Antiapoptotic activity of rwt APC and anticoagulantly impaired APC variants. (A) Inhibition of staurosporine (STS)-induced apoptosis by APC (see “Materials and methods”). Percentage of apoptotic endothelial cells observed in the absence of added APC (18% of all cells) was taken as 100%. Each point represents the mean ± SEM from at least 3 independent experiments. □ indicates rwt APC; ○, 229/230-APC; ⋄, 3K3A-APC; ▪, S360A-APC; and •, no staurosporine. (B-C) Reduction of activated caspase-3-positive cells by rwt APC and APC variants (25 nM, 5 hours) upon induction of apoptosis by staurosporine (2 μM, 4 hours). (B) Activated caspase-3-positive cells expressed as a percentage of the total number of cells present. As indicated by the “no staurosporine” thin line, approximately 2% of the endothelial cells were positive for activated caspase-3 in the absence of staurosporine. Each bar represents the mean ± SEM of 2 to 4 independent experiments. (C) Immunofluorescence analysis of activated caspase-3-positive cells using an activated caspase-3 specific antibody (red) and DAPI nuclear staining (blue). Columns represent identical fields. Original magnification × 200.