Legends
Figure 1. Platelet responses to strong activation in KHE . (A)
Platelet size and shape change (FSC-H mean fluorescence intensity
(MFI)). (B) GPIIb/IIIa activation upon platelet stimulation measured by
PAC1 binding. (C) Platelet GPIb shedding upon activation (resting GPIb
ratio to activated GPIb). (D,E) Platelet α (D) and dense (E) granule
release upon activation. (H) Percentage of annexin V-positive
(procoagulant) platelets. Triangles correspond to healthy pediatric
donors, circles correspond to patients with KHE/KMP. “CRP+TRAP-6”
denotes activation with combination of 10 µg/mL CRP and 12.5 µM SFLLRN.
“2TR+ADP” denotes activation with combination of 12.5 µM SFLLRN, 200
µM AYGPKF and 2 µM ADP. Statistical significance was calculated using
Mann-Whitney U-test, * corresponds to p<0.05.
Figure 2. Collagen-induced platelet thrombus formation under low
shear rate. Thrombus area was measured after 5, 10 and 15 minutes of
whole blood perfusion through fibrillar collagen-coated flow chamber.
Thrombi were identified by fluorescence (highlighted in red). A –
Thrombi of the thrombocytopenic patient with KMP (Patient 1). B –
Thrombi of the typical non-thrombocytopenic patient with KHE. C –
Thrombi of the typical healthy pediatric donor. C – Comparison between
thrombus areas in healthy controls and patients with KHE/KMP at
different timepoints. Statistical significance was calculated using
Mann-Whitney U-test, * corresponds to p<0.05.
Figure 3. Platelet intracellular signaling in KHE. A –
Cytosolic calcium concentration in resting platelets, calcium increase
upon stimulation with fucoidan (100 μg/mL) or rhodocytin (200 nM),
pre-incubation with 2CP and HB125 with or without sequential stimulation
with 200 nM rhodocytin. B,C – Cytosolic calcium concentration (B) and
fibrinogen binding (C) upon platelet stimulation with low doses of ADP
(2 μM), CRP (2 μg/mL) and TRAP-6 (5 μM). Statistical significance was
calculated using Mann-Whitney U-test, * corresponds to p<0.05.
Figure 4. Aggregation of the platelets of the patients with KHE
is impaired. A – Typical aggregation curves for the LaSca assays upon
activation with 800 nM ADP (black) or 20 μg/mL collagen (red). B,C –
Initial velocities of aggregation upon stimulation with ADP (B) were
mostly diminished in patients with KHE, while maximal aggregation (C)
was normal. D,E – Initial velocities of aggregation (D) and maximal
aggregation upon stimulation with collagen were both decreased in most
of the patients. F – Initial velocity of platelet aggregation upon
stimulation with 10 nM rhodocytin was significantly decreased. G –
Maximum platelet aggregation upon stimulation with 10 nM rhodocytin was
not affected by either of the inhibitors. * represents p<0.05
(the Mann-Whitney U test)