Characterization of Monoclonal Anti-human Factor VII Antibody (B101/B1) that Recognized Three-dimensional Structures near Arg at Position 79 in the First EGF-like Domain

Summary

Murine monoclonal antibodies (designated hVII-B101/B1, hVIIDC2/D4 and hVII-DC6/3D8) directed against human factor VII (FVII) were prepared and characterized, with more extensive characterization of hVII-B101/B1 that did not bind reduced FVIIa. The immunoglobulin of the three monoclonal antibodies consisted of IgG1. These antibodies did not inhibit procoagulant activities of other vitamin K-dependent coagulation factors except FVII and did not cross-react with proteins in the immunoblotting test. hVII-DC2/D4 recognized the light chain after reduction of FVIIa with 2-mercaptoethanol, and hVIIDC6/3D8 the heavy chain. hVII-B101/B1 bound FVII without Ca²⁺, and possessed stronger affinity for FVII in the presence of Ca²⁺. The Kd for hVII-B101/B1 to FVII was 1.75 x 10^–10 M in the presence of 5 mM CaCl₂. The antibody inhibited the binding of FVII to tissue factor in the presence of Ca²⁺. hVII-B101/B1 also inhibited the activation of FX by the complex of FVIIa and tissue factor in the presence of Ca²⁺. Furthermore, immunoblotting revealed that hVII-B101/B1 reacted with non-reduced γ-carboxyglutaminic acid (Gla)-domainless-FVII and/or FVIIa. hVII-B101/B1 showed a similar pattern to that of non-reduced proteolytic fragments of FVII by trypsin with hVII-DC2/D4 on immunoblotting test. hVII-B101/B1 reacted differently with the FVII from the dysfunctional FVII variant, FVII Shinjo, which has a substitution of Gln for Arg at residue 79 in the first epidermal growth factor (1st EGF)-like domain (Takamiya O, et al. Haemosta 25, 89-97,1995) compared with normal FVII, when used as a solid phase-antibody for ELISA by the sandwich method. hVII-B101/B1 did not react with a series of short peptide sequences near position 79 in the first EGF-like domain on the solid-phase support for epitope scanning. These results suggested that the specific epitope of the antibody, hVII-B101/B1, was located in the three-dimensional structure near position 79 in the first EGF-like domain of human FVII.

