A Factor VIII Neutralizing Monoclonal Antibody and a Human Inhibitor Alloantibody Recognizing Epitopes in the C2 Domain Inhibit Factor VIII Binding to von Willebrand Factor and to Phosphatidylserine

Summary

A neutralizing monoclonal antibody, NMC-VIII/5, recognizing the 72 kDa thrombin-proteolytic fragment of factor VIII light chain was obtained. Binding of the antibody to immobilized factor VIII (FVIII) was completely blocked by a light chain-specific human alloantibody, TK, which inhibits FVIII activity. Immunoblotting analysis with a panel of recombinant protein fragments of the C2 domain deleted from the amino-terminal or the carboxy-terminal ends demonstrated binding of NMC-VIII/5 to an epitope located between amino acid residues 2170 and 2327. On the other hand, the epitope of the inhibitor alloantibody, TK, was localized to 64 amino acid residues from 2248 to 2312 using the same recombinant fragments. NMC-VIII/5 and TK inhibited FVIII binding to immobilized von Willebrand factor (vWF). The IC₅₀ of NMC-VIII/5 for the inhibition of binding to vWF was 0.23 μg/ml for IgG and 0.2 μg/ml for F(ab)'₂. This concentration was 100-fold lower than that of a monoclonal antibody NMC-VIII/10 which recognizes the amino acid residues 1675 to 1684 within the amino-terminal portion of the light chain. The IC₅₀ of TK was 11 μg/ml by IgG and 6.3 μg/ml by F(ab)'₂. Furthermore, NMC-VIII/5 and TK also inhibited FVIII binding to immobilized phosphatidylserine. The IC₅₀ for inhibition of phospholipid binding of NMC-VIII/5 and TK (anti-FVIII inhibitor titer of 300 Bethesda units/mg of IgG) was 10 μg/ml.

