PAR2-AP and forskolin stimulate selective release of Weibel-Palade bodies only containing von Willebrand factor. (A) Projections were taken from HUVECs incubated with monoclonal mouse anti-VWF antibodies (VWF) and rabbit anti–P-selectin antibodies. The first columns or the P-selectin column represent images as projected through the FITC filter (548 nm), whereas the VWF columns are images as projected through the Texas Red channel (488 nm) and represent VWF, and the VWF/P-selectin columns are merged images with yellow representing colocalization of VWF and P-selectin. Negative controls consisting of incubation with both second-degree fluorescently labeled Abs only or incubation with only first-degree Abs failed to demonstrate appreciable fluorescence. Images obtained are representative of at least 6 to 8 fields viewed of an experiment that was performed 3 times. Cells were stimulated with indicated agonists for 20 minutes with PAR1-AP (100 μM), PAR2-AP (200 μM), or forskolin (10 μM) and then fixed and are representative of 3 independent experiments. In addition, HUVECs were stimulated with 20 μM histamine (data not shown). (B) The WPBs were counted after stimulation with the above-listed agonists. WPBs counted per cell for each agonist tested were normalized to control and expressed as the percentage of control. At least 10 to 12 fields at × 63 magnification per agonist tested were used to count the WPBs.