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Mechanism of platelet dense granule biogenesis: study of cargo transport and function of Rab32 and Rab38 in a model system.

ABSTRACT: Dense granules are important in platelet aggregation to form a hemostatic plug as evidenced by the increased bleeding time in mice and humans with dense granule deficiency. Dense granules also are targeted by antiplatelet agents because of their role in thrombus formation. Therefore, the molecular understanding of the dense granule and its biogenesis is of vital importance. In this work, we establish a human megakaryocytic cell line (MEG-01) as a model system for the study of dense granule biogenesis using a variety of cell biology and biochemical approaches. Using this model system, we determine the late endocytic origin of these organelles by colocalization of the internalized fluid phase marker dextran with both mepacrine and transmembrane dense granule proteins. By mistargeting of mutant dense granule proteins, we demonstrate that sorting signals recognized by adaptor protein-3 are necessary for normal transport to dense granules. Furthermore, we show that tissue-specific Rab32 and Rab38 are crucial for the fusion of vesicles containing dense granule cargo with the maturing organelle. This work sheds light on the biogenesis of dense granules at the molecular level and opens the possibility of using this powerful model system for the investigation of new components of the biogenesis machinery.

SUBMITTER: Ambrosio AL

PROVIDER: S-EPMC3496959 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Essentials Platelet dense granule (DG) deficiency is a major abnormality in RUNX1 haplodeficiency patients. The molecular mechanisms leading to the platelet DG deficiency are unknown. Platelet expression of PLDN (BLOC1S6, pallidin), involved in DG biogenesis, is regulated by RUNX1. Downregulation of PLDN is a mechanism for DG deficiency in RUNX1 haplodeficiency.SummaryBackground Inherited RUNX1 haplodeficiency is associated with thrombocytopenia and platelet dysfunction. Dense granule (DG) deficiency has been reported in patients with RUNX1 haplodeficiency, but the molecular mechanisms are unknown. Platelet mRNA expression profiling in a patient previously reported by us with a RUNX1 mutation and platelet dysfunction showed decreased expression of PLDN (BLOC1S6), which encodes pallidin, a subunit of biogenesis of lysosome-related organelles complex-1 (BLOC-1) involved in DG biogenesis. PLDN mutations in the pallid mouse and Hermansky-Pudlak syndrome-9 are associated with platelet DG deficiency. Objectives We postulated that PLDN is a RUNX1 target, and that its decreased expression leads to platelet DG deficiency in RUNX1 haplodeficiency. Results Platelet pallidin and DG levels were decreased in our patient. This was also observed in two siblings from a different family with a RUNX1 mutation. Chromatin immunoprecipitation and electrophoretic mobility shift assays with phorbol ester-treated human erythroleukemia (HEL) cells showed RUNX1 binding to RUNX1 consensus sites in the PLDN1 5' upstream region. In luciferase reporter studies, mutation of RUNX1 sites in the PLDN promoter reduced activity. RUNX1 overexpression enhanced and RUNX1 downregulation decreased PLDN1 promoter activity and protein expression. RUNX1 downregulation resulted in impaired handling of mepacrine and mislocalization of the DG marker CD63 in HEL cells, indicating impaired DG formation, recapitulating findings on PLDN downregulation. Conclusions These studies provide the first evidence that PLDN is a direct target of RUNX1 and that its dysregulation is a mechanism for platelet DG deficiency associated with RUNX1 haplodeficiency.

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Mechanism of platelet dense granule biogenesis: study of cargo transport and function of Rab32 and Rab38 in a model system.

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