Platelet mass microarchitecture as a regulator of thrombin production

血小板质量微结构作为凝血酶产生的调节剂

• 批准号: 10218337
• 负责人: Talid Sinno
• 金额: $ 23.66万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-15 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10218337
• 关键词: 3-Dimensional; Address; Anatomy; Anticoagulants; Architecture; Area; Artificial Intelligence; Biochemical; Biochemical Reaction; Biochemistry; Blood Coagulation Factor; Blood Platelets; Blood Vessels; Brain; Cardiovascular system; Cessation of life; Clinical; Coagulants; Coagulation Process; Collaborations; Collection; Complex; Confined Spaces; Data; Deposition; Diffusion; Dimensions; Drug Design; Electron Microscopy; Elements; Feedback; Fibrin; Fluorescence; Generations; Geometry; Goals; Heart; Hemophilia A; Hemorrhage; Hemostatic Agents; Hemostatic Disorders; Hemostatic function; Heterogeneity; Image; Injury; Ions; Kinetics; Knowledge; Letters; Mediating; Methods; Microscopy; Modeling; Molecular; Monitor; Monte Carlo Method; Movement; Mus; Myocardial Infarction; Pathologic; Pennsylvania; Physical environment; Physiological; Plasma; Platelet Activation; Process; Production; Regulation; Research; Research Project Grants; Resolution; Role; Scanning Electron Microscopy; Site; Stroke; Structure; Structure of jugular vein; System; Testing; Therapeutic Agents; Thrombin; Thrombosis; Thrombus; Time; Tissues; Training; Translating; Transmission Electron Microscopy; Transport Process; Uncertainty; Universities; Work; base; clinically relevant; high resolution imaging; improved; in vivo; instrument; microscopic imaging; multi-photon; nanometer; nanometer resolution; novel; particle; pressure; prevent; reaction rate; reconstruction; response; simulation; success; therapy design; thrombotic; vascular injury

Project summary Thrombin is a critical element of the hemostatic/thrombotic response, as evidenced by the large number of clinically relevant pro- and anti-coagulant therapies designed to regulate its generation or activity. Thrombin regulation is not a purely biochemical matter, but rather it emerges from the interaction of the biochemical cascade with the evolving physical microenvironment (i.e., platelet deposition). As such, in order to determine how reaction rates of the coagulation cascade may be impacted inside of a hemostatic (or thrombotic) mass we need to study the tightly-woven interaction between the biochemical reactions responsible for thrombin generation and the physical environment in which they occur. Our primary objective is to answer a fundamental question: can the narrow pores of a hemostatic mass operate as a ‘molecular barrier’ and terminate thrombin generation? If so, this would represent an understudied mechanism mediated by platelets and/or fibrin, and the structure they form following accumulation, at a site of injury. The hypothesized molecular barrier results from the hindered movement of soluble species through the evolving hemostatic mass microenvironment. Hemostatic masses are defined by a complex network of mesoscopic scale pores with dimensions of a few to tens of nanometers, and as a result, biochemical reactions relevant to clotting occur in extremely confined spaces. Previous studies explored the idea that the physical environment of a hemostatic plug may contribute to regulating the hemostatic response, but an accurate knowledge of the microstructure of a hemostatic mass remains elusive. Our proposed studies will address this bottleneck by combining novel volume imaging electron microscopy methods of hemostatic masses with artificial intelligence methods to create anatomically realistic domains for simulations of coagulation biochemistry. In Aim #1, in collaboration with Dr. Weisel (letter attached), we will acquire sequential image stacks of hemostatic masses formed in vivo using correlative multi-photon fluorescence and Focused Ion Beam Scanning Electron microscopy. In Aim #2, we employ artificial intelligence methods to perform accurate image-driven 3D reconstruction of hemostatic mass microarchitectures, using the image stacks generated in Aim #1. As part of a related research project, we have already acquired an initial set of transmission electron microscopy images of hemostatic thrombi at single-platelet resolution to guide our initial computational efforts. In Aim #3, we will use the reconstructions obtained to examine how the hemostatic mass microarchitecture impedes molecular transport. We will evolve simulations to systematically evaluate how pore size and molecule size interact to regulate molecular diffusion. Finally, we will ask whether limitations in molecular transport through the hemostatic mass are responsible for the termination of thrombin production at a local level. If confirmed, this mechanism will represent a fundamental shift in the way we understand the role of platelet activation and accumulation, the hallmarks of hemostasis and thrombosis.

项目摘要 凝血酶是止血/血栓形成反应的关键元素，大量 旨在调节其发电或活性的临床相关的促凝疗法和抗凝剂疗法。凝血酶 调节不是纯粹的生化物质，而是从生化的相互作用中出现的 具有不断发展的物理微环境（即血小板沉积）的级联反应。因此，为了确定 凝血级联反应速率如何在止血（或血栓形成）质量内部影响 需要研究负责凝血酶的生化反应之间的紧密编织的相互作用 产生和发生的物理环境。我们的主要目标是回答基本 问题：止血质量算子的狭窄孔作为“分子屏障”并终止凝血酶 一代？如果是这样，这将代表由血小板和/或纤维蛋白介导的一种理解的机制，以及 结构它们在累积后，在受伤部位积累。假设的分子屏障是由 固体物种通过不断发展的止血质量微环境的阻碍运动。止血 质量由一个复杂的介质量孔的网络定义 纳米，结果，与关闭相关的生化反应发生在极限的空间中。 先前的研究探讨了止血塞的物理环境可能有助于 调节止血反应，但要准确了解止血质量的微观结构 仍然难以捉摸。我们提出的研究将通过组合新型体积成像电子设备来解决这一瓶颈 具有人工智能方法的止血肿块的显微镜方法，以创建解剖学现实 凝血生物化学模拟的域。在AIM＃1中，与Weisel博士合作（附上的信）， 我们将使用相关多光子获取体内形成的止血肿块的顺序图像堆 荧光和聚焦离子束扫描电子显微镜。在AIM＃2中，我们采用人工智能 使用止血质量微体系结构进行准确的图像驱动3D重建的方法，使用 AIM＃1中生成的图像堆栈。作为相关研究项目的一部分，我们已经获得了初始集合 在单板位分辨率下止血性血栓的透射电子显微镜图像，以指导我们的初始 计算工作。在AIM＃3中，我们将使用获得的重建来检查止血质量 微体系结构阻碍了分子运输。我们将进化模拟以系统地评估孔的方式 大小和分子大小相互作用以调节分子扩散。最后，我们将询问是否有限制 通过止血质量的分子转运是造成凝血酶在A处的终止 地方一级。如果得到确认，这种机制将代表我们理解的作用的基本转变 血小板激活和积累，止血和血栓形成的标志。