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

BackgroundGray 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. ObjectivesTo investigate a potential role for NBEAL2 in WPB biogenesis and VWF secretion using NBEAL2 deficient endothelial cells. MethodsInteraction 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. ResultsWe 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. ConclusionsWhile NBEAL2 deficiency causes absence of AGs in GPS patients, it has no impact on WPB functionality in ECs. Our data highlight the difference in regulatory mechanisms between these two hemostatic storage compartments. EssentialsO_LIWe characterized Gray Platelet Syndrome patient-derived endothelial cells with biallelic NBEAL2 mutations ex vivo. C_LIO_LINBEAL2 is not essential for Weibel-Palade body biogenesis, maturation, and Von Willebrand factor secretion from endothelial cells. C_LI