Fig. 6. Platelet C5aR1 mediates release of the paracrine effector CXCL4 in an Akt- and PKC-dependent fashion.

a Washed murine WT platelets carefully isolated with inhibitors of platelet activation 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). b Results of four array repeats were quantified. For most factors, C5a stimulation (gray) induced a slight upregulation compared to vehicle control (black). The strongest increase was observed for CXCL4. Data are shown as the mean ± SEM (n = 4 independent experiments) and are displayed as intensity values. *p < 0.05. Bonferroni’s post hoc analysis was performed; results are displayed in Supplementary Fig. 9. c Single-cell staining revealed that murine platelets are activated upon C5a stimulation and release CXCL4. Upon stimulation with ADP and C5a, a ring-like staining pattern was observed, indicating platelet activation (arrows). However, C5a-stimulated platelets exhibit reduced CXCL4 content after stimulation with C5a, which cannot be observed to the same extent in the ADP-stimulated group. ×630 magnification, scale bars represent 5 µm. Images are representative of four independent experiments. d Quantification of P-selectin intensity in each platelet reveals upregulation, indicating platelet activation upon stimulation with ADP and C5a. However, no significant difference was detected between ADP and C5a. Data are shown as the mean ± SEM (n = 5 images analyzed) and are displayed as the percentage of control. The P-selectin intensity in vehicle control-stimulated WT platelets represents 100%. *p < 0.05 e Quantification of CXCL4 intensity in relation to P-selectin intensity in each platelet reveals relatively stronger secretion of CXCL4 upon C5a stimulation compared to ADP. Data are shown as the mean ± SEM (n = 5 images analyzed) and are displayed as the percentage of control. The CXCL4 intensity in relation to P-selectin intensity in vehicle control-stimulated WT platelets represents 100%. *p < 0.05. f Single platelets from mice were stimulated with C5a or vehicle control. Granules were quantified by calculating the area of predominantly green staining within the platelets (P-selectin-predominant), predominantly red areas (CXCL4-predominant) as well as overlayed yellow areas (P-selectin-CXCL4 double positive). ×630 magnification, scale bars represent 1 µm. Images are representative of >100 analyzed single platelets. g As described in Supplementary Fig. 10, granule area fractions were quantified. We observed a distinct distribution pattern of granules, with a similar amount of CXCL4-predominant as well as P-selectin-CXCL4 double-positive granules and a significantly lower number of P-selectin-predominant granules, which contain P-selectin over CXCL4. Data are shown as the mean ± SEM (n = 112 single platelets analyzed) and are displayed as the area fraction. p (CXCL4 versus double positive) < 0.001, p (CD62P versus double positive) = 0.0022. h Upon stimulation with C5a, analysis revealed a reduction in CXCL4-predominant granules, while the area fraction of double-positive granules remains unchanged, while the relative area fraction of P-selectin predominant granules increases, thus demonstrating C5a-induced secretion of a subset of α-granules, which contain CXCL4 over P-selectin. Data are shown as the mean ± SEM (n = 166 single platelets analyzed) and are displayed as the area fraction. p (CD62P versus CXCL4) < 0.001. i Conventional ELISA confirmed a significant dose-dependent increase in CXCL4 secretion from human platelets after C5a stimulation. Maximum CXCL4 release is reached at a C5a concentration of 2000 ng/ml. Data are shown as the mean ± SEM (n = 8 independent experiments containing separate donors) and are displayed as the percentage of control. The CXCL4 protein level of vehicle-stimulated platelet supernatant represents 100%. *p < 0.05. j Murine megakaryocytes from WT versus C5ar1^−/− mice were assessed for distribution of CXCL4-predominant granules (red). Displayed are representative images, nuclei are shown in green. ×630 magnification, scale bars represent 10 µm. k As described in Supplementary Fig. 10, granule area fractions were quantified in megakaryocytes from WT versus C5ar1^−/− mice. No significant difference could be detected for CXCL4-predominant granules. Data are shown as the mean ± SEM (n = 10–12 single megakaryocytes analyzed) and are displayed as the area fraction. l RT-PCR analysis detected similar mRNA levels of CXCL4 in megakaryocytes from WT versus C5ar1^−/− mice. Data are presented as the mean ± SEM (n = 5 independent experiments) and are shown as relative expression in relation to GAPDH. m Citrated whole blood from WT and C5ar1^−/− mice was stimulated using different concentrations of C5a (20, 200, 2000 ng/ml) and vehicle control and assessed for platelet activation markers by flow cytometry. For the gating strategy, please refer to the “Methods” section. Activated platelets are detected as CD62PJONA⁺⁺, whereas JONA detects activated GPIIbIIIa. C5a induced platelet activation in WT platelets but not in C5ar1^−/− platelets. Data are shown as the mean ± SEM (n = 4 independent experiments) and are displayed as the percentage of control. The percentage gated CD62PJONA⁺⁺ platelets in the vehicle-stimulated group represents 100%. *p < 0.05. n In order to uncover the signaling downstream of C5aR1 leading to CXCL4 secretion, lysates of WT platelets were generated after vehicle control or C5a stimulation and samples were probed at equal protein concentrations for phospho-proteins as well as non-phosphorylated controls. Platelet C5aR1 ligation induced reproducible PKC substrate phosphorylation (PKC phosphor Ser). As a control, we used the PKC activator PMA. PKC activation was quantified by dividing the phosphorylation signal by the total protein signal as well as unphosphorylated PKC α (Supplementary Fig. 23). Displayed are representative images of at least four independent experiments. o We also checked whether PKC activation also entails CXCL4 release by stimulating washed platelets with C5a and PMA. Both C5a and PMA induced significant CXCL4 release. Data are shown as the mean ± SEM (n = 5–16 independent experiments) and are displayed as the percentage of control of CXCL4 concentration in platelet supernatant measured by ELISA. The mean fluorescence intensity (MFI) of platelets in the vehicle-stimulated group represents 100%. *p < 0.01. p Furthermore, C5a induced an upregulation of phosphorylated Akt (Ser473), PI3K (Ser 47), GSK-3β (Ser 9), p44/42 MAPK (Thr202/Tyr204), PLCβ3 (Ser537). Displayed are representative images of at least four independent experiments. For quantification of phosphorylation, please refer to Supplementary Fig. 23. q Interestingly, C5a induced no regulation of phosphorylated PLCγ2 (Tyr1217) and PKA (α/β/γ catalytic subunit phospho T197). Activated Rap1 (Rap1-GTP) was analyzed using a Rap1 pulldown assay. No significantly altered amounts of Rap1-GTP could be detected. Displayed are representative images of at least four independent experiments. One-way ANOVA with Bonferroni’s post hoc test in b, d, e, g–i, m. Two-sided Student’s t test in k, l, o.