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Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4248-4255
Some Anticardiolipin Antibodies Recognize a Combination of
Phospholipids With Thrombin-Modified Antithrombin, Complement
C4b-Binding Protein, and Lipopolysaccharide Binding Protein
Josiane Arvieux,
Gilles Pernod,
Véronique Regnault,
Luc Darnige, and
Jérôme Garin
From the ETS Grenoble, Grenoble, France; Laboratoires
d'Hématologie, CHU Grenoble, Grenoble, France and Faculté
de Médecine Nancy, Nancy, France; the Département de
Biologie Clinique, CH Compiègne, Compiègne, France; and
CEA-Grenoble, Laboratoire de Chimie des Protéines, Grenoble,
France.
The standard enzyme-linked immunosorbent assay (ELISA) for
anticardiolipin antibodies (ACA) detects a heterogenous group of antibodies against cardiolipin on its own,
 2-glycoprotein I (2GPI), 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 2GPI detectable in the 2GPI-ELISA. Three of these samples proved to recognize
2GPI in combination with cardiolipin, but not
2GPI directly immobilized on  -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.
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