Human Platelet Defects in Transcription Factor RUNX1 Haplodeficiency

转录因子 RUNX1 单倍体缺陷中的人血小板缺陷

• 批准号: 8788058
• 负责人: Angara Koneti Rao
• 金额: $ 37.83万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-04 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8788058
• 关键词: Abnormal Platelet; Acute leukemia; Adhesions; Agonist; Albumins; Alpha Granule; Arachidonate 12-Lipoxygenase; Biochemical; Biology; Blood; Blood Cells; Blood Platelets; Blood coagulation; Cardiovascular Diseases; Cell Shape; Cells; Core-Binding Factor; Cytoplasmic Granules; Cytoskeleton; Defect; Development; Disease; Down-Regulation; Endocytosis; Family; Flow Cytometry; Functional disorder; G alpha q Protein; GTP-Binding Proteins; Gene Proteins; Genes; Goals; Guanosine Triphosphate Phosphohydrolases; Health; Hematology; Hematopoietic; Hemorrhage; Human; Immunoblotting; Immunoglobulin G; Inherited; Injury; Knowledge; Lead; Link; Megakaryocytes; Molecular; Molecular Biology; Molecular Profiling; Monomeric GTP-Binding Proteins; Mutation; Myocardial Infarction; Myosin Heavy Chains; Myosin Light Chains; Myosin Regulatory Light Chains; Patients; Phospholipase C; Phosphorylation; Platelet Factor 4; Play; Predisposition; Production; Protein Kinase C; Proteins; Publications; RUNX1 gene; Regulation; Reporting; Research; Role; Signaling Protein; Stroke; Therapeutic Agents; Thrombocytopenia; Thrombopoiesis; Time; Tubulin; base; clinical practice; filamin; insight; interest; mouse model; novel; novel therapeutics; overexpression; platelet protein P47; repository; trafficking; transcription factor

DESCRIPTION (provided by applicant): Patients with inherited platelet function disorders and bleeding manifestations are widely encountered in clinical practice of hematology. In the vast majority of such patients, the molecular mechanisms are completely unknown. The longstanding goals of our studies have been to obtain insights into normal platelet mechanisms and the involved proteins through the study of patients with inherited platelet defects. Our studies to date have been highly successful, leading to the first descriptions of hitherto unrecognized deficiencies in three major signaling proteins, phospholipase C-2, the GTP-binding protein Gq and protein kinase C-, and into the platelet dysfunction associated with haplodeficiency of a major transcription factor, RUNX1. These patients are an invaluable repository of untapped information on normal platelet mechanisms. Specific aim of this project is to obtain insights into the molecular basis of the platelet defects associated with mutation in transcription factor RUNX1 (Core binding factor A2, RUNX1) and into the genes regulated in platelets/ megakaryocytes by RUNX1 through studies in patients characterized with RUNX1 haplodeficiency. RUNX1 haplodeficiency is associated with familial thrombocytopenia, impaired platelet function and predisposition to acute leukemia. Our patient has mild thrombocytopenia, markedly abnormal aggregation and secretion associated with diminished agonist-stimulated phosphorylation of pleckstrin and myosin light chain (MLC), decreased PKC- and impaired activation of GPIIb-IIIa. This patient has a heterozygous mutation in RUNX1. Platelet expression profiling studies in this patient showed downregulation of specific platelet/megakaryocyte (MK) genes in association with RUNX1 haplodeficiency. To date we have established that four genes (platelet factor 4 (PF4), myosin light chain (MYL9), 12-lipoxygenase (ALOX12) and protein kinase C- (PRKCQ) are indeed transcriptional targets of RUNX1, and they provide important insights into the platelet defects in the patient. We are now proposing to study specific other genes that are shown to be downregulated in our patient platelets including a) Pallidin (PLDN): dense and alpha granule defects are a major feature of RUNX1 haplodeficiency and the mechanisms unknown. Pallid (pldn) deficiency is associated with delta-SPD in mouse models. b) Small GTPases, RAB1B and RAB31, and RAPGAP1L that are major players in vesicular trafficking, secretion and granule targeting of proteins; and c) FLNA, TUBB1, related to cytoskeletal system, and play a major role in platelet production, cell shape, adhesion and secretion. To our knowledge a human deficiency in platelet pallidin or filamin has not been reported. For each gene we will a) demonstrate a decrease in the specific protein in platelets from the patient; b) establish through molecular biology studies that the gene is a direct transcriptional target of RUNX1; c) study the effect of downregulation and overexpression of RUNX1 on the gene and associated features (protein level, functional effects, MK development and thrombopoiesis); d) perform functional and biochemical studies in patient platelets/MK based on the specific gene. In addition, we will perform selected studies on regulation of RUNX1 in MK. We will extend these studies to other patients with RUNX1 mutation. These studies will provide information on hitherto unrecognized alterations in platelets/MK in RUNX1 haplodeficiency, the genes/proteins regulated by RUNX1 and on their platelet/MK role. They will advance our understanding of the mechanisms in normal and abnormal platelet function.

