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Plasmin immunization preferentially induces potentially prothrombotic IgG anticardiolipin antibodies in MRLMpJ mice.

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ARTHRITIS & RHEUMATISM
Vol. 60, No. 10, October 2009, pp 3108–3117
DOI 10.1002/art.24818
© 2009, American College of Rheumatology
Plasmin Immunization Preferentially Induces
Potentially Prothrombotic IgG Anticardiolipin
Antibodies in MRL/MpJ Mice
Kaleo Ede, Kwan-Ki Hwang, Chen-Ching Wu, Meifang Wu, Yao-Hsu Yang, Wei-Shiang Lin,
Daniel Chien, Pei-Chih Chen, Betty P. Tsao, Deborah K. McCurdy, and Pojen P. Chen
while 4 of 10 bound to ␤2GPI, 3 of 10 bound to
thrombin, and 4 of 10 bound to the activated coagulation factor X (FXa). Functionally, 4 of the 10 IgG mAb
inhibited plasmin activity, 1 of 10 hindered inactivation
of thrombin by antithrombin III, and 2 of 10 inhibited
inactivation of FXa by antithrombin III.
Conclusion. Plasmin immunization leads to production of IgG antiplasmin, aCL, and anti-␤2GPI in
MRL/MpJ mice, but leads to production of only IgG
antiplasmin in BALB/cJ mice. IgG mAb generated from
plasmin-immunized MRL/MpJ mice bind to various
antigens and exhibit procoagulant activity in vitro.
These results suggest that plasmin may drive potentially
prothrombotic aCL in genetically susceptible individuals.
Objective. To test the hypothesis, utilizing 2 experimental mouse models, that plasmin is an important
autoantigen that drives the production of certain IgG
anticardiolipin (aCL) antibodies in patients with the
antiphospholipid syndrome.
Methods. BALB/cJ and MRL/MpJ mice were immunized with Freund’s complete adjuvant in the presence or absence of human plasmin. The mouse sera were
analyzed for production of IgG antiplasmin, IgG aCL,
and IgG anti–␤2-glycoprotein I (anti-␤2GPI) antibodies.
IgG monoclonal antibodies (mAb) were generated from
the plasmin-immunized MRL/MpJ mice with high titers
of aCL, and these 10 mAb were studied for their binding
properties and functional activity in vitro.
Results. Plasmin-immunized BALB/cJ mice produced high titers of IgG antiplasmin only, while
plasmin-immunized MRL/MpJ mice produced high titers of IgG antiplasmin, IgG aCL, and IgG anti-␤2GPI.
Both strains of mice immunized with the adjuvant alone
did not develop IgG antiplasmin or IgG aCL. All 10 of
the IgG mAb bound to human plasmin and cardiolipin,
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 ␤2-glycoprotein I (␤2GPI) 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
Supported by NIH grant AR-42506. Dr. Ede is recipient of a
fellowship grant from the Southern California Chapter of the Arthritis
Foundation. Dr. Chen-Ching Wu’s work was supported by a Faculty
Development award from Kaohsiung Medical University, Taiwan. Dr.
Yang’s work was supported by a Faculty Development award from
National Taiwan University Hospital, Taiwan. Dr. Lin’s work was
supported by a Research Training grant from the Taiwanese government.
Kaleo Ede, MD, Kwan-Ki Hwang, PhD, Chen-Ching Wu,
MD, Meifang Wu, MD, Yao-Hsu Yang, MD, PhD, Wei-Shiang Lin,
MD, Daniel Chien, BS, Pei-Chih Chen, MD, Betty P. Tsao, PhD,
Deborah K. McCurdy, MD, Pojen P. Chen, PhD: University of
California, Los Angeles.
Address correspondence and reprint requests to Kaleo Ede,
MD, University of California, Los Angeles, Division of Pediatric
Allergy, Immunology and Rheumatology, 10833 Le Conte Avenue,
MDCC 12-430, Los Angeles, CA 90095. E-mail: kaleoe@yahoo.com.
Submitted for publication June 20, 2008; accepted in revised
form June 15, 2009.
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PLASMIN INDUCTION OF POTENTIALLY PROTHROMBOTIC IgG aCL IN MRL/MpJ MICE
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
␤2GPI, 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 mice 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-␤2GPI antibodies. Furthermore, when mAb were
generated from the sera of the MRL/MpJ mice with high
titers of IgG antiplasmin and IgG aCL, these mAb were
found to bind human plasmin, cardiolipin, ␤2GPI,
thrombin, and activated coagulation factor X (FXa), to
varying degrees. Importantly, some of the mAb inhibited
plasmin activity and also hindered the inactivation of
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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.
