DIRECTED MUTAGENESIS IN THE STUDY OF THE REQUIREMENTS FOR FACTOR VIII ACTIVITY IN VITRO AND IN VIVO

Factor VIII is a high molecular weight plasma glycoprotein that functions in the blood clotting cascade as the cofactor for factor DCa proteolytic activation of factor X. Factor VIII does not function proteolytically in this reaction hut itself can be proteolytically activated by other coagulation enzymes such as factor Xa and thrombin. In the plasma, factor VIII exists as a 200 kDa amino-terminal fragment in a metal ion stabilized complex with a 76 kDa carboxy-terminal fragment. The isolation of the cENA for human factor VIII provided the deduced primary amino acid sequence of factor VIIT and revealed three distinct structural domains: 1) a triplicated A domain of 330 amino acids which has homology to ceruloplasmin, a plasma copper binding protein, 2) a duplicated C domain of 150 amino acids, and 3) a unique B domain of 980 amino acids. These domains are arranged as shown below. We have previously reported the B domain is dispensible far cofactor activity in vitro (Toole et al. 1986 Proc. Natl. Acad 5939). The in vivo efficacy of factor VIII molecules harboring the B domain deletion was tested by purification of the wildtype and modified forms and infusion into factor VIII deficient, hemophilic, dogs. The wildtype and the deleted forms of recombinant derived factor VIII exhibited very similar survival curves (Tl/2 = 13 hrs) and the cuticle bleeding times suggested that both preparations appeared functionally equivalent. Sepharose 4B chromatography indicated that both factor VIII molecules were capable of binding canine plasma vWF.Further studies have addressed what cleavages are necessary for activation of factor VIII. The position of the thrombin, factor Xa, and activated protein C (AFC) cleavage sites within factor VIII are presented below, site-directed ENA medicated mutagenesis has been performed to modify the arginine at the amino side of each cleavagesite to an soleucine. In all cases this modification resulted in molecules that were resistant to cleavage by thrombin at the modified site. Modification of the thrombin cleavage sites at 336 and 740 and modification of the factor Xa cleavage site at 1721 resulted in no loss of cofactor activity. Modification of the thrombin cleavage site at either 372 or 1689 destroyed oofactor activity. Modification of the thrombin cleavage site at 336 resulted in a factor VIII having an increased activity, possibly due to resistance to inactivation. These results suggest the requirement of cleavage at residues 372 and 1689 for cofactor activity.