Stimulated release of intraluminal vesicles from Weibel-Palade bodies

Weibel-Palade bodies (WPBs) are secretory granules that contain von Willebrand factor and P-selectin, molecules that regulate hemostasis and inflammation, respectively. The presence of CD63/LAMP3 in the limiting membrane of WPBs has led to their classification as lysosome-related organelles. Many lysosome-related organelles contain intraluminal vesicles (ILVs) enriched in CD63 that are secreted into the extracellular environment during cell activation to mediate intercellular communication. To date, there are no reports that WPBs contain or release ILVs. By light microscopy and live-cell imaging, we show that CD63 is enriched in microdomains within WPBs. Extracellular antibody recycling studies showed that CD63 in WPB microdomains can originate from the plasma membrane. By cryo-electron tomography of frozen-hydrated endothelial cells, we identify internal vesicles as novel structural features of the WPB lumen. By live-cell fluorescence microscopy, we directly observe the exocytotic release of EGFP-CD63 ILVs as discrete particles from individual WPBs. WPB exocytosis provides a novel route for release of ILVs during endothelial cell stimulation.