The Apparent Separation of Factor VIII Related Antigen and Coagulant Activity from von Willebrand Factor Activity by an Immunoadsorbent

Abstract

An antibody was raised in rabbits to the small active fragment of human factor VIII. The antigen was obtained by Ca2+ dissociation of a human factor VIII preparation made from a multidonor pool of plasma. After two adsorptions with 0.1 volume of normal human plasma, the antibody neutralized the F. VIII coagulant activity of normal human plasma, but did not precipitate with any plasma or plasma fractions nor did it neutralize vWF activity as measured by ristocetin aggregation of fixed washed platelets. A solid phase immunochemical reagent was prepared by CNBr binding of the partially purified rabbit antibody to 1% agarose beads. Non-immune beads were similarly prepared with IgG fractions from a normal non-immunized rabbit. Using a batch technique the beads were studied for their ability to remove F. VIII coagulant, F. VIII Ag, and vWF activity from normal human plasma. Assay of the supernatant plasma after 2 hrs, 22°, from 10 replicate experiments gave the following results for residual activity, as per cent of non-immune bead control:F. VIII (37.5±4), F. VIII-Ag (30.8±9.7), and vWF (72.1±16). The experiment was repeated with 6 replicate samples with higher ratio of beads to plasma with essentially similar results. This unexpected separation of F. VIII-Ag from vWF activity prompted further investigation into how these activities are related to the molecular structure of F. VIII and vWF.