The proposed research has two principal objectives: (1) development of hydrophobic adsorption chromatography as a new method of improving factor VIII purification; (2) elucidation of the factor VIII antigen (VIIIAg) structure and function with the aid of a new large-pore sodium dodecyl sulfate (SDS) gel which permits examination of unreduced VIIIAg. Our preliminary experiments with the hydrophobic adsorbant dodecylamine-agarose have shown this material to have a high affinity for fibrinogen and a much lower one for factor VIII. We will extend these initial observations to find the best combination of variables, such as ionic strength, temperature and hydrophobic support medium, to achieve maximum yield and purification of VIII. The improved purification procedure will be helpful in acquiring factor VIII for purposes of research and possibly for therapeutic use. The structure of VIIIAg will be probed by a two dimensional SDS gel electrophoretic technique which permits high resolution of unreduced VIIIAg in the first dimension and separation of its component monomers in the second dimension. The method will employed to compare VIIIAg obtained from normal individuals to that from patients with VIII abnormalities. This analysis may be of direct clinical importance in determining, for example, if certain hemophiliacs are more likely than others to develop circulating inhibitors. The method will also yield new information on the structure of VIIIAg and the relation of that structure to the function of factor VIII in promotion clot formation and platelet adhesion.
NIH ExPorter
NIH ExPorter