Evidence that Factor VIII and the Ristocetin Aggregating Factor (VIII Rist ) are Separate Molecular Entities

Abstract

The most highly purified preparations of human factor VIII have at least three clearly defined properties. They shorten the clotting time of hemophilic plasma, an activity which will for convenience be referred to as VIII_coag; they give a precipitin reaction with a rabbit antibody prepared against the preparation, a property referred to as the factor VIII related-antigen (VIII_R-Ag); and in the presence of the antibiotic, Ristocetin, they correct the defective aggregation of platelets found in von Willebrand's disease (1-4), an activity which will be referred to as VIII_Rist. There are two prevailing concepts: (a) that each property represents a function of the same molecule, (b) that there is more than one molecule, each with one or more properties (5). The purpose of the experiments described below was to determine whether VIII_coag, VIII_Rist, and VIII_R-Ag reside on the same or on different molecules. The addition of a naturally occurring human factor VIII antibody known to be of the IgG class neutralizes VIII_coag without affecting the expression of VIII_Rist or VIII_R-Ag (3). Rabbit antibodies, on the other hand, prepared against highly purified factor VIII preparations neutralize VIII_Rist and VIII_R-Ag (3), but their VIII_coag neutralizing activity may be relatively low (6, 7). These findings are not inconsistent with the one-molecule hypothesis as the antibody could neutralize one side chain carrying an active site without affecting others. If the one-molecule hypothesis is correct, following the neutralization of VIII_coag or VIII_Rist by the appropriate antibody, the addition of a second precipitating antibody directed against the IgG class should precipitate both VIII_coag and VIII_Rist activities bound to the first antibody; as the factor VIII molecule would carry all the active sites, the supernatant will be devoid of all the remaining properties of the molecule.