GENETIC BASIS OF THROMBOTIC DISEASE

血栓性疾病的遗传基础

• 批准号: 6202325
• 负责人: GEORGE L LONG
• 金额: $ 25.4万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-29 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6202325
• 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

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.

这个项目持续的总体目标是更好地理解 维生素K的生物合成、激活和功能特性- 依赖的血浆蛋白参与血液凝结及其后果 这些蛋白质的突变对遗传性血栓形成的影响。这个项目 与计划主题有关，因为它通过研究 蛋白C突变，这些蛋白的基本机制 在他们参与之前被修改、运输和分泌 Ca/2+-膜相关过程。的一个新的附加方向 该项目将是一种染色体定位和鉴定 与血栓性疾病相关的第二基因THROMC 在一个大的I型蛋白C缺乏的美洲原住民血统中。 具体目的是在家族中识别新的蛋白C突变 表现出血栓倾向的；构建、表达和生化的 表征分泌型和非分泌型自然发生的突变形式 蛋白C；并通过遗传连锁分析确定存在和 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提取的常规方法 扩增和定点突变，以及DNA测序 受雇的。重组蛋白C将在人肾293中表达 细胞，离子交换层析纯化。纯化的蛋白质将 具有前肽切割的化学和功能特征； 伽玛-羧化二硫化物配对；凝血酶激活- 血栓调节蛋白；钙/2+和磷脂依赖的失活能力 因子V/a和VIII/a，抑制凝血和增强纤溶，使用 重组-纯化成分和全血系统。脉冲追逐 实验将检查细胞内的处理、运输和 蛋白C突变形式的降解多态标记与连锁 分析将用于对THROMC基因进行染色体定位(最终 到1-2厘米摩根区域)。家庭成员也将在 血栓性疾病及其临床标志物的研究进展 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。无血缘关系的有无血栓形成家系 蛋白C缺乏也将被检查与THROMC的关联 带着疾病。