Page 185 - Haematologica3

Platelet function in genetic immune deficiencies
AB
STIM1, thrombus formation on spot 1 (collagen) was nor- mal in spite of the abolished SOCE. Together, this rein- forced the idea that a low platelet count rather than altered SOCE determines the thrombus formation on col- lagen, but not on the other surfaces.
Control experiments with reconstituted blood con- firmed that, for control donors, lowering of the platelet count to 100x109/L had only a limited effect on particular parameters of thrombus formation limited to spot 1, whereas lowering to 50x109/L affected thrombus forma- tion on all spots 1-3 (Online Supplementary Figure S3). Taken together with the predictive effect of platelet count in the PCA, this suggests that a relatively low count affects thrombus formation under flow conditions more severely when combined with a reduced platelet functionality.
Patterns of aberrant whole-blood thrombus formation at low shear rate
Using remaining samples from patients/relatives P1-6, we went on to perform whole blood flow measurements at a low shear rate of 150 s-1. Subtraction heatmap analy- sis of the normalized parameter values, in comparison to control data from HC1-12 cohort, indicated for all patients with ORAI1 mutations (P1-5) a consistent pattern of reduced thrombus formation parameters on spot 1 (Online Supplementary Figure S4A-C). In particular for patients P2 and P3, markers of platelet activation appeared to be reduced (PS exposure, P-selectin expression and αIIbβ3 acti- vation). Once again, for patient P6 with STIM1 mutation, most parameters were unchanged.
Principal component analysis of the low shear data again pointed to a linkage of spot 1 parameters with platelet count and a linkage of Sp1V5 (PS exposure) with SOCE activity (Online Supplementary Figure S5A and B).
Partial recovery of whole blood thrombus formation after bone marrow transplantation
Blood samples from 2 patients were also obtained after bone marrow transplantation (P1 after 2 months, P7 after 3 months), and used for platelet activation and thrombus
formation studies. Transplantation of patient P1 led to a partially recovered SOCE from 20% to 60% of normal (Figure 2). After transplantation, parameters of thrombus formation were particularly reduced on spot 1 (paralleling a reduction in platelet count from 207 to 74x109/L), but were unchanged for spot 2, and enhanced for spot 3 (Figure 6). Transplantation of patient P7 resulted in an overall improvement of most parameters on all spots (platelet count decreased from 182 to 168x109/L).
Discussion
In the present study, we used a multiparameter test of whole blood thrombus formation under flow conditions, as a proxy measurement of hemostatic activity20 to char- acterize quantitative and qualitative platelet abnormalities in rare patients and their relatives with severe immunode- ficiencies, linked to signaling protein defects and muta- tions in the ORAI1, STIM1 and FERMT3 genes. So far, functional effects of the ORAI1 and STIM1 mutations have only been described for human immune cells or cell lines. Hence, the present data are the first to report on comparative alterations in platelet SOCE and platelet functions in as many as 9 genotyped patients/relatives.
Aberrations in SOCE accompanied by mutations in ORAI1 or STIM1
Earlier work with bone marrow chimeric mice with megakaryocytic deficiency in Orai1 or Stim1 demonstrat- ed a prominent role of these Ca2+-entry regulating proteins in platelet calcium homeostasis and activation, including PS exposure.11,29 Our human data are compatible with these findings in that SOCE after thapsigargin or GPVI stimulation appeared to be impaired in platelets from patient P1 with a homozygous ORAI1W/W mutation. A sim- ilar impairment of SOCE has been reported in platelets from Orai1R93W mice, i.e. a loss-of-function mutation orthologous to the human ORAI1 R91W variant.29 Platelets from the latter mice showed a defect in Ca2+ flux-
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Figure 3. Defective Ca2+ entry in platelets from a patient with a heterozygous R429C mutation in STIM1. Fura-2-loaded platelets from patient P6 (STIM1R/C) and travel control C6, suspended in Hepes buffer with 0.1 mM EGTA, were stim- ulated with convulxin (Cvx, 50 ng/mL), thrombin (Thr, 4 nM) or thapsigargin (Thaps, 1.0 μM), after which CaCl2 (2 mM) was given to induce Ca2 entry. Representative traces (A) and quantification (B) of Ca2+ rises in platelets from control subject and patient P6.