Factor VIII-East Hartford (arginine 1689 to cysteine) has procoagulant activity when separated from von Willebrand...

Factor VIII East Hartford (FVIII-EH) procoagulant activity is reduced because the substitution of cysteine for arginine 1689 abolishes an essential Factor VIII light chain thrombin cleavage site. Incubation of FVIII-EH plasma with penicillamine or DTT causes a five- to sixfold increase in FVIII-EH VIII:C, at 80 and 1 mM, respectively. While there is no FVIII-EH light chain cleavage when thrombin is added in the presence of penicillamine or DTT, these reducing agents disrupt the FVIII-vWf complex. For example, the addition of 5 mM DTT to normal or FVIII-EH plasma causes a 50% reduction in Factor VIII binding to vWf. These observations suggested that DTT increases FVIII-EH VIII:C by partial dissociation of FVIII-EH from vWf. This was verified by showing that vWf-free FVIII-EH had VIII:C activity of 21 U/dl, while the starting plasma level was 2.5 U/dl. Removal of other FVIII-EH plasma proteins by agarose gel filtration had no effect on VIII:C activity. The demonstration that this mutant Factor VIII has cofactor function when separated from vWf indicates that the dissociation of Factor VIII from vWf is an essential effect of Factor VIII light chain cleavage at arginine-1689.