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 cardiolipin on its own, beta2-glycoprotein I (beta2GPI), and, potentially, other 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 beta2GPI detectable in the beta2GPI-ELISA. Three of these samples proved to recognize beta2GPI in combination with cardiolipin, but not beta2GPI directly immobilized on gamma-irradiated polystyrene or agarose beads. In the other cases, the component required for ACA binding was purified from adult bovine serum or plasma by means of ammonium sulfate precipitation and chromatography on Phenyl-Sepharose, diethyl aminoethyl (DEAE)-cellulose, heparin-Ultrogel, and Sephacryl S-300 columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis coupled to N-terminal amino acid microsequencing identified the cofactors of patients no. 4, 5, and 6 ACA as lipopolysaccharide binding protein (LBP), complement C4b-binding protein (C4BP), and the thrombin-antithrombin (AT) complex, respectively. Adsorption 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 (which forms high-affinity complexes with LBP) specifically neutralized the cofactor activity of the LBP preparation in a concentration-dependent manner. Bovine serum and plasma, as well as the C4BP preparation, optimally supported the binding of a rabbit anti-C4BP antiserum to immobilized cardiolipin. The binding of a rabbit anti-AT antiserum to solid-phase cardiolipin was sustained by the thrombin-AT preparation and bovine serum, but neither by bovine 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 generation of such activity in human plasma supplemented with bovine AT or bovine prothrombin before clotting. In these conditions, patient no. 6 antibody binding to cardiolipin required the addition of bovine AT, whereas addition of bovine prothrombin alone was ineffective. We therefore concluded that those ACA targeted bovine AT once it has been modified/cleaved by thrombin. These findings underline the wide heterogeneity of ACA and the links that may exist between various coagulation pathways, inflammation and the complement system.
Arvieux
J
J
ETS Grenoble, Grenoble, France.
Pernod
G
G
Regnault
V
V
Darnige
L
L
Garin
J
J
D000209:Acute-Phase Proteins; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D017153:Antibodies, Anticardiolipin / Q000097:blood / Q000276:immunology*; D000918:Antibody Specificity; D000925:Anticoagulants / Q000276:immunology; D000990:Antithrombin III / Q000276:immunology*; D002352:Carrier Proteins / Q000276:immunology*; D002417:Cattle; D002845:Chromatography; D015935:Complement C4b; D003169:Complement Inactivator Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006023:Glycoproteins / Q000276:immunology; D006801:Humans; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D008875:Middle Aged; D010447:Peptide Hydrolases / Q000276:immunology*; D010743:Phospholipids / Q000276:immunology*; D011951:Receptors, Complement / Q000276:immunology*; D053482:beta 2-Glycoprotein I; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D017153:Antibodies, Anticardiolipin; D000925:Anticoagulants; D000990:Antithrombin III; D002352:Carrier Proteins; D015935:Complement C4b; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D010447:Peptide Hydrolases; D010743:Phospholipids; D011951:Receptors, Complement; D053482:beta 2-Glycoprotein I; D000099231:Lipopolysaccharide-Binding Protein; C046193:antithrombin III-protease complex