THE FUNCTIONAL DOMAINS OF COAGULATION FACTOR VIII

The functional domains of Factor VIII have been investigated using site-directed mutagenesis to probe the effect of thrombin cleavage on pro-cofactor/cofactor activity. We have previously shown that clotting activity is obtained upon coexpression of the amino terminal (92 kDa) heavy chain and carboxyl terminal (80 kDa) light chain proteolytic cleavage products as individual, secreted proteins without the909 amino acid central region (Burke et.al., J. Biol. Chem. 261, 12574, 1986). In the present work the thrombin cleavage sites in the heavy and light chains previously characterized by others (D. Eaton et.al., Biochemistry 25, 505, 1986) have been modified to remove these sites and the mutagenized gene reassembled into separate expression vectors for the two chains. Coexpression of wild type and mutant proteins in COS-7 cells has been characterized by coagulant activity, immunological assays specific for each of the two chains, and radioimmuno-precipitations. Alteration of the thrombin cleavage site in the heavy chain (Arg-372→ΔArg-372) leads to loss of coagulant activity, whereas another mutant Arg-372→Lys-372 shows 20-fold reduced activity. Radioimmunoprecipitations and RIA data show that this is not a reflection of reduced synthesis or increased degradation of the mutant polypeptides. These results suggest that Arg-372 is required for the efficient folding, assembly, or proteolytic activation of Factor VIII.