Imaging of Von Willebrand Factor Remodeling Upon Secretion From Vascular Endothelial Cells

Abstract Abstract 263 In response to vascular injury, endothelial cells rapidly secrete high molecular weight multimers of the coagulation protein Von Willebrand factor (VWF). Once expelled from the cells, VWF unfurls in long strings that bind platelets from the bloodstream to induce primary hemostasis. VWF secreted upon stimulation is released from specialized storage compartments called Weibel Palade bodies (WPB) which have a typical rod or cigar shape. They emerge from the Trans Golgi network in a process driven by the formation of helical tubules consisting of VWF multimers and the VWF propeptide. When WPBs undergo exocytosis and release VWF, rapid structural changes occur which eventually result in platelet capturing VWF strings. It has been postulated that the tubular storage of VWF in WPBs is required for sufficient unfolding of the protein during string formation as agents disrupting the VWF tubules were shown to result in less strings. Recently we described a novel structure involved in VWF exocytosis which is formed only upon stimulation. We refer to this structure as a “secretory pod” as it seemed to derive from multiple WPBs and was identified as a VWF release site where strings seemed to be formed. By transmission electron microscopy (TEM) we identified this structure to be a membrane-delimited organelle containing filamentous material resembling unfurled VWF. The VWF tubules as seen in WPBs are absent in secretory pods suggesting that tubular packaging of VWF is not essential for sufficient release and string formation. To study the formation of secretory pods and the subsequent release and remodeling of VWF, several imaging techniques were used such as live-cell imaging and correlative light and electron microscopy. We expressed propeptide-EGFP in endothelial cells to label the WPBs and stimulated them with PMA. By live-cell imaging we visualized the exocytotic events. We observed, apart from single WPB exocytosis, the formation of secretory pods which occurred by the coalescence of several WPBs. In some cases the individual WPBs rounded up first, before they joined into one round structure while in other cases the coalescence event seemed to happen at once. After coalescence, fusion with the plasma membrane occurred to release the pooled VWF which resulted in the disappearance of the fluorescent signal as the propeptide rapidly diffused into the extracellular medium. How the secreted VWF is remodeled after secretion into VWF strings was studied by correlative light and electron microscopy. We correlated confocal pictures of stimulated endothelial cells, which were stained with VWF specific fluorescent antibodies, to consecutive TEM sections. We found that fluorescently labeled VWF dots that were connected to strings, correlated to secretory pods but also to globular mass of secreted VWF. Interestingly, when we analyzed consecutive EM sections, the globular masses were found to originate from the secretory pods. From the globular masses we also observed deriving strings indicating that once VWF is expelled, remodeling occurs independently from secretion. We hypothesize that fluid flow remodels the secreted globular VWF mass into strings. To study this we stimulated endothelial cells under flow. The intracellular VWF pool in the WPBs was labeled green by transient expression of propeptide-EGFP and the secreted VWF was labeled red with strongly diluted red fluorescent VWF specific antibodies in the perfusate. Using live-cell imaging we observed that upon fusion of EGFP labeled WPBs, the green signal transformed into a red signal revealing dots of labeled secreted VWF. These dots rolled, in the direction of the flow, to the edge of the cell where they aggregated and only then formed strings. In non-transfected cells we performed similar experiments and there we observed the same pattern, confirming even more the VWF aggregation and string formation at the edges of the cell. In conclusion, we demonstrated that several WPBs can fuse with each other to form secretory pods and that VWF is secreted as a globular mass of protein. From these globular masses strings originated indicating that string formation occurs independently from the mechanism of secretion in which the tubular packaging of VWF in WPBs does not seem to be of importance. Disclosures: No relevant conflicts of interest to declare.