Human Platelet Defects in Transcription Factor RUNX1 Haplodeficiency

描述(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临床实践中广泛遇到。在绝大多数此类患者中，分子机制完全未知。我们研究的长期目标是通过对遗传性血小板缺陷患者的研究，深入了解正常的血小板机制和相关蛋白。到目前为止，我们的研究已经非常成功，导致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-的未知缺陷，以及与主要转录因子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无价之宝。该项目的具体目的是通过对具有RUNX1单倍体缺陷特征的患者的研究，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调节的血小板/巨核细胞中的基因。RUNX1单倍体缺陷与家族性血小板减少、血小板功能受损和急性白血病的易感性相关。我们的患者有轻度的血小板减少，明显的异常聚集和分泌，伴随着激动剂刺激的Pleckstrin和肌球蛋白轻链(MLC)的磷酸化减少，PKC--降低和GPIIb-IIIa的激活受损。该患者的RUNX1基因发生杂合突变。该患者的血小板表达谱研究显示，特定的血小板/巨核细胞(MK)基因下调与RUNX1单倍体缺陷有关。到目前为止，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的转录靶点，它们为研究患者的血小板缺陷提供了重要的见解。我们现在提议研究在我们的患者血小板中被证明下调的特定的其他基因，包括a)Pallidin(PLDN)：致密和α颗粒缺陷是RUNX1单倍体缺陷的主要特征，机制尚不清楚。在小鼠模型中，苍白(PLDN)缺乏与Delta-SPD相关。B)小GTP酶，RAB1B和RAB31，以及RAPGAP1L，它们是蛋白质的囊泡运输、分泌和颗粒靶向的主要角色；以及c)与细胞骨架系统相关的Flna，TUBB1，在血小板的产生、细胞形态、黏附和分泌中发挥主要作用。据我们所知，人类缺乏血小板、苍白素或细丝蛋白的情况尚未见报道。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的直接转录靶点；c)研究RUNX1下调和过表达对基因及其相关特征(蛋白质水平、功能效应、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RUNX1在MK中的调控进行选择性研究。我们将把这些研究扩展到其他携带RUNX1突变的患者。这些研究将提供有关RUNX1单倍体缺陷症中迄今未被发现的血小板/MK变化、受RUNX1调控的基因/蛋白质及其在血小板/MK中的作用的信息。它们将促进我们对正常和异常血小板功能的机制的理解。

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.