Mutations in the SEC22B interactor NBEAL2 do not impact Weibel-Palade body biogenesis and Von Willebrand factor secretion in Gray Platelet Syndrome Endothelial Colony Forming Cells

Background Gray Platelet Syndrome (GPS) patients with Neurobeachin-like 2 (NBEAL2) deficiency produce platelets lacking alpha-granules (AGs) and present with lifelong bleeding symptoms. AGs are lysosome-related organelles (LROs) and store the hemostatic protein Von Willebrand factor (VWF) and the transmembrane protein P-selectin. Weibel-Palade bodies (WPBs) are LROs of endothelial cells and also store VWF and P-selectin. In megakaryocytes, NBEAL2 links P-selectin on AGs to the SNARE protein SEC22B on the endoplasmic reticulum (ER), thereby preventing premature release of cargo from AG precursors. In endothelial cells, SEC22B drives VWF trafficking from ER to Golgi and promotes the formation of elongated WPBs, but it is unclear if this requires NBEAL2. Objectives To investigate a potential role for NBEAL2 in WPB biogenesis and VWF secretion using NBEAL2 deficient endothelial cells. Methods Interaction of SEC22B with NBEAL2 in endothelial cells was investigated by interactomic mass spectrometry and pull down analysis. Endothelial Colony Forming Cells (ECFCs) were isolated from healthy controls and 3 unrelated GPS patients with mutations in NBEAL2. Results We show that SEC22B binds to NBEAL2 in ECs. GPS patient-derived ECFCs are deficient of NBEAL2, but reveal normal formation and maturation of WPBs and normal WPB cargo recruitment. Neither basal nor histamine-induced VWF secretion are altered in the absence of NBEAL2. Conclusions While NBEAL2 deficiency causes absence of AGs in GPS patients, it but has no impact on WPB functionality in ECs. Our data highlight the difference in regulatory mechanisms between these two hemostatic storage compartments. Essentials Ex vivo characterization of Gray Platelet Syndrome patient-derived endothelial cells with biallelic NBEAL2 mutations NBEAL2 is not essential for Weibel-Palade body biogenesis, maturation, and Von Willebrand factor secretion from endothelial cells