Identification of molecular pathways that initiate proplatelet formation

鉴定启动前血小板形成的分子途径

基本信息

批准号：
8782548
负责人：
Kellie Rae Machlus
金额：
$ 5.42万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-01-01 至 2015-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8782548
关键词：
Abbreviations Acetates Actins Aplastic Anemia Autologous Blood Transfusion Biological Blood Platelets Blood Vessels Bone Marrow Bovine Serum Albumin Cell physiology Cells Clinical Cytoskeletal Modeling Cytoskeletal Proteins Cytoskeleton Data Development Disease Doctor of Philosophy Environment Extracellular Matrix F-Actin Goals Green Fluorescent Proteins HIV Health Hemorrhage Hemostatic function Immune Immunofluorescence Immunologic Immunoprecipitation Injury Knock-out Knockout Mice Liquid Chromatography Megakaryocytes Microfluidics Modeling Molecular Mus Operative Surgical Procedures Pathway interactions Patients Phosphorylation Physiology Platelet Count measurement Play Polyacrylamide Gel Electrophoresis Pregnancy Process Production Protein Biosynthesis Protein Dephosphorylation Protein Kinase C Protein Overexpression Proteins Proteomics RNA Interference Rest Role Small Interfering RNA Sodium Dodecyl Sulfate Sodium Dodecyl Sulfate-PAGE Spectrin Staging Stem cells System Talin Techniques Testing Thrombocytopenia Thrombocytopenic Purpura Thrombosis Vinculin Virus Diseases base chemotherapy crosslink gel electrophoresis link protein multicatalytic endopeptidase complex myristoylated alanine-rich C kinase substrate new therapeutic target novel novel therapeutics overexpression phorbol-12-myristate precursor cell prevent protein degradation research study tandem mass spectrometry therapeutic target tool two-dimensional ubiquilin

项目摘要

DESCRIPTION (provided by applicant): Circulating blood platelets are specialized cells that function to prevent bleeding and minimize blood vessel injury. As such, platelets play a critical role in both normal and disease physiology. The currently favored model of platelet formation states that large progenitor cells in the bone marrow called megakaryocytes (MKs) release platelets by extending long, branching processes, designated proplatelets, into sinusoidal blood vessels. Despite the importance of platelets in thrombosis and hemostasis, the cellular and molecular basis of the process by which MKs complete differentiation and release platelets is poorly understood. In particular, little is known about what triggers resting, mature MKs in the bone marrow to begin forming and releasing proplatelets. Proteomic analysis of resting versus proplatelet-producing MKs suggests that there is a distinct subset of proteins synthesized in the late stages of MK development that are necessary for proplatelet formation. This proposal will focus on two proteins identified by proteomics that are up-regulated in proplatelet-producing MKs, myristoylated, alanine-rich, C kinase substrate (MARCKS) and ubiquilin. Both proteins are involved in cytoskeletal reorganization; MARCKS cross-links F-actin, while ubiquilin links proteins to the proteasome for degradation. I hypothesize that the dynamic cytoskeletal process of proplatelet formation requires both protein synthesis and degradation, and MARCKS and ubiquilin are integral to cytoskeletal remodeling by modulating actin crosslinking and protein degradation, respectively. We will test this hypothesis using techniques such as siRNA, protein overexpression, and immunoprecipitation to establish the role of MARCKS and ubiquilin in proplatelet formation. We will then use the MARCKS knockout mouse and a novel microfluidics system to explore the role of these proteins in an ex vivo environment. The results of the proposed experiments will significantly advance our understanding of the cellular and molecular basis regulating platelet production. Our long-range goal is to elucidate cell biological and molecular pathways that power platelet production, with the intent of defining novel therapeutic strategies to accelerate platelet production in patients with thrombocytopenia.

描述（由申请人提供）：循环血小板是一种特殊的细胞，其功能是防止出血并最大限度地减少血管损伤。因此，血小板在正常和疾病生理学中都发挥着关键作用。目前受欢迎的血小板形成模型指出，骨髓中称为巨核细胞（MK）的大型祖细胞通过将长的分支过程（称为前血小板）延伸到正弦血管中来释放血小板。尽管血小板在血栓形成和止血中很重要，但人们对 MK 完全分化和释放血小板过程的细胞和分子基础知之甚少。特别是，对于是什么触发骨髓中静止的成熟 MK 开始形成和释放前血小板，我们知之甚少。对静息 MK 与产生前血小板的 MK 的蛋白质组学分析表明，在 MK 发育的后期阶段合成了一个独特的蛋白质子集，这些蛋白质是前血小板形成所必需的。该提案将重点关注通过蛋白质组学鉴定的两种蛋白质，它们在前血小板生成 MK 中上调，即肉豆蔻酰化、富含丙氨酸、C 激酶底物 (MARCKS) 和泛素。这两种蛋白质都参与细胞骨架重组。 MARCKS 交联 F-肌动蛋白，而泛素则将蛋白质连接至蛋白酶体以进行降解。我假设前血小板形成的动态细胞骨架过程需要蛋白质合成和降解，而 MARCKS 和泛素分别通过调节肌动蛋白交联和蛋白质降解而成为细胞骨架重塑的组成部分。我们将使用 siRNA、蛋白质过表达和免疫沉淀等技术来检验这一假设，以确定 MARCKS 和泛素在前血小板形成中的作用。然后，我们将使用 MARCKS 敲除小鼠和新型微流体系统来探索这些蛋白质在离体环境中的作用。所提出的实验结果将显着增进我们对调节血小板产生的细胞和分子基础的理解。我们的长期目标是阐明促进血小板生成的细胞生物学和分子途径，旨在确定新的治疗策略以加速血小板减少症患者的血小板生成。