Fig. 2. Platelets express C5aR1 and platelet C5aR1 inhibits various endothelial functions.

a Isolated washed murine platelets express C5aR1 as assessed by immune fluorescence microscopy. There was only partial colocalization of C5aR1 (red) with the α-granule marker P-selectin (green). ×630 magnification, scale bars represent 5 µm. Images are representative of four independent experiments. b Histogram showing C5aR1 expression on platelets (gray curve); the black curves show the histogram obtained with an IgG isotype. The histogram is representative of the analysis of four independent platelet samples. c Furthermore, flow cytometry revealed that C5aR1 expression on platelets is dynamic. Upon stimulation with CRP, platelet C5aR1 expression increased in WT platelets. In c–e, data are displayed as the mean ± SEM (n = 4 independent experiments) and are shown as the percentage of control. The expression in the unstimulated WT group represents 100%. *p < 0.05. d Upon stimulation with ADP, WT platelets displayed C5aR1 upregulation at higher concentrations. e Upon treatment with C5a at the indicated concentrations, no significant changes in C5aR1 expression on platelets could be detected. f Primary lung endothelial cells (MLECs) were grown to a confluent monolayer, which was wounded by scratching with a plastic pipette and coincubated with C5ar1^−/− or WT platelets. The absence of C5aR1 resulted in increased endothelial migration after 17 h. Data are displayed as the mean ± SEM (n = 6 independent experiments). The area not populated with cells at the start of the experiment minus the area not populated with cells after 17 h of coincubation with WT platelets represents 100%. *p < 0.05. g Endothelial tube formation by MHEC-5T cells in vitro was significantly increased after incubation with platelets isolated from C5ar1^−/− mice compared platelets isolated from WT mice. Data are shown as the mean ± SEM (n = 4 independent experiments) and displayed as the total tube length after 6.5 h. The cumulative length of endothelial tubes after incubation with WT control platelets represents 100%. *p < 0.05. h Similarly, endothelial tube formation by MLECs was increased after exposure to C5ar1^−/− platelets compared with WT platelets. Data are shown as the mean ± SEM (n = 4 independent experiments). Tube formation after coincubation with WT control platelets represents 100%. *p < 0.05. i Representative images of endothelial tube formation of MHEC-5T after coincubation with WT or C5ar1^−/− platelets. j The supernatant of murine WT platelets significantly inhibited tube formation from endothelial spheroids compared with the supernatant of C5ar1^−/− platelets. Data are presented as the mean ± SEM (n = 3 independent experiments) and are shown as the percentage of the total tube length after 24 h in controls. Tube formation after incubation with WT control platelet supernatant represents 100%. *p < 0.05. k Representative images of sprout formation from endothelial spheroids in both groups. Scale bar represents 100 μm. l Human platelets were preincubated with a C5aR1 antagonist (PMX53) or control peptide. Subsequently, platelets were coincubated with human umbilical vein endothelial cells (HUVECs) on Matrigel. Endothelial tube formation was significantly enhanced by preincubation with PMX53 compared with the control. Data are shown as the mean ± SEM (n = 4 independent experiments with separate donors) of the total tube length after 6.5 h. The cumulative length of endothelial tubes after incubation with platelets preincubated with control peptide represents 100%. *p < 0.05. m There was no significant difference in adhesion to endothelial cells under static conditions between WT and C5aR1-deficient platelets under normoxic conditions. Data are shown as the mean ± SEM (n = 4–5 independent experiments) and as the percentage of control. Adherent WT platelets expressed as the area fraction of platelet-specific staining divided by the number of endothelial cells expressed as the DAPI count per area represents 100%. *p < 0.05 compared to control-stimulated platelets. n Representative images of static platelet adhesion of WT and C5ar1^−/− platelets to endothelial cells. Scale bars represent 200 µm. Two-sided Student’s t test in b, f–h, j, l. ANOVA in c–e, m.