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 (α)-granules, dense (δ)-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 δ-granule development, less is known about α-granule biogenesis. Studies in our laboratory have identified the only proteins known to date to be relevant for α-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 α-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 α-granules. Depletion of VPS16B in platelet progenitor cells - human iii megakaryocytes - resulted in absent α-granules, though α-granule proteins were still synthesized. Taken together, these data identify VPS16B as a novel molecule required for platelet α-granule biogenesis.

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 (α)-granules, dense (δ)-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 δ-granule development, less is known about α-granule biogenesis. Studies in our laboratory have identified the only proteins known to date to be relevant for α-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 α-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 α-granules. Depletion of VPS16B in platelet progenitor cells - human iii megakaryocytes - resulted in absent α-granules, though α-granule proteins were still synthesized. Taken together, these data identify VPS16B as a novel molecule required for platelet α-granule biogenesis.

VPS33B is a Sec1/Munc18 protein required for the biogenesis of α-granules in megakaryocytes, which give rise to platelets. Mutations in VPS33B cause arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome. Platelets from ARC patients completely lack α-granules, causing a bleeding disorder. VPS33B plays a role in vesicular fusion events through its interaction with the SNARE proteins, though no such interactions have been identified. Here, it is shown that VPS33B interacts with STX6, a member of the syntaxin subfamily of SNAREs. The introduction of ARC mutations into VPS33B completely abrogated binding to STX6. Confocal microscopy studies revealed STX6 co-localizes well with markers of the α-granule biogenesis pathway. This implies a role for the interaction of VPS33B with STX6 in α-granule biogenesis. Based on the known structure of STX6 and that predicted of VPS33B, suggests a novel and unique mode of binding between VPS33B and STX6 compared to other identified SM-STX pairs.

VPS33B is a Sec1/Munc18 protein required for the biogenesis of α-granules in megakaryocytes, which give rise to platelets. Mutations in VPS33B cause arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome. Platelets from ARC patients completely lack α-granules, causing a bleeding disorder. VPS33B plays a role in vesicular fusion events through its interaction with the SNARE proteins, though no such interactions have been identified. Here, it is shown that VPS33B interacts with STX6, a member of the syntaxin subfamily of SNAREs. The introduction of ARC mutations into VPS33B completely abrogated binding to STX6. Confocal microscopy studies revealed STX6 co-localizes well with markers of the α-granule biogenesis pathway. This implies a role for the interaction of VPS33B with STX6 in α-granule biogenesis. Based on the known structure of STX6 and that predicted of VPS33B, suggests a novel and unique mode of binding between VPS33B and STX6 compared to other identified SM-STX pairs.