MOLECULAR ANALYSIS OF PLATELET INTEGRIN ACTIVATION

血小板整合素激活的分子分析

• 批准号: 6182347
• 负责人: JAMES D CHANG
• 金额: $ 11.69万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6182347
• 关键词: biological signal transduction; expression cloning; genetic library; integrins; nucleic acid sequence; platelet activation; receptor binding; tissue /cell culture; transfection

In the course of previous research activities undertaken in laboratories investigating the molecular biology of megakaryocyte development and platelet function, the applicant has developed an interest in pursuing advanced molecular studies of platelet activation. The objective of the proposed research is to identify, clone, and characterize cDNAs encoding effector molecules that participate in the modulation of integrin function in activated platelets. Activation of blood platelets is prerequisite for their full participation in hemostasis and thrombosis (Kroll and Schafer, 1989), and is therefore a hallmark of differentiated function in the megakaryocytic lineage. The applicant seeks to develop a new system for the molecular analysis of signaling mechanisms that mediate one of the cardinal manifestations of platelet activation; agonist-stimulated induction of the fibrinogen (Fg) receptor function of integrin alphaIIbB3 (platelet glycoprotein IIb-IIIa). This phenomenon is dependent upon intracellular signal transduction involving the activity of protein kinase C (PKC) and other signaling molecules, but details of these processes, including the identity of specific intermediary and effector entities that regulate the affinity state of alphaIIbbeta3, are unknown. Although receptor-ligand interactions made possible by alpha IIbbeta3 activation have been studied in detail with biochemical, biophysical, and immunologic methodology, the fundamental mechanistic problem is unsolved. To address this problem, a novel screening strategy has been developed in consultation with Professor Brian Seed (Department of Molecular Biology, Massachusetts General Hospital and harvard Medical School), who is generally acknowledged as a pioneer in the development of this technique (panning), and who will serve as consultant for this phase of the project. A human platelet cDNA library will be transiently expressed in megakaryoblastic human erythroleukemia (HEL) cells, and immunologically screened with alph11bbetta3 ligands that report on the activation state of this integrin. This approach obviates the need for stable expression and thereby circumvents the potential problem of propagating differentiated cells. Another important feature of this strategy is that it permits identification of effector molecules that do not necessarily interact directly with alpha11bbeta3. Preliminary studies of stable HEL cell transfectants have demonstrated that heterologous expression of PKC alpha (which is absent in native HEL cells but abundant in platelets and therefore in terminally differentiated megakaryocytes) results in enhancement of alpha IIbbeta3 function in a cell adhesion assay, demonstrating the feasibility of employing this cell line (HELalpha) in correlative studies of intracellular signaling and integrin affinity modulation. The methodologies required for implementation of the specific aims include: (i) construction, and expression in HEL cells, of a human platelet cDNA library; (ii) identification, and isolation from this library, of candidate cDNAs by immunologic screening and expression cloning; and (iii) molecular characterization of candidate cDNAs by nucleotide sequence analysis and expression in megakaryoblastic and other nucleated cell lines that express alphaiibbeta3. The research environment in which the proposed project is to be undertaken consists of a laboratory that is active in the characterization of the molecular mechanisms underlying intracellular signaling processes in platelets and cells of megakaryoblastic lineage and is an integral part of the Vascular Medicine univ founded by the preceptor at Beth Israel Hospital.

在实验室以前进行的研究活动过程中 研究巨核细胞发育的分子生物学和 血小板功能，申请者已经形成了追求的兴趣 血小板活化的高级分子研究。该计划的目标是 拟议的研究是识别、克隆和鉴定编码的cDNA 参与整合素功能调控的效应分子 在激活的血小板中。血小板的激活是治疗的前提 他们充分参与止血和血栓形成(Kroll和Schafer， 1989)，因此是区分功能的标志 巨核细胞系。申请者寻求开发一种新的系统来 对信号机制的分子分析，这些信号机制介导了 血小板活化的主要表现；激动剂刺激 整合素αIIbB3纤维蛋白原受体功能的诱导 (血小板膜糖蛋白IIb-IIIa)。这一现象依赖于 涉及蛋白激酶活性的细胞内信号转导 C(PKC)和其他信号分子，但这些过程的细节， 包括特定中介和效应器实体的身份， 调节AlphaIIbbeta3的亲和力状态，目前尚不清楚。虽然 αIIbbeta3激活使受体-配体相互作用成为可能 已经用生化、生物物理和免疫学进行了详细的研究 方法论上，根本的机制问题还没有解决。致信地址 针对这一问题，在咨询中制定了一种新的筛查策略 与Brian Seed教授(马萨诸塞州分子生物学系) 综合医院和哈佛医学院)，这是公认的 作为这种技术(平移)发展的先驱，谁将 担任项目这一阶段的顾问。一种人血小板基因的表达谱 文库将在人巨核细胞中瞬时表达 红白血病(HEL)细胞，免疫筛选 Alph11bbetta3报告该整合素的激活状态的配体。 这种方法消除了对稳定表达的需要，从而 绕过了繁殖分化细胞的潜在问题。 这一策略的另一个重要特征是它允许 识别不一定相互作用的效应器分子 直接用字母11bbeta3。稳定的HEL细胞的初步研究 转染者已证明PKCα的异源表达 (在天然HEL细胞中不存在，但在血小板和 因此在终末分化的巨核细胞中)导致 细胞黏附试验中αIIbbeta3功能的增强， 演示使用该细胞系(HELAlpha)的可行性 细胞内信号转导与整合素亲和力的相关性研究 调制。具体实施所需的方法学 目的包括：(I)构建人的HEL细胞并在其中表达 血小板c DNA文库；(Ii)鉴定和分离 通过免疫学筛选和表达的候选cDNA文库 克隆；和(Iii)候选cDNA的分子特征 巨核细胞及其他细胞的核苷酸序列分析及表达 表达alphaibbeta3的有核细胞系。研究环境 拟议的项目将在其中进行，由实验室组成 这在分子机制的表征中是活跃的 血小板和细胞的潜在细胞内信号转导过程 巨核细胞系是血管医学不可或缺的一部分 这所大学是由贝丝以色列医院的校长创办的。