Figure 2.
C5a and normal human serum (ctr) induces platelet adhesion and aggregation on HMEC-1. (A) Experimental design. HMEC-1, ADP-activated or resting, were exposed for 3 hours to ctr serum with or without the addition of C5a (200 ng/mL) or to aHUS serum, and then perfused for 3 minutes with heparinized whole blood, added with mepacrine, from healthy subjects. C5a-additioned serum was tested in the presence or absence of sCR1 (150 μg/mL) or the C5aR1 antagonist CCX168 (200 ng/mL). Formation of platelet aggregates was evaluated at the end of the blood perfusion. (B-C) Effect of C5a-additioned ctr serum on formation of platelet aggregates on HMEC-1 (B, ADP-activated; C, resting). Results are shown as fold increase of stained surface area after incubation with ctr serum alone run in parallel. Data are mean ± SD; n = 3 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. #P < .05 vs ctr serum. (D, upper panel) Experimental design. HMEC-1 were activated or not with ADP for 10 minutes and then stimulated or not with C5a for 30 minutes, exposed for 3 hours to ctr serum, and perfused for 3 minutes with heparinized whole blood, added with mepacrine, from healthy subjects. On selected slides, ADP-activated cells were exposed to aHUS serum, taken as the positive control. Formation of platelet aggregates was evaluated at the end of the blood perfusion. (D, lower panel) Endothelial surface area covered by platelet aggregates after incubation with ctr serum of resting (no ADP, no C5a), ADP-activated, C5a-stimulated or ADP-activated/C5a-stimulated HMEC-1; or after incubation with aHUS serum. Data are mean ± SD; n = 4 independent experiments. *P < .05 vs the groups indicated by horizontal bar. #P < .05 vs resting and ADP-activated HMEC-1 + ctr serum. (E) Representative images of immunostaining for C5aR1 (red staining) on resting or ADP-activated HMEC-1. Blue: DAPI staining. Original magnification: ×400. Scale bar represents 50 μm. (F, upper panel) Experimental design. HMEC-1 were activated or not with ADP for 10 minutes and then stimulated or not with C5a for 30 minutes, exposed for 3 hours to ctr serum, and perfused for 3 minutes with heparinized whole blood, added with mepacrine, from healthy subjects. In 3 of 4 experiments, additional samples were run in which either sCR1 (150 μg/mL) or the C5aR1 antagonist CCX168 (200 ng/mL) were added during the C5a stimulation and the exposure to serum. Formation of platelet aggregates was evaluated at the end of blood perfusion. (F, lower panel) Endothelial surface area covered by platelet aggregates after incubation with ctr serum of resting, ADP-activated, C5a-stimulated or ADP-activated/C5a-stimulated HMEC-1. Data are mean ± SD; n = 3 to 4 independent experiments. *P < .05 vs the groups indicated by horizontal bar. #P < .05 vs resting. (G, upper panel) Experimental design. HMEC-1 were activated or not with ADP for 10 minutes and then stimulated or not with C5a for 30 minutes and 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. (G, lower panel) Endothelial surface area covered by platelet aggregates on resting, ADP-activated, C5a-stimulated or ADP-activated/C5a-stimulated HMEC-1. Data are mean ± SD; n = 3 independent experiments.

C5a and normal human serum (ctr) induces platelet adhesion and aggregation on HMEC-1. (A) Experimental design. HMEC-1, ADP-activated or resting, were exposed for 3 hours to ctr serum with or without the addition of C5a (200 ng/mL) or to aHUS serum, and then perfused for 3 minutes with heparinized whole blood, added with mepacrine, from healthy subjects. C5a-additioned serum was tested in the presence or absence of sCR1 (150 μg/mL) or the C5aR1 antagonist CCX168 (200 ng/mL). Formation of platelet aggregates was evaluated at the end of the blood perfusion. (B-C) Effect of C5a-additioned ctr serum on formation of platelet aggregates on HMEC-1 (B, ADP-activated; C, resting). Results are shown as fold increase of stained surface area after incubation with ctr serum alone run in parallel. Data are mean ± SD; n = 3 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. #P < .05 vs ctr serum. (D, upper panel) Experimental design. HMEC-1 were activated or not with ADP for 10 minutes and then stimulated or not with C5a for 30 minutes, exposed for 3 hours to ctr serum, and perfused for 3 minutes with heparinized whole blood, added with mepacrine, from healthy subjects. On selected slides, ADP-activated cells were exposed to aHUS serum, taken as the positive control. Formation of platelet aggregates was evaluated at the end of the blood perfusion. (D, lower panel) Endothelial surface area covered by platelet aggregates after incubation with ctr serum of resting (no ADP, no C5a), ADP-activated, C5a-stimulated or ADP-activated/C5a-stimulated HMEC-1; or after incubation with aHUS serum. Data are mean ± SD; n = 4 independent experiments. *P < .05 vs the groups indicated by horizontal bar. #P < .05 vs resting and ADP-activated HMEC-1 + ctr serum. (E) Representative images of immunostaining for C5aR1 (red staining) on resting or ADP-activated HMEC-1. Blue: DAPI staining. Original magnification: ×400. Scale bar represents 50 μm. (F, upper panel) Experimental design. HMEC-1 were activated or not with ADP for 10 minutes and then stimulated or not with C5a for 30 minutes, exposed for 3 hours to ctr serum, and perfused for 3 minutes with heparinized whole blood, added with mepacrine, from healthy subjects. In 3 of 4 experiments, additional samples were run in which either sCR1 (150 μg/mL) or the C5aR1 antagonist CCX168 (200 ng/mL) were added during the C5a stimulation and the exposure to serum. Formation of platelet aggregates was evaluated at the end of blood perfusion. (F, lower panel) Endothelial surface area covered by platelet aggregates after incubation with ctr serum of resting, ADP-activated, C5a-stimulated or ADP-activated/C5a-stimulated HMEC-1. Data are mean ± SD; n = 3 to 4 independent experiments. *P < .05 vs the groups indicated by horizontal bar. #P < .05 vs resting. (G, upper panel) Experimental design. HMEC-1 were activated or not with ADP for 10 minutes and then stimulated or not with C5a for 30 minutes and 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. (G, lower panel) Endothelial surface area covered by platelet aggregates on resting, ADP-activated, C5a-stimulated or ADP-activated/C5a-stimulated HMEC-1. Data are mean ± SD; n = 3 independent experiments.

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