Page 182 - Haematologica3

M. Nagy et al.
Table 1. Subjects’ characteristics.
Subject
HC1-12 (home C) C1-5 (travel C) P1 (child)
P1 (BM)
P2 (mother P1) P3 (father P1) P4 (child)
P5 (mother P4)
P6 (adult) P7 (child)
P7 (BM)
P8 (father P7)
P9 (mother P7)
Mutation
None assumed
None assumed ORAI1 (R91W) W/W#
BM transplanted
ORAI1 (R91W) R/W## ORAI1 (R91W) R/W## ORAI1 (G98S) G/S##
ORAI1 (G98S) G/S##
STIM1 (R429C) R/C## FERMT3 (R573X) X/X#
BM transplanted
FERMT3 (R573X) R/X##
FERMT3 (R573X) R/X##
Platelet count (x 109/L)
166-390 156-291 207
74+
115+ 156+ 114+
70+
150 182
168
126+
142+
MPV Hematocrit (fL) (L/L)
9.1-12.1 0.36-0.49 9.0-11.6 0.36-040 n.d. 0.41
Clinical history
None
None Immunodeficiency, myopathy, anhydrosis
8.9+ 0.23+ Thrombocytopenia, stabilized myopathy
8.8+ 0.36 9.2 0.32+ 7.7+ 0.34+
9.2 0.37
Mild thrombocytopenia
No known history of bleeding Thrombocytopenia (recurrent), myopathy with tubular aggregates, anhydrosis Thrombocytopenia, myopathy with tubular aggregates, anhydrosis
No known history of bleeding
n.d. n.d.
7.5+ 0.31+ Immunodeficiency,
6.0+ 0.33+
8.3 0.42
6.8+ 0.39
severe vaginal bleeding, leukocytosis
No known clinical symptoms No known history of bleeding No known history of bleeding
542
HC: home control; C: travel control; n.d.: not determined; MPV: mean platelet volume; P: patient; BM: bone marrow. +Outside of normal range; #homozygosity in the indicated mutation; ##heterozygosity in the indicated mutation.
Results
Variable aberrances in SOCE in platelets from patients with ORAI1 or STIM1 mutations
Blood samples were obtained from 3 patients with a mutation in the Ca2+ flux-regulating proteins ORAI1 or STIM1, as well as from parents carrying the same muta- tion. For comparison, blood samples were also taken from two cohorts of healthy subjects, i.e. a group of normal home controls (HC1-12) and a group of normal travel con- trols (C1-6). Using Fura-2-loaded platelets, we first evalu- ated the alterations in Ca2+ signaling in response to the GPVI receptor agonist, convulxin, the PAR receptor ago- nist, thrombin, and the sarco/endoplasmic reticulum Ca2+- ATPase (SERCA) inhibitor, thapsigargin. In each case, the platelets were first stimulated with agonist in Ca2+-free medium, after which extracellular CaCl2 was added to measure secondary Ca2+ entry.
In comparison to control platelets, platelets from patient P1, carrying a homozygous ORAI191W/W mutation, responded normally to each agonist, but showed a sub- stantially reduced Ca2+ entry following stimulation with convulxin but not thrombin (Figure 1A and B). Markedly, in this patient's platelets, Ca2+ entry after stimulation with thapsigargin (as a default condition for SOCE) was com- pletely abolished. This pointed to a complete absence of the STIM1-ORAI1 pathway, similar to that established for platelets from STIM1- or ORAI1-deficient- mice.7,8 Flow cytometric evaluation indicated that convulxin-stimulated platelets from P1 showed a reduced phosphatidylserine
(PS) exposure [7±1% vs. 28±2% for control platelets, mean±SEM, n=3; P<0.05], but were not altered in integrin αIIbβ3 activation or P-selectin expression (P>0.10). Platelets from the patient's parents (both with confirmed heterozy- gosity) were diminished in SOCE, but to a different extent (Figure 2). Platelets from the mother (P2) showed a nearly annulled Ca2+ entry, whereas platelets from the father (P3) were less severely reduced when compared to platelets from two cohorts of healthy controls. It was possible to obtain a second blood sample from patient P1 at two months after bone marrow transplantation. The platelets showed a 50% recovery in SOCE signal after thapsigargin stimulation (SOCEP1: 22 nM vs. SOCEP1-BM: 575 nM).
Platelets from one patient (P4), carrying a heterozygous mutation ORAI189G/S in the same protein region, respond- ed differently. These platelets displayed a high SOCE sig- nal (Ca2+ entry after thapsigargin). In contrast, platelets from parent P5 (also carrying the mutation) were greatly reduced in SOCE (Figure 2). This difference was con- firmed for a second set of blood samples (data not shown). A different picture was obtained with patient P6 with a heterozygous mutation in STIM1429R/C.22 In these platelets, increases in Ca2+ evoked by convulxin/CaCl2 or throm- bin/CaCl2 were in the normal range, whereas SOCE after thapsigargin was completely abolished (Figure 3A and B).
Phenotypic analysis of platelets was also performed of a LAD-III patient (P7) who carried a homozygous FERMT3573X/X mutation, and presented with immunodefi- ciency and a history of bleeding.26 Flow cytometry indicat- ed an almost complete lack of integrin αIIbβ3 activation in
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