Requirement of VPS33B, a member of the Sec1/Munc18 protein family, in megakaryocyte and platelet  -granule biogenesis

Abstract

Bleeding problems are associated with defects in platelet ␣-granules, yet little is known about how these granules are formed and released. Mutations affecting VPS33B, a novel Sec1/Munc18 protein, have recently been linked to arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome. We have characterized platelets from patients with ARC syndrome and observed reduced aggregation with arachidonate and adenosine diphosphate (ADP). Structural abnormalities seen in ARC platelets included increased platelet size, a pale appearance in blood films, elevated numbers of ␦-granules, and completely absent ␣-granules. Soluble and membrane-bound ␣-granule proteins were significantly decreased or undetectable in ARC platelets, suggesting that both the releasable protein pools and membrane components of ␣-granules were absent. The role of VPS33B in platelet granule biogenesis was evaluated by immunofluorescence microscopy in normal human megakaryocytes. VPS33B colocalized appreciably with markers of ␣-granules, moderately with late endosomes/lysosomes, minimally with ␦-granules/lysosomes, and not with cis-Golgi complexes. VPS33B protein expression determined by immunoblotting confirmed the presence of VPS33B in control fibroblasts but not in ARC fibroblasts, and in normal megakaryocytes but not in platelets. We conclude that like other Sec1/Munc18 proteins, VPS33B is involved in intracellular vesicle trafficking, being essential for the development of platelet ␣-granules but not for granule secretion. IntroductionCongenital platelet disorders involving abnormalities in ␣ and/or dense (␦) granules are an important cause of inherited bleeding disorders. A number of genes have been linked to the ␦-granule defects associated with the Hermansky-Pudlak and ChediakHigashi syndromes, 1-6 and 16 genes have been identified in mice that regulate vesicle trafficking to platelet ␦-granules and melanosomes; some of these genes encode known vesicle trafficking proteins whereas others encode components of BLOC (biogenesis of lysosome-related organelles complexes) protein complexes. 3,7 Much less is known about the genes or proteins involved in ␣-granule defects, although some transcription factors and cellsurface glycoproteins have been implicated. For example, a family with X-linked macrothrombocytopenia and reduced ␣-granules was found to have an aspartate-to-glycine mutation in the transcription factor GATA1, 8 and hemizygous loss of the FLI1 transcription factor causes the Paris-Trousseau syndrome, characterized by abnormal megakaryocytes and giant ␣-granules. 9,10 In mice, loss of transcription factor NF-E2 expression produces abnormal megakaryocytes with absent ␣-granules, 11 and hematopoietic zinc finger (Hzf)-deficient mice have megakaryocytes and platelets with reduced ␣-granules and ␣-granule proteins. 12 Glycoprotein (GP) Ib␤-deficient mouse platelets contain abnormally large ␣-granules, 13 and giant ␣-granules have also been observed in platelets from a patient with Bernard-Soulier syndrome. 14 A deficiency of both platel...

SPE-39 Family Proteins Interact with the HOPS Complex and Function in Lysosomal Delivery

“…Mutations in the human VPS33B gene cause arthrogryposis-renal dysfunction-cholestasis syndrome (Gissen et al, 2004), which is also usually associated with platelet dysfunction (Eastham et al, 2001). Both VPS33A and VPS33B are required during platelet formation; VPS33A is required for dense granule biogenesis, whereas VPS33B is required for ␣-granule biogenesis (Suzuki et al, 2003;Lo et al, 2005). These data suggest that the two VPS33 homologues participate in different sets of vesicular fusion events.…”

Drosophila14-3-3ε has a crucial role in anti-microbial peptide secretion and innate immunity

“…In mammals, SNAREs are crucial for vesicle degranulation and the subsequent release of inflammatory mediators or cytokines, from the secretory granules in mast cells, eosinophils, neutrophils and platelets (Kay et al, 2006;Logan et al, 2006;Manderson et al, 2007;Stow et al, 2006). Patients with mutations in the SNARE protein Sec1/Vps33B exhibit bleeding problems associated with defects in megakaryocyte and platelet alpha-granule release (Lo et al, 2005). New elements of the vesicular secretion machinery are being realised on the basis of immune-deficiency disorders.…”

Characterization of the Mammalian CORVET and HOPS Complexes and Their Modular Restructuring for Endosome Specificity

“…The organization of these mammalian complexes has only partly been addressed with seemingly conflicting results and hampered the interpretation of functional experiments in which the roles of specific or multiple units are addressed (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). Furthermore, it is not known what drives membrane specificity of these complexes in mammals.…”