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Fluorescence Nanoscopy of Platelets Resolves Platelet-State Specific Storage, Release and Uptake of Proteins, Opening up Future Diagnostic Applications

Authors

  • Daniel Rönnlund,

    1. Department of Applied Physics/Experimental Biomolecular Physics, Royal Institute of Technology (KTH), Albanova University Center, Stockholm 106 91, Sweden
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  • Yang Yang,

    1. Department of Applied Physics/Experimental Biomolecular Physics, Royal Institute of Technology (KTH), Albanova University Center, Stockholm 106 91, Sweden
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  • Hans Blom,

    1. Department of Applied Physics/Experimental Biomolecular Physics, Royal Institute of Technology (KTH), Albanova University Center, Stockholm 106 91, Sweden
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  • Gert Auer,

    1. Department of Oncology and Pathology, Karolinska Institutet, Solna 171 67, Sweden
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  • Jerker Widengren

    Corresponding author
    1. Department of Applied Physics/Experimental Biomolecular Physics, Royal Institute of Technology (KTH), Albanova University Center, Stockholm 106 91, Sweden
    • Department of Applied Physics/Experimental Biomolecular Physics, Royal Institute of Technology (KTH), Albanova University Center, Stockholm 106 91, Sweden.
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Abstract

Dysregulation of how platelets store, sequester and release specific proteins seems to be implicated in many disease states, including cancer. Dual-color immunofluorescence stimulated emission depletion (STED) microscopy with 40 nm resolution is used to map pro-angiogenic VEGF, anti-angiogenic PF-4 and fibrinogen in >300 individual platelets. This reveals that these proteins are stored in a segmented, zonal manner within regional clusters, significantly smaller than the size of an α-granule. No colocalization between the different proteins is observed. Upon platelet activation by thrombin or ADP, the proteins undergo significant spatial rearrangements, including alterations in the size and number of the protein clusters, and specific for a certain protein and the type of activation induced. Following these observations, a simple assignment procedure is used to show that the three distinct states of platelets (non-, ADP- and thrombin-activated) can be identified based on the average size, number and peripheral localization profiles of the regional protein clusters within the platelets. Thus, high-resolution spatial mapping of specific proteins is a useful procedure to detect and characterize deviations in the selective storage, release and uptake of these proteins in the platelets. Since these deviations seem to be specific for, and may even underlie, certain patophysiological states, these findings may have interesting diagnostic and therapeutic implications.

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