MATERIALS AND METHODS
Immunization of mice. All mouse studies were performed following protocols in accordance with our institutional
guidelines. Six-to-seven-week-old female BALB/cJ and MRL/
MpJ mice (The Jackson Laboratory, Bar Harbor, ME) were
immunized subcutaneously with either 30 ␮g of human Lysplasmin (Haematologic Technologies, Essex Junction, VT) in
Freund’s complete adjuvant (CFA; Sigma, St. Louis, MO) or
CFA alone. The mice received booster immunizations subcutaneously with the same amount of plasmin in Freund’s
incomplete adjuvant (Sigma) at month 1 and month 3 after the
first immunization. Serum samples were obtained from the
mice before immunization on day 0 and monthly thereafter.
Analysis of sera for IgG antibodies against human
plasmin, cardiolipin, and ␤2GPI. IgG antiplasmin antibodies
were analyzed as described previously (14). Briefly, highbinding plates were coated with 5 ␮g/ml of human plasmin in
phosphate buffered saline (PBS) (pH 7.4). After incubating the
plates overnight at 4°C, the cultures were blocked with 0.25%
gelatin/PBS. Serum samples at 1:100 dilution in 0.1% gelatin/
PBS were distributed to separate wells and incubated for 1.5
hours at room temperature (RT). Bound IgG antibodies were
then measured in the sera, and those samples considered
positive for IgG antiplasmin were analyzed further at a series
of 1:10 dilutions, up to a 1:105 dilution, to determine the
antibody titers. IgG aCL were analyzed in a similar manner,
using a previously described method (22), except that the
microtiter plates were coated with cardiolipin at 50 ␮g/ml in
ethanol and blocked with 10% bovine serum in PBS. Finally,
IgG anti-␤2GPI antibodies were also analyzed in a similar
manner, as described previously (9), except that high-binding
plates were coated with ␤2GPI at 5 ␮g/ml in PBS. In all of
these experiments, a murine serum sample with high reactivity
for each of the respective antigens (antiplasmin, aCL, or
anti-␤2GPI) was used repeatedly in all of the enzyme-linked
immunosorbent assay (ELISA) experiments as the reference
standard to determine antibody titers or reference units (RU).
Generation of monoclonal IgG antiplasmin/IgG aCL
from the immunized mice. Mice with high titers of both IgG
antiplasmin antibodies and IgG aCL received a booster immunization peritoneally with 30 ␮g of plasmin 3–4 days before
fusion. The spleen cells were fused with the P3X63-AG8.653
mouse myeloma cell line (American Type Culture Collection,
Manassas, VA). The culture supernatants were then screened
for IgG antiplasmin and IgG aCL, and the hybridomas from
the double-positive wells were subcloned twice at 1 cell/well.
Of note, all of the IgG aCL–positive wells were also positive
for IgG antiplasmin. The mAb were affinity-purified using a
HiTrap Protein G–HP column (GE Lifesciences, Piscataway,
NJ) and used for all studies. All mAb were then isotyped using
the Mouse-Typer Isotyping ELISA panel (Bio-Rad, Hercules,
CA), and all were determined to be of the IgG1 subtype
(results not shown).
3110
Analyses of mAb for reactivity with plasmin, cardiolipin, and ␤2GPI, and for cross-reactivity with thrombin and
FXa. Purified mAb were analyzed for reactivity against human
plasmin, cardiolipin, and ␤2GPI by ELISA in a manner similar
to that described above, except that polyclonal mouse IgG and
isotype mouse IgG1 (Chemicon, Temecula, CA) were used as
negative controls, and all IgG were used at a concentration of
10 ␮g/ml. The mAb were also tested for thrombin reactivity as
described previously (12). Briefly, high-binding ELISA plates
were coated with 5 ␮g/ml of human ␣-thrombin (Haematologic
Technologies) in Tris buffered saline (TBS; 0.05M Tris HCl
and 0.15M NaCl, pH 7.5). After incubating the plates overnight
at 4°C, the cultures were blocked with TBS containing 0.3%
gelatin. The ELISA to test cross-reactivity with human FXa
was conducted similarly, except that the plates were coated
with 5 ␮g/ml of human FXa (Haematologic Technologies).
