Heil Hannah S, Aigner Max, Maier Sophia, Gupta Prateek, Evers Luise M C, Göb Vanessa, Kusch Charly, Meub Mara, Nieswandt Bernhard, Stegner David, Heinze Katrin G
Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany.
Institute of Experimental Biomedicine I, University Hospital Würzburg, Würzburg, Germany.
Platelets. 2022 Aug 18;33(6):849-858. doi: 10.1080/09537104.2021.2023735. Epub 2022 Feb 2.
Interrogating platelets and their densely packed, highly abundant receptor landscape is key to understand platelet clotting, a process that can save lives when stopping blood loss after an injury, but also kill when causing heart attack, stroke, or pulmonary embolism. The underlying key receptor distributions and interactions, in particular the relevance of integrin clustering, are not fully understood is because of highly abundant and densely distributed αIIbβ3 receptors. This makes receptor distributions difficult to assess even by super-resolution fluorescence microscopy. Here, we combine dual-color expansion and confocal microscopy with colocalization analysis to assess platelet receptor organization without the need of a super-resolution microscope. We show that 4x expansion is highly straight-forward for super-resolution microscopy of platelets, while 10x expansion provides higher precision at the price of increased efforts in sample preparation and imaging. Quantifying various receptor colocalization scenarios we demonstrate that expansion microscopy can pinpoint receptor distributions and interactions in resting and activated platelets being superior to conventional methods that fail in such dense 3D scenarios with highly abundant receptors. We reveal the presence of αIIbβ3 clusters in resting platelets, as well as in activated platelets, indicating that they contribute to the rapid platelet response during platelet clotting.
研究血小板及其密集排列、高度丰富的受体图谱是理解血小板凝血的关键，这一过程在受伤后止血时可挽救生命，但在引发心脏病发作、中风或肺栓塞时也可能致命。由于αIIbβ3受体高度丰富且分布密集，其潜在的关键受体分布和相互作用，尤其是整合素聚集的相关性，尚未完全明确。这使得即使通过超分辨率荧光显微镜也难以评估受体分布。在此，我们将双色扩展和共聚焦显微镜与共定位分析相结合，无需超分辨率显微镜即可评估血小板受体组织。我们表明，4倍扩展对于血小板的超分辨率显微镜检查非常简单直接，而10倍扩展以增加样品制备和成像的工作量为代价提供了更高的精度。通过量化各种受体共定位情况，我们证明扩展显微镜可以精确确定静息和活化血小板中的受体分布和相互作用，优于在这种具有高度丰富受体的密集三维场景中失效的传统方法。我们揭示了静息血小板以及活化血小板中存在αIIbβ3簇，表明它们在血小板凝血过程中有助于血小板的快速反应。
