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

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 amarked reduction in platelet alpha-granules. We generated a tamoxifen-inducible mouse model of VPS33B deficiency, Vps33b(fl/fl)-ERT2, and studied the platelet phenotype and alpha-granule biogenesis. Ultrastructural analysis of Vps33b(fl/fl)-ERT2 platelets identified a marked reduction in alpha-granule count and the presence of small granule-like structures in agreement with the platelet phenotype observed in ARC patients. A reduction of similar to 65% to 75% was observed in the alpha-granule proteins von Willebrand factor and P-selectin. Although platelet aggregation responses were not affected, a defect in delta-granule secretion was observed. Under arteriolar shear conditions, Vps33b(fl/fl)-ERT2 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 alpha-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 alpha-granule deficiency in Vps33b(fl/fl)-ERT2 mouse and ARC patients.