Studies on human antihemophilic factor. Evidence for a covalently linked subunit structure.

Abstract

When purified antihemophilic factor (Factor VIII) was rechromatographed on 4% agarose in 0.15 M NaCl or 1.0 M NaCl, a single protein peak, containing both procoagulant activity and von Willebrand factor activity, as defined by ristocetin-induced platelet aggregation, was eluted in the void volume. Purified Factor VIII immediately lost about 30% of its procoagulant activity when dissolved in 0.25 M CaCl2, and when rechromatographed on 4% agarose in 0.25 M CaCl2, the protein peak and von Willebrand factor activity remained coincident in the void volume; however, most of the remaining procoagulant activity was eluted after the void volume. The elution position of Factor VIII procoagulant activity from 4% agarose in 0.25 M CaCl2, and hence its apparent molecular weight, varied with the protein concentration applied to the column; at low protein concentrations it was eluted close to the inner volume. Yet on Sephadex G-200 in 0.25 M CaCl2, the protein and procoagulant activity were eluted together in the void volume. These observations suggested that the Factor VIII procoagulant activity was not eluting according to size or shape, but was adsorbing to some extent to the agarose. Isolated activity peak material from the 0.25 M CaCl2 columns contained protein and had a typical ultraviolet spectrum. Even at high concentrations, the protein contained no thrombin, Factors IX, X, or Xa activity, or detectable phospholipid. In addition to Factor VIII procoagulant activity, which could be inactivated by a human antibody to Factor VIII, the activity peak protein also contained von Willebrand factor activity. Like native Factor VIII and the void volume protein, the activity peak contained protein that did not enter a sodium dodecyl sulfate 5% polyacrylamide gel in the absence of reducing reagent. After reduction of disulfide bonds, several subunits ranging from 195,000 to 30,000 daltons were observed. These results indicate that the protein in the shifted Factor VIII procoagulant activity peak is large and that its anomalous elution pattern from 4% agarose in 0.25 M CaCl2 results from interaction with the agarose. The Factor VIII-like properties of the activity peak protein and its electrophoretic pattern on sodium dodecyl sulfate gels suggest that it is a species of Factor VIII modified by proteolytic cleavage. These results allow an interpretation that is different from the recently proposed "carrier protein-small active subunit" hypotheses for the structure-function relationships of the Factor VIII molecule.