Plasmin Immunization Preferentially Induces IgG-Anticardiolipin Antibodies That Are Potentially Prothrombotic in MRL/MpJ Mice

The antiphospholipid syndrome (APS) is characterized by clinical manifestations of vascular thrombosis and pregnancy loss associated with the presence of persistently and significantly increased titers of antiphospholipid antibodies (aPL) (1–6). The antigenic specificities of aPL have been the subject of a number of studies, and these studies have shown that aPL represent a heterogeneous group of immunologically and functionally distinct antibodies that recognize various phospholipids, phospholipid-binding plasma proteins, and phospholipid–protein complexes (1,3,7,8). These plasma proteins include β2-glycoprotein I (β2GPI) and various factors involved in hemostasis, such as prothrombin, protein C, and protein S (7,8). Although aPL have been shown to promote thrombosis and miscarriage in animal studies, the etiology and pathogenic mechanisms remain unclear. To characterize pathogenic aPL in APS, we previously generated 7 monoclonal IgG–anticardiolipin (aCL) antibodies from 2 patients with APS (9,10). Of these monoclonal antibodies (mAb), 5 were prothrombotic in an in vivo pinch–induced thrombosis model in mice (11). Importantly, we found that 4 of these 5 aCL directly bind to the key enzymes involved in hemostasis, namely, thrombin, activated protein C, tissue-type plasminogen activator, and plasmin (12–15). These enzymes belong to the trypsin family and are homologous in their enzymatic domains (16–19). Interestingly, these enzyme-reactive aCL bind to plasmin with relative Kd values in the range of 10−7M (14), which are 30–100-fold higher than the affinities of known IgG-aCL toward β2GPI, the major autoantigen in APS (20). These findings, in combination, suggest that plasmin may be an important autoantigen that drives the activities of certain IgG-aCL in some patients with APS. Indeed, Chen et al, in a study in China, found that plasmin could induce IgG-aCL in immunized BALB/cJ mice, and that one of the mAb generated from these mice, IgG1-aCL, displayed lupus anticoagulant activity and induced fetal loss when injected into pregnant mice (21). However, the titers and kinetics of the plasmin-induced IgG-aCL were not given; the IgG-aCL values were only expressed as the fold change (in SD) above the mean value for control mice. Furthermore, although 2 of the mAb inhibited plasmin activity, the effects of the mAb on other cross-reacting target proteases (such as thrombin) were not explored. To address these issues, we immunized BALB/cJ mice with human plasmin, which resulted in only transient and very low titers of IgG-aCL. Therefore, in addition to BALB/cJ mice, we also immunized MRL/MpJ mice with plasmin and analyzed the immune sera for IgG-antiplasmin antibodies and IgG-aCL. The MRL/MpJ strain was chosen because mild immunologic defects (i.e., the presence of low-titer anti–double-stranded DNA autoantibodies and low levels of glomerulonephritis) have been observed in older mice (>1 year of age) in this strain, and MRL/MpJ are the parent and control strain for the well-studied spontaneous lupus model in MRL/lpr mice. The results showed that immunized MRL/MpJ mice, as compared with control BALB/cJ mice, produced high titers of both IgG-antiplasmin antibodies and IgG-aCL. Moreover, the immunized MRL/MpJ mice also produced high titers of IgG–anti-β2GPI antibodies. Furthermore, when mAb were generated from the serum of the MRL/MpJ mice with high titers of IgG-antiplasmin and IgG-aCL, these mAb were found to bind human plasmin, cardiolipin, β2GPI, thrombin, and the activated coagulation factor X (FXa), to varying degrees. Importantly, some of the mAb inhibited plasmin activity and also hindered the inactivation of thrombin and FXa by antithrombin III (AT). Thus, these findings show that plasmin may serve as a driving autoantigen for certain prothrombotic IgG-aCL that have functional significance in vitro.