In the event of a bleeding a blood vessel immediately responds by locally releasing hemostatic proteins into the circulation. The endothelial cells forming the innerlining of the vessel secrete von Willebrand factor (VWF), a protein that will form a sticky mesh covering the area of vascular damage to which platelets can adhere, which will eventually result in the formation of a thrombus. To ensure that sufficient quantities of VWF are available to support the arrest of bleeding, endothelial cells store VWF in intracellular storage vesicles, the so-called Weibel-Palade bodies, which can be released within seconds when a bleeding occurs. These Weibel-Palade bodies have an elongated cigar-like shape, which is very important for the functionality of the VWF stored within and for the smooth escape of VWF from the vesicle when it is released. Insufficient or defective VWF in the circulation is associated with an increased risk of bleeding, such as in the inherited bleeding disorder von Willebrand Disease.
In this project we have studied the mechanisms through which endothelial cells store VWF in Weibel- Palade bodies, how they determine the elongated shape of these vesicles and how Weibel-Palade bodies are being released. For this we first mapped the internal machinery that traffics VWF through the endothelial cell as it is being stored in the Weibel-Palade body. Using advanced mass spectrometry we have identifed the proteins that are within the close proximity of an important regulator of this process, the protein Sec22B. In order to study the function an relevance of the identified proteins in the trafficking of VWF we have used several in vitro cell models in the lab, including endothelial cells in which the expression of selected hits from the proxomity screen was silenced using RNAi. We also used endothelial cells that were isolated from blood samples from patients with genetic variants in genes that encode for important components of these pathways.
In ~30-50% of the patients with Von Willebrand Disease with reduced circulating VWF levels, there are no pathogenic variants in the VWF gene that can explain their low VWF levels. In these patients the underlying disease mechanism is currrently unclear. By using endothelial cells derived from patients with genetic variants in the MADD gene and who have unexplained low VWF levels in plasma, we have been able to determine the role of MADD in hemostasis. Using live imaging of patient-derived endothelial
cells that lack MADD we observed that following activation the secretion of their Weibel-Palade bodies is severely impaired. Our study is the first to identify a gene outside the VWF locus, MADD, that is causally linked to Von Willebrand Disease. More broadly, it shows that defective Weibel-Palade body secretion is a novel disease mechanism that can result in Von Willebrand Disease.