Fig. 1. Complement anaphylatoxin receptor C5aR1 associated with platelets is abundant in ischemic tissue.

WT mice were subjected to hindlimb ischemia as described in the “Methods” section. a One week after the induction of hindlimb ischemia, ischemic muscles revealed abundant complement activation (left side), as demonstrated by the presence of the C3 cleavage product C3b (red). IB4 staining (green) depicts vascular structures; DAPI (blue) depicts nuclei. The nonischemic contralateral control muscle only displayed minimal complement C3b deposition (right side). ×200 magnification, scale bars represent 200 µm. Image is representative of at least four analyzed muscles. b Furthermore, ischemia resulted in significantly increased mRNA levels of the anaphylatoxin receptor C5ar1 compared with nonischemic contralateral hindlimb muscles as internal controls. Data are the mean ± SEM (n = 4 independent experiments) and are shown as the percentage of control. mRNA levels in the contralateral control muscles of WT animals represent 100%. *p < 0.05. c In the ischemic hindlimb tissue, more platelets could be detected compared to the nonischemic control limb using CD42b (red). Displayed is a representative image, IB4 staining (green) depicts vascular structures; DAPI (blue) depicts nuclei. Scale bars represent 10 µm. Image is representative of 11–12 whole-muscle sections per group. d RT-PCR analysis detected significantly increased mRNA levels of the platelet marker Cd42b as well as the platelet marker Cd41 compared with the contralateral unaffected muscles. Data are presented as the mean ± SEM (n = 4 independent experiments) and are shown as the percentage of control. mRNA levels in the contralateral control muscles of WT animals represent 100%. *p < 0.05. e In a static adhesion assay of platelets on endothelial cells (MHEC), the ADP-induced increase in adhesion was significantly greater under ischemic conditions with an ischemically preconditioned endothelial monolayer compared to normoxic endothelial cells. Data are presented as the mean ± SEM (n = 4 independent experiments) and are shown as the percentage of control. ADP-induced platelet adhesion to normoxic endothelial cells expressed as the area fraction of platelet-specific staining represents 100%. *p < 0.05. f Immunofluorescent costaining of murine hindlimb muscle sections 4 as well as 14 days after the induction of ischemia at ×630 magnification (left side) showing colocalization of the platelet markers CD42b (red) and C5aR1 (green). Representative images confirm that the cells are anuclear (DAPI negative, blue). Control staining of muscles from C5ar1^−/− mice (right side) verifies specificity of C5aR1 staining. Scale bars represent 5 µm. Image is representative of at least four analyzed muscles. Two-sided Student’s t test in b, d, e.