GENETIC BASIS OF THROMBOTIC DISEASE

血栓性疾病的遗传基础

• 批准号: 6358055
• 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: 2000
• 资助国家: 美国
• 起止时间: 2000-09-28 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6358055
• 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.

这个项目的持续总体目标是更好地理解