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...

Summary. Background: In vascular endothelial cells, high molecular weight multimers of von Willebrand factor (VWF) are folded into tubular structures for storage in Weibel-Palade bodies. On stimulation, VWF is secreted and forms strings to induce primary hemostasis. The structural changes composing the transition of stored tubular VWF into secreted unfurled VWF strings are still unresolved even though they are vital for normal hemostasis. The secretory pod is a novel structure that we previously described in endothelial cells. It is formed on stimulation and has been postulated to function as a VWF release site. In this study, we investigated the actual formation of secretory pods and the subsequent remodeling of VWF into strings. Methods: Human umbilical vein endothelial cells were stimulated and studied using various imaging techniques such as live-cell imaging and correlative light and electron microscopy. Results: We found by using live-cell imaging that secretory pods are formed through the coalescence of multiple Weibel-Palade bodies without involvement of other large structures. Secreted VWF expelled from secretory pods was found to adopt a globular conformation. We visualized that VWF strings derive from those globular masses of VWF. Flow experiments showed that, on secretion, the globular masses of VWF move to the edge of the cell, where they anchor and generate VWF strings. Conclusion: On secretion, VWF adopts a globular conformation that remodels into strings after translocation and anchoring at the edge of the cell. This finding reveals new pathophysiological mechanisms that could be affected in patients with von Willebrand disease.

Our data suggest that erythrocyte retention in venous thrombi is mediated by erythrocyte-VWF or erythrocyte-VWF-fibrin interactions. Targeting erythrocyte retention could be a new strategy in the treatment or prevention of venous thrombosis.

Key Points• WPBs stay connected to the Golgi apparatus until vesicle formation is completed.• During biogenesis at the Golgi, WPBs increase in size through the addition of nontubular VWF.Weibel-Palade bodies (WPBs) comprise an on-demand storage organelle within vascular endothelial cells. It's major component, the hemostatic protein von Willebrand factor (VWF), is known to assemble into long helical tubules and is hypothesized to drive WPB biogenesis. However, electron micrographs of WPBs at the Golgi apparatus show that these forming WPBs contain very little tubular VWF compared with mature peripheral WPBs, which raises questions on the mechanisms that increase the VWF content and facilitate vesicle growth. Using correlative light and electron microscopy and electron tomography, we investigated WPB biogenesis in time. We reveal that forming WPBs maintain multiple connections to the Golgi apparatus throughout their biogenesis. Also by volume scanning electron microscopy, we confirmed the presence of these connections linking WPBs and the Golgi apparatus. From electron tomograms, we provided evidence that nontubular VWF is added to WPBs, which suggested that tubule formation occurs in the WPB lumen. During this process, the Golgi membrane and clathrin seem to provide a scaffold to align forming VWF tubules. Overall, our data show that multiple connections with the Golgi facilitate content delivery and indicate that the Golgi appears to provide a framework to determine the overall size and dimensions of newly forming WPBs. (Blood. 2015;125(22):3509-3516) IntroductionRapid secretion of the endothelial storage organelles, the WeibelPalade bodies (WPBs), 1 is fundamental for hemostasis. WPBs contain the hemostatic glycoprotein von Willebrand factor (VWF), which recruits platelets to sites of injury to arrest bleeding.2 Within WPBs, VWF is packed into helical tubules that give the organelle an elongated shape with a length of 1 to 5 mm and a width of 100 to 300 nm. 1,[3][4][5] The formation of WPBs is dependent on VWF and also occurs on VWF expression in nonendothelial cells. 6,7 VWF is synthesized in the endoplasmic reticulum as a pre-proprotein consisting of a signal peptide, a propeptide, and mature VWF.2 On removal of the signal peptide, VWF dimers are formed that are transported to the Golgi apparatus. At the Golgi, the propeptide is cleaved from mature VWF to guide multimerization and tubule formation. 3,8,9 VWF tubule formation is crucial for the development of mature, densely packed elongated WPBs. Mutations in the VWF gene, as found in patients with the bleeding disorder von Willebrand disease, were shown to result in altered WPB morphology. 2,[10][11][12] In vitro studies on VWF tubule formation demonstrated that the core of the VWF tubules is formed by the propeptide and the N-terminal D9 and D3 assembly of mature VWF.3 However, it is still poorly understood how the formation of the VWF tubules is related to WPB biogenesis.Electron microscopy studies have revealed several stages in the WPB formation pro...