IntroductionRegulated exocytosis from vascular endothelial cells forms the first line of repair following tissue damage and inflammation. 1 Endothelial-specific secretory granules, the so-called Weibel-Palade bodies (WPBs), 2 release their contents in response to various physiologic stimuli such as physical trauma, mediators of inflammation, and hypoxia. The major secretory product of WPBs, von Willebrand Factor (VWF), assembles into remarkably long strings (up to several millimeters long) that capture flowing platelets and bind to connective tissue at the site of vascular injury to form a hemostatic plug. [3][4][5] WPBs have a distinctive elongated shape of 0.1 to 0.2 m wide and up to 5 m long, with a uniform pattern of striations running along the longitudinal axis. [6][7][8] These striations represent VWF filaments that have assembled into helical tubules. 9 Packing of VWF multimers into tubules requires both the N-terminal domains of mature VWF and the cleaved VWF propeptide, while the maintenance of the tubules in WPBs depends on a pH-sensitive interaction between mature VWF and the propeptide. 4,10,11 Microscopic imaging techniques have been instrumental in advancing our knowledge of WPB biogenesis and exocytosis. In particular, live-cell imaging studies using genetically labeled WPB cargo proteins have stressed the extraordinary plasticity of the regulated secretory pathway leading to WPB exocytosis. [12][13][14][15][16] Thus secretagogues that elevate intracellular cAMP levels cause a subset of WPBs to cluster at the level of the microtubuleorganizing center so that they do not partake in exocytosis. 12 Secretagogues that elevate intracellular Ca 2ϩ levels, on the other hand, do not elicit this effect. On the basis of these findings, and taking into account evidence for the existence of WPB subpopulations that except for VWF differ in their content of cargo molecules, it has been suggested that WPB clustering allows for the differential release of bioactive molecules from WPBs. 17 Further modulation of the release of WPB constituents is possible during the exocytosis process itself, as it has been shown that WPBs can engage in 2 modes of exocytosis, full-collapse and a slow form of kiss-and-run (lingering kiss). 14 In the latter mode, a 10-to 12-nm fusion pore is formed that acts as a molecular sieve allowing for the selective release of smaller molecules (interleukin-8, CD63) while larger molecules such as VWF are retained.In the present report, we expand the palette of exocytosis modes of WPBs by providing evidence for multigranular exocytosis, that is, the homotypic fusion of secretory granules prior to exocytosis. Using confocal, live-cell, correlative, scanning electron, and electron tomographic imaging techniques applied to human umbilical vein endothelial cells (HUVECs), we identified a novel structure, which we termed secretory pod, and which represents a secretory intermediate resulting from the coalescence of WPBs. In addition, our data suggest that fusion of WPBs with secretory pods is mediate...
