Actin structure formation, membrane dynamics, and force generation during blood platelet spreading
by Anna Zelena
Date of Examination:2022-03-22
Date of issue:2023-03-17
Advisor:Prof. Dr. Sarah Koester
Referee:Prof. Dr. Sarah Koester
Referee:Dr. Florian Rehfeldt
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EnglishBlood platelets are essential to the formation of blood clots and prevent uncontrolled bleeding. They are non-nucleated fragments of larger bone marrow cells known as megakaryocytes. Platelets undergo fast morphological changes during blood flow recovery when they change their shape from a discoid to a flat structure. The platelet cytoskeleton is reorganized during this process of activation and, ultimately, forms a strong adhesion bond to the extracellular matrix. In this thesis, we show two studies. The first study focuses on the investigation of single platelet in vitro by combining traction force microscopy with silicon-rhodamine actin probes in a time-resolved manner. Thus we can spatially and temporally correlate the force generation with the emerging acting structures. Additionally, we test this mechanism for different thrombin concentrations for platelet activation as well as different fibrinogen coverages on the substrate. Our results show, that the local hot spots in the force fields align well with the visualized end points of fibrous actin. Besides, the force generation is a very robust mechanism independent of changes in the amount of thrombin or fibrinogen. The second study focuses on membrane adhesion in combination with metal-induced energy transfer imaging in static and rapid modes. The static MIET mode reveals three-dimensional structures of the basal membrane with areas that are particularly close to the metal substrate. The spatial distribution of these areas likely corresponds to focal adhesion spots. The rapid mode shows the temporal evolution of the membrane-to-surface distance during adhesion and spreading with nanometer resolution. In conclusion, we demonstrate that platelets are indeed very strong with a fast and robust spreading mechanism.
Keywords: human blood plateles; Traction Force Microscopy; fluorescence microscopy; single cells; actin; Metal-induced Energy Transfer