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

Patients with platelet alpha or dense delta-granule defects have bleeding problems. Although several proteins are known to be required for delta-granule development, less is known about alpha-granule biogenesis. Our previous work showed that the BEACH protein NBEAL2 and the Sec1/Munc18 protein VPS33B are required for alpha-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 alpha-granules, whereas delta-granules were observed. Soluble and membrane-bound alpha-granule proteins were reduced or undetectable, suggesting that both releasable and membrane-bound alpha-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 alpha-granules. We conclude that VPS16B, similar to its binding partner VPS33B, is essential for megakaryocyte and platelet alpha-granule biogenesis. (Blood. 2012; 120(25): 5032-5040)