GENETIC BASIS OF THROMBOTIC DISEASE
血栓性疾病的遗传基础
基本信息
- 批准号:6110089
- 负责人:
- 金额:$ 25.4万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1998
- 资助国家:美国
- 起止时间:1998-09-01 至 1999-08-31
- 项目状态:已结题
- 来源:
- 关键词:Native Americans blood coagulation disorders clinical research congenital blood protein disorder disease /disorder proneness /risk family genetics fibrinolysis gene mutation hemostasis human genetic material tag human subject intracellular transport linkage mapping polymerase chain reaction protein C protein sequence recombinant proteins thrombosis transfection vitamin K
项目摘要
The continuing overall objective of this project is to better understand
the biosynthesis, activation, and functional properties of the vitamin K-
dependent plasma proteins involved in blood clotting, and the consequences
of mutations in these proteins on hereditary thrombosis. This project
relates to the Program theme in that it explores, through the study of
protein C mutations, the fundamental mechanisms by which these proteins
are modified, transported, and secreted prior to their involvement in
Ca/2+-membrane associated processes. A new additional direction of the
project will be the chromosomal localization and identification of a
putative second gene, THROMC, that is associated with thrombotic disease
in a large type I protein C deficient kindred of Native American descent.
Specific aims are to identify new protein C mutations in families
exhibiting thrombophilia; construct, express, and biochemically
characterize secreted and non-secreted naturally occurring mutant forms of
protein C; and determine by genetic linkage analysis the existence and
location of a second gene (THROMC) associated with thrombosis.
Conventional methods of peripheral blood cell DNA and RNA isolation, PCR
amplification and site-directed mutagenesis, and DNA sequencing will be
employed. Recombinant protein C will be expressed in human kidney 293
cells and purified by ion exchange chromatography. Purified protein will
be chemically and functionally characterized as to propeptide cleavage;
gamma-carboxylation disulfide pairing; activation by thrombin-
thrombomodulin; Ca/2+ and phospholipid dependent ability to inactivate
factors V/a and VIII/a, inhibit clotting, and enhance fibrinolysis, using
reconstituted-purified component and whole-blood systems. Pulse-chase
experiments will examine the intracellular processing, transport, and
degradation of mutant forms of protein C. Polymorphic markers and linkage
analysis will be used to chromosomally locate the THROMC gene (ultimately
to a 1-2 centiMorgan region). Family members will also be phenotyped in
regard to thrombotic disease and several clinical markers of the
prothrombotic state, including protein C, prothrombin fragment 1.2, and
fibrin D-dimer levels. Unrelated thrombophilic families with and without
protein C deficiency will also be examined for the association of THROMC
with disease.
该项目的持续总体目标是更好地了解
维生素 K-的生物合成、活化和功能特性
参与血液凝固的依赖性血浆蛋白及其后果
这些蛋白质的突变对遗传性血栓形成的影响。 这个项目
与该计划的主题相关,因为它通过研究
蛋白 C 突变,这些蛋白的基本机制
在参与之前被修改、运输和分泌
Ca/2+-膜相关过程。 一个新的附加方向
该项目将是染色体定位和鉴定
假定的第二个基因 THROMC,与血栓性疾病相关
存在于美洲原住民血统的大型 I 型蛋白 C 缺陷家族中。
具体目标是识别家族中新的蛋白 C 突变
表现出血栓形成倾向;构建、表达和生化
表征分泌型和非分泌型自然发生的突变形式
蛋白质C;并通过遗传连锁分析确定其存在和
与血栓形成相关的第二个基因(THROMC)的位置。
外周血细胞DNA和RNA分离的常规方法、PCR
扩增和定点突变,以及 DNA 测序
受雇。 重组蛋白C将在人肾中表达293
细胞并通过离子交换层析纯化。 纯化的蛋白质将
对前肽裂解进行化学和功能表征;
γ-羧化二硫键配对;凝血酶激活-
血栓调节蛋白; Ca/2+ 和磷脂依赖性失活能力
因子 V/a 和 VIII/a,抑制凝血,并增强纤维蛋白溶解,使用
重组纯化成分和全血系统。 脉冲追踪
实验将检查细胞内的加工、运输和
蛋白质 C 突变体形式的降解。多态性标记和连锁
分析将用于染色体定位 THROMC 基因(最终
到 1-2 厘摩根区域)。 家庭成员也将进行表型分析
关于血栓性疾病和一些临床标志物
血栓前状态,包括蛋白 C、凝血酶原片段 1.2 和
纤维蛋白 D-二聚体水平。 无关的血栓形成倾向家族有或没有
还将检查蛋白 C 缺乏是否与 THROMC 相关
患有疾病。
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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GEORGE L LONG其他文献
GEORGE L LONG的其他文献
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{{ truncateString('GEORGE L LONG', 18)}}的其他基金
NHLBI SHARED RESEARCH FACILITY FOR MOLECULAR BIOLOGY
NHLBI 分子生物学共享研究设施
- 批准号:
3003475 - 财政年份:1987
- 资助金额:
$ 25.4万 - 项目类别:
STRUCTURE AND MOLECULAR BIOLOGY OF THE PROTEIN S GENE
蛋白质 S 基因的结构和分子生物学
- 批准号:
2219099 - 财政年份:1987
- 资助金额:
$ 25.4万 - 项目类别: