Characterization of the human factor VIII procoagulant protein with a heterologous precipitating antibody.

Abstract

The human factor VIII procoagulant protein (VIIIC) was purified from the VIIIC-factor VIII-related antigen (VIIIRAg) complex in commercial factor VIII concentrate by immunoadsorbent chromatography with a monoclonal anti-VIIIRAg antibody bound to Sepharose. In this complex, VIIIC is noncovalently bound to factor VIII-related antigen. The VIIIC eluted from the complex was free of VIIIRAg as determined by immunoassay and had a specific activity of 2294 VIIIC U/mg of protein, representing a 164,000-fold purification from plasma. Electrophoresis of this VIIIC preparation in reduced sodium dodecyl sulfate containing 5% polyacrylamide slab gels and subsequent staining with Coomassie blue showed the VIIIC to be a strongly staining doublet of MW 79,000 and 80,000 and more faintly staining bands of up to MW 188,000. Treatment of the VIIIC with catalytic amounts of thrombin resulted in diminution or complete disappearance of all of these bands and appearance of a doublet of MW 71,000 and 72,000, a band at MW 54,000, and material of lower MW. The purified VIIIC was used to produce a precipitating heterologous anti-VIIIC antibody, which was monospecific for VIIIC after absorption with factor VIII-depleted commercial concentrate. Use of this antibody in crossed immunoelectrophoresis positively identified the VIIIC bands in the sodium dodecyl sulfate/polyacrylamide gels. These techniques have allowed the identification of VIIIC protein and description of its extensive MW heterogeneity.