Runx1 Haplodeficiency, Endocytosis and Vesicle transport

项目摘要 RUNX 1是一种主要的造血转录因子，RUNX 1单倍缺陷（RHD） 以家族性血小板减少症和血小板功能受损为特征。我们有一个 遗传性血小板功能障碍的分子基础，特别是 与RHD有关。在RHD中已经描述了许多血小板异常， 包括致密颗粒（DG）和α-颗粒（AG）缺陷。我们的血小板 对风湿性心脏病患者的表达谱研究表明， 调节（MYL 9，PRKCQ，ALOX 12，PF 4，PLDN，PCTP）;我们已经表明，这些 是RUNX 1的直接转录靶点，影响血小板/巨核细胞（MK） 生物学本提案的总体目标是获得对 血小板/MK中的内吞作用、囊泡运输和α颗粒形成机制 通过研究风心病的表型异常和基因失调。这 该项目建立在我们在RHD中识别的独特异常的基础上。我们在我们的 患者血小板白蛋白和IgG（通过大量内吞作用并入AG） 降低我们的表达谱研究表明，血小板RAB 1B，RAB 31和 与囊泡运输密切相关的DNM 3-三种GTP酶减少。之甚少 目前已知的关于调节血小板/MK中的内吞作用的机制， 这些GTP酶的作用。我们的假设是内吞和囊泡的机制 贩运在RHD中受损。目的1是深入了解调节 血小板/MK中的内吞作用，通过研究导致血小板减少的机制， 血小板白蛋白和IgG。我们将研究白蛋白和IgG的摄取和转运 在正常和RHD血小板中，研究RUNX 1，RAB 1B， RAB 31和DNM 3对MK中这些过程的影响，使用以下标记进行研究： 囊泡运输的分泌和内吞途径。目标2：了解 导致RHD AG缺乏的机制。我们将研究：血小板AG在正常和 RHD血小板集中于选定的AG蛋白; RUNXI下调的作用， RAB 1B、RAB 31和DNM 3对AG和AG蛋白在MK中的转运的影响。这些研究 将在5-6例RHD患者中进行，并使用诱导多能干细胞 （IPSC）已经从RHD患者发展而来。他们将提供重要的新见解 血小板/MK中的内吞作用和囊泡运输，目前对此知之甚少 知道的 相关性 血小板在止血、血栓形成、炎症、动脉粥样硬化和血栓形成中起主要作用。 处理病原体。我们的研究将提供关于以下基本方面的新信息： 通过对人类RUNX 1单倍缺陷的研究，血小板/MK功能， 信息的储存库。这些信息将为新的治疗方法奠定基础。 血栓性和出血性疾病的治疗方法。

Project Summary RUNX1 is a major hematopoietic transcription factor and RUNX1 haplodeficiency (RHD) is characterized by familial thrombocytopenia and impaired platelet function. We have a longstanding interest in the molecular basis of inherited platelet dysfunction, particularly related to RHD. Numerous platelet abnormalities have been described in RHD, several by us, and include deficiencies of dense granule (DG) and α-granules (AG). Our platelet expression profiling studies in a patient with RHD showed that several genes are down regulated (MYL9, PRKCQ, ALOX12, PF4, PLDN, PCTP); and we have shown that these are direct RUNX1 transcriptional targets and impact platelet/megakaryocyte (MK) biology. The overall objective of this proposal is to obtain new insights into the mechanisms of endocytosis, vesicle trafficking, and α-granule formation in platelets/MK through the study of phenotypic abnormalities and genes dysregulated in RHD. This project builds on unique abnormalities identified by us in RHD. We have reported in our patient that platelet albumin and IgG (incorporated by bulk endocytosis into AG) are decreased. Our expression profiling studies show that platelet RAB1B, RAB31 and DNM3 – three GTPases closely linked to vesicle trafficking are decreased. Little is currently known regarding the mechanisms regulating endocytosis in platelets/MK or the role of these GTPases. Our hypothesis is that mechanisms of endocytosis and vesicle trafficking are impaired in RHD. Aim 1 is to obtain insights into mechanisms regulating endocytosis in platelets/MK through the study of mechanisms leading to the decreased platelet albumin and IgG in RHD. We will study uptake and transport of albumin and IgG in normal and RHD platelets, study the effect of downregulation of RUNX1, RAB1B, RAB31 and DNM3 on these processes in MK, perform studies using markers of secretory and endocytic pathways of vesicle trafficking. Aim 2 is to understand the mechanisms leading to AG deficiency in RHD. We will study: platelet AG in normal and RHD platelets focusing on selected AG proteins; the effect of downregulation of RUNXI, RAB1B, RAB31and DNM3 on AG and trafficking of AG proteins in MK. These studies will be performed on 5-6 patients with RHD and using induced pluripotent stem cells (IPSCs) already developed from a RHD patient. They will provide important new insights into endocytosis and vesicle trafficking in platelets/MK, about which little is presently known. Relevance Platelets play a major role in hemostasis, thrombosis, inflammation, atherosclerosis and handling of pathogens. Our studies will provide new information on the basic aspects of platelet/MK function through studies in human RUNX1 haplodeficiency, a unique reservoir of information. This information will lay the foundation for new therapeutic approaches for both thrombotic and bleeding disorders.