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-...
