Page 186 - Haematologica3

M. Nagy et al.
es and other responses, when triggered with low concen- trations of (thrombin or collagen) receptor agonists. These platelets displayed normal aggregation under flow condi- tions, but a decreased procoagulant activity (PS exposure).29
Relatives P2 and P3, heterozygous for the R91W muta- tion, showed approximately 20-50% residual SOCE activity, which is compatible with co-expression of the non-mutated allele in the platelets. Interestingly, Ca2+ entry after thrombin stimulations remained unaffected in patients/relatives P1-3, which is explained by involvement of the SOCE-independent Ca2+ entry mechanism through TRPC6 channels.30
On the other hand, the R98S mutation in ORAI1 carried by patient P4, with assumed gain-of-function,31 was not accompanied by altered SOCE activity after thapsigargin or GPVI stimulation, whereas SOCE was substantially reduced in the relative P5 (confirmed in two independent blood samples). The difference in SOCE activity between P4 and P5 might be explained by a different 'penetration' of the mutated allele in ORAI1 expression in megakary-
ocytes and platelets. However, an alternative explanation is the presence of other modifying genetic or acquired fac- tors between P4 and P5, with possible effects on platelet SOCE activity, on which we can only speculate.
Concerning patient P6 with a heterozygous R429C mutation in STIM1, Ca2+ entry in platelets was impaired after thapsigargin, but not after GPVI or PAR1/4 stimula- tion. In mammalian cell lines, the R429 mutation modu- lates the C-terminal oligomerization and puncta forma- tion of STIM1 with ORAI1.32 Our results suggest that, in platelets, this mutation strongly inhibits the interaction of STIM1 with ORAI1 after full Ca2+ store depletion, such as that provoked by thapsigargin.
Altered platelet functions in thrombus formation accompanied by mutations in ORAI1, STIM1 or FERMT3
Previous murine studies have pointed out that deficien- cy in platelet ORAI1 or STIM1 led to a moderately reduced collagen-dependent thrombus formation with minimal effect on bleeding,8,11 whereas murine deficiency
Figure 4. Altered thrombus formation of blood from patients with ORAI1W/W, ORAI1G/S or STIM1R/C mutations. Whole blood from indicated control subjects and patients was perfused over three microspots [(Sp1, collagen type I; Sp2, von Willebrand Factor (VWF)/rhodocytin; Sp3, VWF/fibrinogen; downstream → upstream)] at wall-shear rate of 1600 s-1. After 3.5 minutes of perfusion, brightfield images were taken from thrombi on all microspots, and platelets were stained for phos- phatidylserine (PS) exposure (AF568-annexin A5), P-selectin expression (AF647 anti-CD62P mAb), and integrin αIIbβ3 activation (FITC anti-fibrinogen mAb). Shown are representative brightfield and fluorescence images at spot 1, obtained with blood from control C2 and patients P1, P4 and P6 (bars, 50 μm).
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haematologica | 2018; 103(3)