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

ABSTRACT: RUNX1 haplodeficiency is associated with thrombocytopenia, platelet dysfunction and a predisposition to acute leukaemia. Platelets possess three distinct types of granules and secretory processes involving dense granules (DG), ?-granules and vesicles or lysosomes containing acid hydrolases (AH). Dense granules and granule deficiencies have been reported in patients with RUNX1 mutations. Little is known regarding the secretion from AH-containing vesicles.We studied two related patients with a RUNX1 mutation, easy bruising, and mild thrombocytopenia. Platelet aggregation and 14 C serotonin in platelet-rich plasma (PRP) were impaired in response to ADP, epinephrine, collagen and arachidonic acid. Contents of DG (ATP, ADP), ?-granules (?-thromboglobulin) and AH-containing vesicles (?-glucuronidase, ?-hexosaminidase, ?-mannosidase) were normal or minimally decreased. Dense granules secretion on stimulation of gel-filtered platelets with thrombin and divalent ionophore A23187 (4-12 ?mol L-1 ) were diminished. ?-thromboglobulin and AH secretion was impaired in response to thrombin or A23187. We studied thromboxane-related pathways. The incorporation of 14 C -arachidonic acid into phospholipids and subsequent arachidonic acid release on thrombin activation was normal. Platelet thromboxane A2 production in whole blood serum and on thrombin stimulation of PRP was normal, suggesting that the defective secretion was not due to impaired thromboxane production.These studies provide the first evidence in patients with a RUNX1 mutation for a defect in AH (lysosomal) secretion, and for a global defect in secretion involving all three types of platelet granules that is unrelated to a granule content deficiency. They highlight the pleiotropic effects and multiple platelet defects associated with RUNX1 mutations.

SUBMITTER: Rao AK

PROVIDER: S-EPMC5623153 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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

Rao A K AK Poncz M M

Haemophilia : the official journal of the World Federation of Hemophilia 20170629 5

<h4>Background</h4>RUNX1 haplodeficiency is associated with thrombocytopenia, platelet dysfunction and a predisposition to acute leukaemia. Platelets possess three distinct types of granules and secretory processes involving dense granules (DG), α-granules and vesicles or lysosomes containing acid hydrolases (AH). Dense granules and granule deficiencies have been reported in patients with RUNX1 mutations. Little is known regarding the secretion from AH-containing vesicles.<h4>Methods and results ...[more]

PMID: 28662545

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Project description:AbstractPlatelet α-granules have numerous proteins, some synthesized by megakaryocytes (MK) and others not synthesized but incorporated by endocytosis, an incompletely understood process in platelets/MK. Germ line RUNX1 haplodeficiency, referred to as familial platelet defect with predisposition to myeloid malignancies (FPDMMs), is associated with thrombocytopenia, platelet dysfunction, and granule deficiencies. In previous studies, we found that platelet albumin, fibrinogen, and immunoglobulin G (IgG) were decreased in a patient with FPDMM. We now show that platelet endocytosis of fluorescent-labeled albumin, fibrinogen, and IgG is decreased in the patient and his daughter with FPDMM. In megakaryocytic human erythroleukemia (HEL) cells, small interfering RNA RUNX1 knockdown (KD) increased uptake of these proteins over 24 hours compared with control cells, with increases in caveolin-1 and flotillin-1 (2 independent regulators of clathrin-independent endocytosis), LAMP2 (a lysosomal marker), RAB11 (a marker of recycling endosomes), and IFITM3. Caveolin-1 downregulation in RUNX1-deficient HEL cells abrogated the increased uptake of albumin, but not fibrinogen. Albumin, but not fibrinogen, partially colocalized with caveolin-1. RUNX1 KD resulted in increased colocalization of albumin with flotillin and fibrinogen with RAB11, suggesting altered trafficking of both proteins. The increased uptake of albumin and fibrinogen, as well as levels of caveolin-1, flotillin-1, LAMP2, and IFITM3, were recapitulated by short hairpin RNA RUNX1 KD in CD34+-derived MK. To our knowledge, these studies provide first evidence that platelet endocytosis of albumin and fibrinogen is impaired in some patients with RUNX1-haplodeficiency and suggest that megakaryocytes have enhanced endocytosis with defective trafficking, leading to loss of these proteins by distinct mechanisms. This study provides new insights into mechanisms governing endocytosis and α-granule deficiencies in RUNX1-haplodeficiency.

