MOLECULAR ANALYSIS OF PLATELET INTEGRIN ACTIVATION
血小板整合素激活的分子分析
基本信息
- 批准号:2771148
- 负责人:
- 金额:$ 8.26万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1996
- 资助国家:美国
- 起止时间:1996-09-30 至 2001-08-31
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
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.
在以前的实验室研究活动中,
研究巨核细胞发育的分子生物学,
血小板功能,申请人已经产生了追求
血小板活化的先进分子研究。 的目的
建议的研究是鉴定、克隆和表征编码
参与整联蛋白功能调节的效应分子
在活化的血小板中。 血小板的活化是
它们完全参与止血和血栓形成(Kroll和Schafer,
1989),因此是一个标志的分化功能,在
巨核细胞谱系。 申请人寻求开发一种新系统,
对介导其中一种信号传导机制的分子分析,
血小板活化的主要表现;激动剂刺激的
诱导整联蛋白α IIbB 3的纤维蛋白原(Fg)受体功能
(血小板糖蛋白IIb-IIIa)。 这种现象取决于
涉及蛋白激酶活性的细胞内信号转导
蛋白激酶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实体的身份,
调节α IIb β 3的亲和状态,是未知的。 虽然
受体-配体相互作用通过α IIb β 3激活成为可能
已经详细研究了生物化学,生物物理学和免疫学
方法论,基本的机械问题尚未解决。 解决
这个问题,一个新的筛选策略已经制定了协商
Brian Seed教授(马萨诸塞州分子生物学系
总医院和哈佛医学院),谁是公认的
作为这项技术(平移)发展的先驱,谁将
担任该项目这一阶段的顾问。 人血小板cDNA
文库将在巨核细胞人类中瞬时表达
红白血病(HEL)细胞,并用
报告该整联蛋白活化状态的β 11 β 3配体。
这种方法消除了对稳定表达的需要,从而
避免了增殖分化细胞的潜在问题。
该策略的另一个重要特征是,
识别不一定相互作用的效应分子
直接与α 11b β 3结合。 稳定HEL细胞的初步研究
转染子已经证明PKC α的异源表达
(其在天然HEL细胞中不存在,但在血小板中丰富,
因此在终末分化的巨核细胞中)导致
在细胞粘附试验中增强α IIb β 3功能,
证明了将该细胞系(HELalpha)用于
细胞内信号传导与整合素亲和力的相关研究
调变 执行《公约》具体规定所需的方法
目的包括:(i)构建并在HEL细胞中表达人
血小板cDNA文库;(ii)鉴定,并从中分离
通过免疫学筛选和表达的候选cDNA文库
克隆;和(iii)候选cDNA的分子表征,
核苷酸序列分析和表达在巨核细胞和其他
表达α 1B β 3的有核细胞系。 研究环境
其中拟开展的项目包括一个实验室,
它在分子机制的表征中很活跃
血小板和血小板膜细胞中潜在的细胞内信号传导过程
巨核细胞谱系,是血管医学的组成部分
这所大学由贝斯以色列医院的导师创立。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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JAMES D CHANG其他文献
JAMES D CHANG的其他文献
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{{ truncateString('JAMES D CHANG', 18)}}的其他基金
MOLECULAR ANALYSIS OF PLATELET INTEGRIN ACTIVATION
血小板整合素激活的分子分析
- 批准号:
2027060 - 财政年份:1996
- 资助金额:
$ 8.26万 - 项目类别:
MOLECULAR ANALYSIS OF PLATELET INTEGRIN ACTIVATION
血小板整合素激活的分子分析
- 批准号:
6056067 - 财政年份:1996
- 资助金额:
$ 8.26万 - 项目类别:
MOLECULAR ANALYSIS OF PLATELET INTEGRIN ACTIVATION
血小板整合素激活的分子分析
- 批准号:
6182347 - 财政年份:1996
- 资助金额:
$ 8.26万 - 项目类别:
MOLECULAR ANALYSIS OF PLATELET INTEGRIN ACTIVATION
血小板整合素激活的分子分析
- 批准号:
2519186 - 财政年份:1996
- 资助金额:
$ 8.26万 - 项目类别:
ROLE OF PROTEIN KINASE C IN PLATELET ACTIVATION
蛋白激酶 C 在血小板激活中的作用
- 批准号:
2213388 - 财政年份:1993
- 资助金额:
$ 8.26万 - 项目类别: