Mutations in Neurobeachin-like 2 do not impact Weibel-Palade body biogenesis and von Willebrand factor secretion in gray platelet syndrome Endothelial Colony Forming Cells

Abstract

Background: Patients with gray platelet syndrome (GPS) and Neurobeachin-like 2 (NBEAL2) deficiency produce platelets lacking alpha-granules (AGs) and present with lifelong bleeding symptoms. AGs are lysosome-related organelles and store the hemostatic protein von Willebrand factor (VWF) and the transmembrane protein P-selectin. Weibel-Palade bodies (WPBs) are lysosome-related organelles 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, thereby preventing premature release of cargo from AG precursors. In endothelial cells, SEC22B drives VWF trafficking from the endoplasmic reticulum to Golgi and promotes the formation of elongated WPBs, but it is unclear whether this requires NBEAL2.

Objectives: To investigate a potential role for NBEAL2 in WPB biogenesis and VWF secretion using NBEAL2-deficient endothelial cells.

Methods: The interaction of SEC22B with NBEAL2 in endothelial cells was investigated by interatomic mass spectrometry and pull-down analysis. Endothelial colony forming cells were isolated from healthy controls and 3 unrelated patients with GPS and mutations in NBEAL2.

Results: We showed that SEC22B binds to NBEAL2 in ECs. Endothelial colony forming cells derived from a patient with GPS are deficient in NBEAL2 but reveal normal formation and maturation of WPBs and normal WPB cargo recruitment. Neither basal nor histamine-induced VWF secretion is altered in the absence of NBEAL2.

Conclusions: Although NBEAL2 deficiency causes the absence of AGs in patients with GPS, it does not impact WPB functionality in ECs. Our data highlight the differences in the regulatory mechanisms between these 2 hemostatic storage compartments.

Keywords: NBEAL2 protein; SEC22B; Weibel-Palade bodies; endothelial cells; gray platelet syndrome; von Willebrand factor.

Figure 1

Mutations in the SEC22B interactor NBEAL2 are associated with loss of NBEAL2 expression in endothelial cells with NBEAL2 mutations derived from a patient with GPS. (A) Label-free quantification (LFQ) of SEC22B and NBEAL2 protein levels in GFP pull-down samples of mEGFP or mEGFP-SEC22B baits expressed in HUVECs determined by mass spectrometry analysis [22]. (B) Pull-down analysis of mEGFP or mEGFP-SEC22B bait expressed in HUVECs using GFP or control (CTRL) beads and Western blot analysis of GFP, NBEAL2, and P-selectin. The molecular weights of protein ladder marker bands are indicated in kDa on the left. (C) Domain structure of full length (2754 a.a.) NBEAL2 showing the position of the mutations present in patients C, D, and E. (D) Table showing NBEAL2 gene mutations, changes at protein level, and type of mutation in patients with GPS included in this study (m: male, f: female). Gene transcript identifier: NM_015175/Protein identifier: NP_055990. (E) Western blot of NBEAL2 expression in ECFCs from 3 patients with GPS (C, D, and E; 2 clones were analyzed for patient D) compared to ECFCs isolated from healthy control donors (CTRL 1-3). α-Tubulin is shown as a loading control. The molecular weights of protein ladder marker bands are indicated in kDa on the left.

Figure 2

Endothelial cells with NBEAL2 mutations derived from a patient with GPS generate WPBs despite NBEAL2 deficiency. Representative maximal projections of confocal immunofluorescent analysis of (A) VWF (red) and nuclei (Hoechst, blue) and (B) SEC22B (red), P-selectin (green) and nuclei (blue) in CTRL and GPS patient-derived ECFCs. Grayscale images for SEC22B and P-selectin channels are shown below. Scale bars represent 20 μm.

Figure 3

NBEAL2 is dispensable for WPB cargo sorting and maturation and for basal and secretagogue-induced VWF secretion. Representative maximal projections of confocal immunofluorescent analysis of (A) VWF (red), CD63 (green) and Angiopoietin 2 (Ang-2, blue) or (B) VWF (red), Rab27A (green), and nuclei (Hoechst, blue) in CTRL and GPS ECFCs. Boxed areas are shown below in grayscale. Yellow arrowheads indicate example WPBs. Scale bars represent 20 μm. (C) Western blot analysis of VWF content in lysates and media from CTRL and GPS patient-derived ECFCs. (D) VWF multimer analysis of 24-hour basal media from CTRL and GPS patient-derived ECFCs. UL: ultra-large, HMW: high molecular weight, LMW: low molecular weight, D: dimer. (E) Percentage of stored and secreted VWF in 24 hours under basal conditions (n = 7, mean ± SEM, 2-way analysis of variance, not significant). (F) Secreted VWF in 30 minutes under unstimulated conditions (vehicle) or in the presence of 100 μM histamine expressed as a percentage of the total intracellular VWF levels in unstimulated cells (n = 4, mean ± SEM, 2-way analysis of variance, not significant).