• References

• 1 Broze GJ, Majerus PW. Purification and properties of human coagulation factor VII. J Biol Chem 1980; 255: 1242-7.
• 2 Bajaj SP, Rapaport SI, Brown SF. Isolation and characterization of human factor VII. Activation of factor VII by factor Xa. J Biol Chem 1981; 256: 253-9.
• 3 Bach R, Gentry R, Nemerson Y. Factor VII binding to tissue factor in reconstituted phospholipid vesicles: Induction of cooperativity by phosphatidylserine. Biochemistry 1986; 25: 4007-20.
• 4 Masys DR, Bajaj SP, and Rapaport SI. Activation of human factor VII by activated factors IX and X. Blood 1982; 60: 1143-50.
• 5 Kisiel W, Fujikawa K, and Davie EW. Activation of bovine factor VII (proconvertin) by factor XIIa (activated Hageman factor). Biochemistry 1977; 16: 4189-94.
• 6 Nakagaki T, Foster DC, Berkner KL, Kisiel W. Initiation of the extrinsic pathway of blood coagulation: Evidence for the tissue factor dependent autoactivation of human coagulation factor VII. Biochemistry 1991; 30: 10819-24.
• 7 Wildgoose P, Kisiel W. Activation of human factor VII by factors IXa and Xa on human bladder carcinoma cells. Blood 1983; 73: 1888-95.
• 8 Hougie C. Hemophilia and related conditions – congenital deficiencies of prothrombin (factor II), factor V, and factors VII to XII. In: Disorders of hemostasis – congential disorders of blood coagulation factors: Hematology. Williams WJ, Ernest B, Allan JE, Marshall AL. (eds). Third edication. McGraw-Hill; New York: 1994. pp 1393-4.
• 9 Takamiya O, Abe S, Yoshioka A, Nakajima K, McVey JH, Tuddenham EG.D.. Factor VII Shinjo: A dysfunctional factor VII variant homozygous for the substitution Gln for Arg at position 79. Haemosta 1995; 25: 89-97.
• 10 Takamiya O, Kinoshita S, Yoshioka K. Electroimmunoassay of factor VII antigen. Thromb Res 1986; 42: 847-53.
• 11 Galfre G, Milstein C. Preparation of monoclonal antibodies: Strategies and procedures. Methods Enzymol 1981; 73: 3-46.
• 12 Kasper CK, Aledort LM, Counts RB, Edson JR, Fratantoni J, Green D, Hampton JW, Hilgartner MW, Lazerson J, Levine PH, McMillan CW, Pool JG, Shapiro SS. A more uniform measurement of factor VIII inhibitors. Thromb Diath Haemorrh 1975; 34: 869-72.
• 13 Kimoto H. Measurement of affinity for monoclonal antibody – solid assay –. Saibokogaku 1985; 100-4 (in Japanese).
• 14 Takamiya O, Nakamura S, Yoshioka A. A solid-phase assay for the binding ability of factor VII to human tissue factor. Special reference to 8 patients with congential factor VII abnormalities. Jap J Thromb Hemostas 1994; 5: 254-61 (in Japanese).
• 15 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
• 16 Turkington PT, Cathepsin G. a regulator of human vitamin K dependent clotting factors and inhibitors. Thromb Res 1992; 67: 147-55.
• 17 Kazama Y, Pastuszyn A, Wildgoose P, Hamamoto T, Kisiel W. Isolation and characterization of proteolytic fragments of human factor VIIa which inhibit the tissue factor-enhanced amidolytic activity of factor VIIa*. J Biol Chem 1993; 268: 16231-40.
• 18 Hagen FS, Gray CL, O’Hara P, Grant FJ, Saari GC, Woodbury RG, Hart CE, Insley M, Kisiel W, Kurachi K, Davie EW. Characterization of a cDNA coding for human factor-VII. Proc Natl Acad Sci USA 1986; 83: 2412-16.
• 19 Coppola R, Tombesi S, Valentini F, Alborali S, Albertini A, Mannucci PM. Characterization of a Ca (II)-independent monoclonal antibody that lacks reactivity with gla domainless human factor VII. Thromb Res 1992; 68: 269-81.
• 20 Cheung W-F, Stafford DW. Localization of an epitope of a calcium-dependent monoclonal antibody to the N-terminal region of the gla domain of human factor VII. Thromb Res 1995; 79: 199-206.
• 21 Higashi S, Kawabata S, Nishimura H, Funasaki H, Ohyama S, Miyamoto S, Funatsu A, Iwanaga S. Monoclonal antibody (VII-M13) to bovine factor VII: A specific epitope in the γ-carboxyglutamic acid domain. J Biochem 1990; 108: 654-62.
• 22 Takamiya O, Kemball-Cook G, Martin DMA, Cooper DN, von Felten A, Meile E, Hann I, Prangnell DR, Lumley H, Tuddenham EGD, McVey JH. Detection of missense mutations by single-strand conformational polymorphism (SSCP) analysis of coagulation variants of coagulation factor VII. Human Mol Genet 1988; 2: 1355-9.
• 23 Church WR, Messier T, Howard PR, Amiral J, Meyer D, Mann KD. A conserved epitope on several human vitamin K-dependent proteins. Location of the antigenic site and influence of metal ions on antibody binding. J Biol Chem 1988; 263: 6259-67.
• 24 Schiodt J, Harrit N, Christensen U, Petersen C. Two different Ca²⁺ ion binding sites in factor VIIa and in des(1-38) factor VIIa. FEBS Lett 1992; 306: 265-8.
• 25 Wildgoose P, Foster D, Schiodt J, Wiberg FC, Birktoft J, Petersen LC. Identification of a calcium binding site in the protease domain of human blood coagulation factor VII: evidence for its role in factor VII-tissue factor interaction. Biochemistry 1993; 32: 114-9.