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Engelke, Hanna C. (2010): Coagulation protein FVIII binding to phospholipid membranes investigated by Fluorescence Correlation Spectroscopy. Dissertation, LMU München: Faculty of Physics
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Abstract

Fluorescence Correlation Spectroscopy (FCS) allows one to measure protein-membrane binding, self-assembly and other molecular reactions and parameters quantitatively in buffer as well as in complex media. Subject of this thesis was to investigate protein-membrane interactions within blood coagulation in buffer as well as in their biological environment with FCS. Binding of Factor VIII (FVIII) to phosphatidylserine (PS)-expressing platelets is a key process in the intravascular pathway of the blood coagulation cascade. Representing a complex component of the highly regulated network of the coagulation cascade, this protein-membrane interaction is influrenced by many cofactors, such as annexin, which binds to PS-containing membranes as well. Since defects in coagulation, particularly in FVIII binding to membranes lead to severe bleeding disorders, a better understanding of the underlying biophysical and biochemical mechanisms and regulatory influences of this interaction could boost diagnosis and therapy of such diseases, especially when used in combination with an improved systems biology description of the cascade. This thesis investigates the mechanism of FVIII binding to PS-containing model membranes and its regulation by annexin using FCS. Activated FVIII, in contrast to inactivated FVIII, was found to exhibit a striking binding anomaly, consisting in a sharply peaked dependence of the binding constant K(PS) as a function of the PS content. It exceeds the binding of inactivated FVIII in a regime around 12% PS, including physiological concentrations. Furthermore, the regulatory influence of annexin, which can both, increase as well as decrease the binding of activated FVIII, was explained based on this binding anomaly. A quantitative model of this regulatory mechanism assuming efficient shielding of charges by annexin was developed, which allows for the reconstruction of the full three-dimensional phase diagram of FVIII binding to membranes as a function of their PS-content and the concentration of annexin. In order to prove the relevance of these results for coagulation, the experiments were repeated in plasma. Since plasma is a scattering medium, which is crowded by macromolecules and hence strongly affects FCS experiments, a procedure to analyze measurements performed in such complex media was developed. To this end, the influences of scattering and crowding on FCS were investigated using a model system of GFP in highly concentrated vesicle solutions. Scattering was found to enhance and distort the focal volume, whereas crowding slows down diffusion. Taking both effects into account, corrections could be applied, which were demonstrated to allow for artifact-free analysis of binding measurements in complex soft matter systems. To further improve the performance of FCS in complex media and, particularly, in cells, a two-photon FCS microscope was set up. Based on the results of the investigations on scattering and crowding, FCS experiments on living cells were performed. The effective viscosity in dictyostelium discoideum cells was probed and compared to values obtained in lysate. The enhancement of viscosity in the cytoplasm was found to be due to crowding by polydisperse macromolecules, whereas the viscosity of the actin cortex was determined by actin polymerization. Drug treatment allowed for regulation of the polymerization level in the cytoplasm and for detection and determination of the viscosity of actin waves. A project in close colaboration with the groups of Prof. Bein and Prof. Bräuchle succeded in the design, characterization and testing of a drug delivery system employing colloidal mesoporous silica nanoparticles efficiently coated by lipids with a solvent exchange method. Using cross-correlation spectroscopy the lipids were shown to form a close and dense bilayer around the nanoparticles. In-vitro drug delivery experiments gave evidence of the capping-mechanism of the lipids and in-vivo studies proved the effcient delivery and release of drugs by the lipid-coated nanoparticles.