Effect of CCE on WPB exocytosis. (A, B, D, and E) HUVECs coexpressing GFP-VWF and the mCherry–P-selectinLum domain were stimulated with 100 ng/ml PMA for 10 min with or without a 15-min preincubation with 1 µM CCE (A, B, D, and E) or 0.5 µM jasplakinolide (B). Individual WPB fusion events (marked by loss of mCherry–P-selectinLum [P-sel.lum.mcherry] fluorescence) and release of its GFP-VWF contents (marked by loss of GFP fluorescence into the medium) in live cells were assayed as described in Fig. 1. To compare individual WPBs, fusion is assigned to 0 s (see Results for explanation). (A) Mean number of individual WPB fusion events in 300 s (n = 11 cells for each condition). Note a more than threefold increase in fusion events in CCE-treated, PMA-stimulated cells. **, P = 0.004; t test. Error bars show SDs. (B) Duration of release of VWF measured as the lag between WPB fusion and full release of GFP-VWF for individual WPBs in live PMA-stimulated cells (83 fusion events in 11 cells) or in PMA-stimulated cells treated with CCE (107 fusion events in five cells) or jasplakinolide (66 fusion events from six cells). 300+ s indicates that VWF failed to release during 600 s of time-lapse filming. (C, control) Still images from a video of individual WPB fusion and release of VWF in live cells. (D, CCE treated) Note that in CCE-treated cells, WPB fusion occurs (0 s), yet GFP-VWF fails to release even after 470 s after fusion. (E) Whole-cell view of untreated or CCE-treated, PMA-stimulated live cells. Asterisks indicate WPBs that fully exocytose during 300 s of imaging. The boxed regions are the regions shown at higher magnification in C and D at a number of time points. Images shown (C–E) represent maximum intensity projections of z stacks. (F) Cells were fixed and labeled for released VWF. Note that markedly less VWF strings are apparent in CCE-treated compared with untreated, PMA-stimulated cells. The arrows denote the presence of the strings of VWF. Bars: (C and D) 2 µm; (E) 10 µm; (F) 100 µm.