Epitope mapping of human factor VIII inhibitor antibodies by deletion analysis of factor VIII fragments expressed in Escherichia coli.

Abstract

Epitopes for antibodies that inhibit factor VIII procoagulant protein were analyzed by deletion mapping of factor VIII protein fragments expressed in Escherichia coli. A human factor VIII cDNA clone was used to generate E. coli expression vectors encoding fragments containing the 80-kDa factor VIII light chain (A3, C1, and C2 domains) and the 44-kDa carboxyl-terminal half of the factor VIII heavy chain (A2 domain). A series of deletions of each fragment was constructed and tested by immunoblotting for the binding of alloantibody and autoantibody inhibitors. Analysis of derivatives of the 80-kDa fragment showed that six inhibitors recognized a major epitope(s) within the carboxyl-terminal 17.3 kDa of factor VIII. These inhibitors also recognized weaker epitopes nearby and one inhibitor recognized epitopes scattered throughout the 80-kDa fragment. Deletions within the heavy chain fragment revealed one epitope-containing region confined to the amino-terminal 18.3 kDa recognized by six inhibitors. Bacterially produced factor VIII fragments containing the major epitopes were capable of neutralizing inhibitors in vitro but fragments containing weaker or no epitopes did not. These data suggest a potential therapeutic use of factor VIII fragments for neutralization of inhibitor antibodies.

Molecular basis of factor VIII inhibition by human antibodies. Antibodies that bind to the factor VIII light chain prevent the interaction of factor VIII with phospholipid.

“…6 A) and have demonstrated that the protective effect of phosphatidylserine is most marked for those inhibitors that bind factor VIII light chain determinants in immunoblotting studies (4,5 (12). The epitopes recognized by the light chain inhibitors have been previously localized within the factor VIII C2 domain (7). Since PS-factor VIII interaction is prevented by these inhibitors, it is likely that the phospholipid-binding site is located within the C2 domain ofhuman factor VIII.…”

“…Dr. Carol Fulcher kindly determined the epitope specificities ofthe inhibitors by immunoblotting with human factor VIII and thrombin cleaved factor VIII, as previously described (4,5). The factor VIII epitopes bound by the inhibitors and by the murine monoclonal antibody C8 were localized further by immunoblotting with the factor VIII protein deletion fragments described by Scandella et al (7). These fiagments were derived from a cDNA clone of factor VIII, and they were produced in Escherichia coli.…”

Influence of the type of factor VIII concentrate on the incidence of factor VIII inhibitors in previously untreated patients with severe hemophilia A

“…40 Regarding von Willebrand factor, this factor indeed has a protective action against inhibitor development, as demonstrated in an animal model. 41 It binds to the C2 domain of FVIII, which is frequently the target of anti-FVIII antibodies 42 and contains the binding site for phospholipids (PLs). The thrombin cleavage within the C2 domain leads to the release of FVIIIa from its complex and increase in FVIIIa affinity for PLs.…”

Circulating factor VIII immune complexes in patients with type 2 acquired hemophilia A and protection from activated protein C–mediated proteolysis

“…9 In contrast, most hemophilic alloantibodies appear to recognize both domains. 10 Most inhibitor antibodies with a C2 epitope inhibit binding of FVIII to both vWF and phospholipid (PL). 11,12 However, a new dominant epitope in the A3-C1 domains has been identified, and some antibodies that recognize this epitope also prevent the association between FVIII and factor IXa (FIXa).…”