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: 8602856
• 负责人: Angara Koneti Rao
• 金额: $ 37.64万
• 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 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8602856
• 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; public health relevance; 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（申请人提供）：血液学临床实践中广泛遇到患有遗传性血小板功能障碍和出血表现的患者。对于绝大多数此类患者，分子机制完全未知。我们研究的长期目标是通过研究患有遗传性血小板缺陷的患者来深入了解正常血小板机制和相关蛋白质。迄今为止，我们的研究非常成功，首次描述了三种主要信号蛋白（磷脂酶 C-2、GTP 结合蛋白 Gq 和蛋白激酶 C-）迄今为止未被认识的缺陷，并首次描述了与主要转录因子 RUNX1 单倍体缺陷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资源。该项目的具体目的是通过对 RUNX1 单倍体缺陷患者的研究，深入了解与转录因子 RUNX1（核心结合因子 A2，RUNX1）突变相关的血小板缺陷的分子基础，以及 RUNX1 在血小板/巨核细胞中调节的基因。 RUNX1 单倍体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易感性相关。该患者患有轻度血小板减少症、与激动剂刺激的普莱克斯特林和肌球蛋白轻链 (MLC) 磷酸化减弱相关的明显异常聚集和分泌、PKC- 降低以及 GPIIb-IIIa 激活受损。该患者的 RUNX1 存在杂合突变。该患者的血小板表达谱研究显示特定血小板/巨核细胞 (MK) 基因的下调与 RUNX1 单倍体缺陷相关。迄今为止，我们已经确定四个基因（血小板因子 4 (PF4)、肌球蛋白轻链 (MYL9)、12-脂氧合酶 (ALOX12) 和蛋白激酶 C- (PRKCQ) 确实是 RUNX1 的转录靶标，它们为了解患者血小板缺陷提供了重要见解。我们现在提议研究在我们的患者血小板中显示下调的特定其他基因，包括： Pallidin (PLDN)：致密和α颗粒缺陷是RUNX1单倍体缺陷的主要特征，其机制未知。 Pallid (pldn) 缺陷与小鼠模型中的 delta-SPD 相关。 b) 小 GTP 酶、RAB1B 和 RAB31 以及 RAPGAP1L，它们在蛋白质的囊泡运输、分泌和颗粒靶向中起主要作用； c) FLNA、TUBB1，与细胞骨架系统相关，在血小板生成、细胞形状、粘附和分泌中起主要作用。据我们所知，尚未有人类缺乏血小板苍白蛋白或细丝蛋白的报道。对于每个基因，我们将 a) 证明患者血小板中特定蛋白质的减少； 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的下调和过表达对该基因和相关特征（蛋白质水平、功能影响、MK发育和血小板生成）的影响； d) 根据特定基因对患者血小板/MK 进行功能和生化研究。此外，我们还将对 MK 中 RUNX1 的调控进行选择性研究。我们将把这些研究扩展到其他具有 RUNX1 突变的患者。这些研究将提供有关 RUNX1 单倍体缺陷中血小板/MK 迄今为止未被识别的改变、受 RUNX1 调节的基因/蛋白质及其血小板/MK 作用的信息。它们将增进我们对正常和异常血小板功能机制的理解。