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.

项目摘要/摘要 拟议的研究的广泛长期目标是确定调节的机制 纤维蛋白和纤维蛋白原功能，目的是改善心血管的诊断和治疗 疾病。这样，我们计划用跨学科技能培训下一代科学家 解决医学，物理和生物化学方面的问题。 在这个具体建议中，我们将研究聚合，机械性能和酶消化 纤维蛋白纤维。纤维蛋白纤维形成血凝块的结构支架，并且非常弹性，通常是 与橡皮筋相比，在伤口愈合后被纤溶酶消化之前。理解 纤维蛋白如何像橡胶一样起作用，对于临床诊断和发展都很重要 许多心血管疾病的治疗方法，因为纤维蛋白弹性的改变通常与 中风，心脏病发作和其他病理。但是，我们目前对 纤维蛋白的哪种结构特性使其弹性。根据先前的研究和间接证据，我们 假设这些弹性特性起源于αc连接器区域纤维蛋白中的特定区域。在 该研究项目，我们将检验该假设，还将确定αc连接器区域是否为 参与纤维蛋白聚合，纤维蛋白结构和纤维蛋白纤维蛋白消化。这 跨学科的工作将严重依赖学生研究人员，提供分子生物学培训， 生物化学，生物物理学和血液凝结。 特定目标1：确定αc区域对机械和结构特性的重要性 纤维蛋白纤维和纤维蛋白布。使用蛋白质工程，我们将用截短的αC产生纤维蛋白分子 连接器区域。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，浊度和浊度法，扫描电子显微镜以及渗透率 测定并将其与原生纤维蛋白制成的纤维进行比较。我们将测量 这些纤维使用原子力显微镜。此外，我们将使用分子张力来设计纤维蛋白分子 传感器（基于FBRSTER共振能量转移）嵌入αC连接器区域。在一起， 这些实验将揭示该区域调节纤维蛋白聚合的程度 性质和纤维结构。 具体目标2：确定纤维蛋白纤维纤维蛋白水解速率的机械和结构调节剂。小的 知道各个纤维的机械和结构特性如何影响其对其对 纤溶酶裂解，即使血凝块的溶解是通过纤维消化发生的。我们将确定如何 内部纤维结构，纤维张力和纤维之间的间距会使用天然纤维蛋白影响纤溶酶消化 以及AIM 1中描述的工程纤维蛋白分子。