Effects of mAb on plasmin activity. The effects of the
mAb on plasmin activity were studied using the chromogenic
substrate S-2251 (Sigma) as described previously (21). Briefly,
plasmin (20 mM) in 50 ␮l of HEPES buffer was incubated with
50 ␮l of mAb (200 ␮g/ml) for 1 hour at RT in microtiter wells.
S-2251 at a concentration of 100 ␮l (250 ␮g/ml final concentration) was then added, and the plate was incubated in a
humidified environment for 1 hour. The optical density (OD)
value at 405 nm was measured.
Effects of mAb on the inactivation of thrombin by AT.
The effects of the thrombin-reactive mAb on the inactivation
of thrombin by AT were studied in a functional assay for the
thrombin activity in the presence of AT and heparin, as
described previously (12). Briefly, 25 ␮l of thrombin was
incubated with 25 ␮l of either test mAb, normal polyclonal
mouse IgG, or the isotype control monoclonal mouse IgG1, in
duplicate for 1 hour at RT. Fifty microliters of AT (Enzyme
Research Laboratories, South Bend, IN) was then added to
each reaction mixture in buffer containing heparin, and finally,
200 ␮l of the chromogenic substrate S-2238 (150 ␮M; Chromogenix, Molndal, Sweden) was added, and after ⬃1 minute of
incubation, the OD at 405 nm was measured. The percentage
of thrombin inactivation by AT was calculated as (1 ⫺ [residual
thrombin activity with AT]/[initial thrombin activity without
AT]) ⫻ 100.
Effects of mAb on the inactivation of FXa by AT. A
functional assay for the effects of the FXa-reactive mAb on
FXa inactivation by AT was done using a previously described
method (23). The assay used was similar to the thrombin
functional assay described above, except that 25 ␮l of FXa was
incubated separately with 25 ␮l of test mAb or control IgG for
1 hour at RT. Fifty microliters of AT was then added in the
buffer containing heparin, and subsequently, 50 ␮l of the
chromogenic substrate S-2765 (660 ␮M; DiaPharma, West
Chester, OH) was added, and the OD at 405 nm was measured
over time. The percentage of FXa inactivation by AT was
calculated as (1 ⫺ [residual FXa activity with AT]/[initial FXa
activity without AT]) ⫻ 100.
Statistical analysis. Differences in the titers of IgG
antiplasmin antibodies and IgG aCL between the 2 immunization groups (the control group and plasmin-immunized
group) were analyzed with the Kruskal-Wallis test followed by
Dunn’s multiple comparison test. Of note, when a data point in
a group was outside of the mean ⫾ 3 SD of the remaining data
points of the same group, it was considered an outlier and was
EDE ET AL
excluded from all analyses. Differences in reactivity to ␤2GPI
(OD values) in the sera between the 2 groups were analyzed
with the unpaired t-test. Relationships between 2 of the
variables (aCL titers versus anti-␤2GPI antibody titers) were
assessed by Pearson’s correlation analyses using logtransformed values of aCL and anti-␤2GPI titers (both expressed in RU ⫻ 103). Differences in the plasmin activity and
percentages of either thrombin or FXa inactivation by AT
between the mAb-treated and untreated control groups were
determined by one-way analysis of variance followed by Dunnett’s multiple comparison test.
RESULTS
Induction by plasmin immunization of mainly
IgG antiplasmin antibodies in BALB/cJ mice, but both
IgG antiplasmin and IgG aCL in MRL/MpJ mice. When
BALB/cJ and MRL/MpJ mice were immunized with
human plasmin, both strains produced high titers of IgG
antiplasmin antibodies (Figures 1A and B). The IgG
antiplasmin antibody titers peaked at month 2 and
remained high up to month 5 in both strains of mice.
Importantly, immunized MRL/MpJ mice also
displayed significantly raised titers of IgG aCL. From
month 2 to month 5, the titers of IgG aCL in the
plasmin-immunized mice were significantly higher than
those in the corresponding control mice (Figure 1D). Of
note, MRL/MpJ mouse sera were also analyzed for the
presence of aCL of both the IgA and IgM isotypes, but
no significant differences in the levels of IgA or IgM
aCL were found in comparison with control mice (results not shown).
Immunized BALB/cJ mice also developed titers
of IgG aCL that were significantly higher (P ⬍ 0.05)
than those in the control mice at month 2, although
the significance of this difference was not sustained
throughout the remainder of the experiment (Figure
1C). However, the levels of IgG aCL in the BALB/cJ
mice were much lower than in their MRL/MpJ counterparts (Figure 1C). The failure of induction of aCL in
plasmin-immunized BALB/cJ mice might imply that
plasmin can drive aCL production only in autoimmuneprone individuals.
