Fast actomyosin-independent VWF expulsion from WPBs during Ca²⁺-mediated exocytosis. In the absence of flow, secreted VWF concatemers form irregular-shaped patches on the cell surface and disperse slowly into solution. Importantly, the initial expulsion of VWF from WPBs and the subsequent dispersal from the cell surface constitute separate processes. (Ai) Montages of individual WPB exocytotic events taken from live-cell videos of histamine-stimulated human umbilical vein endothelial cells (HUVECs) expressing VWF-EGFP (top) or VWFpp-EGFP (bottom). Images were acquired at 10 frames per second. The first frame in which an increase in EGFP fluorescence due to fusion was detected is set to t = 0 seconds. Bars represent 2 μm. (Aii) Histograms of the pooled times, from t = 0 seconds to expulsion, indicated by formation of irregular patches of cell surface VWF-EGFP (gray bars) or VWFpp-EGFP (black bars) from individual WPBs after stimulation with histamine or ionomycin. For VWF-EGFP, n = 543 fusion events (100 μM histamine, n = 183, 10 cells; 1 µM ionomycin, n = 310, 11 cells); for VWFpp-EGFP, n = 402 fusion events (100 μM histamine, n = 92, 7 cells; 1 μM ionomycin, n = 310, 13 cells). (Bi) Dual-color imaging of a single HUVEC coexpressing VWFpp-EGFP and TagRFP-actin. Bar represents 10 μm. (Bii) Upper: Image montage of a single WPB undergoing exocytosis during ionomycin stimulation; EGFP fluorescence (top), RFP fluorescence (middle), merge image (bottom). Images were acquired at 30 frames per second, and selected frames (times indicated) are shown. Bar represents 2 µm. Lower: Mean fluorescence intensity (FI) within the color-coded regions of interest indicated on the first frame of the upper panel top row, plotted against time. WPB fusion is associated with a sharp increase in EGFP fluorescence due to EGFP-dequenching.⁸  Note that there was no evidence of RFP-actin accumulation prior to or during WPB exocytosis (n = 136 fusion events, 12 cells). (Ci-ii) Same as for panel Bi-ii, but in HUVECs coexpressing MyoIIB-GFP and VWFpp-mRFP. Note that there was no evidence of MyoIIB-EGFP accumulation prior to or during WPB exocytosis (n = 24 fusion events, 5 cells). Also note that RFP fluorescence is not pH sensitive, and exocytosis, therefore, is marked only by a fall in WPB associated RFP fluorescence. (D) Blebbistatin (Blebb) treatment (25 µM for 20 minutes; red bars) does not alter basal (−), histamine-evoked (100 μM; +), or ionomycin-evoked (1 µM; +) VWF secretion. Plots show data pooled from 3 independent experiments each carried out in triplicate (mean ± standard error of the mean). (E) Examples of individual WPB current spikes recorded by amperometry in control (black) and Blebb-pretreated (red) HUVECs. The kinetics of current spike foot signals and main spike rise times provide information about fusion pore formation and expansion. Mean (± standard error of the mean) pre–foot spike parameters, including foot signal duration (t_foot; control: 13.72 ± 3.31 ms, n = 216, 44 cells; Blebb: 9.85 ± 1.09 ms, n = 256, 50 cells) and total foot signal charge (Q_foot; control: 0.057 ± 0.013 pC; Blebb: 0.0636 ± 0.0147 pC), were no different (Student t test). Mean main spike parameters, including spike rise time (t_rise; control: 3.22 ± 0.17 ms, n = 317 spikes, 44 cells; Blebb: 3.26 ± 0.15 ms, n = 384 spikes, 50 cells), peak amplitude (I_max; control: 29.14 ± 1.31 pA; Blebb: 27.96 ± 1.11 pA), and spike decay time (t_fall; control: 15.08 ± 1.09 ms; Blebb: 13.64 ± 0.84 ms), were not different (Student t test). a.u., arbitrary unit; GFP, green fluorescent protein; RFP, red fluorescent protein.