We have used immunoblotting to localize major epitopes for 86 FVIII inhibitor alloantibodies and autoantibodies to the amino- terminal half of the 44 kDa thrombin fragment of the FVIII heavy chain and the carboxy-terminal one fourth of the 72 kDa FVIII light chain. Two of these inhibitors, which were reactive with 44 kDa thrombin fragment, were also strongly reactive with the 54 kDa thrombin fragment. We have further localized a different epitope(s) for each of these two inhibitors by the ability of three FVIII synthetic peptides from the 54 kDa thrombin fragment to partially neutralize their anti-FVIII activity. We have also used immunoblotting to analyze FVIII inhibitor immunoglobulin class and subclass content and to detect changes in FVIII epitope specificity during the course of an inhibitor. Further studies proposed in this application include: epitope mapping of FVIII inhibitors using proteolytic or recombinant DNA produced fragments of FVIII and antibodies of defined FVIII epitope specificity; epitope mapping of FVIII inhibitors using competitive immunoassays between FVIII inhibitors and heterologous antibodies of defined FVIII epitope specificity; and definition of FVIII inhibitor epitopes using synthetic peptides of FVIII. To achieve these aims we have developed and characterized an extensive collection of immunologic reagents, including 10 monoclonal anti-FVIII antibodies whose epitopes are known to within 6-40 amino acid residues of the FVIII sequence; 104 FVIII synthetic peptides; 47 rabbit antibodies to FVIII synthetic peptides 15 amino acid residues in length, which also recognize FVIII; and a collection of 86 FVIII inhibitor plasma samples. The proposed work is directed toward the long term goal of developing new therapeutic products for FVIII inhibitors.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL035090-04
Application #
3348643
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1988-09-30
Project End
1993-09-29
Budget Start
1988-09-30
Budget End
1989-09-29
Support Year
4
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
San Diego
State
CA
Country
United States
Zip Code
92037
Ware, J; MacDonald, M J; Lo, M et al. (1992) Epitope mapping of human factor VIII inhibitor antibodies by site-directed mutagenesis of a factor VIII polypeptide. Blood Coagul Fibrinolysis 3:703-16
Scandella, D; Mattingly, M; de Graaf, S et al. (1989) Localization of epitopes for human factor VIII inhibitor antibodies by immunoblotting and antibody neutralization. Blood 74:1618-26
Foster, P A; Fulcher, C A; Houghten, R A et al. (1988) Localization of the binding regions of a murine monoclonal anti-factor 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 J Clin Invest 82:123-8
Fulcher, C A; Lechner, K; de Graaf Mahoney, S (1988) Immunoblot analysis shows changes in factor VIII inhibitor chain specificity in factor VIII inhibitor patients over time. Blood 72:1348-56
Shima, M; Fulcher, C A; de Graaf Mahoney, S et al. (1988) Localization of the binding site for a factor VIII activity neutralizing antibody to amino acid residues Asp1663-Ser1669. J Biol Chem 263:10198-203
Foster, P A; Fulcher, C A; Houghten, R A et al. (1988) An immunogenic region within residues Val1670-Glu1684 of the factor VIII light chain induces antibodies which inhibit binding of factor VIII to von Willebrand factor. J Biol Chem 263:5230-4
Fulcher, C A; de Graaf Mahoney, S; Zimmerman, T S (1987) FVIII inhibitor IgG subclass and FVIII polypeptide specificity determined by immunoblotting. Blood 69:1475-80
Foster, P A; Fulcher, C A; Marti, T et al. (1987) A major factor VIII binding domain resides within the amino-terminal 272 amino acid residues of von Willebrand factor. J Biol Chem 262:8443-6