PROTEASE-ACTIVATED RECEPTORS IN EMBRYONIC DEVELOPMENT

胚胎发育中的蛋白酶激活受体

• 批准号: 6390869
• 负责人: SHAUN R. COUGHLIN
• 金额: $ 36.88万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-30 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6390869
• 关键词: G protein coupled receptor kinase; biological signal transduction; blood coagulation; blood vessels; cell type; developmental genetics; electron microscopy; embryo /fetus tissue /cell culture; embryogenesis; fibrinogen; gene targeting; genetic enhancer element; genetic promoter element; genetically modified animals; histochemistry /cytochemistry; histogenesis; in situ hybridization; laboratory mouse; messenger RNA; protein structure function; receptor expression; serine proteinases; thrombin receptor; thrombomodulin; vascular endothelium

DESCRIPTION: (Adopted from the Applicant's abstract.) The long-term goal of this research is to understand the role of the coagulation system in development. Considerable evidence exists from knock-outs of components of that system that it has an important role in embryogenesis, including evidence from PAR-1 knock-out, which leads to the demise of approximately half of PAR1-deficient embryos at aboutE9.5. Death of these embryos was associated with bleeding but could not be attributed to defects in platelet function, implying that thrombin signaling may have a role in embryonic development unrelated to its role in hemostasis, at least in the usual sense. In situ hybridization revealed PAR1 mRNA expression in endocardial and endothelial cells at E9.5, suggesting that PAR1 signaling might be important for normal blood vessel development and vascular integrity. The Principal Investigator postulates that the coagulation cascade functions in part as a "leak detector" that monitors and regulates the integrity of developing blood vessels. To test these hypotheses he proposes the following specific aims. Aim 1 will ask the question: In which cell type(s) is PAR1 signaling important for embryonic development? To address this question they will: a) Use a lacZ knock-in to identify the cell types that express PAR1 and determine their fate in PAR1-deficient embryos; b) Characterize vascular development in PAR1-deficient embryos; c) determine by transgenic rescue whether lack of PAR1 function in endothelial cells is the primary defect in PAR1-deficient embryos; d) Use knock-in of gain-of-function mutations to identify the cells in which PAR1 activation normally occurs during embryonic development; e) Test the hypothesis that disinhibited thrombin production and PAR1 signaling are responsible for the death of thrombomodulin-deficient embryos. In Aim 2, they ask the question: Beyond PAR1, what are the other effectors of the coagulation cascade during embryonic development? They will: a) Test the hypothesis that fibrinogen becomes important in the absence of PAR1, b) Determine whether PAR4 and/or as yet unidentified receptors provide "back-up" thrombin signaling in embryonic development, c) Determine the effect of PAR2 deficiency in combination with other PAR-deficiencies on embryonic development.

说明：（摘自申请人摘要）的长期目标 本研究旨在了解凝血系统在 发展相当多的证据存在于敲除的组件， 系统，它有一个重要的作用，在胚胎发育，包括证据， PAR-1敲除，导致大约一半的 PAR 1缺陷胚胎在E9.5左右。这些胚胎的死亡与 出血，但不能归因于血小板功能缺陷，这意味着 凝血酶信号可能在胚胎发育中起作用， 它在止血中的作用，至少在通常意义上。原位杂交 显示E9.5时内皮细胞和内皮细胞中PAR 1 mRNA表达， 提示PAR 1信号可能对正常血管 发育和血管完整性。主要研究者假设， 凝结级联部分地起到“泄漏检测器”的作用， 并调节发育中血管的完整性。测试这些 他提出了以下具体目标。目标1将询问 问题：在哪种细胞类型中，PAR 1信号对胚胎发育重要 发展？为了解决这个问题，他们将：a）使用lacZ敲入， 鉴定表达PAR 1的细胞类型，并确定它们在 PAR 1缺陷型胚胎; B）表征PAR 1缺陷型胚胎中的血管发育 c）通过转基因拯救来确定是否缺乏PAR 1功能， 内皮细胞是PAR 1缺陷胚胎的主要缺陷; 敲入功能获得性突变以鉴定其中PAR 1 激活通常发生在胚胎发育期间; e）测试假设 凝血酶的产生和PAR 1信号的释放是导致 缺乏血栓调节蛋白的胚胎死亡。在目标2中，他们提出了这样一个问题： 除了PAR 1，在凝血过程中凝血级联的其他效应物是什么？ 胚胎发育？他们将：a）检验纤维蛋白原 B）确定PAR 4和/或作为 尚未鉴定的受体在胚胎中提供“备份”凝血酶信号传导， c）确定PAR 2缺陷与以下组合的影响： 胚胎发育的其他PAR缺陷。