C5a treatment of platelets results in activation and mediator release. (A-B) Human whole blood was stimulated with ADP (A) or CRP (B), and the expression of C5aR1 on platelets was analyzed by flow cytometry. Data are shown as the mean ± SEM (n = 5) and are displayed as geometric mean fluorescence intensity (geo. MFI). (C-D) Citrated whole blood from WT and C5aR1^−/− mice was stimulated using ADP, C5a, or vehicle control and assessed for the platelet activation marker P-selectin by flow cytometry. C5a-induced platelet activation in WT platelets (C) but not in C5aR1^−/− platelets (D). Data are shown as the mean ± SEM (n = 5) and are displayed as % of control. The % gated CD62⁺ platelets in the vehicle-stimulated group represents 100%. ∗∗P < .001, ∗P < .05 in comparison to the IgG control. (E) Human isolated platelets were incubated on von Willebrand factor (vWF)-coated wells. After careful washing, adherent platelets were quantified. We observed an increase in adhesion upon ADP stimulation; however, no additional effect by C5a stimulation. There was no significant difference between WT and C5aR1^−/− platelets. Data represent mean ± SEM. n = 4 to 8. ∗P < .05. ns = no statistically significant difference could be observed in between WT compared with C5aR1^−/− platelets. (F) Murine platelets were isolated and stimulated using different concentrations of C5a. C5a induced a significant increase in platelet fibrinogen binding. Data are shown as the mean ± SEM (n = 5) and are displayed as geometric mean fluorescence intensity. ∗P < .05. (G) To uncover a potential signaling mechanism downstream of C5aR1 ligation, lysates of WT platelets were generated after vehicle control or C5a stimulation and samples were probed at equal protein concentrations for phosphoproteins as well as nonphosphorylated controls. C5a induced reproducible PKC phosphorylation as detected by a phospho-PKC substrate antibody, which detects phosphorylation of various PKC isoforms when phosphorylated at serine residues surrounded by Arg or Lys at the −2 and +2 positions and a hydrophobic residue at the +1 position.⁸² As control, we used PKC α. Displayed are representative images of at least 4 independent experiments. (H) Quantification of the phosphorylated signal relative to PKC α control signal is displaed. n = 4. The group of control-stimulated supernatant of WT platelets represents 100%. ∗P < .05. (I) Analysis of phosphorylated Akt revealed involvement in C5a-induced signaling in platelets. Displayed are representative images of at least 4 independent experiments. (J) C5aR1^−/− mice were applied in the Matrigel plug in vivo assay, and platelets isolated from WT or C5aR1^−/− mice were coinjected with the Matrigel solution. We observed a reversed phenotype, when WT platelets were used, but not when C5aR1^−/− platelets or WT platelets treated with the small-molecule inhibitor PMX53 were applied. n = 5 to 10 plugs were analyzed. The group of C5aR1^−/− mice reinjected with WT platelets represents 100%. ∗∗P < .01, ∗P < .05. (K) Washed murine WT platelets were stimulated with C5a. The supernatant was analyzed by a membrane-based antibody array. Specific mediators such as CXCL4 (red circles), were upregulated after stimulation with C5a (bottom) compared with the vehicle control (top). The strongest increase was observed for CXCL4. (L) Conventional enzyme-linked immunosorbent assay (ELISA) confirmed a significant dose-dependent increase in CXCL4 secretion from platelets after C5a stimulation. Maximum CXCL4 release is reached at a C5a concentration of 200 ng/mL, which was similar at 500 ng/mL. Data are shown as the mean ± SEM (n = 4-9) and are displayed as percentage of control. The CXCL4 protein level of unstimulated platelet supernatant represents 100%. ∗P < .05.