Figure 5.
Terminal complement activation, and role of C5a/C5aR1 signaling in COVID-19 serum-induced platelet adhesion and aggregation on HMEC-1. (A) Endothelial surface area covered by staining for C5b-9 after incubation of ADP-activated HMEC-1 for 2 hours with ctr serum, or serum from patients with COVID-19 or aHUS, taken as positive controls. Results are shown as fold increase of stained surface area after incubation with ctr serum run in parallel. Data are mean ± SD n = 5 independent experiments. Red points represent fold increase values of single experiments. Purple lines are the upper and lower limits of normal range (calculated as shown in supplemental Figure 2). *P < .05 vs control. (B) C5a levels in plasma from healthy subjects (n = 10) or patients with COVID-19 (n = 4). Data are mean ± SD. *P < .05 vs healthy subjects. (C, upper panel) Experimental design. ADP-activated HMEC-1 were exposed for 2 hours to ctr serum or serum from patients with COVID-19 or aHUS taken as positive control, and then perfused for 3 minutes with heparinized whole blood, added with mepacrine, from healthy subjects. COVID-19 serum was added or not with the C5aR1 antagonist CCX168 (200 ng/mL). 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 COVID-19 serum (n = 6) or aHUS serum (n = 4) 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 the groups indicated by horizontal bar. #P < .05 vs ctr serum. (D) Representative images of experiments (green staining) relative to Figure 5C. Original magnification ×200. Scale bar represents 50 μm. (E, upper panel) Experimental design. ADP-activated HMEC-1 were exposed for 2 hours to ctr serum or serum from patients with COVID-19 and then perfused for 3 minutes with heparinized whole blood, added with mepacrine, from healthy subjects. COVID-19 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. (E, lower panel) Endothelial surface area covered by platelet aggregates. Results are shown as fold increase of stained surface area after incubation with COVID-19 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. (F, 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 2 hours to ctr serum or COVID-19 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. (F, lower panel) Endothelial surface area covered by platelet aggregates. Results are shown as fold increase of stained surface area after incubation with COVID-19 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.

Terminal complement activation, and role of C5a/C5aR1 signaling in COVID-19 serum-induced platelet adhesion and aggregation on HMEC-1. (A) Endothelial surface area covered by staining for C5b-9 after incubation of ADP-activated HMEC-1 for 2 hours with ctr serum, or serum from patients with COVID-19 or aHUS, taken as positive controls. Results are shown as fold increase of stained surface area after incubation with ctr serum run in parallel. Data are mean ± SD n = 5 independent experiments. Red points represent fold increase values of single experiments. Purple lines are the upper and lower limits of normal range (calculated as shown in supplemental Figure 2). *P < .05 vs control. (B) C5a levels in plasma from healthy subjects (n = 10) or patients with COVID-19 (n = 4). Data are mean ± SD. *P < .05 vs healthy subjects. (C, upper panel) Experimental design. ADP-activated HMEC-1 were exposed for 2 hours to ctr serum or serum from patients with COVID-19 or aHUS taken as positive control, and then perfused for 3 minutes with heparinized whole blood, added with mepacrine, from healthy subjects. COVID-19 serum was added or not with the C5aR1 antagonist CCX168 (200 ng/mL). 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 COVID-19 serum (n = 6) or aHUS serum (n = 4) 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 the groups indicated by horizontal bar. #P < .05 vs ctr serum. (D) Representative images of experiments (green staining) relative to Figure 5C. Original magnification ×200. Scale bar represents 50 μm. (E, upper panel) Experimental design. ADP-activated HMEC-1 were exposed for 2 hours to ctr serum or serum from patients with COVID-19 and then perfused for 3 minutes with heparinized whole blood, added with mepacrine, from healthy subjects. COVID-19 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. (E, lower panel) Endothelial surface area covered by platelet aggregates. Results are shown as fold increase of stained surface area after incubation with COVID-19 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. (F, 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 2 hours to ctr serum or COVID-19 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. (F, lower panel) Endothelial surface area covered by platelet aggregates. Results are shown as fold increase of stained surface area after incubation with COVID-19 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.

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