When artificial surfaces are exposed to blood, a single or relatively few molecular signals from non-specific attachment of protein molecules to the surface can cause a fast and precipitous response, such as in the surface activation of the coagulation and complement systems. Blood proteins reach surfaces before the cellular blood components; cellular blood components collide with the surface already covered with a (multi)layer of proteins. This hierarchy of events causes the effects of nonspecific interactions between proteins and the surface to be propagated to a higher, cellular level by mediating the specific attachment of cells to the protein layer. The reason for this is that the attachment of cells to surfaces is controlled by the spatial pattern of proteins which expose their specific amino acid sequence for specific cell membrane receptors. This is equally true for the attachment of platelets to the surfaces of subendothelial matrix and to artificial materials. Direct observation of proteins binding to surfaces by scanning force microscopy (SFM) is now possible. It is also possible to deliberately manipulate surface-bound proteins in ordered arrays using the same scanning probe technique. Our preliminary experiments indicate that the attachment and spreading of platelets can be directly observed in situ with high resolution (10 nm). In this competing continuation application we propose to continue development of SFM as an in situ, high resolution research tool for direct observation of cell interactions with protein-coated biomaterials and model surfaces. In particular, we propose to apply the scanning force microscopy technique to the following studies: 1. The in situ mechanism of platelet adhesion and spreading onto patterned, protein-coated surfaces and onto the surfaces of microphase-separated biomaterials. 2. Characterization of microphase-separated biomaterial surfaces in aqueous environment using SFM adhesion and elasticity maps. 3. Characterization of the homogeneity of biomaterial coatings on the submicron scale using SFM probes with specific ligands attached.
