aPC, independent of its anticoagulant property, inhibits apoptosis in human podocytes, which predominantly express PAR-2 and PAR-3. (A-C) aPC reduces dose-dependently and independent of its anticoagulant effect PAN-induced apoptosis in podocytes. Bar graphs summarizing the frequency of apoptosis in PAN-stressed human podocytes treated with different concentrations of aPC (A) or treated with aPC preincubated 1:1 with the antibody HAPC1573, which specifically blocks the anticoagulant properties of aPC (B). Representative images of TUNEL assay with fluorescent-labeled nucleotides (green) and Hoechst 33258 nuclear counterstain (blue, C). (D) PAR-2 and PAR-3 are expressed in human podocytes. Representative images show expression of PARs and EPCR in human podocytes and HUVECs (positive control) as determined by semiquantitative RT-PCR (left panel) and Western blot (right panel). (E) Immunofluorescence images showing strong colocalization (yellow) of PAR-2 and PAR-3, but not of PAR-1 or EPCR (all red) with the podocyte specific marker synaptopodin (green) in normal human kidney samples. PAR-2, conventional fluorescence microscopy on frozen section; PAR-1, PAR-3, and EPCR, confocal microscopy on paraffin sections; Hoechst 33258 nuclear counterstain (blue). Top: Bar represents 50 μm. Bottom: Bar represents 10 μm. Data are mean ± SEM of at least 3 independent experiments performed in duplicates. *P < .001 vs control. ^XP < .01 vs control (ANOVA).