Construction and characterization of an active factor VIII variant lacking the central one-third of the molecule

• Published in: Biochemistry (Easton) 1986-12, Vol.25 (26), p.8343-8347
• Main Authors: Eaton, Dan L, Wood, William I, Eaton, Debbie, Hass, Philip E, Hollingshead, Philip, Wion, Karen, Mather, Jennie, Lawn, Richard M, Vehar, Gordon A, Gorman, Cornelia
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Biological and medical sciences; Blood coagulation. Blood cells; Coagulation factors; DNA Restriction Enzymes; Factor VIII - genetics; Factor VIII - metabolism; Fundamental and applied biological sciences. Psychology; Genetic Variation; Hemophilia A - blood; Humans; Molecular and cellular biology; Plasmids; Thrombin - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00374a001

The primary structure of factor VIII consists of 2332 amino acids that exhibit 3 distinct structural domains, including a triplicated region (A domains), a unique region of 909 amino acids (B domain), and a carboxy-terminal duplicated region (C domains), that are arranged in the order A1-A2-B-A3-C1-C2. The B domain (residues 741-1648) of factor VIII is lost when factor VIII is activated by thrombin, which proteolytically processes factor VIII to active subunits of Mr 50,000 (domain A1), 43,000 (domain A2), and 73,000 (domains A3-C1-C2). To determine if the B domain is required for factor VIII coagulant activity, a variant was constructed by using recombinant DNA techniques in which residues 797-1562 were eliminated. This shortened the B domain from 909 to 142 amino acids. This variant factor VIIIdes-797-1652 was expressed in mammalian cells and was found to be functional. The factor VIIIdes-797-1562 protein was purified and shown to be processed by thrombin in the same manner as full-length factor VIII. The factor VIIIdes-797-1562 variant also bound to von Willebrand factor (vWF) immobilized on Sepharose. These results indicate that most of the highly glycosylated B domain of factor VIII is not required for the expression of factor VIII coagulant activity and its interaction with vWF.