Structural origin of fibrin clot mechanical properties

描述（由申请人提供）：虽然我们对纤维蛋白（原）的结构和许多方面的功能了解很多，但我们对纤维蛋白凝块力学特性的微观和分子结构起源知之甚少。由于体内凝血本质上是一项机械任务，因此确定凝块和血栓对高动态条件（如血流、血管壁拉伸和伤口等）施加的机械应力的反应是很重要的。在本申请中提出的研究中，纤维蛋白生物聚合物的弹性和粘性特性的结构基础将采用综合方法进行研究，包括不同水平的分析，分子水平，单个纤维，纤维网络和整个凝块，并确定这些不同结构水平之间的关系。具体目标1：在纳米尺度上，纤维蛋白（原）的微观力学将通过单分子原子力显微镜在牵拉工程寡聚物结构时强制展开其分子域来检查，并通过广角x射线衍射或傅立叶变换红外光谱观察纤维蛋白凝块拉伸时的结构转变。具体目标2：在微观尺度上，将通过原子力显微镜或光学镊子对不同凝块中的单个纤维进行弯曲和拉伸来研究纤维的机械性能，并通过小角度x射线衍射图在磁性取向凝块拉伸过程中研究分子和分子堆积的潜在伸长。纤维网络重排的结构变化，如纤维的排列和束状，与凝块变形将通过扫描和透射电子显微镜检查。具体目标3：在宏观层面上，将使用旋转和拉伸流变仪测量从患者冠状动脉中提取的各种完整血块和血栓的粘弹性特性，并与量化血块和血栓结构的参数相关联。为了建立凝块力学结构起源的一般理论，我们将开发本构模型，利用从所有结构水平的实验中获得的定量信息。在生物学术语中，纤维蛋白（原）可能是强迫蛋白质展开生理功能的第一个明确例子之一。在临床方面，了解纤维蛋白变形的机制将解释和预测与止血、血栓形成和伤口愈合相关的不同生理或病理生理条件下的凝块行为，并可能导致新的预防、诊断或治疗方法。该建议代表了一个新的和有前途的生物医学研究领域，即止血和血栓形成的生物力学。公共卫生相关性：本拨款申请中提出的研究重点将放在纤维蛋白（原）分子、纤维和网络的特征上，这些特征会引起血凝块的机械特性，并利用各种生物物理技术确定这些不同结构水平之间的关系。这些研究的结果具有临床意义，因为低弹性和高可塑性的血块往往与出血有关，而非常坚硬的血块则与血栓形成和血栓栓塞有关，从而导致心脏病发作和中风。在生物学术语中，纤维蛋白（原）可能是蛋白质展开生理功能的第一个明确例子之一。更一般地说，这项研究涉及到确定分子结构和一种非凡的生物材料——血凝块的机械特性之间的关系。

DESCRIPTION (provided by applicant): Although we know a great deal about the structure and many aspects of the functions of fibrin(ogen), we still know very little about the microscopic and molecular structural origins of the fibrin clot's mechanical properties. Since blood clotting in vivo is essentially a mechanical task, it is important to determine how clots and thrombi respond to mechanical stresses imposed by highly dynamic conditions, such as blood flow, stretching a vessel wall and wounds, etc. In the research proposed in this application, the structural basis of the elastic and viscous properties of fibrin biopolymers is going to be examined using an integrated approach, which includes different levels of analysis, the molecular level, individual fibers, fiber network, and the whole clot, and the determination of relationships between these different levels of structure. Specific Aim 1: At the nano scale, the micromechanics of fibrin(ogen) will be examined by forced unfolding of its molecular domains during pulling on engineered oligomeric constructs by single-molecule atomic force microscopy, and observing the structural transitions by wide angle X-ray diffraction or Fourier Transform infrared spectroscopy while stretching of fibrin clots. Specific Aim 2: At the microscopic scale, the mechanical properties of fibers will be studied by bending and stretching of individual fibers in different clots by atomic force microscopy or optical tweezers, and investigating potential elongation of molecules and molecular packing by means of the small angle X-ray diffraction pattern during stretching of magnetically oriented clots. Structural changes in fiber network rearrangement, such as alignment and bundling of fibers, with clot deformation will be examined by scanning and transmission electron microscopy. Specific Aim 3: At the macro level, the viscoelastic properties of a variety of whole clots and thrombi extracted from patients' coronary arteries will be measured using rotational and extensional rheometry and correlated with parameters quantifying clot and thrombi structure. To build a general theory of the structural origin of clot mechanics, we will develop constitutive models that take advantage of the quantitative information derived from experiments at all the structural levels. In biological terms, fibrin(ogen) may represent one of the first clear examples of the physiological function of forced protein unfolding. On the clinical side, understanding mechanisms of fibrin deformation would explain and predict clot behavior in different physiological or pathophysiological conditions related to hemostasis, thrombosis, and wound healing and may lead to new methods of prophylaxis, diagnosis, or treatment. The proposal represents a new and promising field of biomedical research, namely biomechanics of hemostasis and thrombosis. PUBLIC HEALTH RELEVANCE: The focus of the research proposed in this grant application will be on the characteristics of fibrin(ogen) molecules, fibers, and networks that give rise to blood clot mechanical properties and the determination of relationships between these different levels of structure, using a variety of biophysical techniques. The results of these studies have clinical significance since clots with low elasticity and high plasticity tend to be associated with bleeding, while very stiff clots have been associated with thrombosis and thromboembolism, which cause heart attacks and strokes. In biological terms, fibrin(ogen) may represent one of the first clear examples of the physiological function of protein unfolding. More generally, this research involves the determination of relationships between molecular structure and the mechanical properties of a remarkable biological material, the blood clot.