FVIII is secreted from stimulated GMVECs bound to ULVWF strings.
GMVECs were stimulated with 100 μM histamine for 2 min. Cells were then stained with rabbit anti-VWF plus chicken anti-rabbit IgG AF-488 (green), washed and fixed. Following fixation, the cells were stained with mouse monoclonal anti-FVIII plus goat anti-mouse IgG AF-647 (red). Panels (A, C and E) show representative ULVWF strings with bound FVIII from merged images. Dashed lines (that were moved away to not obscure the string image) indicate the measured portions of the string. Corresponding graphs (B from image A, D from image C, and F from image E) show the intensities from the 488-nm (VWF, green) and 647-nm (FVIII, red) channels measured along the ULVWF string (in pixels) in the merged image. In the 60× images (A and E) 100 pixels = 11.4 μm and in the 100× image (C) 200 pixels = 11.8 μm. The ratio of FVIII intensity/VWF intensity is shown for each ULVWF string. Images are representative of 9–11 experiments.