrinogen) in a transparent par- allel-plate perfusion chamber. Perfusion was at high wall-shear rate of 1600 s-1 for 3.5 minutes (min), or at a low shear rate of 150 s-1 for 6.0 min. The thrombi formed on the microspots were immediately post stained with FITC-labeled anti-fibrinogen mAb (1:100), FITC-anti-CD62P mAb (25 μg/mL) and AF647- annexin A5 (0.25 μg/mL). Representative brightfield and fluores- cence images were captured from each microspot in real-time without fixation. Duplicate runs were performed whenever pos- sible. Analysis of brightfield and fluorescence images, giving 7 parameters per microspot, was performed with pre-defined scripts in Fiji software.25 (Un)supervised heatmaps using scaled parameters (range 0-10) of thrombus formation were construct- ed using R software.
Statistical analysis
Significance of differences was determined with the paired sample t-test (intervention effects) and by principal component analysis (PCA), using the statistical package for social sciences (SPSS, v.11.0).
A detailed description of the methods used is available in the
Online Supplementary Appendix.
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