Scanning transmission X-ray microscopy study of subcellular granules in human platelets at the carbon K- and calcium L2,3-edges

Abstract

Platelets and their subcellular components (e.g., dense granules) are essential components in hemostasis. Understanding their chemical heterogeneities at the sub-micrometer scale, particularly their activation during hemostasis and production of platelet-derived extracellular vesicles, may provide important insights into their mechanisms; however, this has rarely been investigated, mainly owing to the lack of appropriate chemical characterization tools at nanometer scale. Here, the use of scanning transmission X-ray microscopy (STXM) combined with X-ray absorption near edge structure (XANES) to characterize human platelets and their subcellular components at the carbon K-edge and calcium L_2,3-edge, is reported. STXM images can identify not only the spatial distribution of subcellular components in human platelets, such as dense granules (DGs) with sizes of ~200 nm, but also their granule-to-granule chemical heterogeneities on the sub-micrometer scale, based on their XANES spectra. The calcium distribution map as well as the principal component analysis of the STXM image stacks clearly identified the numbers and locations of the calcium-rich DGs within human platelets. Deconvolution of the carbon K-edge XANES spectra, extracted from various locations in the platelets, showed that amide carbonyl and carboxylic acid functional groups were mainly found in the cytoplasm, while ketone-phenol-nitrile-imine, aliphatic, and carbonate functional groups were dominant in the platelet DGs. These observations suggest that platelet DGs are most likely composed of calcium polyphosphate associated with adenosine triphosphate (ATP) and adenosine diphosphate (ADP), with significant granule-to-granule variations in their compositions, while the cytoplasm regions of platelets contain significant amounts of proteins.

Keywords:

• Bioimaging
• calcium biological material
• dense granule
• elemental mapping
• platelet
• XANES spectroscopy
• X-ray microscopy

Acknowledgements

This research was supported by the Ministry of Trade, Industry and Energy (MOTIE) and Korea Institute for Advancement of Technology (KIAT) through the International Cooperative R&D program (N053100009, “Horizon2020 Kor-EU collaborative R&BD on ACEnano Toolbox”) as part of the European Commission Horizon 2020 Program (H2020) project ACEnano grant agreement No 720952. The STXM measurements were performed at the Canadian Light Source, a national research facility at the University of Saskatchewan, which is supported by the Canada Foundation for Innovation (CFI), the Natural Sciences and Engineering Research Council (NSERC), the National Research Council (NRC), the Canadian Institutes of Health Research (CIHR), the Government of Saskatchewan, and the University of Saskatchewan.

Disclosure statement

No potential conflict of interest was reported by the author(s).

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Funding

This work was supported by the European Commission Horizon 2020 Program [NMBP-26-2016-720952]; Ministry of Trade, Industry and Energy (MOTIE) and Korea Institute for Advancement of Technology (KIAT) through the International Cooperative R&D program [N053100009].