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因素，如大量的凝血酶抑制剂所证明的。 临床相关的促凝血和抗凝治疗，旨在调节其产生或活性。凝血酶 调节不是一个纯粹的生物化学问题，而是从生物化学的相互作用中出现的。 与演进的物理微环境级联（即，血小板沉积）。因此，为了确定 凝血级联反应速率如何在止血（或血栓形成）肿块内受到影响， 我们需要研究产生凝血酶的生物化学反应之间紧密的相互作用 它的产生和发生的物理环境。我们的主要目标是回答一个基本的 问题：止血物质的狭窄孔隙是否可以作为“分子屏障”并终止凝血酶 一代？如果是这样，这将代表血小板和/或纤维蛋白介导的未充分研究的机制， 它们在损伤部位积聚后形成的结构。假设的分子屏障是由 阻碍可溶性物质通过演变的止血物质微环境的运动。止血 质量由介观尺度孔隙的复杂网络限定，其尺寸为几个到几十个， 因此，与凝血相关的生化反应发生在极其狭窄的空间中。 以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，但止血块微观结构的准确知识 仍然难以捉摸我们提出的研究将通过结合新的体积成像电子 止血块的显微镜方法与人工智能方法，以创建解剖逼真 用于模拟凝血生物化学的域。在目标1中，与Weisel博士合作（随附信函）， 我们将使用相关多光子技术获得体内形成的止血块的连续图像堆栈 荧光和聚焦离子束扫描电子显微镜。在目标2中，我们采用人工智能 方法进行准确的图像驱动的3D重建止血质量微架构，使用 目标#1中生成的图像堆栈。作为相关研究项目的一部分，我们已经获得了一套初步的 止血血栓的透射电子显微镜图像在单血小板分辨率，以指导我们的初步 计算的努力。在目标#3中，我们将使用获得的重建来检查止血块如何 微结构阻碍分子运输。我们将发展模拟来系统地评估孔隙如何 尺寸和分子尺寸相互作用以调节分子扩散。最后，我们会问， 通过止血物质的分子运输负责凝血酶产生的终止， 地方一级如果得到证实，这一机制将代表着我们对人类作用的理解方式的根本转变。 血小板活化和聚集，止血和血栓形成的标志。