Page 183 - Haematologica3

Platelet function in genetic immune deficiencies
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response to receptor agonists, as observed in another patient carrying this mutation.27 In platelets from the 2 heterozygous parents (P8, P9), αIIbβ3 integrin activation was in the normal range (data not shown).
Lower range blood cell counts in patients with ORAI1, STIM1 or FERMT3 mutations
Table 1 provides an overview of the clinical histories of patients P1-9 including hematologic parameters deter- mined in freshly isolated blood samples. In all patients, with the exception of P1 and P7, platelet counts were between 70 and 150x109/L, which are slightly below the normal ranges presented in both control cohorts. In the majority of patients, also hematocrit levels were in the lower range of normal, i.e. between 0.31 and 0.42 L/L. After bone marrow transplantation (2-3 months), platelet count in P1 and P7 had restored to 74 and 168x109/L, respectively.
Different patterns between patients of aberrant whole-blood thrombus formation at high shear rate
To obtain detailed insight into the hemostatic potential of the patients' platelets, whole blood samples were used for multiparameter assessment of thrombus formation under flow at high wall-shear rate of 1600 s-1. Samples from corresponding control subjects (C1-6) and home con- trol subjects (HC1-12) were again used for comparison. This high-throughput method, previously established,20 allowed simultaneous examination of platelet adhesion, aggregation and activation at three microspots: spot 1 with coated type I collagen (involving platelet receptors: GPIb-V-IX, GPVI and integrin α2β1); spot 2 with coated VWF/rhodocytin (receptors GPIb-V-IX and CLEC-2); and spot 3 with coated VWF/fibrinogen (receptors GPIb-V-IX and αIIbβ3). From each microspot, microscopic brightfield images were recorded to assess platelet adhesion and aggregation (parameters V1-4); in parallel, fluorescence images were recorded in three colors for detection of PS
Figure 1. Defective Ca2+ entry in platelets from a patient with a homozygous R91W mutation in ORAI1. Fura-2-loaded platelets from patient P1 (ORAI1W/W) and travel control C1 were used, suspended in Hepes buffer with 0.1 mM EGTA. Rises in cytosolic Ca2+ were measured in time upon stimulation with convulxin (Cvx, 50 ng/mL), thrombin (Thr, 4 nM) or thapsigargin (Thaps, 1.0 μM). After a defined time, CaCl2 (2 mM) was added to induce Ca2+ entry. Representative traces (A) and quantification (B) of Ca2+ increases in platelets from control sub- ject and patient P1. Note that the maximal Ca2+ rise in response to CaCl2 follow- ing thapsigargin was used as a measure of store-operated Ca2+ entry (SOCE) capacity.
exposure, P-selectin expression and αIIbβ3 activation (para- meters V5-7).
Representative examples of brightfield and fluorescence images from spot 1 for blood samples of control C2 and immunodeficient patients with ORAI1 or STIM1 muta- tion (P1, P4, and P6) are shown in Figure 4. Regarding the patient blood samples, the images show patterns of platelet adhesion, aggregation and activation that vary from from normal to reduced, as was also observed for spot 2 (VWF/rhodocytin) and spot 3 (VWF/fibrinogen) (see below).
A heatmap was constructed with data from all analyzed images (3 spots × 7 parameters) for the cohort of 12 nor- mal home controls (HC1-12), the 6 travel controls (C1-6), and the 9 individual patients/relatives with mutations in ORAI1 (P1-5), STIM1 (P6) or FERMT3 (P7-9), in which all values were normalized to a scale of 0-10 per parameter (Figure 5A). In the derived subtraction heatmap of Figure 5B, the patient data were expressed relative to those from the home controls. This subtraction confirmed an overall high similarity between the values of the two control groups (HC1-12 and C1-6), with the exception of a param- eter reflecting platelet deposition on spot 2. Datasets for individual subjects C1-6 were within the normal ranges (data not shown). This confirmed the usefulness of the multi-parameter test,20 and underscored the quality of the analyzed blood samples. For each patient/relative, we arbitrarily set a relevant effect threshold, i.e. when outside the range of mean±2 SD of the control group (HC1-12). This filter produced a 'relevant' subtraction heatmap, indi- cating distinct patterns of altered parameters of thrombus formation for individual patients P1-9 (Figure 5C).
Figure 5C underlines that, overall, multiple parameters on spot 1 were reduced for patients P2-5, whereas espe- cially parameters on spots 2-3 were reduced for patients P1 and P5. Interestingly, patient P4 carrying the assumed gain-of-function mutation ORAI1G98S (but not relative P5 with low SOCE) showed a typical increase in platelet PS
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