Some anticardiolipin antibodies recognize a combination of phospholipids with thrombin-modified antithrombin, complement C4b-binding protein, and lipopolysaccharide binding protein

The standard enzyme-linked immunosorbent assay (ELISA) for anticardiolipin antibodies (ACA) detects a heterogenous group of antibodies against cardiol ipin on its own, beta(2)-glycoprotein I (beta(2)GPI), and, potentially, oth er phospholipid-binding plasma proteins from bovine or human origin. In an attempt to identify new proteic targets of ACA, we selected 6 patients who possessed cofactor-dependent ACA but no antibody to human or bovine beta(2) GPI detectable in the beta(2)GPI-ELISA. Three of these samples proved to re cognize beta(2)GPI in combination with cardiolipin, but not beta(2)GPI dire ctly immobilized on gamma-irradiated polystyrene or agarose beads. In the o ther cases, the component required for ACA binding was purified from adult bovine serum or plasma by means of ammonium sulfate precipitation and chrom atography on Phenyl-Sepharose, diethyl aminoethyl (DEAE)-cellulose, heparin -Ultrogel, and Sephacryl S-300 columns. Sodium dodecyl sulfate-polyacrylami de gel electrophoresis (SDS-PAGE) analysis coupled to N-terminal amino acid microsequencing identified the cofactors of patients no. 4, 5, and 6 ACA a s lipopolysaccharide binding protein (LBP), complement C4b-binding protein (C4BP), and the thrombin-antithrombin (AT) complex, respectively. Adsorptio n of each of these cofactor preparations with cardiolipin liposomes led to suppression of ACA reactivity, concomitant with the loss of bands from SDS gels corresponding to sequenced material. Bacterial lipopolysaccharide (whi ch forms high-affinity complexes with LBP) specifically neutralized the cof actor activity of the LBP preparation in a concentration-dependent manner. Bovine serum and plasma, as well as the C4BP preparation, optimally support ed the binding of a rabbit anti-C4BP antiserum to immobilized cardiolipin. The binding of a rabbit anti-AT antiserum to solid-phase cardiolipin was su stained by the thrombin-AT preparation and bovine serum, but neither by bov ine plasma nor by native AT,thus reproducing the behavior of patient no. 6 ACA. Taking advantage of the restricted recognition by the latter ACA of a cofactor from bovine origin appearing upon clotting, we studied the generat ion of such activity in human plasma supplemented with bovine AT or bovine prothrombin before clotting. In these conditions, patient no. 6 antibody bi nding to cardiolipin required the addition of bovine AT,whereas addition of bovine prothrombin alone was ineffective. We therefore concluded that thos e ACA targeted bovine AT once it has been modified/cleaved by thrombin. The se findings underline the wide heterogeneity of ACA and the links that may exist between various coagulation pathways, inflammation and the complement system. (C) 1999 by The American Society of Hematology.