PF804 SEC22B‐DEPENDENT TRAFFICKING IS ESSENTIAL FOR VWF MULTIMERIZATION AND FORMATION OF ELONGATED WPBS

Abstract

Background:Von Willebrand factor (VWF) is a key hemostatic glycoprotein that is predominantly synthesized in endothelial cells. Upon synthesis, the pro‐VWF precursor dimerizes in the ER and is subsequently transported to the Golgi where it undergoes proteolytical processing, multimerization and finally is stored in newly emerging secretory organelles, known as Weibel‐Palade bodies (WPBs). Assembly of VWF into high molecular weight (HMW) multimers, packaging in elongated WPBs and regulated release to the extracellular environment as ultra‐long VWF strings is crucial for its ability to recruit platelets to sites of vascular injury. The adhesive properties of VWF are strongly tied to the length of the secretory organelles in which it's contained in, however, how endothelial cells control the dimensions of their WPBs is still unclear.Aims:In this study, we investigated the crucial role of SNARE‐mediated ER/Golgi trafficking in VWF maturation and WPB biogenesis.Methods:In a shRNA screen of longin‐containing SNAREs in primary endothelial cells, we identified the proteinsinvolved in ER/Golgi trafficking. Protein expression was assessed by Western Blot and the morphology of intracellular compartment was studied using confocal microscopy.Results:We found that silencing of Sec22b resulted in the loss of the typically elongated morphology of WPBs. VWF was partially retained in the ER, resulting in a significantly higher ratio of proVWF:VWF. Decreased VWF influx to the Golgi affected the morphology of both WPBs, that were short and “stubby”, as well as the trans‐Golgi network (TGN). In addition, downmodulation of Sec22b expression resulted in the absence of HMW VWF multimers. Overexpression of a dominant‐negative Sec22b variant lacking its SNARE domain confirmed these phenotypes, revealing that correct localization and/or function of Sec22B is important for VWF trafficking and WPB formation.Summary/Conclusion:Our data suggest that Sec22b is a novel regulator of VWF trafficking and WPB biogenesis that modulates VWF multimerization and the elongated shape of WPBs by controlling ER/Golgi VWF influx.