Ingmar Dorn

Diffusion and molecular binding in crowded vesicle solutions measured by fluorescence correlation spectroscopy

Fluorescence Correlation Spectroscopy (FCS) in concentrated complex media such as vesicle or colloidal suspensions is obscured by light scattering and hydrodynamic crowding effects. Nevertheless, quantitative measurements are desirable for intracellular live cell measurements and studies on pharmaceutical or food products. Here we study the diffusion of green fluorescent protein (GFP) and fluorescently labeled antibodies (IGG) in suspensions of PEGylated liposomes. As a function of vesicle concentration the apparent particle number in the focal volume, as well as the structure parameter increase. This finding is consistent with a distortion of the focal volume due to scattering. We also find that the diffusion times of the proteins increase in agreement with theory of diffusion in concentrated solutions of hard spheres. Taking both effects into account we are able to demonstrate using Annexin V binding to PS vesicles that FCS binding experiments yield the same binding constant in buffer and in concentrated liposome solutions.

FVIII Binding to PS Membranes Differs in the Activated and Non-Activated Form and Can Be Shielded by Annexin A5

Binding of Factor VIII to phosphatidylserine (PS)-expressing platelets is a key process in the intravascular pathway of the blood coagulation cascade. Activated by thrombin, FVIIIa acts as a cofactor on the surface of platelets. It is under debate whether and how annexin A5 influences FVIIIa binding to platelets. Here, we investigate FVIII binding to PS-containing vesicles as model platelets and its interplay with annexin A5 in buffer using fluorescence correlation spectroscopy (FCS). We find that activated FVIIIa, in contrast to inactivated FVIII, exhibits a striking binding anomaly as a function of PS content, marked by a sharp maximum of the binding constant around 11% PS, which is close to the natural PS content of platelets. Furthermore, we show that the addition of annexin A5 can both increase or decrease this FVIIIa binding depending on whether the relative PS content is lower or higher than the maximum binding value. We demonstrate in theory that the observed binding diagram supports the hypothesis that annexin shields PS, indicating a possible indirect regulatory role of annexin A5 in blood coagulation. The overall PS- and annexin-dependent binding behavior of activated FVIIIa is preserved in experiments in blood plasma, confirming the validity of our results under more physiological conditions.