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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要大量凝血酶证明了凝血酶是止血/血栓反应的关键要素临床相关的促凝血和抗凝血疗法，旨在调节凝血酶的生成或活性。调节不是纯粹的生化物质，而是生化物质相互作用的结果。与不断变化的物理微环境（即血小板沉积）级联，以确定。我们如何影响止血（或血栓）团块内部的凝血级联反应速率需要研究负责凝血酶的生化反应之间紧密交织的相互作用我们的首要目标是回答一个基本问题。问题：止血物质的窄孔能否作为“分子屏障”发挥作用并终止凝血酶如果是这样，这将代表血小板和/或纤维蛋白介导的尚未研究的机制，以及它们在受伤部位积累后形成的结构。通过不断变化的止血微环境阻碍可溶性物质的运动。质量由尺寸为几到几十的介观尺度孔隙的复杂网络定义。纳米级，因此，与凝血相关的生化反应发生在极其有限的空间内。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，但准确了解止血块的微观结构我们提出的研究将通过结合新型体积成像电子来解决这个瓶颈。止血肿块的显微方法与人工智能方法创建解剖学上的真实感在目标 #1 中，与 Weisel 博士合作（附信），我们将使用相关多光子获取体内形成的止血团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，我们采用了人工智能。方法对止血块微结构进行精确的图像驱动 3D 重建，使用在目标 #1 中生成的图像堆栈作为相关研究项目的一部分，我们已经获得了初始集。单血小板分辨率的止血血栓的透射电子显微镜图像来指导我们的初步研究在目标 #3 中，我们将使用获得的重建来检查止血质量如何。我们将发展模拟来系统地评估孔隙如何阻碍分子传输。最后，我们会问是否存在限制。通过止血物质的分子运输负责终止凝血酶的产生如果得到证实，这一机制将代表我们理解其作用的方式发生根本性转变。血小板活化和积聚是止血和血栓形成的标志。