The immunization experiments were repeated
separately on 2 occasions for the BALB/cJ mice and 3
times for the MRL/MpJ mice. Analysis of the sera was
completed on all samples, and the results shown in
Figure 1 are representative data from 1 experiment. The
human Lys-plasmin used in these experiments was tested
by Western blotting for contamination with ␤2GPI and
was found to have ⬍0.25% ␤2GPI, which suggests that
PLASMIN INDUCTION OF POTENTIALLY PROTHROMBOTIC IgG aCL IN MRL/MpJ MICE
3111
Figure 1. Effects of plasmin immunization on production of IgG antiplasmin antibodies (Ab) (A and B) and IgG anticardiolipin antibodies (aCL)
(C and D) in BALB/cJ and MRL/MpJ mice. Mice were immunized (denoted by arrows) with either 30 ␮g human plasmin in adjuvant (P) or adjuvant
alone (control [C]) on day 0 and 1 month and 3 months later. Serum samples were obtained before immunization on day 0 and monthly thereafter
and were analyzed at 1:102–1:105 dilutions for IgG antiplasmin and at a 1:102 dilution for IgG aCL. Results are expressed either in titers (for
antiplasmin) or in relative units (RU) (for aCL). The experiments were repeated twice in BALB/cJ mice and 3 times in MRL/MpJ mice, and similar
results were obtained in both strains of mice. Representative data from 1 experiment are shown. Horizontal bars indicate the geometric mean for
each group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01 versus controls treated with adjuvant alone.
there was little to no contamination (results are available
from the corresponding author upon request).
The induced IgG aCL did not react with cardiolipin in the absence of bovine serum (results not shown),
suggesting that these murine mAb have the characteristics of IgG aCL in APS. Taken together, these results
showed that plasmin could drive IgG aCL production and
suggested that genetic factors could play a significant role
in the production of the plasmin-driven IgG aCL.
Induction of IgG anti-␤2GPI antibodies in MRL/
MpJ mice, but not in BALB/cJ mice. ␤2GPI is recognized as the major autoantigen in APS, and the reactivity of antibodies against ␤2GPI and its complexes with
cardiolipin may account for most of the positive findings
on aCL activity in APS (24). Therefore, it is of interest
to investigate whether the plasmin-induced aCL could
also react with ␤2GPI in these murine models. To this
end, the immune sera that displayed peak titers of IgG
aCL (i.e., at the fifth month in MRL/MpJ mice) were
analyzed for reactivity with ␤2GPI.
As shown in Figure 2B, the immune MRL/MpJ
mouse sera displayed significantly higher titers of IgG
anti-␤2GPI antibodies as compared with the control
MRL/MpJ mouse sera (P ⬍ 0.001). Moreover, the
corresponding immune sera from BALB/cJ mice had no
IgG anti-␤2GPI antibodies compared with the sera from
control (nonimmunized) mice (Figure 2A). Thus, these
results confirm that the plasmin-induced aCL in MRL/
MpJ mice mimic the characteristics of aCL in APS.
Association of IgG aCL and IgG anti-␤2GPI
antibodies with IgG antiplasmin antibodies. To further
test our hypothesis that plasmin is a driving autoantigen
for some IgG aCL, we studied the association of the
production of IgG aCL with that of IgG antiplasmin
antibodies in the immune sera of mice obtained at
month 5 after the first immunization. As can be seen in
Figure 2C, the levels of IgG aCL were highly correlated
with the levels of antiplasmin antibodies (Pearson’s r ⫽
0.64, P ⫽ 0.026).
Moreover, we analyzed the relationship between
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EDE ET AL
Figure 2. Serologic analyses of immune sera for IgG anti–␤2-glycoprotein I (anti-␤2GPI) antibodies (Ab) in BALB/cJ and MRL/MpJ mice (A and
B) and analyses of association of anticardiolipin (aCL) and anti-␤2GPI antibodies with antiplasmin antibodies in immunized MRL/MpJ mice (C and
D). Serum samples at month 5 after the first immunization were analyzed for IgG anti-␤2GPI antibodies at a 1:102 dilution (A and B). Murine IgG
anti-␤2GPI antibodies at a 1:300 dilution were used as the reference serum, and results are expressed in relative units (RU). Horizontal bars indicate
the geometric mean (n ⫽ 2 experiments). Associations of antiplasmin antibodies with aCL (C) and with anti-␤2GPI antibodies (D) were assessed
by Pearson’s correlation coefficient analyses, using log-transformed values of antiplasmin antibodies (expressed as the optical density [OD] ⫻ 103),
aCL (expressed as RU ⫻ 103), and anti-␤2GPI antibodies (expressed as RU ⫻ 103). For antiplasmin antibodies, mouse sera were analyzed at a 1:104
dilution in order to express the values in OD (as a continuous variable). ⴱⴱⴱ ⫽ P ⬍ 0.001.