• References

• 1 Van Dieijin G, Tans G, Rosing J, Hemker HC. The role of phospholipid and factor VIIIa in the activation of bovine factor X. J Biol Chem 1981; 256: 3433-3442
• 2 Wood WI, Capon DJ, Simonsen CC, Eaton DL, Gitschier J, Keyt B, Seeburg PH, Smith DH, Hollingshead P, Wion KL, Delwart E, Tuddenham EGD, Vehar GA, Lawn RM. Expression of active human factor VIII from recombinant DNA clones. Nature 1984; 312: 330-337
• 3 Toole JJ, Knopf JL, Wozney JM, Sultzman LA, Buecker JL, Pittman DD, Kaufman RJ, Brown E, Shoemaker C, Orr EC, Amphlett GW, Foster WB, Coe ML, Knutson GJ, Fass DN, Hewick RM. Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature 1984; 312: 342-347
• 4 Vehar GA, Keyt B, Eaton D, Rodriguez H, O’Brien DP, Rotblat F, Oppermann H, Keck R, Wood WI, Harkins RN, Tuddenham EGD, Lawn RM, Capon DJ. Structure of human factor VIII. Nature 1984; 312: 337-342
• 5 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: 505-512
• 6 Lollar P, Parker ET. Structural basis for the decreased procoagulant activity of human factor VIII compared to the porcine homolog. J Biol Chem 1991; 266: 12481-12486
• 7 Tschopp TB, Weiss HJ, Baumgartner HR. Decreased adhesion of platelets to subendothelium in von Willebrand’s disease. J Lab Clin Med 1974; 83: 296-300
• 8 Weiss HJ, Sussman II, Hoyer LW. Stabilization of factor VIII in plasma by the von Willebrand factor. J Clin Invest 1977; 60: 390-404
• 9 Nishino M, Girma JP, Rothshild C, Fressinaud E, Meyer D. New variant of von Willebrand disease with defective binding to factor VIII. Blood 1989; 74: 1591-1599
• 10 Koedam JA, Meijers JCM, Sixma JJ, Bouma B. Inactivation of human factor VIII by activated protein C. Cofactor activity of protein S and protective effect of von Willebrand factor. J Clin Invest 1988; 82: 1236-1243
• 11 Hill-Eubanks DC, Lollar P. Von Willebrand factor is a cofactor for thrombin-catalyzed cleavage of the factor VIII light chain. J Biol Chem 1990; 265: 17854-17858
• 12 Kaufman RJ, Wasley LC, Dorner AJ. Synthesis, processing and secretion of recombinant human factor VIII expressed in mammalian cells. J Biol Chem 1988; 263: 6352-6362
• 13 Foster PA, Fulcher CA, Houghten RA, Zimmerman TS. An immunogenic region within residues Va11670-Glu1684 of the factor VIII light chain induces antibodies which inhibit binding of factor VIII to von Willebrand factor. J Biol Chem 1988; 263: 5230-5234
• 14 Leyte A, Mertens K, Distel B, Evers RF, De Keyzer-Nellen MJM, Groenen-van Dooren MMCL, de Bruin JDE, Pannekoek H, van Mourik JA. Verbeet MP Inhibition of human coagulant factor VIII by monoclonal antibodies. Mapping of functional epitopes with recombinant factor VIII fragments. Biochem J 1989; 263: 189-194
• 15 Precup JW, Kline BC, Fass DN. A monoclonal antibody to factor VIII inhibits von Willebrand factor binding and thrombin cleavage. Blood 1991; 77: 1929-1936
• 16 Shima M, Yoshioka A, Nakai H, Tanaka I, Sawamoto Y, Kamisue S, Terada S, Fukui H. Epitope localization of monoclonal antibodies against factor VIII light chain which inhibit complex formation by factor VIII and von Willebrand factor. Int J Hematol 1991; 54: 515-522
• 17 Leyte A, van Schijndel HB, Niehrs C, Huttner WB, Verbeet MP, Mertens K, van Mourik JA. Sulfation of Tyr 1680 of human blood coagulation factor VIII is essential for the interaction of factor VIII with von Willebrand factor. J Biol Chem 1991; 266: 740-746
• 18 Gilbert GE, Furie BC, Furie B. Binding of human factor VIII to phospholipid vesicles. J Biol Chem 1990; 265: 815-822
• 19 Foster PA, Fulcher CA, Houghten RA, Zimmerman TS. Synthetic factor VIII peptides with amino acids sequences contained within the C2 domain of factor VIII inhibit factor VIII binding to phosphatidylserine. Blood 1990; 75: 1999-2004
• 20 Arai M, Scandella D, Hoyer LW. Molecular basis of factor VIII inhibition by human antibodies. Antibodies that bind to factor VIII light chain prevent the interaction of factor VIII with phospholipid. J Clin Invest 1989; 83: 1978-84
• 21 Lajmanovich A, Hudry-Clergeon G, Freyssinet JM, Marguerie G. Human factor VIII procoagulant activity and phospholipid interaction. Biochim Biophys Acta 1981; 678: 132-136
• 22 Andersson LO, Brown JE. Interaction of factor VIII-von Willebrand factor with phospholipid vesicles. Biochem J 1981; 200: 161-167
• 23 Hardisty RM, Macpherson JC. A one-stage factor VIII (antihemophilic globulin) assay and its use on venous and capillary plasma. Thromb Diathes Haemorrh 1962; 7: 215-228
• 24 Shima M, Yoshioka A, Yoshikawa N, Tanaka I, Imai S, Tsubura Y, Fukui H. Enzyme-linked immunosorbent assay (ELISA) for von Willebrand factor antigen (vWF:AG) using monoclonal antibodies to von Willebrand factor. J Nara Med Assoc 1986; 31: 672-679
• 25 Scandella DS, Mahoney SdeG, Mattingly D, Roeder L, Timmons L, Fulcher CA. Epitope mapping of human factor VIII inhibitor antibodies by deletion analysis of factor VIII fragments expressed in Escherichia coli. Proc Natl Acad Sci USA 1988; 85: 6152-6156
• 26 Kasper CK, Aledort LM, Counts RB, Edson JR, Frantatoni J, Gree D, Hampton JW, Hilgartner MW, Lazerson J, Levine PH, McMillan CW, Pool JG, Shapiro SS, Shulman NR, van Eyes J. A more uniform measurement of factor VIII inhibitors. Thromb Diathes Haemorrh 1975; 34: 869-872
• 27 Shima M, Yoshioka A, Nakajima M, Nakai H, Fukui H. A monoclonal antibody (NMC-VIII/10) to factor VIII light chain recognizing Glu¹⁶⁷⁵-Glu¹⁶⁸⁴ inhibits factor VIII binding to endogenous von Willebrand factor in human umbilical vein endothelial cells. Br J Haematol 1992; 81: 533-538
• 28 Foster PA, Fulcher CA, Houghten RA, Mahoney SdeG, Zimmerman TS. Localization of the binding regions of a murine monoclonal antifactor VIII antibody and a human anti-factor VIII alloantibody, both of which inhibit factor VIII procoagulant activity, to amino acid residues threonine³⁵¹-serine³⁶⁵ of the factor VIII heavy chain. J Clin Invest 1988; 82: 123-128
• 29 Fulcher CA, Mahoney SdeG, Roberts JR, Kasper CK, Zimmerman TS. Localization of human factor VIII inhibitor epitopes of two polypeptide fragments. Proc Natl Acad Sci USA 1985; 82: 7728-7732
• 30 Fay PJ, Coumans J-V, Walker FJ. von Willebrand factor mediates protection of factor VIII from activated protein C-catalyzed inactivation. J Biol Chem 1991; 266: 2172-2177
• 31 Lollar P, Hill-Eubanks DC, Parker CG. Association of the factor VIII light chain with von Willebrand factor. J Biol Chem 1988; 263: 10451-10455