Dysregulation of PLDN (pallidin) is a mechanism for platelet dense granule deficiency in RUNX1 haplodeficiency

Abstract

Summary Background Inherited RUNX1 haplodeficiency is associated with thrombocytopenia and platelet dysfunction. Dense granule (DG) deficiency is 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 for pallidin, a subunit of BLOC-1 (biogenesis of lysosome-related organelles complex-1) involved in granule biogenesis. PLDN mutations in the pallid mouse and the Hermansky-Pudlak syndrome (HPS)-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 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 using 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 over-expression enhanced and RUNX1 downregulation decreased PLDN1 promoter activity and protein expression. RUNX1 downregulation resulted in impaired handling of mepacrine and mislocalization of DG marker CD63 in HEL cells, indicating impaired DG formation, with recapitulation of findings on PLDN downregulation. Conclusions These studies provide the first evidence that PLDN is a direct target of RUNX1 and its dysregulation is a mechanism for platelet DG deficiency associated with RUNX1 haplodeficiency.

Defective acid hydrolase secretion in RUNX1 haplodeficiency: Evidence for a global platelet secretory defect

“…Moreover, our expression profiling studies in the same patient showed that a wide array of genes involved in platelet function and formation are downregulated [8]. We have shown that several relevant genes including PRKQ [9] , MYL9 [10], ALOX12 [12], PLDN [24] and PCTP [25] are direct transcriptional targets of RUNX1. Thus, we postulate that the secretory abnormalities result from aberrations in common pathways critical to the general process of exocytosis, for example, involving vesicle transport mechanisms, Rab proteins, and SNARE proteins [16, 26].…”

Transcription factor defects causing platelet disorders

“…44 Two preliminary studies reported that the genes encoding RAB1B (RAB1B), a low molecular weight GTPase that is essential for vesicle transport between the endoplasmic reticulum and the Golgi apparatus, and Pallidin (PLDN), which is involved in granule vesicle biogenesis, were both downregulated in RUNX1 haplodeficiency, and shown to be direct targets for RUNX1 regulation, providing possible mechanisms for the defective granule biogenesis and secretion observed in RUNX1 deficiency. 45,46…”

Inherited platelet dysfunction and hematopoietic transcription factor mutations

“…26 PLDN encodes for pallidin, a protein that is involved in biogenesis of dense granules and is decreased in the pallid mouse and the human Hermansky-Pudlak syndrome. 27–29 There is also evidence that patients with RUNX1 mutations may have global defects that compromise agonist-stimulated secretion of contents of the α- and dense-granules, as well as from the acid hydrolase containing vesicles, which is unrelated to a deficiency in the granule contents.…”

“…In our platelet transcript profiling studies of a patient with RUNX1 mutation numerous genes relevant to multiple platelet biological pathways were downregulated. 33 Several genes have been shown to be direct transcriptional targets of RUNX1, including 12-lipoxygenase ( ALOX12 ), 25 platelet factor 4 ( PF4 ), 34 platelet myosin light chain ( MYL9 ), 35 protein kinase C-θ ( PRKCQ ), 36 palladin ( PLDN ), 26 and the thrombopoietin receptor ( c-MPL ). 37 These impact various aspects of MK biology, platelet production, structure, signaling, and responses.…”