Figure 3.
Role of vWF and P-selectin in C5a-induced platelet adhesion and aggregation on HMEC-1. (A) Endothelial surface area stained for vWF or P-selectin on resting or C5a-stimulated HMEC-1. Results are shown as fold increase of stained area on resting HMEC-1. Data are mean ± SD; n = 4 (vWF) or 3 (P-selectin) independent experiments. *P < .05 vs no stimulation. (B, upper panel) Experimental design. Before the experiment, HMEC-1 were left for 2 hours or 16 hours with medium added or not with the RalA inhibitor BQU57 (10 µM). Thereafter, HMEC-1 were activated or not for 10 minutes with ADP and then stimulated or not for 30 minutes with C5a (200 ng/mL). vWF staining was performed at the end of C5a stimulation. (B, lower panel) Endothelial surface area stained for vWF. Data are mean ± SD of 13 fields; n = 1 to 2 experiments. *P < .05 vs the groups indicated by horizontal bar. (C, upper panel) Experimental design. HMEC-1 were activated or not with ADP and then stimulated or not with C5a and exposed or not for 3 hours to ctr serum. vWF staining was performed at the end of C5a stimulation or exposure to serum. (C, lower panel) Endothelial surface area stained for vWF. Mean ± SD of 13 fields. *P < .05. #P < .05 vs the corresponding condition with serum. (D, upper panel) Experimental design. HMEC-1 were activated or not with ADP and then stimulated or not with C5a, exposed for 3 hours to ctr serum and then perfused for 3 minutes with heparinized whole blood, added with mepacrine, from healthy subjects. In 3 of 4 experiments, either an anti-vWF (10 μg/mL) or an anti-P-selectin (20 μg/mL) antibody was added during the C5a stimulation and the exposure to serum. Formation of platelet aggregates was evaluated at the end of blood perfusion. (D, lower panel) Endothelial surface area covered by platelet aggregates on resting (no ADP, no C5a), C5a-stimulated, or ADP-activated/C5a-stimulated HMEC-1 incubated with ctr serum. Results are shown as fold increase of stained surface area on resting HMEC-1 incubated with ctr serum alone run in parallel. Data are mean ± SD; n = 3 to 4 independent experiments. Red points represent fold increase values of single experiments. Purple lines are the upper and lower limits of normal range. *P < .05 vs the groups indicated by horizontal bar.

Role of vWF and P-selectin in C5a-induced platelet adhesion and aggregation on HMEC-1. (A) Endothelial surface area stained for vWF or P-selectin on resting or C5a-stimulated HMEC-1. Results are shown as fold increase of stained area on resting HMEC-1. Data are mean ± SD; n = 4 (vWF) or 3 (P-selectin) independent experiments. *P < .05 vs no stimulation. (B, upper panel) Experimental design. Before the experiment, HMEC-1 were left for 2 hours or 16 hours with medium added or not with the RalA inhibitor BQU57 (10 µM). Thereafter, HMEC-1 were activated or not for 10 minutes with ADP and then stimulated or not for 30 minutes with C5a (200 ng/mL). vWF staining was performed at the end of C5a stimulation. (B, lower panel) Endothelial surface area stained for vWF. Data are mean ± SD of 13 fields; n = 1 to 2 experiments. *P < .05 vs the groups indicated by horizontal bar. (C, upper panel) Experimental design. HMEC-1 were activated or not with ADP and then stimulated or not with C5a and exposed or not for 3 hours to ctr serum. vWF staining was performed at the end of C5a stimulation or exposure to serum. (C, lower panel) Endothelial surface area stained for vWF. Mean ± SD of 13 fields. *P < .05. #P < .05 vs the corresponding condition with serum. (D, upper panel) Experimental design. HMEC-1 were activated or not with ADP and then stimulated or not with C5a, exposed for 3 hours to ctr serum and then perfused for 3 minutes with heparinized whole blood, added with mepacrine, from healthy subjects. In 3 of 4 experiments, either an anti-vWF (10 μg/mL) or an anti-P-selectin (20 μg/mL) antibody was added during the C5a stimulation and the exposure to serum. Formation of platelet aggregates was evaluated at the end of blood perfusion. (D, lower panel) Endothelial surface area covered by platelet aggregates on resting (no ADP, no C5a), C5a-stimulated, or ADP-activated/C5a-stimulated HMEC-1 incubated with ctr serum. Results are shown as fold increase of stained surface area on resting HMEC-1 incubated with ctr serum alone run in parallel. Data are mean ± SD; n = 3 to 4 independent experiments. Red points represent fold increase values of single experiments. Purple lines are the upper and lower limits of normal range. *P < .05 vs the groups indicated by horizontal bar.

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