ULVWF strings are secreted from HUVECs stimulated by histamine or by the binding of Stx-2 to Gb3. Green VWF granules (A,F) or strings (B-E) were visualized by fluorescence microscopy using rabbit anti-human VWF IgG and goat anti-rabbit IgG-FITC or IgG-Alexa Fluor 488. (A) Unstimulated HUVECs permeabilized by Triton-X and fixed with paraformaldehyde contain cytoplasmic VWF-containing granules. (B) HUVECs stimulated under flowing conditions with the known VWF secretagogue, histamine (0.1 mM), secrete long ULVWF strings. HUVEC nuclei were stained by propidium iodide (red). In panels A and B, the cells were fixed with 1% paraformaldehyde in PBS and made permeable with Triton-X (in panel B after cell stimulation). The cells were unfixed in panels C-F. HUVECs stimulated by Stx-2 (1 nM) secrete ULVWF strings (C), which often become entangled (D-E). HUVEC nuclei in panels C, D, and are stained blue with DAPI. Original magnification × 1000 for all panels, except panel E, which is × 200. (F) Preincubation of HUVECs with monoclonal mouse anti-human Gb3 (CD77; 5 ng/mL) blocks Stx-2 stimulated ULVWF multimeric string secretion under flowing conditions. Only intracellular VWF granules in clumps near the cell surface are demonstrated in the unfixed cells with the VWF stimulation blocked. HUVECs in all experiments were either in primary culture or passage 1. Each of these photos is representative of 3 to 20 experiments.