| Fibrinogen is a plasma glycoprotein playing an important role in blood coagulation, and platelet adhesion and aggregation. It also mediates thrombus formation occurring on blood-contacting surfaces like implantable cardiovascular devices or atherosclerotic lesions. However, the detailed mechanisms of fibrinogen/surface interaction are still mostly unknown. Atomic force microscopy (AFM) provides molecular scale information on hydrated structures of biomolecules, and surface-dependent cellular and molecular events. The main objective of this thesis is thus to elucidate the molecular level structure/function relationship of surface-bound fibrinogen by AFM under aqueous conditions.; Hydrated fibrinogen displays a characteristic trinodular structure on all three tested surfaces: hydrophobic octadecyltrichlorosilane (OTS), positively charged 3-aminopropyltriethoxysilane (APTES) and negatively charged mica. The hydrated structure of adsorbed fibrinogen can be represented by three connecting ellipsoids. Tip contribution to the measured lateral dimensions of adsorbed fibrinogen is substantially reduced by mathematical morphology, an image processing technique. Dimensional measurements indicate that surface-dependent structural deformation of hydrated fibrinogen, manifested as molecular spreading, decreases according to the following order: OTS > APTES > mica. Additionally, increasing D and E domain widths, and molecular length of fibrinogen correspond with decreasing heights.; Functional studies were performed to correlate structural results. Monitoring by time-lapse AFM under aqueous conditions, fibrin assembly is observed on hydrophobic graphite but not mica. Fibrin monomers initially assemble into short linear fibrin strands and then undergo time-dependent conformational rearrangement and ordering leading to an extensive fibrin network. Two-stranded protofibril is the predominant structure of fibrin strands which resemble ‘beads-on-a-string’ structures with a periodicity of 22.5 nm, half the molecular length of fibrinogen, between adjacent globular domains. Branch points and eyelet structure are two characteristic features observed within the fibrin network. Moreover, surface-dependent exposure of monoclonal antibody (mAb) 9F9 epitope, γ112–119 (EIYNSNNQ), is directly visualized by AFM as gold bead-mAb conjugates bind to fibrinogen adsorbed on OTS but not mica, suggesting the epitope is available only on the former surface.; Taken together, the results demonstrate that the structures and functions of fibrinogen are dependent on the physiochemical properties of the interacting surfaces and thus the nature of intermolecular interactions, notably non-specific hydrophobic and electrostatic interactions. |
