Prothrombin Binds to the Surface of Apoptotic, But Not Viable, Cells and Serves as a Target of Lupus Anticoagulant Autoantibodies

Abstract

Anti-phospholipid Ab (aPL) are a heterogeneous group of autoantibodies directed against various combinations of phospholipids (PL) and PL-binding proteins. Lupus anticoagulant (LA) Ab, a subset of aPL, exhibit anticoagulant properties in vitro, but are procoagulant in vivo. Most LA Ab are specific for either β2-glycoprotein I (β2GPI) or prothrombin (PT), two PL-binding proteins. We have previously shown that β2GPI and β2GPI-dependent aPL bind specifically to apoptotic, but not viable, thymocytes. In this study, we demonstrate that PT, like β2GPI, binds selectively to the surface of apoptotic, but not viable, Jurkat cells. Furthermore, PT supports the binding of systemic lupus erythematosus-derived polyclonal and murine monoclonal LA Ab to apoptotic cells. Two LA mAb, which differed dramatically in their relative affinities for PT, were studied. Although one mAb (29J3-62) had a high affinity for PT alone, the other (29I4-24) showed minimal reactivity with PT alone and required PL for elevated binding. Monovalent fragments of 29I4-24 reacted with PL-bound PT with high affinity, suggesting that this mAb recognizes a PL-dependent epitope. Despite these differences, PT-dependent binding of both mAb to apoptotic cells was 30-fold greater than that to viable cells. Moreover, binding of PT to apoptotic cells was, itself, increased in the presence of bivalent, but not monovalent, forms of either mAb. In summary, our data demonstrate the following: 1) specific binding of PT to apoptotic cells, an effect enhanced by PT-dependent LA Ab; 2) heterogeneity of PT-dependent LA Ab; and 3) potential pathogenicity of Ab of either low or high affinity for PT.

Anti-phospholipid antibodies (aPL) and apoptosis: prothrombin-dependent aPL as a paradigm for phospholipid-dependent interactions with apoptotic cells

“…These mAb were derived from mice immunized with phospholipid/PT complexes, comprised of either dioleoylphosphatidylserine (DOPS) and PT, or DOPS/ dioleoylphosphatidylcholine (DOPC) and PT, respectively. Both mAb exhibited similar and strong LA activity ( Table 1) that was dose-dependent and specifically inhibited by hexagonal (II) phase PE [27]. Neither mAb reacted with cardiolipin, DOPS, DOPC, or β2GPI in enzyme-linked immunoassay (ELISA) ( Table 1).…”

“…Recently, we have also shown that this paradigm for recognition of apoptotic cells by aPL can be extended to prothrombin (PT)-dependent aPL [27]. We review some of these findings here, and propose that this paradigm may apply to other aPL.…”

“…We evaluated whether antibody bivalency was necessary to observe the enhanced binding of PT. IgG, IgG′, and F(ab′) 2 fragments of both LA mAb enhanced binding of FITC-PT to apoptotic cells (maximal MFI of F(ab′) 2 =36.27 and 14.70 for 29J3-62 and 29I4-24, respectively), while monovalent Fab′ fragments of the mAb had no effect on FITC-PT binding (maximal MFI=6.97) [27]. These data show that antibody bivalency is essential for the enhancement of PT binding to apoptotic cells by LA mAb.…”

“…PT is a phospholipid-binding protein known to interact with PS in the presence of calcium. We used as our model Jurkat T cells induced to undergo apoptosis in the presence of staurosporine [27]. Fluorescein-labeled PT (FITC-PT) was found to bind to the surface of apoptotic, but not viable, Jurkat cells in the presence, but not the absence, of calcium chloride (CaCl 2 ) (Fig.…”

“…In contrast, IgG from two patients demonstrated slightly greater binding to viable than to apoptotic cells, irrespective of the presence of PT. PT-dependent binding to apoptotic cells correlated with binding of the same IgG fractions to phosphatidylethanolamine (PE)-bound PT [11], but not with the strength of LA activity or with clinical features of the patients [27].…”