The Biological Roles of Phoshadylinositol Transfer Proteins in Platelets

血小板中磷脂肌醇转移蛋白的生物学作用

• 批准号: 8257824
• 负责人: CHARLES S. ABRAMS
• 金额: $ 42.74万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-15 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8257824
• 关键词: Actins; Adhesions; Anabolism; Atherosclerosis; Binding; Biochemical; Biological; Biology; Blood Platelets; Blood Vessels; Cells; Coagulation Process; Country; Cytoplasm; Cytoplasmic Granules; Cytoskeleton; Data; Defect; Development; Disease; Dose; Enzymes; Event; Family; Family member; Fatty Acids; Focal Adhesions; Funding; Generations; Genes; Genetically Engineered Mouse; Goals; Hand; Heart Diseases; Hematopoietic; Hemostatic function; In Vitro; Individual; Integrins; Knock-out; Knockout Mice; Lead; Link; Mammalian Cell; Mediating; Megakaryocytes; Membrane; Metabolism; Morbidity - disease rate; Mus; Phenotype; Phosphatidylinositol Phosphates; Phosphatidylinositol Transfer Protein; Phosphatidylinositols; Phospholipids; Phosphorylation; Platelet Activation; Play; Process; Property; Protein Family; Protein Isoforms; Proteins; Regulation; Role; Second Messenger Systems; Signal Pathway; Signal Transduction; Signaling Protein; Site; Specificity; Stroke; Testing; Thrombin; Thrombocytopenia; Thrombosis; Tissues; Transmembrane Transport; Vesicle; Work; Yeasts; aqueous; design; in vivo; member; monomer; mortality; novel therapeutic intervention; null mutation; prevent; protein expression; research study; response; second messenger; trafficking

DESCRIPTION (provided by applicant): Phosphorylated phosphatidylinositols (phosphoinositides) are a type of membrane bound phospholipid that contributes to multiple diverse processes required during platelet activation. PhosphatidylInositol Transfer Proteins (PITPs) are a small family of proteins that have been demonstrated in vitro to bind and transfer phosphoinositide monomers from one cellular compartment to another in an energy independent manner during vesicle trafficking and phospholipid signaling. Although there are no studies on the role of PITPs in hematopoietic cells, there is evidence in yeast cells that these proteins are essential for the biosynthesis and metabolism of phosphoinositides. Platelets have two dominant PITP family members, PITP1 and PITP2. The overall hypothesis of this proposal is that these individual PITP isoforms have non- overlapping functions that are each essential for the generation and spatial localization of discrete species of phosphoinositides within platelets. A secondary hypothesis is that the enzymatic activities of both PITP isoforms are necessary for normal platelet adhesion, aggregation, and granule secretion. To understand the unique and discrete roles of these individual PITP isoforms in platelet biology, we have generated mice containing conditional null mutations within the PITP1 and PITP2 genes. This R01 is to request funds that will allow us to characterize PITP1fl/fl PF4Cre+, PITP2fl/fl PF4Cre+, and PITP1fl/fl PITP2fl/fl PF4Cre+ (double knockout) mice. These mice have platelets and mature megakaryocytes lacking either PITP1 or PITP2, but they have normal expression of these proteins in all other tissues. Loss of either isoform results in thrombocytopenia. Our preliminary data indicates that platelets lacking PITP1 have a complete loss in the second messenger, Ins(3,4,5)P3 (also known as IP3) following stimulation by maximal doses of thrombin. I plan to perform a comprehensive and systematic study of the function of these individual PITP isoforms in platelets in order to understand their unique biochemical role and biologic importance within platelets. In Aim 1, we will determine the link between PITP isoforms and polyphosphoinositide synthesis, as well as analyze the contribution of PITPs to platelet signaling. In Aim 2, I propose experiments designed to understand the distinct biochemical functions of the individual PITP isoforms. In the final Aim, we will identify the role of PITP isoforms in platelet activation ex vivo and in vivo. PUBLIC HEALTH RELEVANCE: Heart disease and strokes are among the leading causes of morbidity and mortality in this country. Activated blood platelet cells can form clots at sites of atherosclerosis, and are often the precipitating event for both of these diseases. This work is focused on two platelet proteins, PITP1 and PITP2. I believe that a better understanding of the events that regulate platelet activation, including those mediated by PITP1 and PITP2, will lead to new therapeutic approaches to prevent vascular occlusion, as well as lead to a better understanding of platelet biology.

性状（由申请方提供）：磷酸化磷脂酰肌醇（磷酸肌醇）是一种膜结合磷脂，有助于血小板活化期间所需的多种不同过程。磷脂酰肌醇转移蛋白（PITP）是一个小家族的蛋白质，其已经在体外被证明在囊泡运输和磷脂信号传导期间以能量不依赖的方式结合并将磷酸肌醇单体从一个细胞区室转移到另一个细胞区室。 虽然还没有关于PITPs在造血细胞中作用的研究，但在酵母细胞中有证据表明，这些PITPs在造血细胞中的作用可能与它们在造血细胞中的作用有关。 蛋白质对于磷酸肌醇的生物合成和代谢是必需的。 血小板有两个主要的PITP家族成员，PITP 1和PITP 2。该提议的总体假设是这些单独的PITP同种型具有非重叠的功能，其各自对于血小板内磷酸肌醇的离散种类的产生和空间定位是必需的。第二个假设是两种PITP亚型的酶活性对于正常的血小板粘附、聚集和颗粒分泌是必需的。为了了解这些个体PITP亚型在血小板生物学中的独特和离散作用，我们产生了在PITP 1和PITP 2基因内含有条件无效突变的小鼠。 本R 01旨在申请资金，以使我们能够表征PITP 1fl/fl PF 4Cre+、PITP 2fl/fl PF 4Cre+和PITP 1fl/fl PITP 2fl/fl PF 4Cre+（双敲除）小鼠。这些小鼠的血小板和成熟巨核细胞缺乏PITP 1或PITP 2，但它们在所有其他组织中正常表达这些蛋白质。任何一种亚型的缺失都会导致血小板减少症。我们的初步数据表明，缺乏PITP 1的血小板在最大剂量的凝血酶刺激后，第二信使Ins（3，4，5）P3（也称为IP 3）完全丧失。我计划对血小板中这些单独的PITP亚型的功能进行全面和系统的研究，以了解它们在血小板中独特的生化作用和生物学重要性。在目的1中，我们将确定PITP亚型和多磷酸肌醇合成之间的联系，以及分析PITP对血小板信号传导的贡献。在目标2中，我提出了旨在了解个体PITP亚型的独特生化功能的实验。在最后的目的，我们将确定的作用，PITP亚型在血小板活化离体和体内。 公共卫生相关性：心脏病和中风是该国发病率和死亡率的主要原因。激活的血小板细胞可以在动脉粥样硬化部位形成血栓，并且通常是这两种疾病的诱发事件。 这项工作的重点是两个血小板蛋白，PITP 1和PITP 2。 我相信，更好地了解调节血小板活化的事件，包括PITP 1和PITP 2介导的事件，将导致新的治疗方法来预防血管闭塞，并导致更好地了解血小板生物学。