Abstract Number: PB1502
Meeting: ISTH 2020 Congress
Background: Transcription factor RUNX1 regulates genes critical for megakaryocyte (MK)/platelet formation and function. RUNX1haplodeficiency (RHD) is associated with thrombocytopenia, and defective platelet granules and function. Platelet expression profiling of a RHD patient revealed decreased RAB31expression. RAB GTPases regulate vesicle trafficking, granule biogenesis and secretion. The role of RAB31 in MK/platelets is unknown.
Aims: To study the regulation of RAB31 by RUNX1and role of RAB31 in platelets/MK.
Methods: We performed studies in platelets from RHD patients and in PMA-treated megakaryocytic HEL cells.
Results: By real-time PCR, platelet RAB31 mRNA was decreased by 60-80% compared to 5 healthy controls in our RHD patient (P1) and in two additional patients (P2 and P3). Platelet RAB31 protein was decreased in P2 and P3. RAB31 promoter region revealed 4 RUNX1 consensus sites. CHiP and EMSA revealed RUNX1 binding to sites II and IV; their mutation decreased promoter activity. RUNX1 downregulation (siRNA) and overexpression altered RAB31 promoter activity and protein expression in HEL cells. RAB31 regulates vesicular trafficking between the Golgi/TGN and endosomes, and from early endosomes (EE) to late endosomes (LE). We investigated RUNX1 and RAB31 role in endosomal dynamics using immunofluorescent staining for markers of EE (EEA1) and LE/multivesicular bodies (LE/MVB, CD63). Either RUNX1 or RAB31 siRNA yielded a striking enlargement of EE (marker EEA1), partially reversed by RAB31 reconstitution. Mannose-6-phosphate receptors (MPRs) regulate lysosomal protein trafficking; RAB31 regulates transport of MPRs from TGN to endosomes. In HEL cells, siRNA RUNX1knockdown increased MPR (immunoblotting). In preliminary studies RAB31 deletion in HEL cells (5 independent knockout clones) increased MPR suggesting that RUNX1 and RAB31 downregulation alters MPR handling.
Conclusions: RAB31 is a direct transcriptional target of RUNX1. RAB31 or RUNX1 downregulation results in impaired endosomal maturation/trafficking and handling of proteins.These effects may underlie the α-granule protein defects in RHD patient.
To cite this abstract in AMA style:Jalagadugula G, Mao G, Goldfinger L, Wurtzel J, Lambert M, Rao AK. Defective RAB31-mediated Endosomal Trafficking in RUNX1 Haplodeficiency [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/defective-rab31-mediated-endosomal-trafficking-in-runx1-haplodeficiency/. Accessed January 21, 2022.
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