The role of Nbeal2 in the homeostasis and retention of granules in haematopoietic cells

Abstract

Gray platelet syndrome (GPS) is a rare disorder primarily characterised by the absence of α-granules in platelets. Typical clinical features of GPS include macrothrombocytopenia, abnormal or excessive bleeding, splenomegaly, and bone marrow fibrosis. GPS is caused by biallelic mutations in the NBEAL2 gene, where the deficiency of platelet α-granules is attributed to a loss of function in the Nbeal2 protein. Thus, the study of Nbeal2 can provide insight into both the clinical manifestations of GPS and the essential processes that contribute to the homeostasis of platelet α-granules. To this end, this work investigates the function of Nbeal2 through the study of a large collection of GPS patients through the NIHR BioResource-Rare Diseases (NBR-RD) programme, the Nbeal2−/− mouse model, and gene-edited Nbeal2−/− cell lines to understand its role in essential granule-related processes in haematopoietic cells. Through the NBR-RD GPS study, detailed clinical phenotypes were compared and patient blood samples were examined by Sysmex, RNA-Seq, and mass spectrometry analysis. The study revealed novel clinical phenotypes, including an array of autoimmune diseases and a prevalence of autoantibodies, differences in the transcriptomes and proteomes of GPS platelets, neutrophils, monocytes, and CD4+ lymphocytes, compared to healthy blood donors, as well as a pro-inflammatory signature in GPS plasma that may be mediated by changes in the liver. Loss of Nbeal2 function in GPS patients also affected the granularity and presence of granule-related proteins in leukocytes, which provides evidence that Nbeal2’s function is not exclusive to the regulation of platelet α-granules. This work also aimed to evaluate the mechanism by which Nbeal2 controls granule- related processes in haematopoietic cells. Subcellular fractionation of human and murine platelets revealed that Dock7, a known Nbeal2 binding partner, is mislocalised and has reduced expression in the platelets of GPS patients and Nbeal2−/− mice. The interaction of Nbeal2 with Dock7, a protein that regulates cytoskeletal rearrangements, prompted an investigation of actin-related processes in megakaryocytes (MKs) and platelets, which showed that MKs from Nbeal2−/− mice exhibit appropriate proplatelet formation, but thrombin- activated Nbeal2−/− platelets do not appropriately express F-actin. The reduced abundance and mislocalisation of Dock7, and the lack of α-granule release, may contribute to the defective platelet shape change observed in GPS patients. Lastly, Nbeal2−/− cell models were generated in the haematopoietic CHRF and K562 cell lines. These gene-edited cells were compared with induced pluripotent stem cell-derived MKs and Nbeal2−/− murine platelets to assess differences in their morphology, granularity, and size. Additionally, RNA-Seq and mass spectrometry analysis of these cell lines revealed differences in the respective transcriptomes and proteomes, which were used to define cellular processes associated with Nbeal2. These results were compared to the transcriptome and proteome results of additional data sets derived from GPS patient, Nbeal2−/− mouse, and cell line samples, to pinpoint novel genes and proteins that may be essential to the Nbeal2 mechanism.