Identifying the function of the Fibrin(ogen) alpha-C connector region

确定纤维蛋白（原）α-C 连接器区域的功能

• 批准号: 9813281
• 负责人: Nathan Hudson
• 金额: $ 43.83万
• 依托单位: EAST CAROLINA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9813281
• 关键词: Affect; Amino Acids; Area; Atomic Force Microscopy; Biochemistry; Biological Assay; Biophysics; Blood; Blood Platelets; Blood coagulation; Blood flow; Cardiovascular Diseases; Cleaved cell; Coagulation Process; Crystallization; Cytolysis; Data; Development; Diagnosis; Diffuse; Digestion; Elasticity; Engineering; Enzymes; Erythrocytes; Exhibits; Fiber; Fibrin; Fibrinogen; Fibrinolysis; Fluorescence Resonance Energy Transfer; Goals; Hemorrhage; Hemostatic function; Human; Individual; Injury; Interdisciplinary Study; Ischemic Stroke; Kinetics; Label; Length; Lytic; Measures; Mechanics; Mediating; Medicine; Molecular; Molecular Biology; Molecular Structure; Mutation; Myocardial Infarction; Nomenclature; Pathology; Patients; Peptide Hydrolases; Permeability; Physics; Plasma; Plasmin; Play; Plug-in; Polymers; Predisposition; Process; Property; Protein Engineering; Recombinant Proteins; Recombinants; Reporting; Research; Research Personnel; Research Project Grants; Role; Rubber; Scanning Electron Microscopy; Science; Scientist; Series; Site; Stretching; Stroke; Structure; Students; Technology; Testing; Thromboembolism; Thrombosis; Training; Turbidimetry; Universities; Variant; Venous; Work; Wound Healing; base; blood vessel occlusion; clinical Diagnosis; crosslink; density; experience; experimental study; fibrinopeptide; flexibility; forest; graduate student; improved; mechanical properties; molecular scale; network architecture; next generation; polymerization; prevent; scaffold; sensor; skills; therapy development; undergraduate student

Project Summary/Abstract The broad, long-term objective of the proposed research is to determine the mechanisms regulating fibrin and fibrinogen function, with the goal of improving the diagnosis and treatment of cardiovascular disease. In so doing, we plan to train the next generation of scientists with an interdisciplinary skill set to tackle problems in medicine, physics, and biochemistry. In this specific proposal, we will study the polymerization, mechanical properties, and enzymatic digestion of fibrin fibers. Fibrin fibers form the structural scaffold for blood clots and are remarkably elastic, often being compared to rubber bands, before being digested by plasmin after wound healing terminates. Understanding how fibrin can act like a rubber is potentially important for both clinical diagnoses and the development of treatment approaches for many cardiovascular diseases, because altered fibrin elasticity is often associated with strokes, heart attacks, and other pathologies. However, we currently do not have a complete understanding of which structural properties of fibrin enable its elasticity. Based on previous studies and indirect evidence, we hypothesize that these elastic properties originate from a specific region in fibrin, the αC connector region. In this research project, we will test this hypothesis and will also determine whether the αC connector region is involved in fibrin polymerization, fibrin structure, and the digestion of fibrin by the enzyme plasmin. This interdisciplinary work will rely heavily on student researchers, providing training in molecular biology, biochemistry, biophysics, and blood coagulation. Specific Aim 1: Determine the importance of the αC region on the mechanical and structural properties of fibrin fibers and fibrin clots. Using protein engineering, we will generate fibrin molecules with truncated αC connector regions. We will test the polymerization and structure of fibrin fibers composed of these molecules using fibrinopeptide release assays, turbidity and turbidimetry, scanning electron microscopy, and permeability assays and compare it to fibers made of native, human fibrin. We will measure the mechanical properties of these fibers using atomic force microscopy. Additionally, we will engineer fibrin molecules with molecular tension sensors (based on Fӧrster Resonance Energy Transfer) embedded in the αC connector region. Taken together, these experiments will reveal the extent to which this region regulates fibrin polymerization, mechanical properties, and fiber structure. Specific Aim 2: Determine the mechanical and structural regulators of fibrin fiber fibrinolytic rates. Little is known about how the mechanical and structural properties of individual fibers influence their susceptibility to plasmin lysis, even though the lysis of a blood clot occurs through the digestion of fibers. We will determine how internal fiber structure, fiber tension, and the spacing between fibers impacts plasmin digestion using native fibrin and the engineered fibrin molecules described in Aim 1.

项目概要/摘要 拟议研究的广泛、长期目标是确定调节机制 纤维蛋白和纤维蛋白原功能，以改善心血管疾病的诊断和治疗为目标 为此，我们计划培养具有跨学科技能的下一代科学家。 解决医学、物理和生物化学方面的问题。 在这个具体提案中，我们将研究聚合、机械性能和酶消化 纤维蛋白纤维形成血凝块的结构支架，并且具有异常的弹性，通常具有弹性。 与橡皮筋相比，在伤口愈合终止后被纤溶酶消化之前。 纤维蛋白如何像橡胶一样发挥作用对于临床诊断和开发具有潜在的重要意义 许多心血管疾病的治疗方法，因为纤维蛋白弹性通常与 然而，我们目前还没有完全了解。 根据先前的研究和间接证据，我们发现纤维蛋白的结构特性使其具有弹性。 坚持认为这些弹性特性源自纤维蛋白中的特定区域，即 αC 连接器区域。 在这个研究项目中，我们将测试这个假设，并将确定 αC 连接器区域是否是 参与纤维蛋白聚合、纤维蛋白结构以及纤溶酶对纤维蛋白的消化。 跨学科工作将在很大程度上依赖学生研究人员，提供分子生物学培训， 生物化学、生物物理学和凝血。 具体目标 1：确定 αC 区域对机械和结构性能的重要性 利用蛋白质工程，我们将生成具有截短的 αC 的纤维蛋白分子。 我们将测试由这些分子组成的纤维蛋白纤维的聚合和结构。 使用纤维蛋白肽释放测定、浊度和浊度测定法、扫描电子显微镜和渗透性 分析并将其与天然人类纤维蛋白制成的纤维进行比较，我们将测量其机械性能。 此外，我们将利用原子力显微镜对这些纤维进行工程改造，使其具有分子张力。 嵌入在 αC 连接器区域的传感器（基于 Førster 共振能量传输）合在一起， 这些实验将揭示该区域调节纤维蛋白聚合、机械力学的程度。 性能和纤维结构。 具体目标 2：确定纤维蛋白纤维纤溶速率的机械和结构调节因子。 已知单根纤维的机械和结构特性如何影响其对 纤溶酶裂解，即使血凝块的裂解是通过纤维的消化发生的，我们将确定如何进行。 内部纤维结构、纤维张力和纤维之间的间距影响使用天然纤维蛋白的纤溶酶消化 以及目标 1 中描述的工程纤维蛋白分子。