Determining intramolecular binding sites on surface-bound von Willebrand factor under aqueous conditions

The results described in this report demonstrate the feasibility of using AFM in combination with immuno-gold labeling to investigate the accessibility of various binding sites on vWF and to localize the binding site within a vWF multimer. With the aid of monoclonal antibodies [5, 11 and 23] it should be possible to use this approach to perform a quantitative assessment of the differential accessibility of various binding sites on vWF. This should allow localization and quantification of binding sites within the observed tertiary structure of the vWF, providing a measure of the accessibility, a point of reference with which the tertiary structure of vWF could be correlated to the primary sequence, and a means to determine the structural features of the antibody binding regions under physiologic buffer conditions. There are a number of obvious questions that are not addressed here: The role of different biologic and artificial surfaces; time-dependent effects; vWF orientation with respect to different thrombogenic surfaces; and the location of critical binding sites, such as for platelet GPIalpha and GPIIb-IIIa binding regions in the hydrated tertiary structure of vWF. Nevertheless, the work described in this report provides essential groundwork that should provide a novel basis on which to explore the molecular steps, both structural and functional, of vWF in thrombus development. In a wider sense, this experimental approach is applicable to structure-function studies on a wide variety of proteins in physiologic environments.