Page 184 - Haematologica3

M. Nagy et al.
Table 2. Overall comparison of defective store-operated Ca2+ entry (SOCE) and impairment in thrombus formation of individual patients.
For indicated patients, measurements of store-operated Ca2+ entry (SOCE) (see Figure 2) and differential heatmap data relative to control cohort HC1-12 (see Figure 5C) were tabled to obtain an overview of the changes in platelet function.For thrombi (1600 s-1) formed on the three microspots (Sp1-3),all parameters (V1-7) were scored as within nor- mal range (0), decreased (-1) or increased (+1). Per patient, summed scores are indicated (column 'Sum'). In addition, fractions of altered scores are indicated for three phases of thrombus formation.Color intensity corresponds with the relative changes in parameters.Phase 1 refers to platelet adhesion and aggregation (Sp1-3,V1-4),Phase 2 to platelet integrin activation and granule secretion (Sp1-3,V6-7), and Phase 3 to platelet procoagulant activity (Sp1-3,V5). n.d.: not determined; #homozygosity in the indicated mutation; ##heterozygosity in the indicated mutation.
# ## ##
## ##
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exposure. For the patient with homozygous FERMT3 mutation (P7), a more severe reduction on all three spots was seen in comparison to the 2 heterozygous relatives (P8 and P9).
Effects of low platelet count
Considering that whole blood thrombus formation can be influenced by not only the inherited platelet disorder, but also a low platelet count,28 the present heatmaps may reflect both platelet-related properties. To examine this in more detail, the subtraction heatmap data were extended with values of platelet count and SOCE. Unsupervised clustering of the extended dataset indicated that particu- larly spot 1, parameters (V1-4) clustered with platelet count, while spot 2 parameters (V3-5) and PS exposure (Sp1V5, Sp2V5) clustered with altered SOCE (Online Supplementary Figure S1).
As an alternative approach, we used the primary (non- normalized) data of thrombus formation (Sp1-3, V1-7), platelet count and SOCE for PCA. This revealed a similar pattern, in that the majority of spot 1 parameters together with platelet count determined component 1, whereas most spot 2 parameters determined component 2 (Online Supplementary Figure S2A). Pearson regression coefficient confirmed a correlation of spot 1 parameters with platelet count, and also a correlation of platelet PS exposure (Sp1V5, Sp3V5) with SOCE (Online Supplementary Figure S2B).
This information was then used to interpret the alter- ations in thrombus formation for individual patients (see clustered heatmap in Online Supplementary Figure S1). Concerning the patient (P1) with homozygous R91W mutation in ORAI1, with normal platelet count (207x109/L) and near abolished SOCE, thrombus forma- tion was near normal on spot 1 (collagen), but markedly reduced on spot 2 (VWF/rhodocytin) and spot 3 (VWF/fib- rinogen). The impaired Ca2+ signal was linked to a low PS exposure on all spots (Online Supplementary Table S1). On the other hand, for the patients P2 and P3 with heterozy- gous R91W mutation in ORAI1 (relatively low platelet counts of 115-156x109/L, and 20-60% of normal SOCE,
Figure 2. Variably defective Ca2+ entry in platelets from patients with ORAI1W/W, ORAI1G/S or STIM1R/C mutations. Fura-2-loaded platelets in Hepes buffer with 0.1 mM EGTA were stimulated with thapsigargin (1.0 μM), and after a defined time with CaCl2 (2 mM). Platelets were analyzed from 12 healthy home controls (HC1-12), 6 healthy travel controls (C1-6), and the indicated patients/relatives (P1-6). Platelets from patient P1 were also analyzed after bone marrow (BM) transplantation. Shown are maximal increases in Ca2+. Dotted lines indicate
range of store-operated Ca
entry (SOCE) levels (CaCl2-induced Ca
2+
rise after
2+
thapsigargin) for platelets from home controls (mean ± SD, for HC1-12).
respectively), particularly parameters for spot 1 were below normal, with a reduced PS exposure.
Concerning patients P4 and P5, heterozygous for the G98S mutation in ORAI1 (low platelet counts of 70 and 114x109/L; 100% and 30% of normal SOCE, respectively), thrombus formation on spot 1 was reduced, while param- eters of thrombus formation on spots 2-3 (including PS exposure) were only reduced for patient P5. In P4 (but not P5), a gain-of-function of Ca2+ channel activity was appar- ent from an increased PS exposure on spots 2-3. For patient P6, heterozygous for the R429C mutation in
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