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)