title: Lupus-derived antiprothrombin autoantibodies from a V gene phage display library are specific for the kringle 2 domain of prothrombin.
abstract: Autoantibodies to prothrombin are common in patients with systemic lupus erythematosus. Although their presence is a risk factor for thrombosis, neither their origin nor their precise role in inducing the procoagulant state is known. We have developed a phage-display antibody library from patients with systemic lupus erythematosus with antiprothrombin antibodies, and we have selected two single-chain Fv antibody fragments (ScFvs) by panning on a prothrombin-coated surface. In prothrombin activation assays using purified components, these antibodies promoted prothrombin activation. These ScFvs, termed AN78 and AN129, bound to immobilized prothrombin in a concentration-dependent specific manner but not to other anionic phospholipid binding proteins such as beta2-glycoprotein I or annexin V. Phosphatidylserine-bound prothrombin, but not soluble prothrombin, inhibited the binding suggesting that the epitope is available only on immobilized prothrombin. To localize the epitope, prothrombin was treated with thrombin or factor Xa and various prothrombin activation fragments were subsequently isolated and tested in ELISA with the ScFvs. Both AN78 and AN129 bound to prethrombin I (the fragment lacking the Gla domain and the first kringle domain), to fragment 1.2 (containing Gla and the two kringle domains only) and to fragment 2 but not to thrombin, thus localizing the cognate epitope to the kringle 2 domain in prothrombin. Analysis of the cDNA sequences of these antibodies show clustered mutational patterns in the complementarity determining region, suggesting that variable domains are the products of antigen-driven B cell clonal maturation.
authors.affiliation: Department of Pathology, Baylor College of Medicine, Houston, Texas, USA.
authors.forename: Anhquyen
authors.initials: A
authors.lastname: Le
authors.forename: Swapan
authors.initials: S
authors.lastname: Dasgupta
authors.forename: Stephanie
authors.initials: S
authors.lastname: Planque
authors.forename: Sudhir
authors.initials: S
authors.lastname: Paul
authors.forename: Perumal
authors.initials: P
authors.lastname: Thiagarajan
mesh_terms: D000595:Amino Acid Sequence; D000918:Antibody Specificity; D001323:Autoantibodies / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D017104:Bacteriophage M13 / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001666:Binding Sites, Antibody; D000939:Epitopes / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D018082:Kringles / Q000276:immunology*; D008180:Lupus Erythematosus, Systemic / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D019151:Peptide Library; D011516:Prothrombin / Q000276:immunology* / Q000378:metabolism*
chemical_list: D001323:Autoantibodies; D000939:Epitopes; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; C055966:immunoglobulin Fv; D011516:Prothrombin