REGIONS IN DOMAIN 1 OF b2GPI THAT ARE IMPORTANT IN THE BINDING OF ANTI-CARDIOLIPIN AUTOANTIBODIES.

Eric M. Smith, Ed Victoria and David Marquis.

La Jolla Pharmaceutical Company, San Diego, CA 92121, USA

b2GPI is a serum protein comprised of five complement control protein (CCP or "Sushi") domains that has been shown to play an important role in the antigenic specificity of anti-cardiolipin autoantibodies. These autoantibodies are associated with thrombosis, thrombocytopenia and recurrent fetal loss in humans. The location of the antigenic site on the b2GPI molecule has been the subject of considerable research. Our results obtained using deletion mutants of b2GPI (see abstract by Iverson et al., b2GPI-Dependent Anticardiolipin Autoantibodies Recognize an Epitope on the First Domain of b2GPI), suggest that domain 1 is important in the binding of human affinity purified anti-cardiolipin antibodies. Using phage display technology, domain 1 or domain 5 were displayed as pIII fusion proteins on the surface of filamentous phage. Anti-cardiolipin autoantibodies were shown to bind preferentially to phage bearing domain 1. To further define the antigenic site on domain 1, error-prone PCR was used to generate a library of domain 1 mutants. From several hundred individual clones, 22 were identified with single point mutations in domain 1. These mutants were tested for their ability to bind to a panel of nine human affinity-purified anti-cardiolipin antibodies. The antibodies bound mutant phage in a similar, but not identical pattern. Our results suggest that there are multiple, overlapping regions on domain 1 which contribute to the binding of anti-cardiolipin antibodies.

Presented at the 8th International Symposium on Antiphospholipid Antibodies, Sapporo, Japan, Oct 6-9, 1998.