anti-␤2GPI antibody production and IgG antiplasmin
antibody production. As is evident in Figure 2D, the
levels of anti-␤2GPI antibodies were highly correlated
with the levels of antiplasmin antibodies (Pearson’s r ⫽
0.61, P ⫽ 0.048).
Reactivity of some plasmin-induced murine
monoclonal IgG aCL with human ␤2GPI. To further
characterize the plasmin-induced IgG aCL, we initiated
efforts to generate and analyze monoclonal IgG aCL
from the immunized MRL/MpJ mice with high titers of
IgG aCL. The hybridomas were screened for IgG antiplasmin antibodies and IgG aCL, and only the doublepositive hybridomas were utilized, resulting in the generation of 10 test mAb. These mAb clones were
designated M2-11, M4-0, M9-6, M9-12, M9-14, M9-15,
M9-16, M9-17, M9-43, and M20-48 (Figures 3A and B).
As noted in the preceding experiments, the
plasmin-induced aCL were observed to bind to ␤2GPI
and were thus shown to mimic the major characteristic
of aCL in APS. To explore this finding further, we
analyzed the test mAb for reactivity with ␤2GPI. As
shown in Figure 3C, 4 (40%) of the 10 mAb (i.e., M2-11,
M4-0, M9-6, and M9-12) bound to ␤2GPI, providing
further support that a subset of plasmin-induced aCL
mimic the main characteristic of aCL in APS.
Reactivity of some plasmin-induced murine
monoclonal IgG aCL with the serine protease thrombin
and FXa. To test our hypothesis that some of the
plasmin-driven aCL cross-react with certain serine proteases that share homologous enzymatic domains with
plasmin, we analyzed our mAb for cross-reactivity with
thrombin and FXa. Figure 3D demonstrates that several
of the mAb bound to thrombin, including M2-11, M4-0,
M9-12, and M9-43. In addition, as shown in Figure 3E,
some of the mAb also bound to FXa, namely, M2-11,
M4-0, and M9-43. Taken together, these results indicate
that a significant subset of the plasmin-induced aCL
cross-react with other serine proteases.
PLASMIN INDUCTION OF POTENTIALLY PROTHROMBOTIC IgG aCL IN MRL/MpJ MICE
3113
Figure 3. Binding of plasmin-induced murine monoclonal anticardiolipin antibodies (aCL) to different antigens, including human plasmin (A),
cardiolipin (B), ␤2-glycoprotein I (␤2GPI) (C), thrombin (D), and activated coagulation factor X (FXa) (E). Splenocytes from plasmin-immunized
MRL/MpJ mice with high titers of IgG aCL were used to generate murine hybridoma cells that were positive for both IgG antiplasmin antibodies
and IgG aCL. The resulting 10 murine monoclonal antibodies (mAb) were analyzed at a concentration of 10 ␮g/ml for binding to the various
antigens. A polyclonal mouse IgG antibody (mIgG) was included as a control on each plate. In addition, buffer alone was used as the untreated
control. Bars show the mean and SEM optical density (OD) (n ⫽ 2 experiments).
Murine mAb inhibition of the amidolytic activity
of plasmin. To assess the pathologic significance of the
plasmin-driven aCL, we first studied the effects of the mAb
on the amidolytic activity of plasmin, using the chromogenic substrate S-2251. The results showed that 4 mAb
(M2-11, M9-16, M9-17, and M20-48) significantly inhibited the amidolytic activity of plasmin, with the extent of
inhibition between 18% and 39% (P ⬍ 0.01 versus
untreated controls) (Figures 4A and B). These results
indicate that the plasmin-driven aCL may promote
thrombosis by directly interfering with the fibrinolytic
function of plasmin, similar to the previously described
effects of patient-derived CL15 monoclonal IgG aCL
(14).
