VPS33B regulates protein sorting into and maturation of α-granule progenitor organelles in mouse megakaryocytes

Blood. 2015 Jul 9;126(2):133-43. doi: 10.1182/blood-2014-12-614677. Epub 2015 May 6.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arthrogryposis / genetics
  • Cells, Cultured
  • Cholestasis / genetics
  • Cytoplasmic Granules / metabolism*
  • Gray Platelet Syndrome / genetics
  • Gray Platelet Syndrome / metabolism
  • Humans
  • Megakaryocytes / cytology
  • Megakaryocytes / metabolism*
  • Megakaryocytes / physiology
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Organelles / metabolism
  • Platelet Count
  • Polymorphism, Single Nucleotide
  • Protein Transport / genetics
  • Renal Insufficiency / genetics
  • Vesicular Transport Proteins / genetics
  • Vesicular Transport Proteins / physiology*

Substances

  • VPS33B protein, human
  • VPS33B protein, mouse
  • Vesicular Transport Proteins

Supplementary concepts

  • Arthrogryposis renal dysfunction cholestasis syndrome