描述（由申请人提供）：遗传性血小板功能障碍和出血表现的患者在血液学的临床实践中广泛遇到。在绝大多数此类患者中，分子机制是完全未知的。我们研究的长期目标是通过研究遗传性血小板缺陷的患者来了解正常血小板机制和所涉及的蛋白质。迄今为止，我们的研究非常成功，导致在三种主要信号蛋白（磷脂酶C-2，GTP结合蛋白GQ和蛋白激酶C-）中首次描述迄今无法识别的缺陷，并进入了与主要转录因子的单倍障碍因子相关的血小板功能障碍。这些患者是关于正常血小板机制的未开发信息的宝贵存储库。该项目的具体目的是了解与转录因子RUNX1突变相关的血小板缺陷的分子基础（核心结合因子A2，RUNX1），并通过Runx1单位缺陷表征的患者的unlx1中的runx1在血小板/巨核细胞中调节的基因。 RUNX1单位缺陷与家族性血小板减少症，血小板功能受损和急性白血病的易感性有关。我们的患者患有轻度的血小板减少症，明显异常的聚集和分泌，与激动剂刺激的PLECKSTRIN和肌球蛋白轻链磷酸化（MLC）相关，PKC-降低和GPIIB-IIIA的激活降低。该患者在RUNX1中具有杂合突变。该患者中的血小板表达分析研究表明，特异性血小板/巨核细胞（MK）基因的下调与RUNX1单倍缺陷相关。迄今为止，我们已经确定，四个基因（血小板因子4（PF4），肌球蛋白光链（MYL9），12-脂氧合酶（ALOX12）和蛋白激酶C-（PRKCQ）（PRKCQ）确实是Runx1的转录靶标，确实是Runx1的转录靶标，它们在患者中提供了一些特定的基因，我们正在为我们提供了一些重要的洞察力。 Pallidin（PLDN）：致密和α颗粒缺陷是Runx1单倍缺陷的主要特征，而机制未知。 pallid（PLDN）缺陷与鼠标模型中的Delta-SPD有关。 b）小gtpases，rab1b和rab31以及rapgap1l，是蛋白质靶向囊泡，分泌和颗粒的主要参与者； c）flNA，TUBB1，与细胞骨架系统有关，在血小板产生，细胞形状，粘附和分泌中起主要作用。据我们所知，尚未报道人血小板或丝蛋白缺乏。对于每个基因，我们将a）证明患者血小板中特异性蛋白的降低； b）通过分子生物学研究确定该基因是Runx1的直接转录靶标； c）研究runx1下调和过表达对基因和相关特征的影响（蛋白质水平，功能效应，MK发育和血小板）； d）基于特定基因对患者血小板/MK进行功能和生化研究。此外，我们将对MK中的Runx1调节进行选定的研究。我们将将这些研究扩展到其他RUNX1突变患者。这些研究将提供有关迄今未识别的runx1单倍缺陷（由Runx1调节的基因/蛋白质及其血小板/MK角色）的血小板/MK的尚无法识别的变化的信息。他们将促进我们对正常和异常血小板功能中机制的理解。