Figure 5.
Regulation of CXCL4 secretion from platelets by C5a. (A) To determine the secretion of CXCL4 from platelets upon exposure to different C5a concentrations (20-500 ng/mL), lysates were prepared from the stimulated platelets, and levels of remaining intracellular CXCL4 were determined by ELISA. Data are shown as the mean ± SEM (n = 4-7) and are displayed as % of control. The CXCL4 protein level of unstimulated platelet supernatant represents 100%. ∗P < .05 in comparison to control. (B) Murine megakaryocytes from WT vs C5aR1−/− mice were assessed for the content of CXCL4-predominant granules (red). Displayed are images representative of 10 to 12 single megakaryocytes analyzed; nuclei are shown in green. 630× original magnification, scale bars represent 10 μm. (C) We quantified CXCL4 staining in 11 vs 14 images of megakaryocytes from WT or C5aR1−/− mice. This quantification showed no difference in the area of the CXCL4 signal in relation to the area of the whole cell between both genotypes. Data represent mean ± SEM. n = 11 to 14. n.s. = no statistically significant difference was observed. (D-G) Serotonin levels (indicating dense granules) or β-hexosaminidase levels (indicating lysosomes) were analyzed in the supernatant of isolated murine platelets after stimulation with activators, as indicated in the figure. We observed no relevant secretion after C5a stimulation, in contrast to classical platelet activators. Data are shown as the mean ± SEM (n = 4-9) and are displayed as % of control. The respective protein level of unstimulated platelet supernatant represents 100%. ∗∗∗P < .001, ∗∗P < .01, ∗P < .05 in comparison to control. ns = no significant difference was measured. (H) Single platelets from WT mice were stimulated with C5a or vehicle control. Granules were stained green for P-selectin and red for CXCL4, yellow areas represent overlay, that is, P-selectin-CXCL4 double-positive α granules. 630× original magnification, scale bars represent 1 μm. Images are representative of >100 analyzed single platelets. (I) Washed murine platelets from WT or C5aR1−/− mice were stimulated with C5a. The supernatant was analyzed by ELISA for the level of CXCL4. C5a stimulation yielded significant CXCL4 secretion only in WT platelets but not in C5aR1−/− platelets. Data are shown as the mean ± SEM. n = 8. ns = no significant difference was measured; ∗P < .05; ∗∗P < .01.

Regulation of CXCL4 secretion from platelets by C5a. (A) To determine the secretion of CXCL4 from platelets upon exposure to different C5a concentrations (20-500 ng/mL), lysates were prepared from the stimulated platelets, and levels of remaining intracellular CXCL4 were determined by ELISA. Data are shown as the mean ± SEM (n = 4-7) and are displayed as % of control. The CXCL4 protein level of unstimulated platelet supernatant represents 100%. ∗P < .05 in comparison to control. (B) Murine megakaryocytes from WT vs C5aR1−/− mice were assessed for the content of CXCL4-predominant granules (red). Displayed are images representative of 10 to 12 single megakaryocytes analyzed; nuclei are shown in green. 630× original magnification, scale bars represent 10 μm. (C) We quantified CXCL4 staining in 11 vs 14 images of megakaryocytes from WT or C5aR1−/− mice. This quantification showed no difference in the area of the CXCL4 signal in relation to the area of the whole cell between both genotypes. Data represent mean ± SEM. n = 11 to 14. n.s. = no statistically significant difference was observed. (D-G) Serotonin levels (indicating dense granules) or β-hexosaminidase levels (indicating lysosomes) were analyzed in the supernatant of isolated murine platelets after stimulation with activators, as indicated in the figure. We observed no relevant secretion after C5a stimulation, in contrast to classical platelet activators. Data are shown as the mean ± SEM (n = 4-9) and are displayed as % of control. The respective protein level of unstimulated platelet supernatant represents 100%. ∗∗∗P < .001, ∗∗P < .01, ∗P < .05 in comparison to control. ns = no significant difference was measured. (H) Single platelets from WT mice were stimulated with C5a or vehicle control. Granules were stained green for P-selectin and red for CXCL4, yellow areas represent overlay, that is, P-selectin-CXCL4 double-positive α granules. 630× original magnification, scale bars represent 1 μm. Images are representative of >100 analyzed single platelets. (I) Washed murine platelets from WT or C5aR1−/− mice were stimulated with C5a. The supernatant was analyzed by ELISA for the level of CXCL4. C5a stimulation yielded significant CXCL4 secretion only in WT platelets but not in C5aR1−/− platelets. Data are shown as the mean ± SEM. n = 8. ns = no significant difference was measured; ∗P < .05; ∗∗P < .01.

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