Human Platelet Defects in Transcription Factor RUNX1 Haplodeficiency

摘要 我们对遗传性血小板功能障碍的分子基础有着长期的研究兴趣。 转录因子RUNX1是造血、巨核和血小板生成的主要调节因子。 这个项目的总体目标是深入了解血小板功能障碍的分子基础。 与人类RUNX1突变相关的基因及其在血小板/巨核细胞(MK)中的调控 通过对以家族性血小板减少为特征的RUNX1单倍体缺陷(RHD)患者的研究， 血小板功能障碍和急性白血病的易感性。他们的血小板颗粒和 血小板反应受损。我们对一例风湿性心脏病患者的血小板表达谱，这是最早的血小板之一 对一名遗传性血小板功能障碍患者的图谱研究显示，几个基因下调 受调控，有些是直接的RUNX1靶点，并影响血小板/MK生物学的特定方面。RHD是一种 重要的人体模型和尚未开发的血小板/MK生物学的信息库。研究 在本届R01奖的支持下取得了极大的成功，并扩大了RUNX1的相关性 从出血性疾病到心血管疾病的基因调控。我们建议对2的RUNX1调控进行研究 风湿性心脏病血小板表达降低的基因：凝血因子XIIIA(基因F13A)和 磷酸二酯酶E5A(PDE5A)；对其调节知之甚少。目标1是研究其机制。 风湿性心脏病患者MK/血小板F13A、PDE5A表达降低的影响。F13A由MK合成， (~3%的血小板蛋白)，并调节凝块回缩。PDE5A调节cGMP水平，cGMP是 血小板反应。我们将在RHD患者的血小板和培养细胞(HEL细胞和MK)中进行研究 由风湿性心脏病患者的IPSCs产生)。我们将评估RUNX1对这些基因的调控在 并与心脏病患者未来的死亡和心肌梗死有关。RUNX1由两个表达式表示 交替的启动子--远端的P1和近端的P2启动子，导致两个不同的蛋白质，RUNX1C和 RUNX1B，具有不同的效果。目的2研究MK/血小板基因的差异调控。 RUNX1B和RUNX1C。最近的证据表明，RUNX1C是由RUNX1自动调节的。目标3是 研究RUNX1B和RUNX1C对RUNX1的自动调节作用。我们的研究将提供重要的、新的 风湿性心脏病血小板/MK异常机制及RUNX1对差异基因调控的研究 变异及其下游影响，以及与心血管疾病的关系。他们将奠定基础， 开发新的抗血栓策略。

Summary We have a longstanding research interest into the molecular basis of inherited platelet dysfunction. Transcription factor RUNX1 is a master regulator of hematopoiesis, megakaryopoiesis and thrombopoiesis. The overall goal of this project is to obtain insights into the molecular basis of the platelet dysfunction associated with human RUNX1 mutations and into the genes regulated by it in platelets/megakaryocytes (MK) through studies in patients with RUNX1 haplodeficiency (RHD), characterized by familial thrombocytopenia, platelet dysfunction and a predisposition to acute leukemia. They have abnormalities in platelet granules and impaired platelet responses. Our platelet expression profiling of a RHD patient, one of the first platelet profiling studies in a patient with inherited platelet dysfunction, showed several genes are down regulated, some are direct RUNX1 targets, and affect specific aspects of platelet/MK biology. RHD is an important human model and untapped reservoir of information into platelet/ MK biology. Studies supported by the current R01 Award have been highly successful and extend the relevance of RUNX1 regulation of genes from bleeding disorder to CV disease. We propose studies on the RUNX1 regulation of 2 genes whose expression is decreased in RHD platelets: coagulation factor XIIIa (gene F13A) and phosphodiesterase E5A (PDE5A); little is known regarding their regulation. Aim 1 is to study the mechanisms and effects of decreased MK/platelet expression of F13A and PDE5A in RHD. F13A is synthesized by MK, (~3% of platelet protein) and regulates clot retraction. PDE5A regulates cGMP levels, a major regulator of platelet responses. We will perform studies in RHD patient platelets and in cultured cells, (HEL cells and MK generated from IPSCs from a RHD patient). We will assess association of RUNX1 regulation of these genes in vivo and in relation to future death and MI in patients with heart disease. RUNX1 is expressed from two alternate promoters – a distal P1 and a proximal P2 promoter, leading to two distinct proteins, RUNX1C and RUNX1B, with differential effects. Aim 2 is to study the differential regulation of MK/platelet genes by the RUNX1B and RUNX1C. Recent evidence suggests that RUNX1C is autoregulated by RUNX1. Aim 3 is to study the autoregulation of RUNX1 by RUNX1B and RUNX1C. Our studies will provide important, new information into the aberrant platelet/MK mechanisms in RHD, the differential gene regulation by RUNX1 variants and their downstream effects, and the relationship to CV disease. They will lay the basis for developing newer antithrombotic strategies.