Previously, Chen et al showed that plasmin could induce both IgG-antiplasmin antibodies and limited IgG-aCL in BALB/cJ mice, and that one monoclonal aCL had lupus anticoagulant activity and induced fetal loss in mice (21
). To characterize further the plasmin-driven IgG-aCL, we used plasmin to immunize BALB/cJ mice and MRL/MpJ mice. We found that plasmin immunization in MRL/MpJ mice induced IgG-aCL and IgG-antiplasmin antibodies, whereas only persistent IgG-antiplasmin antibodies were induced in BALB/cJ mice (). Moreover, the antibodies produced in the plasmin-immunized MRL/MpJ mice reacted with human β2
GPI (), and the IgG-antiplasmin antibody levels were highly correlated with those of IgG-aCL and IgG–anti-β2
GPI (), demonstrating that the plasmin-driven aCL displayed a major characteristic of the diagnostic aCL in APS patients. Of note, to rule out the possibility that the observed anti-β2
GPI activity might be due to β2
GPI contamination in plasmin, we used a Western blotting analysis with 40 µg plasmin; the results (not shown) indicated that there was no detectable β2
Taken together, these results suggest that aCL are tightly regulated in normal mice with a normal immune system, but are more ready to escape immune regulation and persist in autoimmune-prone mice that probably have deficient/defective immune regulation. Moreover, these findings imply that plasmin could drive aCL production more readily in certain genetically susceptible individuals who are prone to autoimmune disorders.
Subsequently, we generated 10 monoclonal IgG-aCL from the plasmin-immunized MRL/MpJ mice. Importantly, 4 (40%) of these monoclonal IgG-aCL reacted with β2GPI (), and some of monoclonal IgG-aCL cross-reacted with other human serine proteases, namely, thrombin and FXa (). Of note, the sera from the sixth blood withdrawal (i.e., 2 months after the third plasmin immunization) were analyzed for reactivity with thrombin and FXa, and these sera were found to contain antithrombin activity but not anti-FXa activity (P = 0.02 and P = 0.16, respectively, versus control mice immunized with adjuvants only) (results not shown).
Finally, the pathologic significance of the plasmin-induced IgG-aCL was assessed by studying the effects of the mAb on the function and regulation of the reactive target proteases. The results showed that 4 of the mAb (M2-11, M9-16, M9-17, and M20-48) could directly inhibit the amidolytic activity of plasmin, with the extent of inhibition ranging from 18% to 39% (). Interestingly, the strength of binding of the mAb to plasmin () did not directly correlate with the profiles of plasmin inhibition (), which is likely a reflection of the fact that the mAb bind to different regions on plasmin, and that only the mAb that bind to or near the active site of plasmin may inhibit the activity of plasmin.
In addition, the thrombin-reactive monoclonal IgG-aCL M2-11 was also found to reduce the inactivation of thrombin by AT down to 29% (), and 2 mAb, M2-11 and M4-0, reduced the inactivation of FXa by AT to 3% and 38%, respectively (). These latter data suggest that ~20% of the plasmin-driven IgG-aCL may interfere with feedback regulation of FXa, resulting in unchecked activation of FXa and a proco-agulant state. Taken together, these data indicate that the plasmin-driven IgG-aCL may promote thrombosis from 2 ends, via the unregulated conversion of fibrinogen to fibrin and the reduced rate of fibrin resolution.
As reported in previous studies, 5 of 7 monoclonal IgG-aCL derived from 2 patients with APS reacted with plasmin (9
). Furthermore, these 5 plasmin-reactive aCL were observed to bind to plasmin with relative Kd
values in the range of 10−7
), which are 30–100-fold higher than the affinities of known IgG-aCL toward β2
GPI, the major autoantigen in APS (20
). Taken together with the present findings, these data strongly suggest that plasmin is an important autoantigen that drives the production of certain prothrombotic IgG-aCL in APS patients.
In this context, it is interesting to note that many bacteria use human plasmin to dissolve the surrounding fibrin clots that function as a host defense mechanism to prevent spreading of bacteria via the circulation (28
). For example, group A streptococcus
(GAS) uses its plasminogen receptors to recruit and activate human plasminogen to plasmin on the bacterial surface (28
). Consequently, plasmin is presented to the host together with streptococci, and thus may induce the immune response to plasmin, according to the “danger signal” hypothesis (30
). In addition to GAS, several other common pathogens, including Staphylococcus aureus
and Yersinia pestis
, use the human plasminogen system for survival (28
). Although the majority of aCL generated in the postinfection period are transient in nature (32
), it is possible that repeated exposure to common pathogenic bacteria may allow the development of antiplasmin antibodies in genetically susceptible individuals, which may, in turn, lead to the development of aCL, anti-β2
GPI antibodies, and APS. Future studies are warranted to test the above hypothesis.
Alternatively, we recently reported that some aPL in APS patients recognize conformational epitopes shared by β2
GPI and the homologous enzymatic domains of several serine proteases, including plasmin (33
). Specifically, 2 patient-derived IgG anti-β2
GPI mAb bound to thrombin and plasmin, and 1 antithrombin mAb reacted with β2
GPI. In addition, the binding of a cross-reactive mAb to β2
GPI was inhibited by α-thrombin, which contains only the catalytic domain of thrombin. Taken together with the results of the present study, these findings suggest that plasmin may induce and/or drive the production of aCL and anti-β2
GPI antibodies in genetically susceptible individuals via epitope spreading.
Thus, the present study shows that plasmin immunization induces significantly and persistently raised titers of disease-relevant IgG-aCL in MRL/MpJ mice, but not in BALB/cJ mice, suggesting that plasmin-driven aCL production is under genetic control. Therefore, plasmin is more likely to serve as a driving autoantigen in certain genetically susceptible individuals.