GMVECs. The cells were passage 4, grown on glass coverslips. Bright field images at different magnifications show elongated cells (A; original magnification × 400) and the rounded central portion of a single cell (B; original magnification × 1000). (C-D) GMVECs were stained with rabbit anti-VWF IgG and goat anti-rabbit-IgG-Alexa 488 (green), as well as the nuclear stain, DAPI (blue). The unstimulated GMVECs contain cytoplasmic VWF-containing granules (C). The granules are secreted as long ULVWF multimeric strings (D) in response to stimulation of the GMVECs for 3 minutes with Stx-2 (1 nM). The ULVWF strings frequently become entangled, as at the top of the frame (arrow). In some Stx-1- or Stx-2-stimulated GMVECs at passage number 4 (or higher), circular conglomerates of surface VWF (^*) that do not unfurl into strings in response to stimulation are also seen. (E) VWF multimers enriched in ULVWF are also released slowly into the culture medium in soluble form by unstimulated GMVECs over the course of 24 hours. The VWF multimers were separated by 1% agarose/SDS gel electrophoresis, and then detected by membrane transfer, a polyclonal rabbit anti-VWF IgG, goat anti-rabbit IgG-HRP and chemiluminescence. VWF released from unstimulated HUVECs over 24 hours, or present in normal human platelet-poor plasma (NP), are shown for comparison. The vertical line indicates the gel location of ULVWF forms. These photos are representative of 3 experiments.