Figure 1.
Serum from patients with aHUS induces platelet adhesion and aggregation on microvascular endothelial cells through C5a/C5aR1 signaling. (A, upper panel) Experimental design. HMEC-1 were activated with ADP, exposed for 3 hours to ctr serum or serum from patients with aHUS in remission, and then perfused for 3 minutes with heparinized whole blood, added with mepacrine, from healthy subjects. aHUS serum was added or not with the pan complement inhibitor sCR1 (150 μg/mL). Formation of platelet aggregates was evaluated at the end of blood perfusion (through confocal microscopy). (A, lower panel) Endothelial surface area with positive staining for platelet aggregates. Results are shown as fold increase of stained surface area after incubation with ctr serum run in parallel. Data are mean ± SD; n = 9 independent experiments. Red points represent fold increase values of single experiments. Purple lines are the upper and lower limits of normal range. (B) Representative images of the ultrastructure of aggregates of platelets adhered on HMEC-1 preexposed to aHUS serum, evaluated with scanning electron microscopy at low magnification (left panels), with the corresponding high magnification insets (right panels). Insets show high-power view of same platelet aggregate. Scale bars represent 10 µm (n = 2 experiments). (C, upper panel) Experimental design. HMEC-1 were activated 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. aHUS serum was added or not, with an anti-C5 antibody (135 μg/mL), or the C5aR1 antagonist CCX168 (200 ng/mL), or an anti-C7 antibody (350 μg/mL). Formation of platelet aggregates was evaluated at the end of the 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 ctr serum run in parallel. Data are mean ± SD; n = 4 independent experiments for +anti-C5; n = 3 independent experiments for +C5aR1a and +anti-C7. Red points represent fold increase values of single experiments. Purple lines are the upper and lower limits of normal range. (D, 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 anti-C5 antibody (135 μg/mL). Serum C5a levels were measured before and after incubation. (D, lower panel) C5a concentration in ctr or aHUS serum before and after incubation with ADP-activated HMEC-1. Data are mean ± SD; n = 3 independent experiments. (E) Scheme of treatment with the C5a receptor antagonist CCX168 and serum sample collection in patients with aHUS enrolled in the ACCESS study. (F) Experimental design. HMEC-1 were activated with ADP, exposed for 3 hours to ctr serum or serum from patients with aHUS (taken before treatment with CCX168, day 0, and after 14 days of treatment), and then analyzed for C5b-9 deposits or 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-H) Endothelial surface area with positive staining for platelet aggregates (G) or C5b-9 (H) after incubation of ADP-activated HMEC-1 with ctr serum or serum from patients with aHUS treated with CCX168 collected at days 0 and 14 of the study. Data are mean ± SD of cumulative data from 5 patients. (I) C5a levels in ctr serum or in serum from patients with aHUS taken before treatment with CCX168, day 0, and after 14 days of treatment (n = 5). *P < .05 vs the groups indicated by horizontal bar or, if not indicated, vs all groups. **P < .05 vs +C5aR1a. #P < .05 vs ctr serum.

Serum from patients with aHUS induces platelet adhesion and aggregation on microvascular endothelial cells through C5a/C5aR1 signaling. (A, upper panel) Experimental design. HMEC-1 were activated with ADP, exposed for 3 hours to ctr serum or serum from patients with aHUS in remission, and then perfused for 3 minutes with heparinized whole blood, added with mepacrine, from healthy subjects. aHUS serum was added or not with the pan complement inhibitor sCR1 (150 μg/mL). Formation of platelet aggregates was evaluated at the end of blood perfusion (through confocal microscopy). (A, lower panel) Endothelial surface area with positive staining for platelet aggregates. Results are shown as fold increase of stained surface area after incubation with ctr serum run in parallel. Data are mean ± SD; n = 9 independent experiments. Red points represent fold increase values of single experiments. Purple lines are the upper and lower limits of normal range. (B) Representative images of the ultrastructure of aggregates of platelets adhered on HMEC-1 preexposed to aHUS serum, evaluated with scanning electron microscopy at low magnification (left panels), with the corresponding high magnification insets (right panels). Insets show high-power view of same platelet aggregate. Scale bars represent 10 µm (n = 2 experiments). (C, upper panel) Experimental design. HMEC-1 were activated 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. aHUS serum was added or not, with an anti-C5 antibody (135 μg/mL), or the C5aR1 antagonist CCX168 (200 ng/mL), or an anti-C7 antibody (350 μg/mL). Formation of platelet aggregates was evaluated at the end of the 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 ctr serum run in parallel. Data are mean ± SD; n = 4 independent experiments for +anti-C5; n = 3 independent experiments for +C5aR1a and +anti-C7. Red points represent fold increase values of single experiments. Purple lines are the upper and lower limits of normal range. (D, 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 anti-C5 antibody (135 μg/mL). Serum C5a levels were measured before and after incubation. (D, lower panel) C5a concentration in ctr or aHUS serum before and after incubation with ADP-activated HMEC-1. Data are mean ± SD; n = 3 independent experiments. (E) Scheme of treatment with the C5a receptor antagonist CCX168 and serum sample collection in patients with aHUS enrolled in the ACCESS study. (F) Experimental design. HMEC-1 were activated with ADP, exposed for 3 hours to ctr serum or serum from patients with aHUS (taken before treatment with CCX168, day 0, and after 14 days of treatment), and then analyzed for C5b-9 deposits or 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-H) Endothelial surface area with positive staining for platelet aggregates (G) or C5b-9 (H) after incubation of ADP-activated HMEC-1 with ctr serum or serum from patients with aHUS treated with CCX168 collected at days 0 and 14 of the study. Data are mean ± SD of cumulative data from 5 patients. (I) C5a levels in ctr serum or in serum from patients with aHUS taken before treatment with CCX168, day 0, and after 14 days of treatment (n = 5). *P < .05 vs the groups indicated by horizontal bar or, if not indicated, vs all groups. **P < .05 vs +C5aR1a. #P < .05 vs ctr serum.

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