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Browsing by Subject "platelets"

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  • Puutio, Johanna (2020)
    Extracellular vesicles (EVs) are phospholipid bilayer-enclosed nanoparticles that are secreted by eukaryotic and prokaryotic cells. EVs carry macromolecules and signalling molecules to adjacent cells and play an important role in intercellular communication under both pathologic and homeostatic conditions. Therefore, they have become of significant interest for their therapeutic, diagnostic and prognostic potential. EVs are small and highly heterogeneous in size, shape, cargo and membrane composition, posing several challenges for establishing analytical and clinical guidelines. Therefore, EV research requires standardized and robust methods for their separation and characterization. In this study physical and immunochemical methods were employed to characterize human platelet-derived EVs (pEVs) generated from platelets activated with different external biochemical stimuli. The platelet-activating effect of the pro-inflammatory S100A8/A9 protein complex and a combination of thrombin and collagen were studied with nano flow cytometry. The size distribution of pEVs was studied with nanoparticle tracking analysis (NTA) and asymmetrical flow field-flow fractionation (AF4), which represents a newly emerging method on the EV field. Finally, fluorescent labelling and co-localization analysis were employed to characterize membrane marker composition of pEVs and assess its usefulness as an analytic tool for EV research. We succeeded in providing new hints towards meaningful discoveries in platelet biology by characterizing the way platelets respond to inflammatory and hemostatic signals by shedding pEVs. When platelet activation markers are characterized with flow cytometry, the S100A8/A9 protein appeared to cause a shift in membrane activation markers when compared to the thrombin- collagen mix and the baseline control. Increased TLT-1 translocation and decreased integrin αIIbβ3 expression on pEV surfaces suggests that S100A8/A9 induced pEV secretion through differently packed platelet α-granules, rather than from the plasma membrane. An increase in TLT-1 expression compared to decreased P-selectin and αIIbβ3 suggests that S100A8/A9 stimulation shifts platelet phenotype towards secretion rather than aggregation. A protocol for small pEV separation with AF4-MALS was set up. With this method, subtle differences between small pEV populations were seen that were not distinguishable with NTA or flow cytometry. When investigated with AF4-MALS, S100A8/A9 induced pEVs appeared larger than those produced with thrombin- collagen activation. The mean particle sizes of the pEV populations obtained from activated platelets were generally also larger than those produced without an activator. We tested novel methods to detect subtle differences in small EV population sizes that are easily missed with conventional methods due to their technical limitations. A well-optimised AF4 protocol can detect different pEV subpopulations and is a promising tool for EV. In the future, when AF4 is combined with a MALS detector and a fraction collector, nanoimaging of fluorescently labelled EVs could be combined with it as a downstream application to obtain information on their versatile biological functions.
  • Pessi, Emilia (2024)
    Platelets originate from megakaryocytes and therefore contain the same receptors. This also applies to the extracellular vesicles (EVs) they release into the bloodstream. Glycoprotein VI (GPVI) is an activating collagen receptor on platelets. It plays an essential role in platelet biology by binding to collagen and activating platelets, leading to generation of EVs. Regulation of hemostasis involves shedding of GPVI from activated platelets, leading to a soluble fragment of GPVI. Soluble GPVI is used as a biomarker for diseases. According to current literature, GPVI is present on megakaryocyte-derived EVs but not on platelet-derived EVs (pEVs), as it is considered that activation of platelets leads to proteolytic cleavage of GPVI. Research on the presence of GPVI on pEVs is so far limited and the results are inconsistent. Based on alternate finding on the presence of GPVI on pEVs using proteomics (Palviainen et al. 2024), the aims of this project were to investigate the presence of GPVI on pEVs and to compare the presence of GPVI on megakaryocyte-derived EVs and pEVs. The presence of GPVI on pEVs was investigated by using multiple set of samples which could express GPVI differently. Platelets from platelet concentrate were isolated, activated, removed after activation and the samples were analysed with flow cytometry. Isolated pEVs were analysed with dot blot and western blot. To obtain megakaryocyte-derived EVs, K562 cell line was differentiated to megakaryocyte-like cells and EVs were isolated from cell conditioned media. The presence of GPVI on pEVs and megakaryocyte-derived EVs was compared with western blot. GPVI was found on pEVs. However, an expected difference in the presence of GPVI between pEVs from activated and unstimulated platelets was not observed. The results also indicated a higher amount of GPVI in megakaryocyte-derived EVs compared to pEVs, but further optimization of the methods used is required for more reliable results. GPVI, previously thought to exist only on megakaryocyte-derived EVs in the circulation or in soluble form cleaved from activated platelets, may actually be present on pEVs. Distinguishing the presence of GPVI between megakaryocyte-derived EVs and pEVs, is relevant when using the receptor as a biomarker. The results of this study are a foundation for further investigation of GPVI on pEVs to elucidate this exciting discrepancy.