Human Platelet Defects in Transcription Factor RUNX1 Haplodeficiency

描述（由申请人提供）：在血液学临床实践中，广泛遇到遗传性血小板功能障碍和出血表现的患者。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，深入了解正常血小板机制和相关蛋白质。迄今为止，我们的研究已经非常成功，导致迄今未被认识到的缺陷，在三个主要的信号蛋白，磷脂酶C-2，GTP结合蛋白Gq和蛋白激酶C-的第一个描述，并进入血小板功能障碍与单倍缺陷的主要转录因子，RUNX 1。这些患者是一个宝贵的知识库，未开发的信息正常血小板机制。本项目的具体目的是通过对RUNX 1单倍缺陷患者的研究，深入了解与转录因子RUNX 1（核心结合因子A2，RUNX 1）突变相关的血小板缺陷的分子基础，以及RUNX 1在血小板/巨核细胞中调控的基因。RUNX 1单倍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。我们的病人有轻度血小板减少症，明显异常的聚集和分泌与减少激动剂刺激的磷酸化pleckstrin和肌球蛋白轻链（MLC），降低PKC-和受损的激活GPIIb-IIIa。该患者在RUNX 1中存在杂合突变。该患者的血小板表达谱研究显示，与RUNX 1单倍缺陷相关的特定血小板/巨核细胞（MK）基因下调。迄今为止，我们已经确定了四个基因（血小板因子4（PF 4），肌球蛋白轻链（MYL 9），12-脂氧合酶（ALOX 12）和蛋白激酶C-（PRKCQ）确实是RUNX 1的转录靶点，它们为患者血小板缺陷提供了重要的见解。我们现在建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a）Pallidin（PLDN）：致密和α颗粒缺陷是RUNX 1单倍缺陷的主要特征，其机制未知。在小鼠模型中，苍白（pldn）缺乏与delta-SPD相关。B）小GTP酶，RAB 1 B和RAB 31，以及RAPGAP 1 L，它们是蛋白质的囊泡运输、分泌和颗粒靶向的主要参与者;以及c）FLNA，TUBB 1，与细胞骨架系统相关，并且在血小板产生、细胞形状、粘附和分泌中起主要作用。据我们所知，人类缺乏血小板苍白球蛋白或细丝蛋白尚未报道。对于每个基因，我们将a）证明患者血小板中特异性蛋白质的减少; B）通过分子生物学研究确定该基因是RUNX 1的直接转录靶; c）研究RUNX 1的下调和过表达对该基因和相关特征的影响（蛋白质水平、功能作用、MK发育和血小板生成）; d）基于特定基因在患者血小板/MK中进行功能和生物化学研究。此外，我们还将对MK中RUNX 1的调节进行选定的研究。我们将这些研究扩展到其他RUNX 1突变患者。这些研究将提供关于RUNX 1单倍缺陷症中血小板/MK的迄今未被认识的改变、由RUNX 1调节的基因/蛋白质及其血小板/MK作用的信息。他们将促进我们对正常和异常血小板功能机制的理解。

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.