Platelet mass microarchitecture as a regulator of thrombin production

项目总结 凝血酶是止血/血栓反应的关键成分，大量的 临床上相关的促凝血和抗凝治疗，旨在调节其生成或活性。凝血酶 调控不是纯粹的生物化学问题，而是生物化学相互作用的结果。 与不断演变的物理微环境(即，血小板沉积)级联。因此，为了确定 凝血级联的反应速度如何在止血(或血栓)团块内受到影响 需要研究导致凝血酶的生化反应之间紧密交织的相互作用 以及它们产生的物理环境。我们的首要目标是回答一个基本问题 问：止血物质的狭窄毛孔能否起到分子屏障的作用，从而终止凝血酶 一代人？如果是这样的话，这将是一种由血小板和/或纤维蛋白介导的未被充分研究的机制，而 它们在受伤部位堆积后形成的结构。假设的分子屏障是由 通过不断演变的止血物质微环境阻碍了可溶性物质的移动。止血药 质量是由介观尺度的气孔组成的复杂网络定义的，这些气孔的尺寸从几个到几十个 因此，与凝血相关的生化反应在极其有限的空间发生。 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，但准确了解止血物质的微观结构 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来解决这一瓶颈 用人工智能方法创造解剖真实感的止血肿块的显微方法 用于混凝生物化学模拟的领域。在Aim#1中，与Weisel博士合作(附信)， 我们将使用相关的多光子技术获取体内形成的止血肿块的序列图像堆栈 荧光和聚焦离子束扫描电子显微镜。在目标2中，我们使用了人工智能 方法对止血质量微结构进行精确的图像驱动三维重建，使用 在AIM#1中生成的图像堆栈。作为相关研究项目的一部分，我们已经获得了一个初始集 在单血小板分辨率下的止血血栓的透射电子显微镜图像来指导我们的初始 计算的努力。在目标3中，我们将使用所获得的重建来检查止血块如何 微体系结构阻碍了分子的运输。我们将发展模拟来系统地评估毛孔如何 大小和分子大小相互作用，调节分子扩散。最后，我们将询问是否存在以下限制 通过止血物质的分子运输负责凝血酶的产生在 地方层面。如果得到证实，这一机制将代表着我们对 血小板活化和积聚是止血和血栓形成的标志。

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.