ANTIPHOSPHOLIPID ANTIBODIES RECOGNIZE AN EPITOPE IN DOMAIN 1 OF b2-GLYCOPROTEIN 1.

Matthew D. Linnik, G. Michael Iverson, Edward J. Victoria, David M. Marquis, Patricia A. McNeeley, Jacqueline F. Crisologo, Darlene C. Tuyay and Eric M. Smith.

La Jolla Pharmaceutical Co., San Diego, CA 92121

Antiphospholipid syndrome (APS) is an hypercoagulative disorder that has been implicated in stroke, deep vein thrombosis, thrombocytopenia and recurrent fetal loss. APS is characterized by the presence of pathogenic autoantibodies directed against the circulating protein b2-glycoprotein 1 (b2-GPI). b2-GPI is composed of 5 short consensus repeat domains and the current study was designed to determine if the APS epitope could be localized to a single domain in b2-GPI. A series of recombinant domain deletion mutants of b2-GPI were analyzed for their ability to compete with affinity purified antibodies for binding to immobilized b2-GPI. Eleven antibodies were tested against 8 deletion mutants and only those proteins containing domain 1 of b2-GPI effectively competed with antibody for binding. Domain 1 alone also competed with ACA antibodies for binding to full length b2-GPI. Similar results were obtained in direct binding experiments, which implicated domain 1 as the critical domain for antibody binding. Surface plasmon resonance was utilized to assess specificity of binding to b2-GPI in 76 APS patient plasma samples and 57 controls. 87% of the patient samples demonstrated a greater than 3 fold preference for b2-GPI containing domains 1-5 vs. b2-GPI containing domains 2-5. Site specific mutagenesis studies of b2-GPI domain 1 suggest that there are key amino acids in domain 1 that are necessary for antibodies to bind to domain 1. These results demonstrate that the binding epitope(s) for the pathogenic antibodies in APS are localized to domain 1 of b2-GPI.

Presented at the 24th Annual Conference of the La Jolla Immunologists, Nov. 17-18, 1998, La Jolla, CA.