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

Danai Bem; Holly Smith; Blerida Banushi; Jemima J Burden; Ian J White; Joanna Hanley; Nadia Jeremiah; Frédéric Rieux-Laucat; Ruth Bettels; Gema Ariceta; Andrew D Mumford; Steven G Thomas; Steve P Watson; Paul Gissen

doi:10.1182/blood-2014-12-614677

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

Danai Bem, Holly Smith, Blerida Banushi, Jemima J Burden, Ian J White, Joanna Hanley, Nadia Jeremiah, Frédéric Rieux-Laucat, Ruth Bettels, Gema Ariceta, Andrew D Mumford, Steven G Thomas, Steve P Watson, Paul Gissen

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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.

Original language	English
Pages (from-to)	133-143
Number of pages	11
Journal	Blood
Volume	126
Issue number	2
Early online date	6 May 2015
DOIs	https://doi.org/10.1182/blood-2014-12-614677
Publication status	Published - 9 Jul 2015

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Bem, D., Smith, H., Banushi, B., Burden, J. J., White, I. J., Hanley, J., Jeremiah, N., Rieux-Laucat, F., Bettels, R., Ariceta, G., Mumford, A. D., Thomas, S. G., Watson, S. P., & Gissen, P. (2015). VPS33B regulates protein sorting into and maturation of α-granule progenitor organelles in mouse megakaryocytes. Blood, 126(2), 133-143. https://doi.org/10.1182/blood-2014-12-614677

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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.",

author = "Danai Bem and Holly Smith and Blerida Banushi and Burden, {Jemima J} and White, {Ian J} and Joanna Hanley and Nadia Jeremiah and Fr{\'e}d{\'e}ric Rieux-Laucat and Ruth Bettels and Gema Ariceta and Mumford, {Andrew D} and Thomas, {Steven G} and Watson, {Steve P} and Paul Gissen",

note = "{\textcopyright} 2015 by The American Society of Hematology.",

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doi = "10.1182/blood-2014-12-614677",

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T1 - VPS33B regulates protein sorting into and maturation of α-granule progenitor organelles in mouse megakaryocytes

AU - Bem, Danai

AU - Smith, Holly

AU - Banushi, Blerida

AU - Burden, Jemima J

AU - White, Ian J

AU - Hanley, Joanna

AU - Jeremiah, Nadia

AU - Rieux-Laucat, Frédéric

AU - Bettels, Ruth

AU - Ariceta, Gema

AU - Mumford, Andrew D

AU - Thomas, Steven G

AU - Watson, Steve P

AU - Gissen, Paul

PY - 2015/7/9

Y1 - 2015/7/9

N2 - 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.

AB - 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.

U2 - 10.1182/blood-2014-12-614677

DO - 10.1182/blood-2014-12-614677

M3 - Article

C2 - 25947942

SN - 0006-4971

VL - 126

SP - 133

EP - 143

JO - Blood

JF - Blood

IS - 2

ER -

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

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