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

项目总结/摘要 拟议研究的广泛和长期目标是确定调节 纤维蛋白和纤维蛋白原功能，目的是提高心血管疾病的诊断和治疗 疾病通过这样做，我们计划培养具有跨学科技能的下一代科学家 来解决医学、物理学和生物化学方面的问题。 在这个具体的建议，我们将研究聚合，机械性能，和酶消化的 纤维蛋白纤维纤维形成血块的结构支架，并且具有显著的弹性，通常被 与橡皮筋相比，在伤口愈合终止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，对于临床诊断和开发 许多心血管疾病的治疗方法，因为改变纤维蛋白弹性往往与 中风、心脏病发作和其他疾病。然而，我们目前还没有完全了解 纤维蛋白的结构特性使其具有弹性。根据以前的研究和间接证据，我们 假设这些弹性特性起源于纤维蛋白中的特定区域，αC连接器区域。在 在本研究项目中，我们将测试这一假设，并确定αC连接器区域是否 参与纤维蛋白聚合、纤维蛋白结构和纤维蛋白溶酶对纤维蛋白的消化。这 跨学科工作将在很大程度上依赖于学生研究人员，提供分子生物学方面的培训， 生物化学、生物物理学和血液凝固。 具体目标1：确定αC区域对机械和结构性能的重要性 纤维蛋白纤维和纤维蛋白凝块。利用蛋白质工程，我们将产生具有截短αC的纤维蛋白分子， 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试验、浊度和比浊法、扫描电子显微镜和渗透性 并将其与天然人纤维蛋白制成的纤维进行比较。我们将测量 用原子力显微镜观察这些纤维。此外，我们将设计具有分子张力的纤维蛋白分子 传感器（基于F rster共振能量转移）嵌入αC连接器区域。综合起来看， 这些实验将揭示这一区域调节纤维蛋白聚合、机械聚合 性能和纤维结构。 具体目标2：确定纤维蛋白纤维溶解速率的机械和结构调节剂。小 已知单个纤维的机械和结构特性如何影响它们对 纤溶酶溶解，即使血凝块的溶解通过纤维的消化发生。我们将决定如何 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 和目标1中描述的工程化纤维蛋白分子。

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.