Interaction networks of Weibel-Palade body regulators syntaxin-3 and syntaxin binding protein 5 in endothelial cells

The endothelium stores the hemostatic protein Von Willebrand factor (VWF) in endothelial storage organelles called Weibel-Palade bodies (WPBs). During maturation, WPBs recruit a complex of Rab GTPases and effectors that associate with components of the SNARE machinery that control WPB exocytosis. Recent genome wide association studies have found links between genetic variations in the SNAREs syntaxin-2 (STX2) and syntaxin binding protein 5 (STXBP5) and VWF plasma levels, suggesting a role for SNARE proteins in regulating VWF release. Moreover, we have previously identified the SNARE proteins syntaxin-3 and STXBP1 as regulators of WPB release. In this study we used an unbiased iterative interactomic approach to identify new components of the WPB exocytotic machinery. An interactome screen of syntaxin-3 identifies a number of SNAREs and SNARE associated proteins (STXBP2, STXBP5, SNAP23, NAPA and NSF). We show that the VAMP-like domain (VLD) of STXBP5 is indispensable for the interaction with SNARE proteins and this capacity of the VLD could be exploited to identify an extended set of novel endothelial SNARE interactors of STXBP5. In addition, an STXBP5 variant with an N436S substitution, which is linked to lower VWF plasma levels, does not show a difference in interactome when compared with WT STXBP5. SIGNIFICANCE: The hemostatic protein Von Willebrand factor plays a pivotal role in vascular health: quantitative or qualitative deficiencies of VWF can lead to bleeding, while elevated levels of VWF are associated with increased risk of thrombosis. Tight regulation of VWF secretion from WPBs is therefore essential to maintain vascular homeostasis. We used an unbiased proteomic screen to identify new components of the regulatory machinery that controls WPB exocytosis. Our data expand the endothelial SNARE protein network and provide a set of novel candidate WPB regulators that may contribute to regulation of VWF plasma levels and vascular health.