Project description:Essentials Platelet dense granule (DG) deficiency is a major abnormality in RUNX1 haplodeficiency patients. The molecular mechanisms leading to the platelet DG deficiency are unknown. Platelet expression of PLDN (BLOC1S6, pallidin), involved in DG biogenesis, is regulated by RUNX1. Downregulation of PLDN is a mechanism for DG deficiency in RUNX1 haplodeficiency.SummaryBackground Inherited RUNX1 haplodeficiency is associated with thrombocytopenia and platelet dysfunction. Dense granule (DG) deficiency has been 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 pallidin, a subunit of biogenesis of lysosome-related organelles complex-1 (BLOC-1) involved in DG biogenesis. PLDN mutations in the pallid mouse and Hermansky-Pudlak syndrome-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 levels 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 with 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 overexpression enhanced and RUNX1 downregulation decreased PLDN1 promoter activity and protein expression. RUNX1 downregulation resulted in impaired handling of mepacrine and mislocalization of the DG marker CD63 in HEL cells, indicating impaired DG formation, recapitulating findings on PLDN downregulation. Conclusions These studies provide the first evidence that PLDN is a direct target of RUNX1 and that its dysregulation is a mechanism for platelet DG deficiency associated with RUNX1 haplodeficiency.

Project description:BackgroundPCTP (phosphatidylcholine transfer protein) regulates the intermembrane transfer of phosphatidylcholine. Higher platelet PCTP expression is associated with increased platelet responses on activation of protease-activated receptor 4 thrombin receptors noted in black subjects compared with white subjects. Little is known about the regulation of platelet PCTP. Haplodeficiency of RUNX1, a major hematopoietic transcription factor, is associated with thrombocytopenia and impaired platelet responses on activation. Platelet expression profiling of a patient with a RUNX1 loss-of-function mutation revealed a 10-fold downregulation of the PCTP gene compared with healthy controls.MethodsWe pursued the hypothesis that PCTP is regulated by RUNX1 and that PCTP expression is correlated with cardiovascular events. We studied RUNX1 binding to the PCTP promoter using DNA-protein binding studies and human erythroleukemia cells and promoter activity using luciferase reporter studies. We assessed the relationship between RUNX1 and PCTP in peripheral blood RNA and PCTP and death or myocardial infarction in 2 separate patient cohorts (587 total patients) with cardiovascular disease.ResultsPlatelet PCTP protein in the patient was reduced by ≈50%. DNA-protein binding studies showed RUNX1 binding to consensus sites in ≈1 kB of PCTP promoter. PCTP expression was increased with RUNX1 overexpression and reduced with RUNX1 knockdown in human erythroleukemia cells, indicating that PCTP is regulated by RUNX1. Studies in 2 cohorts of patients showed that RUNX1 expression in blood correlated with PCTP gene expression; PCTP expression was higher in black compared with white subjects and was associated with future death/myocardial infarction after adjustment for age, sex, and race (odds ratio, 2.05; 95% confidence interval 1.6-2.7; P<0.0001). RUNX1 expression is known to initiate at 2 alternative promoters, a distal P1 and a proximal P2 promoter. In patient cohorts, there were differential effects of RUNX1 isoforms on PCTP expression with a negative correlation in blood between RUNX1 expressed from the P1 promoter and PCTP expression.ConclusionsPCTP is a direct transcriptional target of RUNX1. PCTP expression is associated with death/myocardial infarction in patients with cardiovascular disease. RUNX1 regulation of PCTP may play a role in the pathogenesis of platelet-mediated cardiovascular events.

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

Publications

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

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