The association of factor VIII with von Willebrand factor (vWF) is important for protection of factor VIII against proteolytic degradation in plasma. Recently developed factor VIII preparations, namely monoclonal antibody-purified factor VIII (M-factor VIII) and recombinant factor VIII, contain free form of factor VIII that is expected to form complexes with endogenous vWF after venous injection in the patients with hemophilia A. To clarify the difference of free factor VIII and factor VIII-vWF complex in the commercial factor VIII concentrates, we developed a quantitative assay for free factor VIII in which the sample was added to the wells of vWF-coated microtiter plates. Bound factor VIII was detected by monoclonal antibody against factor VIII, followed by incubation with peroxidase conjugated anti-mouse IgG, then the substrate ABTS for measuring absorbance at 405nm. When the amount of free factor VIII antigen in one unit factor VIII activity of recombinant factor VIII was defined as one arbitrary unit, the ELISA detected free factor VIII as low as 0.016 unit/ml. Factor VIII was incubated with vWF or plasma from a patient with severe Hemophilia A for various concentrations, prior to the free factor VIII assay. At saturation, the stoichiometry was one factor VIII molecule per 50vWF monomers. Of the products of factor VIII-vWF complex concentrate, no free factor VIII was detected. While a M-factor VIII concentrate contained free factor VIII comprising 24% of total factor VIII activity. In a gel filtration experiment of M-factor VIII, 15% of the total factor VIII was eluted as free factor VIII and 77% of that was co-eluted with vWF in the void volume. The free factor VIII and the complex form of factor VIII were immunoisolated and were analyzed after SDS-PAGE by immunoblotting. The factor VIII molecular structure and the susceptibility of thrombin cleavage were indistinguishable between the two forms of factor VIII. These results suggested that free factor VIII present in the M-factor VIII concentrate forms complexes with vWF in hemophilic plasma and is involved in physiologic hemostasis.
