Microheterogeneity of Human Factor VIII
Human factor VIII is a glycoprotein and tends to form a variety of large aggregates, the presence of which was demonstrated by a number of techniques including the use of large pore gel chromatography and electrophoresis using a polyacrylamide gel system designed for high molecular weight aggregates. Factor VIII aggregates can be fractionated in part according to molecular size. Reduction of these polymers by 2-mercaptoethanol results in presumably identical fragments suggesting that all polymers, though differing in size, are composed of identical subunits.Factor VIII aggregates are particularly sensitive to proteolytic breakdown by trypsin and plasmin as judged by large pore polyacrylamide gel electrophoresis. Short-term incubation of factor VIII with trace quantities (equivalent to plasmin- or trypsin-like activities present in normal plasma) of these enzymes respectively results in substantial fragmentation with concurrent loss of both factor VIII procoagulant and von Willebrand factor activity. However, factor VIII activity is lost prior to discernible protein fragmentation whereas von Willebrand factor inactivation is associated with advanced protein degradation. When the ionic strength of the medium is lowered the susceptibility of factor VIII to proteolytic breakdown by trypsin is increased dramatically. Interestingly, degradation of factor VIII by plasmin is not affected by the ionic strength. These data, then, provide conclusive evidence of microheterogeneity of normal human factor VIII and may account for the observed heterogeneity of factor VIII on crossed immunoelectrophoresis.