Figure 4.
Role of vWF and exocytosis of WPBs in aHUS serum-induced platelet adhesion and aggregation on HMEC-1. (A, upper panel) Experimental design. HMEC-1 were activated with ADP and then exposed for 3 hours to ctr or aHUS serum. aHUS serum was added or not with sCR1 (150 μg/mL) or the C5aR1 antagonist CCX168 (200 ng/mL). vWF staining was performed at the end of exposure to serum. (A, lower panel) Endothelial surface area stained for vWF. Results are shown as fold increase of stained area after incubation, with aHUS serum (n = 4) vs ctr serum run in parallel. Data are mean ± SD. *P < .05 vs all groups. (B, upper panel) Experimental design. ADP-activated HMEC-1 were exposed for 3 hours to ctr or aHUS serum and then perfused for 3 minutes with heparinized whole blood, added with mepacrine, from healthy subjects. aHUS serum was added or not with an anti-vWF antibody (10 μg/mL). Formation of platelet aggregates was evaluated at the end of blood perfusion. (B, lower panel) Endothelial surface area covered by platelet aggregates. Results are shown as fold increase of surface area covered by platelet aggregates after incubation with aHUS serum (n = 3) vs ctr serum run in parallel. Data are mean ± SD. Red points represent fold increase values of single patients. Purple lines are the upper and lower limits of normal range. *P < .05 vs all groups. (C, upper panel) Experimental design. Before the experiment, HMEC-1 were left for 16 hours in medium with or without the RalA inhibitor BQU57 (10 µM). Thereafter, HMEC-1 were activated for 10 minutes with ADP, exposed for 3 hours to ctr or aHUS serum, and then perfused for 3 minutes with heparinized whole blood, added with mepacrine, from healthy subjects. Formation of platelet aggregates was evaluated at the end of blood perfusion. (C, lower panel) Endothelial surface area covered by platelet aggregates. Results are shown as fold increase of stained surface area after incubation with aHUS serum (n = 3) vs ctr serum run in parallel. Data are mean ± SD. Red points represent fold increase values of single patients. Purple lines are the upper and lower limits of normal range. *P < .05 vs all groups. (D) Representative images of experiments (green staining) relative to Figure 4C. Original magnification ×200. Scale bar represents 50 μm.

Role of vWF and exocytosis of WPBs in aHUS serum-induced platelet adhesion and aggregation on HMEC-1. (A, upper panel) Experimental design. HMEC-1 were activated with ADP and then exposed for 3 hours to ctr or aHUS serum. aHUS serum was added or not with sCR1 (150 μg/mL) or the C5aR1 antagonist CCX168 (200 ng/mL). vWF staining was performed at the end of exposure to serum. (A, lower panel) Endothelial surface area stained for vWF. Results are shown as fold increase of stained area after incubation, with aHUS serum (n = 4) vs ctr serum run in parallel. Data are mean ± SD. *P < .05 vs all groups. (B, upper panel) Experimental design. ADP-activated HMEC-1 were exposed for 3 hours to ctr or aHUS serum and then perfused for 3 minutes with heparinized whole blood, added with mepacrine, from healthy subjects. aHUS serum was added or not with an anti-vWF antibody (10 μg/mL). Formation of platelet aggregates was evaluated at the end of blood perfusion. (B, lower panel) Endothelial surface area covered by platelet aggregates. Results are shown as fold increase of surface area covered by platelet aggregates after incubation with aHUS serum (n = 3) vs ctr serum run in parallel. Data are mean ± SD. Red points represent fold increase values of single patients. Purple lines are the upper and lower limits of normal range. *P < .05 vs all groups. (C, upper panel) Experimental design. Before the experiment, HMEC-1 were left for 16 hours in medium with or without the RalA inhibitor BQU57 (10 µM). Thereafter, HMEC-1 were activated for 10 minutes with ADP, exposed for 3 hours to ctr or aHUS serum, and then perfused for 3 minutes with heparinized whole blood, added with mepacrine, from healthy subjects. Formation of platelet aggregates was evaluated at the end of blood perfusion. (C, lower panel) Endothelial surface area covered by platelet aggregates. Results are shown as fold increase of stained surface area after incubation with aHUS serum (n = 3) vs ctr serum run in parallel. Data are mean ± SD. Red points represent fold increase values of single patients. Purple lines are the upper and lower limits of normal range. *P < .05 vs all groups. (D) Representative images of experiments (green staining) relative to Figure 4C. Original magnification ×200. Scale bar represents 50 μm.

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