Secretory pod formation induced by thrombin. HUVECs were secretagogue challenged for 10 minutes with thrombin at a concentration of 1 U/mL. (A-B) Examples of VWF dots revealed by labeling with 2 distinct VWF antibodies, the first applied during secretagogue challenge (green channel, left panels) and the second after fixation and permeabilization (red channel, middle panels). The right panels depict the overlays of the green and red channels. Closed arrowheads indicate VWF dots that were not accessible to the extracellular anti-VWF antibodies (ie, nonfused SPs), open arrowheads indicate partially accessible VWF dots (ie, fused SPs), and arrows indicate fully accessible VWF dots. Scale bar is 2 μm. (C) Example of VWF strings and VWF dots induced by thrombin stimulation in the absence of VWF antibodies. VWF labeling was performed only after fixation and permeabilization. Scale bar represents 5 μm. Microscopy and fluorochromes used were as described in Figure 1 legend. (D) Electron micrograph of a thrombin-induced SP. Asterisks indicate WPBs clustering around or making close contact with the pod. Notice in particular the top WPB that appears to be docking lengthwise onto the pod (arrowheads). Scale bar represents 0.5 μm. Microscopy was as described in Figure 2 legend. (E) Hypothetical model summarizing the data presented herein. Secretagogue challenge of HUVECs induces multigranular exocytosis of WPBs. The initial event leading to this process may be WPB rounding (1), although WPB rounding has also been implicated in lingering-kiss exocytosis. Coalescence of WPBs is mediated by nanovesicles and leads to the formation of intracellular secretory pods (2). Subsequent fusion of the secretory pods with the plasma membrane results in secretory pod exocytosis (3). VWF is released via the fusion pore of the SPs. The delimiting membrane of the SPs is reinternalized via clathrin-mediated compensatory membrane retrieval. WPBs may continue to fuse with multigranular aggregates that have already undergone exocytosis.