MECHANISMS OF THROMBIN STIMULATION OF ENDOTHELIUM

凝血酶刺激内皮的机制

• 批准号: 6109796
• 负责人: ANDREW J CONNOLLY
• 金额: $ 32.32万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6109796
• 关键词: enzyme activity; expression cloning; fluorescence microscopy; genetically modified animals; immunocytochemistry; immunofluorescence technique; in situ hybridization; laboratory mouse; northern blottings; platelet activation; receptor coupling; receptor expression; southern blotting; thrombin; thrombomodulin; vascular endothelial growth factors; vascular endothelium

Thrombin is a multifunctional serine protease generated at sites of vascular injury, inflammation, or endothelial activation. In addition to its activity as a coagulation factor, it is a potent agonist for many cell types, including platelets, leukocytes, and endothelium. Its varied cellular activities suggest that in addition to its role in hemostasis, thrombin may play a role in inflammatory and proliferative responses to injury. A G protein- couple receptor that mediates thrombin activation of human platelets ha been cloned. Previous studies by our group had demonstrated that this receptor, protease-activated receptors-1 (PAR-1), is activated by a novel proteolytic mechanism. Another protease-activated receptor, PAR-2, subsequently was cloned by homology to PAR-1 and is activated by trypsin but not thrombin. Recently, the applicant disrupted the gene for PAR-1 in mice, which revealed PAR-1 plays an unexpected role in vasculogenesis in mouse embryonic development and that a second thrombin receptor exists in mouse platelets. We cloned the second thrombin receptor, termed PAR-3, which is homologous to PAR-1 and -2. The applicant is particularly interested in thrombin induced signaling in endothelial cells, because of their location at the blood/tissue interface and their diverse roles in vessel wall pathobiology. Preliminary studies for this project that demonstrated that all three PARs are expressed in endothelium, and the applicant has identified pharmacologically a third thrombin receptor that is expressed in a transformed endothelial cell line. The overall objective of this project is to elucidate the roles of the various protease-activated receptors in endothelial responses to thrombin in normal biology and disease. We propose to combine cellular and molecular biologic approaches in in vitro and in vivo studies as follows: Specific Aim 1: To clarify the molecular mechanisms of thrombin activation of endothelial cells in vitro: a) Expression: We will identify which thrombin receptors are expressed in various endothelial cells by Norther blot, Western blot, and immunofluorescence microscopy; b) Sufficient role: We will use specific agonists for PAR-1, -2, and -3 to identify which endothelial responses to thrombin can be mediated by activation of the PARs; c) Necessary roles: To identify which receptors are necessary for thrombin effects, we will use pharmacologic desensitization experiments, antibody blocking studies, and cultures of microvascular endothelial cells from mice with disruption of par-1, -2, or -3 genes. Specific Aim 2: To identify the roles of protease-activated receptors in endothelial responses to thrombin in vivo: a) Expression: We will identify which PARs are expressed in various endothelial cells in normal and diseased tissues by in situ hybridization and immunohistochemistry; b) Sufficient roles: We will use specific agonists for PARs to identify which vascular responses to thrombin can be mediated by activation of the PARs; c) Necessary roles: We will examines the vascular responses to thrombin and VEGF in par-1 -/- mice. We will further characterize the embryonic lethal phenotype in par-1 -/- mice, including an attempted rescue by transgenic expression of PAR-1 in endothelium. Specific Aim 3: To characterize and clone other protease-activated receptors that may mediate thrombin activation of endothelium: a) We will clone a third thrombin receptor from bEND.3 transformed mouse brain endothelial cells by expression cloning or homology-based approaches; b) We will characterize the expression pattern, protease sensitivity, and signaling properties of new thrombin receptors. Our hope is that a more complete understanding of thrombin stimulation of endothelium can be coupled with our increasing knowledge of the pathophysiology of the endothelium, in order to develop new strategies for therapeutic interventions in cardiovascular disease.

凝血酶是一种多功能丝氨酸蛋白酶，产生于 血管损伤、炎症或内皮激活。此外 其作为凝血因子的活性，是许多细胞的有效激动剂 类型，包括血小板、白细胞和内皮细胞。其变化多样 细胞活性表明，除了止血作用外， 凝血酶可能在炎症和增殖反应中发挥作用 受伤。 介导人类凝血酶激活的 G 蛋白偶联受体 血小板已被克隆。我们小组之前的研究表明 该受体，蛋白酶激活受体-1 (PAR-1)，被激活 通过一种新的蛋白水解机制。另一种蛋白酶激活受体， 随后通过与 PAR-1 同源性克隆 PAR-2，并通过 胰蛋白酶但不是凝血酶。最近，申请人破坏了该基因 小鼠体内的 PAR-1，揭示了 PAR-1 在 小鼠胚胎发育中的血管发生和第二凝血酶 受体存在于小鼠血小板中。我们克隆了第二个凝血酶 受体，称为 PAR-3，与 PAR-1 和-2 同源。 申请人对凝血酶诱导的信号传导特别感兴趣 内皮细胞，因为它们位于血液/组织界面 及其在血管壁病理学中的不同作用。初步研究 对于这个项目，证明所有三个 PAR 均以 内皮细胞，并且申请人已经在药理学上鉴定出第三种 在转化的内皮细胞中表达的凝血酶受体 线。该项目的总体目标是阐明 内皮细胞反应中的各种蛋白酶激活受体 正常生物学和疾病中的凝血酶。我们建议将蜂窝和 体外和体内研究中的分子生物学方法如下： 具体目标1：阐明凝血酶激活的分子机制 体外内皮细胞：a) 表达：我们将鉴定哪些 凝血酶受体在多种内皮细胞中表达 印迹、蛋白质印迹和免疫荧光显微镜； b) 足够的角色： 我们将使用 PAR-1、-2 和 -3 的特定激动剂来确定哪些 内皮细胞对凝血酶的反应可以通过激活 PAR； c) 必要的作用：确定哪些受体对于 凝血酶的作用，我们将使用药理脱敏实验， 抗体阻断研究和微血管内皮细胞培养 来自 par-1、-2 或 -3 基因破坏的小鼠。 具体目标 2：确定蛋白酶激活受体在 体内内皮细胞对凝血酶的反应： a) 表达：我们将鉴定 哪些 PAR 在正常和不同的内皮细胞中表达 通过原位杂交和免疫组织化学检测病变组织； b) 足够的作用：我们将使用 PAR 的特定激动剂来确定哪些 血管对凝血酶的反应可以通过 PAR 的激活来介导； c) 必要的作用：我们将检查血管对凝血酶的反应 和 VEGF 在 par-1 -/- 小鼠中。我们将进一步表征胚胎 par-1 -/- 小鼠的致死表型，包括尝试营救 内皮细胞中 PAR-1 的转基因表达。 具体目标 3：表征和克隆其他蛋白酶激活的 可能介导内皮凝血酶激活的受体：a）我们将 从 bEND.3 转化的小鼠大脑中克隆第三个凝血酶受体 通过表达克隆或基于同源性的方法获得内皮细胞； b) 我们将表征表达模式、蛋白酶敏感性和 新凝血酶受体的信号传导特性。 我们希望能够更全面地了解凝血酶刺激 内皮细胞可以与我们不断增加的知识相结合 内皮的病理生理学，以便制定新的策略 心血管疾病的治疗干预。