| Platelets are small, anucleate cells that fulfill a central role in hemostasis through their ability to adhere to sites of vessel injury, where they change shape, secrete molecules, and aggregate to stop blood leakages. Their secretory organelles - alpha (alpha)-granules, dense (delta)-granules and lysosomes - are critical in mediating the formation of a hemostatic plug, and interference with their biogenesis leads to bleeding disorders. Although several proteins are known to be required for delta-granule development, less is known about alpha-granule biogenesis. Studies in our laboratory have identified the only proteins known to date to be relevant for alpha-granule biogenesis: the BEACH protein NBEAL2 and the Sec1/Munc18 protein VPS33B. Mutations in VPS33B have been associated with arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome. ARC platelets are pale and agranular in appearance, and display a complete absence of alpha-granule structures and contents. Using a yeast two-hybrid screen, mass spectrometry, co-immunoprecipitation, and bioinformatics studies, VPS16B was identified as a VPS33B-binding protein. Platelets from a patient with ARC syndrome containing mutations in C14orf133 encoding VPS16B recapitulate the phenotype observed in VPS33B-null platelets. Immunofluorescence microscopy of Dami cells stably expressing GFP-VPS16B revealed that similar to VPS33B, GFP-VPS16B co-localized with markers of the trans-Golgi network, late endosomes and alpha-granules. Depletion of VPS16B in platelet progenitor cells - human megakaryocytes - resulted in absent ?-granules, though alpha-granule proteins were still synthesized. Taken together, these data identify VPS16B as a novel molecule required for platelet alpha-granule biogenesis. |
