title: VPS33B regulates protein sorting into and maturation of α-granule progenitor organelles in mouse megakaryocytes.
abstract: Arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome is caused by deficiencies in the trafficking proteins VPS33B or VIPAR, and is associated with a bleeding diathesis and a marked reduction in platelet α-granules. We generated a tamoxifen-inducible mouse model of VPS33B deficiency, Vps33b(fl/fl)-ER(T2), and studied the platelet phenotype and α-granule biogenesis. Ultrastructural analysis of Vps33b(fl/fl)-ER(T2) platelets identified a marked reduction in α-granule count and the presence of small granule-like structures in agreement with the platelet phenotype observed in ARC patients. A reduction of ∼65% to 75% was observed in the α-granule proteins von Willebrand factor and P-selectin. Although platelet aggregation responses were not affected, a defect in δ-granule secretion was observed. Under arteriolar shear conditions, Vps33b(fl/fl)-ER(T2) platelets were unable to form stable aggregates, and tail-bleeding measurement revealed a bleeding diathesis. Analysis of bone marrow-derived megakaryocytes (MKs) by conventional and immuno-electron microscopy from Vps33b(fl/fl)-ER(T2) mice revealed a reduction in mature type-II multivesicular bodies (MVB II) and an accumulation of large vacuoles. Proteins that are normally stored in α-granules were underrepresented in MVB II and proplatelet extensions. These results demonstrate that abnormal protein trafficking and impairment in MVB maturation in MKs underlie the α-granule deficiency in Vps33b(fl/fl)-ER(T2) mouse and ARC patients.
authors.affiliation: Centre for Cardiovascular Sciences, School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom;
authors.forename: Danai
authors.initials: D
authors.lastname: Bem
authors.affiliation: Centre for Cardiovascular Sciences, School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom; Medical Research Council, Laboratory for Molecular Cell Biology, University College London, London, United Kingdom;
authors.forename: Holly
authors.initials: H
authors.lastname: Smith
authors.affiliation: Medical Research Council, Laboratory for Molecular Cell Biology, University College London, London, United Kingdom;
authors.forename: Blerida
authors.initials: B
authors.lastname: Banushi
authors.affiliation: Medical Research Council, Laboratory for Molecular Cell Biology, University College London, London, United Kingdom;
authors.forename: Jemima J
authors.initials: JJ
authors.lastname: Burden
authors.affiliation: Medical Research Council, Laboratory for Molecular Cell Biology, University College London, London, United Kingdom;
authors.forename: Ian J
authors.initials: IJ
authors.lastname: White
authors.affiliation: Medical Research Council, Laboratory for Molecular Cell Biology, University College London, London, United Kingdom; University College London Institute of Child Health, Gene Therapy Laboratory, London, United Kingdom;
authors.forename: Joanna
authors.initials: J
authors.lastname: Hanley
authors.affiliation: Laboratory of Immunogenetics of Pediatric Autoimmune Diseases, Paris, France;
authors.forename: Nadia
authors.initials: N
authors.lastname: Jeremiah
authors.affiliation: Laboratory of Immunogenetics of Pediatric Autoimmune Diseases, Paris, France;
authors.forename: Frédéric
authors.initials: F
authors.lastname: Rieux-Laucat
authors.affiliation: Department of General Pediatrics, University Children's Hospital, Muenster, Germany;
authors.forename: Ruth
authors.initials: R
authors.lastname: Bettels
authors.affiliation: Department of Pediatric Nephrology, University Hospital Vall d'Hebron, Barcelona, Spain;
authors.forename: Gema
authors.initials: G
authors.lastname: Ariceta
authors.affiliation: School of Cellular and Molecular Medicine, University of Bristol, Bristol, United Kingdom; and.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Mumford
authors.affiliation: Centre for Cardiovascular Sciences, School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom;
authors.forename: Steven G
authors.initials: SG
authors.lastname: Thomas
authors.affiliation: Centre for Cardiovascular Sciences, School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom;
authors.forename: Steve P
authors.initials: SP
authors.lastname: Watson
authors.affiliation: Medical Research Council, Laboratory for Molecular Cell Biology, University College London, London, United Kingdom; Inherited Metabolic Diseases, Great Ormond Street Hospital, London, United Kingdom.
authors.forename: Paul
authors.initials: P
authors.lastname: Gissen
mesh_terms: D000818:Animals; D001176:Arthrogryposis / Q000235:genetics; D002478:Cells, Cultured; D002779:Cholestasis / Q000235:genetics; D003594:Cytoplasmic Granules / Q000378:metabolism*; D055652:Gray Platelet Syndrome / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D015388:Organelles / Q000378:metabolism; D010976:Platelet Count; D020641:Polymorphism, Single Nucleotide; D021381:Protein Transport / Q000235:genetics; D051437:Renal Insufficiency / Q000235:genetics; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000502:physiology*
chemical_list: C411584:VPS33B protein, human; C000601834:VPS33B protein, mouse; D033921:Vesicular Transport Proteins