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.
Raghavachari
M
M
Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106-7207, USA.
Kottke-Marchant
K
K
Marchant
R E
RE
D000327:Adsorption; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D005240:Feasibility Studies; D018056:Hemorheology; D006801:Humans; D007150:Immunohistochemistry; D018625:Microscopy, Atomic Force; D010973:Platelet Adhesiveness; D011487:Protein Conformation; D012996:Solutions; D014867:Water; D014841:von Willebrand Factor / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism
D012996:Solutions; D014841:von Willebrand Factor; D014867:Water