Mechanism of platelet factor 4 (PF4) deficiency with RUNX1 haplodeficiency: RUNX1 is a transcriptional regulator of PF4

Background: Platelet factor 4 (PF4) is an abundant protein stored in platelet alpha-granules. Several patients have been described with platelet PF4 deficiency, including the gray platelet syndrome, characterized by a deficiency of alpha-granule proteins. Defective granule formation and protein targeting are considered to be the predominant mechanisms. We have reported on a patient with thrombocytopenia and impaired platelet aggregation, secretion, and protein phosphorylation, associated with a mutation in the transcription factor RUNX1. Platelet expression profiling showed decreased transcript expression of PF4 and its non-allelic variant PF4V1. Objectives: To understand the mechanism leading to PF4 deficiency associated with RUNX1 haplodeficiency, we addressed the hypothesis that PF4 is a transcriptional target of RUNX1. Methods/results: Chromatin immunoprecipitation and gel-shift assays with phorbol 12-myristate 13-acetate-treated human erythroleukemia (HEL) cells revealed RUNX1 binding to RUNX1 consensus sites at -1774/-1769 and -157/-152 on the PF4 promoter. In luciferase reporter studies in HEL cells, mutation of each site markedly reduced activity. PF4 promoter activity and PF4 protein level were decreased by small interfering RNA RUNX1 knockdown and increased by RUNX1 overexpression. Conclusions: Our results provide the first evidence that PF4 is regulated by RUNX1 and that impaired transcriptional regulation leads to the PF4 deficiency associated with RUNX1 haplodeficiency. Because our patient had decreased platelet albumin and IgG (not synthesized by megakaryocytes) levels, we postulate additional defects in RUNX1-regulated genes involved in vesicular trafficking. These studies advance our understanding of the mechanisms in alpha-granule deficiency.