True anti‐anionic phospholipid immunoglobulin M antibodies can exert lupus anticoagulant activity

Abstract

Summary. True (cofactor-independent) anticardiolipin antibodies (aCL) are thought to lack lupus anticoagulant (LA) activity and pathogenic potential. A serum monoclonal immunoglobulin Mk (mIgMk) with aCL and LA activities found in a man with a splenicIgMk + B-cell lymphoplasmacytic lymphoma (LPL) without thrombotic events has been characterized. LPL-derived hybridoma clones (designated HY-FRO) producing the serum mIgMk were obtained. mIgMk secreted by HY-FRO grown in protein-free culture medium, like that purified from serum, (i) showed binding, in a cofactor-free system, to solid-phase CL and phosphatidylserine (PS) and to the membrane of PS-expressing cells (apoptotic cells and activated platelets); (ii) failed to bind neutral phospholipids (PL), b 2 Glycoprotein, histone, ssDNA, dsDNA, human IgG and umbilical vein endothelial cells. Absorption with apoptotic cells abolished its binding to anionic plate-bound CL and PS. IgMk-FRO used poorly mutated VH and Vk region genes, with a pattern that was inconsistent with an antigen-driven selection. Basic amino acids were present in the IgH complementarity determining region 3 (CDR3), which can be important for binding to anionic PL. These findings demonstrate unequivocally that true anti-anionic PL IgM antibodies can exert LA and indicate this anti-PL type does not involve thrombophilia.

Biochemical characterization of recombinant and circulating human Spα

“…Two‐dimensional protein analysis of cryoglobulins has recently revealed the presence of Spα in IgM but not in IgG or IgA serum/plasma fractions (8). To confirm the nature of the possible IgM–Spα interaction, we tested the binding of rSpα to human IgM from two sources: polyclonal IgM (PoIgM) from human serum and monoclonal IgM (MoIgM) produced from the HY‐FRO hybridoma cell line (14). rSpα or the negative control BSA was coupled to CNBr‐activated Sepharose and used to affinity chromatography purify PoIgM and MoIgM, which had been partially purified by precipitation by extensive dialysis in deionized water.…”

“…Protein‐free culture supernatants from hybridoma cells producing human monoclonal IgM (MoIgM) HY‐FRO were a gift from Dr T. Gallart. The HY‐FRO hybridoma cell line was derived from spleen cells of a patient producing anti‐cardiolipin IgMλ (14).…”

Effects of β2‐glycoprotein I and monoclonal anticardiolipin antibodies in platelet interaction with subendothelium under flow conditions

“…In addition, a human monoclonal IgM aCL (FRO) showing both aCL and LA activity, but not anti‐β 2 GPI activity (26), was used. This monoclonal antibody was obtained from a patient who had a splenic IgMλ‐positive B cell lymphoplasmacytic lymphoma without thrombotic events.…”

“…Elution fractions were concentrated (Centriprep‐30; Amicon, Lexington, MA) and the monoclonal IgMλ further purified by preparative electrophoresis over cellulose acetate blocks (Cellogel; Chemetron, Milan, Italy) (27, 29). In its characterization, FRO proved to be a true anti‐anionic β 2 GPI‐independent aPL (26). FRO showed high binding to cardiolipin and phosphatidylserine but failed to bind phosphatidylethanolamine (26).…”

Hyperhomocysteinemia Relates to the Subtype of Antiphospholipid Antibodies in Non-SLE Patients

“…For example, the aCL, (17) aβGPI, (18,19) or β 2 -glycoprotein I-dependent aCL (20) would exhibit LAC activity in vitro, or aβGPI would show a positive result similar to LAC in the diluted Russell's viper venom test (21). These phenomena are because of a nonspecific competition on the phospholipid surface by APAbs, a low calcium level in a tested reagent, or multiple APAbs contemporarily.…”