Coagulation factor VIII (FVIII) is a heterodimer consisting of a light chain of 80 kDa (domains A3-C1-C2) in a metal ion-dependent association with a 220-kDa heavy chain (domains A1-A2-B). The nature of the metal iondependent association between the heavy and light chains was investigated using atomic absorption spectroscopy, electron paramagnetic resonance spectroscopy (EPR), and site-directed mutagenesis and expression of the FVIII cDNA. Whereas copper ion was not detected in intact recombinant FVIII, EDTA dissociation of the chains yielded an EPR signal consistent with 1 mol of Cu(I)/mol of active protein, supporting the hypothesis that a single molecule of reduced copper ion is buried within intact FVIII and is released and oxidized upon treatment with EDTA. Cu(I), and not Cu(II), was able to reconstitute FVIII activity from dissociated chains, demonstrating a requirement for Cu(I) in FVIII function. Three potential copper ion binding sites exist within FVIII: one type-2 site and two type-1 sites. The importance of these potential copper ion ligands was tested by studying the effect of site-directed mutants. Of the two histidines that compose the type-2 binding site, the His-1957 3 Ala mutant displayed secretion, light and heavy chain assembly, and activity similar to wild-type FVIII, while mutant His-99 3 Ala was partially defective for secretion and had low levels of heavy and light chain association and activity. In contrast, FVIII having the mutation Cys-310 3 Ser within the type-1 copper binding site in the A1 domain was inactive and partially defective for secretion from the cell, and the heavy and light chains of the secreted protein were not associated. Mutant Cys-2000 3 Ser within the A3 domain displayed secretion, assembly, and activity similar to that for wildtype FVIII. These results support the hypothesis that Cu(I) is buried within the type-1 copper binding site within the A1 domain and is required for FVIII chain association and activity.Factor VIII (FVIII) 1 is the X-chromosome-linked gene product that is deficient or defective in the bleeding disorder hemophilia A. FVIII functions in the blood-clotting cascade as the cofactor for factor IXa proteolytic activation of factor X. FVIII has a domain organization of A1-A2-B-A3-C1-C2 and is synthesized as a single chain polypeptide of 2351 amino acids, from which a 19-amino acid signal peptide is cleaved upon translocation into the lumen of the endoplasmic reticulum (ER) (1, 2). Upon secretion from the cell, FVIII is further processed by cleavage after residue 1648 to yield a heterodimer consisting of a 220-kDa amino-terminal-derived heavy chain (domains A1-A2-B) associated in a metal ion-dependent manner with a carboxyl-terminal-derived light chain (domains A3-C1-C2) (3).The FVIII heterodimer circulates in plasma in a complex with von Willebrand factor (vWF) in an inactive form that requires proteolytic cleavage by thrombin or factor Xa for release from vWF and generation of coagulant activity. Cleavage within the heavy chain after Arg-740 gener...
