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
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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.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/44139 |
| Date | 02 April 2014 |
| Creators | Urban, Denisa |
| Contributors | Kahr, Walter |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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