Browsing by Subject "GPVI"

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.