Defective RAB1B-related megakaryocytic ER-to-Golgi transport in RUNX1 haplodeficiency: impact on von Willebrand factor.

Patients with RUNX1 haplodeficiency have thrombocytopenia, platelet dysfunction, and deficiencies of α-granules and dense granules. Platelet expression profiling of a patient with a heterozygous mutation (c.969-323G>T) revealed decreased , which encodes a small G protein. RAB GTPases regulate vesicle trafficking, and RAB1B is implicated in endoplasmic reticulum (ER)-to-Golgi transport in nonhematopoietic cells, but its role in megakaryocytes (MK) is unknown. We addressed the hypothesis that is a transcriptional target of RUNX1 and that RAB1B regulates ER-to-Golgi transport in MK cells. Chromatin immunoprecipitation studies and electrophoretic mobility shift assay using phorbol 12-myristate 13-acetate (PMA)-treated human erythroleukemia cells revealed RUNX1 binding to promoter region RUNX1 consensus sites, and their mutation reduced the promoter activity. promoter activity and protein expression were inhibited by siRNA and enhanced by RUNX1 overexpression. These indicate that is a direct RUNX1 target, providing a mechanism for decreased in patient platelets. Vesicle trafficking from ER to Golgi in PMA-treated human erythroleukemia cells was impaired along with Golgi disruption on siRNA downregulation of or The effects of knockdown were reversed by reconstitution. Trafficking of von Willebrand factor (vWF), an α-granule MK synthesized protein, was impaired with or downregulation and reconstituted by ectopic RAB1B expression. Platelet vWF was decreased in patients with mutations. Thus, ER-to-Golgi transport, an early critical step in protein trafficking to granules, is impaired in megakaryocytic cells on downregulation, secondary to decreased expression. Impaired mediated ER-to-Golgi transport contributes to platelet α-granule defects in haplodeficiency.