A Synthetic Factor VIII Peptide of Eight Amino Acid Residues (1677-1684) Contains the Binding Region of an Anti-Factor VIII Antibody which Inhibits the Binding of Factor VIII to von Willebrand Factor

Summary

The monoclonal anti-factor VIII (FVIII) antibody C4 has previously been reported to inhibit the binding of purified FVIII to immobilized von Willebrand factor (vWF). The binding area of C4 was identified to be within fifteen amino acid residues (1670-1684) based on the ability of a synthetic FVIII peptide consisting of amino acid residues 1670-1684 to completely inhibit the binding of C4 to FVIII. We now report the further localization of the binding region of C4 to within eight amino acid residues (1677-1684) of FVIII light chain. Nine new overlapping FVIII peptides were synthesized based on the amino acid sequence of the acidic region of FVIII light chain and tested, along with seven previously tested peptides, for the ability to inhibit C4 binding to FVIII in an ELISA assay. Three synthetic FVIII peptides 1670-1684, 1675-1690, and 1677-1684 demonstrated dose dependent inhibition of C4 binding to FVIII. The three reactive peptides contain residues 1677-1684 in common. Since C4 can completely inhibit the binding of FVIII to vWF, this report further localizes an eight amino acid residue region of FVIII which may be important in the mediation of vWF binding.

• References

• 1 Van Dieijen G, Tans G, Rosing J, Hemker HC. The role of phospholipid and factor Villa in the activation of bovine factor X. J Biol Chem 1981; 256: 3433-42
• 2 Hultin MB, Nemerson Y. Activation of factor X by factors IXa and VIII; a specific assay for factor IXa in the presence of thrombin-activated factor VIII. Blood 1978; 52: 928-40
• 3 Owen WG, Wagner RH. Antihemophilic factor: separation of an active fragment following dissociation by salts or detergents. Thromb Diath Haemorrh 1972; 27: 502-15
• 4 Ngo KY, Trifard Glotz V, Koziol JA, Lynch DG, Gitschier J, Ranieri P, Ciavarella N, Ruggeri ZM, Zimmerman TS. Homozygous and heterozygous deletions of the von Willebrand factor gene in patients and carriers of severe von Willebrand disease. Proc Natl Acad Sci (USA) 1988; 85: 2753-7
• 5 Weiss HJ, Sussman II, Hoyer LW. Stabilization of factor VIII in plasma by the von Willebrand factor. Studies on posttransfusion and dissociated factor VIII and in patients with von Willebrand’s disease. J Clin Invest 1977; 60: 390-404
• 6 Kaufman RJ, Wasley LC, Dorner AJ. Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells. J Biol Chem 1988; 263: 6352-62
• 7 Tuddenham EG D, Lane RS, Rotblat F, Johnson AJ, Snape TJ, Middleton S, Kernoff PB A. Response to infusions of polyelectrolyte fractionated human factor VIII concentrate in human haemophilia A and von Willebrand’s disease. Br J Haematol 1982; 52: 259-67
• 8 Brinkhous KM, Sandberg H, Garris JB, Mattsson C, Palm M, Griggs T, Read MS. Purified human factor VIII procoagulant protein: comparative hemostatic response after infusion into hemophilic and von Willebrand disease dogs. Proc Natl Acad Sci (USA) 1985; 82: 8752-6
• 9 Foster PA, Fulcher CA, Houghten RA, Zimmerman TS. An immunogenic region within amino acid residues Val1670-Glu1684 of the factor VIII light chain induces antibodies which binding of FVIII to von Willebrand factor. J Biol Chem 1988; 263: 5230-4
• 10 Fulcher CA, Zimmerman TS. Characterization of the human factor VIII procoagulant protein with a heterologous precipitating antibody. Proc Natl Acad Sci (USA) 1982; 79: 1648-52
• 11 Fulcher CA, Roberts JR, Holland LZ, Zimmerman TS. Human factor VIII piucoagulanl pro Lem. Monoclonal antibodies define pre-cursor-product relationships and functional epitopes. J Clin Invest 1985; 76: 117-24
• 12 Peterson GL. Determination of total protein. Methods Enzymol 1983; 91: 95-119
• 13 Houghten RA. General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc Natl Acad Sci (USA) 1985; 82: 5131-5
• 14 Eaton D, Rodriguez H, Vehar GA. Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity. Biochemistry 1986; 25: 505-12
• 15 Pittman DD, Kaufman RJ. Proteolytic requirements for thrombin activation of anti-hemophilic factor (factor VIII). Proc Natl Acad Sci (USA) 1988; 85: 2429-33
• 16 Leyte A, Verbeet MP, Brodniewicz-Proba T, van Mourik JA, Mertens K. The interaction between human blood-coagulation factor VIII and von Willebrand factor. Biochem J 1989; 257: 679-83
• 17 Precup JW, Sikkink A, Kline BC, Fass DN. Monoclonal antibody to a factor VIII peptide that inhibits von Willebrand factor (vWF) binding can prevent thrombin cleavage at residue 1689. Blood 1988; 72: 306
• 18 Hamer RJ, Koedam JA, Beeser-Visser NH, Sixma JJ. The effect of thrombin on the complex between factor VIII and von Willebrand factor. Eur J Biochem 1987; 167: 253-9
• 19 Sewerin K, Larsen K, Sandberg H, Andersson L-O. The binding between factor VIII and von Willebrand factor. Ric Clin Lab 1986; 16: 235
• 20 Lollar P, Hill-Eubanks DC, Parker CG. Association of the FVIII light chain with von Willebrand factor. J Biol Chem 1988; 263: 10451-5
• 21 Pittman DD, Wasley LC, Murray BL, Wang JH, Kaufman RJ. Analysis of structural requirements for factor VIII function using site directed mutagenesis. Thromb Hacmostas 1987; 58: 344
• 22 Pittman DD, Kaufman RJ. Structure-function relationships of factor Vlll elucidated through recombinant DNA technology. Thromb Haemostas 1989; 61: 161-5
• 23 Mikkelsen J, Thomsen J, Kongerslev L, Christiansen M, Ezban M. Heterogeneity in the tyrosine sulfation of Chinese hamster ovary cell produced recombinant FVIII. Thromb Haemostas 1989; 62: 197