The VPS33B-binding protein VPS16B is required in megakaryocyte and platelet α-granule biogenesis

Abstract

Patients with platelet ␣ or dense ␦-granule defects have bleeding problems. Although several proteins are known to be required for ␦-granule development, less is known about ␣-granule biogenesis. Our previous work showed that the BEACH protein NBEAL2 and the Sec1/Munc18 protein VPS33B are required for ␣-granule biogenesis. Using a yeast two-hybrid screen, mass spectrometry, coimmunoprecipitation, and bioinformatics studies, we identified VPS16B as a VPS33B-binding protein. Immunoblotting confirmed VPS16B expression in various human tissues and cells including megakaryocytes and platelets, and also in megakaryocytic Dami cells. Characterization of platelets from a patient with arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome containing mutations in C14orf133 encoding VPS16B revealed pale-appearing platelets in blood films and electron microscopy revealed a complete absence of ␣-granules, whereas ␦-granules were observed. Soluble and membrane-bound ␣-granule proteins were reduced or undetectable, suggesting that both releasable and membranebound ␣-granule constituents were absent. Immunofluorescence microscopy of Dami cells stably expressing GFP-VPS16B revealed that similar to VPS33B, GFP-VPS16B colocalized with markers of the trans-Golgi network, late endosomes and ␣-granules. We conclude that VPS16B, similar to its binding partner VPS33B, is essential for megakaryocyte and platelet ␣-granule biogenesis. (Blood. 2012; 120(25):5032-5040) IntroductionInherited platelet disorders are important causes of abnormal bleeding. Studies of congenital platelet disorders have made major contributions to platelet biology, 1,2 and key insights into platelet granule formation and function have come from investigating patients with deficient or absent ␣ and/or ␦-granules. Most inherited defects involve platelets lacking ␣-granules or ␦-granules but not both, suggesting the existence of distinct granule development pathways.Platelets arise from bone marrow-resident megakaryocytes (MKs), which differentiate from hematopoietic stem cells and produce masses of platelets via the formation of microtubuledependent proplatelet extensions. 3,4 Budding vesicles in the megakaryocyte trans-Golgi network mature into multivesicular bodies (MVBs) and granules 5-7 that are transported into proplatelets 8 to produce mature platelets containing 50 to 80 ␣-granules, 3 to 8 ␦-granules, and a few lysosomes. ␣-granules contain a wide range of proteins that are both endogenously synthesized as well as endocytosed. 5 Several insights into MK and platelet ␦-granule development have come from studying patients with Hermansky-Pudlak syndrome (HPS), and Chediak-Higashi syndrome (CHS; MIM214500) for which mouse models exist. [9][10][11][12] In both of these genetic diseases platelets lack ␦-granules. Several genes/proteins linked to the regulation of vesicle trafficking have been implicated in ␦-granule formation, including components of BLOC (biogenesis of lysosomerelated organelles complex) protein complexes (BLOC-1, 2, 3), known vesicle-...

Sorting protein VPS33B regulates exosomal autocrine signaling to mediate hematopoiesis and leukemogenesis

“…We also evaluated the frequency of Mac-1 + c-Kit + leukemia cells (as reported to be enriched for LICs), which was markedly reduced in primary VPS33B-null recipients (33% vs. 65%; Figure 7, B and C). Deleting Vps33b led to a dramatically delayed onset of leukemogenesis, as shown by tective effect that VPS16B provides VPS33B as shown by Denisa Urban et al (29). Taken together, these data indicate that VPS33B may collaborate with GDI2/RAB11A/RAB27A signaling to control exosome maturation and secretion.…”

“…To determine potential molecules that play important roles in exosome secretion, we screened several potential candidates that previous studies have suggested may participate in vesicle trafficking, including VPS16B, VPS33B, RAB11A, and RAB27A (22,27,29,33). We found that VPS16B and VPS33B, but not RAB11A or RAB27A (Supplemental Figure 1D), were highly enriched in human plasma-derived exosomes ( Figure 1F).…”

“…Denisa Urban and colleagues, as well as other groups, have demonstrated that VPS16B interacts with VPS33B to contribute to the formation of α-granules (29). However, how HSCs and LICs regulate vesicle trafficking for secretory proteins, and whether VPS33B is involved in exosome biogenesis, maturation, and secretion requires elucidation.…”

Dynamin 2–dependent endocytosis is required for normal megakaryocyte development in mice

“…Clathrin heavy chain was punctate in control MKs, consistent with continuous endocytosis, whereas it appeared abnormally clustered in the center of the cell in the absence of DNM2. The early endosome markers EEA1 and APPL1, 34,35 which stained large vesicle structures in control MKs, were mislocalized in Dnm2-null MKs. In contrast, the distribution of the endoplasmic reticulum chaperone calreticulin 36 was normal in Dnm2-null MKs.…”

Defective release of α granule and lysosome contents from platelets in mouse Hermansky-Pudlak syndrome models

“…[33][34][35][36][37] In platelets of HPS patients, the number, morphology, and content levels of a granules and lysosomes are normal. 17,38,39,40 Thrombin-induced secretion of a granule and lysosome contents was impaired in platelets from 1 uncharacterized HPS patient, 41 but has not been systematically analyzed in different HPS subtypes.…”