Evaluation of human platelet granules by structured illumination laser fluorescence microscopy

Many roles of human platelets in health and disease are linked to their ability to transport and secrete a variety of small molecules and proteins carried in dense (δ-) and α-granules. Determination of granule number and content is important for diagnosis of platelet disorders and for studies of platelet structure, function, and development. We have optimized methods for detection and localization of platelet proteins via antibody and lectin staining, imaging via structured illumination laser fluorescence microscopy (SIM), and three-dimension (3D) image analysis. The methods were validated via comparison with published studies based on electron microscopy and high-resolution fluorescence microscopy. The α-granule cargo proteins thrombospondin-1 (TSP1), osteonectin (SPARC), fibrinogen (FGN), and Von Willebrand factor (VWF) were localized within the granule lumen, as was the proteoglycan serglycin (SRGN). Colocalization analysis indicates that staining with fluorescently labeled wheat germ agglutinin (WGA) allows detection of α-granules as effectively as immunostaining for cargo proteins, with the advantage of not requiring antibodies. RAB27B was observed to be concentrated at dense granules, allowing them to be counted via visual scoring and object analysis. We present a workflow for counting dense and α-granules via object analysis of 3D SIM images of platelets stained for RAB27B and with WGA. Abbreviation: SIM: structured illumination microscopy; WGA: wheat germ agglutinin; FGN: fibrinogen; TSP1: thrombospondin 1; ER: endoplasmic reticulum Platelets support blood clotting, wound healing, and other essential processes. These functions rely on the ability of platelets to transport and release small molecules like serotonin carried in dense granules and a wide range of proteins carried in alpha granules. Several conditions have been linked to abnormalities in one or more of platelet granule number, content, structure, and function. These conditions can be difficult to diagnose because platelet granules are so small they cannot be consistently resolved by conventional light microscopy, while higher power electron microscopy is not widely accessible. The goal of this study was to develop a method for counting and examining platelet dense and alpha granules without the need of electron microscopy. Key to this was the discovery that alpha granules can be reliably stained with the plant lectin wheat germ agglutinin, which has the advantages of being a smaller and less expensive molecule than the antibodies commonly used to detect alpha granule cargo proteins. We also establish that dense granules can be detected with high specificity via antibody staining of the membrane-associated protein RAB27B. We used structured illumination laser fluorescence microscopy to obtain high-resolution images of stained platelets. These were assembled into 3D renders using image analysis software, which was used to validate a protocol for rapidly counting granules within individual platelets. Our method supports the relatively rapid, accurate, and cost-effective assessment of platelet granules. We have already shown that it can confirm dense granule deficiency, and we anticipate that this approach will also prove useful in diagnosing and studying alpha granule abnormalities.

人体血小板在健康与疾病状态下的诸多功能，均与其能够转运并分泌致密（δ）颗粒与α颗粒中携带的多种小分子与蛋白质密切相关。精准测定颗粒的数量与组成，对于血小板疾病的诊断以及血小板结构、功能与发育相关研究均具有重要意义。本研究优化了通过抗体与凝集素染色、结构光照明显微激光荧光显微镜（structured illumination laser fluorescence microscopy, SIM）成像以及三维（3D）图像分析来检测并定位血小板蛋白质的方法。我们通过与已发表的基于电子显微镜与高分辨率荧光显微镜的研究结果对比，验证了该方法的可靠性。α颗粒的内容蛋白包括血小板反应蛋白-1（thrombospondin-1, TSP1）、骨连接蛋白（osteonectin, SPARC）、纤维蛋白原（fibrinogen, FGN）以及血管性血友病因子（Von Willebrand factor, VWF），蛋白聚糖丝甘蛋白聚糖（serglycin, SRGN）同样定位于颗粒腔中。共定位分析结果显示，使用荧光标记的小麦胚芽凝集素（wheat germ agglutinin, WGA）进行染色，可实现与内容蛋白免疫染色同等高效的α颗粒检测，且无需使用抗体，具备独特优势。研究发现RAB27B富集于致密颗粒表面，可通过人工计数与目标物分析实现致密颗粒的定量统计。本研究提出了一套基于RAB27B与WGA染色的血小板三维SIM图像目标物分析流程，用于定量统计致密颗粒与α颗粒的数量。

缩写说明：SIM：结构照明显微镜（structured illumination microscopy）；WGA：小麦胚芽凝集素（wheat germ agglutinin）；FGN：纤维蛋白原（fibrinogen）；TSP1：血小板反应蛋白1（thrombospondin 1）；ER：内质网（endoplasmic reticulum）

血小板参与凝血、伤口愈合等诸多生命必需过程，这些功能依赖于血小板转运并释放致密颗粒中携带的5-羟色胺等小分子，以及α颗粒中承载的多种蛋白质的能力。多种临床病症与血小板颗粒的数量、组成、结构或功能异常相关，但此类病症往往难以诊断——血小板颗粒体积过小，常规光学显微镜无法稳定分辨其形态，而高分辨率电子显微镜的普及度较低。本研究的目标即开发一种无需依赖电子显微镜，即可定量计数并分析血小板致密颗粒与α颗粒的实验方法。本方法的核心突破在于发现植物凝集素WGA可稳定染色α颗粒，相较于常用于检测α颗粒内容蛋白的抗体，WGA分子量更小、成本更低。此外，本研究证实通过对膜相关蛋白RAB27B进行抗体染色，可高特异性地识别致密颗粒。我们采用结构光照明显微激光荧光显微镜获取染色后血小板的高分辨率图像，并通过图像分析软件将其重构为三维渲染模型，以此验证了可快速统计单个血小板内颗粒数量的实验流程。本方法可实现对血小板颗粒快速、精准且经济高效的定量评估。本团队已证实该方法可有效验证致密颗粒缺乏症，且预计该方法同样可用于α颗粒异常相关疾病的诊断与研究。