Faulkner Emma L, Pike Jeremy A, Garlick Evelyn, Neely Robert K, Styles Iain B, Watson Stephen P, Poulter Natalie S, Thomas Steven G
Department of Cardiovascular Sciences, School of Medical Sciences, College of Medicine and Health, University of Birmingham, Birmingham, United Kingdom; Centre for Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, Midlands, United Kingdom.
Centre for Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, Midlands, United Kingdom; Research Software Group, Advanced Research Computing, University of Birmingham, Birmingham, United Kingdom.
J Thromb Haemost. 2025 Aug;23(8):2618-2633. doi: 10.1016/j.jtha.2025.04.014. Epub 2025 Apr 25.
Current microscopy approaches applied to platelet aggregates from multiple settings indicate their structure has important implications for efficient hemostasis and in clinical treatment of thrombosis. However, current fluorescence microscopy approaches are not amenable to detailed volumetric imaging of platelet aggregate structures. This is due to the small size of individual platelets and tight packing of platelets within aggregates, resulting in optical opacity.
To address this, we investigated methods of performing superresolution microscopy and image analysis on aggregates of platelets to describe their morphology.
We applied expansion microscopy and custom analysis workflows to extract quantitative information on the structure of platelet aggregates.
We demonstrated that expansion microscopy applied to platelet aggregates revealed multiscale information about their structure. We produced volumetric images at nanoscale resolution of >700 platelet aggregates stained for cytoskeletal and membrane components under normal conditions and following cytoskeletal disruption. We demonstrated that our custom analysis workflow provided quantitative description of platelet numbers, volumes, and morphology within entire platelet aggregates. Additionally, we quantitatively described subcellular organization of F-actin. By comparing these measurements following treatment with actin inhibitors, cytochalasin D, and latrunculin A, we could robustly detect structural disruptions in platelet aggregates.
Together, these data provide a workflow to qualitatively and quantitatively describe the architecture of platelet aggregates at a range of scales (whole aggregates down to subcellular features within individual platelets). This provides an important tool for analysis of platelet aggregates and thrombi in conditions such as hemostasis, immunothrombosis, and cardiovascular disease.
目前应用于多种情况下血小板聚集体的显微镜方法表明，其结构对有效的止血和血栓形成的临床治疗具有重要意义。然而，目前的荧光显微镜方法不适用于对血小板聚集体结构进行详细的体积成像。这是由于单个血小板尺寸小且聚集体内血小板紧密堆积，导致光学不透明。
为解决这一问题，我们研究了对血小板聚集体进行超分辨率显微镜检查和图像分析以描述其形态的方法。
我们应用膨胀显微镜和定制分析工作流程来提取有关血小板聚集体结构的定量信息。
我们证明，应用于血小板聚集体的膨胀显微镜揭示了有关其结构的多尺度信息。我们在纳米级分辨率下生成了体积图像，这些图像显示了在正常条件下以及细胞骨架破坏后，700多个针对细胞骨架和膜成分染色的血小板聚集体。我们证明，我们的定制分析工作流程提供了对整个血小板聚集体内血小板数量、体积和形态的定量描述。此外，我们还定量描述了F-肌动蛋白的亚细胞组织。通过比较用肌动蛋白抑制剂、细胞松弛素D和拉春库林A处理后的这些测量结果，我们能够可靠地检测出血小板聚集体中的结构破坏。
总之，这些数据提供了一个工作流程，用于定性和定量描述一系列尺度（从整个聚集体到单个血小板内的亚细胞特征）下血小板聚集体的结构。这为在止血、免疫血栓形成和心血管疾病等情况下分析血小板聚集体和血栓提供了一个重要工具。