Murine mAb inhibition of the inactivation of
thrombin by AT. To further characterize the thrombinreactive mAb, we investigated the ability of these mAb
to interfere with AT inactivation of thrombin, using a
functional assay containing 0.1 units/ml of heparin. The
addition of heparin is needed to approximate the in vivo
inactivation of thrombin by AT, since AT often binds to
heparin-like glycosaminoglycans (including heparan sulfates on the endothelial cell surface) (25–27).
Under these conditions, AT inactivated 98% of
thrombin activity, and the degrees of thrombin inactivation by AT were not changed by the presence of either
polyclonal mouse IgG or a monoclonal mouse IgG1
isotype control (Figure 5A). In contrast, the mAb M2-11
reduced the degree of thrombin inactivation to 29%
(SEM 1.4 [n ⫽ 2 experiments]; P ⬍ 0.01 versus untreated controls), whereas the remaining 2 mAb (M4-0
and M9-43) did not significantly affect AT inactivation
of thrombin.
Because M2-11 could reduce the thrombin inactivation from 98% in buffer alone to 29% in cultures
with the M2-11 mAb, the resultant increase in thrombin
activity over time could present a significant procoagulant effect, since the residual thrombin may continue to
convert fibrinogen into fibrin at a constant rate. To
visualize this rapid cumulative effect over time, overall
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EDE ET AL
interfere with the FXa inactivation by AT. The final
concentrations of FXa, AT, and IgG were 1.25 nM, 12.5
nM, and 33.3 ␮g/ml, respectively. Under these conditions, AT inactivated 92% of FXa activity (in buffer
alone), and the degrees of FXa inactivation by AT were
not significantly changed by the presence of either
Figure 4. Effects of the 10 murine IgG aCL mAb on plasmin amidolytic activity, as determined using the chromogenic substrate S-2251. A,
Plasmin amidolytic activity was assessed at the 60-minute time point
using substrate S-2251 in the absence or presence of test mAb,
polyclonal mouse IgG control, or murine IgG1 monoclonal isotype
control (mIgG1) (all at 100 ␮g/ml); buffer alone was used as the
untreated control. Bars show the mean and SEM OD (n ⫽ 2
experiments). B, The mAb-mediated inhibition of plasmin activity was
calculated using the data obtained in the activity assay. Bars show the
mean and SEM percentage of inhibition relative to that with buffer
alone (as the untreated control). ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01 versus
untreated control. See Figure 3 for other definitions.
conversion of a thrombin substrate in the absence or
presence of test mAb, polyclonal mouse IgG, or an
isotype control mAb was measured over a period of 5
minutes. As shown in Figure 5B, accumulated substrate
conversion in the presence of M2-11 increased dramatically over that in the absence of M2-11 in the span of 5
minutes.
Murine mAb inhibition of the inactivation of FXa
by AT. In a similar manner, we used a functional assay to
study the FXa–cross-reactive mAb for their ability to
Figure 5. Inhibition of thrombin inactivation by antithrombin III
(AT) with M2-11, one of the murine IgG aCL mAb. A, Thrombin was
preincubated with the test mAb, polyclonal mouse IgG, or monoclonal
mouse IgG1 isotype control (mIgG1), and AT was then added to the
reaction mixtures (in the presence of heparin), which was followed
immediately by the addition of the thrombin chromogenic substrate
S-2238. The OD after 1 minute was measured. Bars show the mean and
SEM percentage of thrombin inactivation by AT (n ⫽ 2 experiments).
ⴱⴱ ⫽ P ⬍ 0.01 versus buffer alone (as untreated control). B, The effect
of M2-11 on accumulated thrombin activity in the presence of AT and
heparin was further assessed, in a manner similar to that described in
A, except that generation of p-nitroaniline was monitored continuously
for 5 minutes. Representative results (expressed as the OD) from 1 of
2 experiments are shown. See Figure 3 for other definitions.
PLASMIN INDUCTION OF POTENTIALLY PROTHROMBOTIC IgG aCL IN MRL/MpJ MICE
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to a mean ⫾ SEM 3 ⫾ 0.7% and 38 ⫾ 4.2%, respectively
(each n ⫽ 2 experiments; P ⬍ 0.01 versus untreated
controls).
As noted, these experiments were done using IgG
at a concentration of 33.3 ␮g/ml (222 nM) and FXa at a
concentration of 1.25 nM, resulting in an IgG:FXa molar
ratio of 178:1. Assuming that the plasma concentration
of total IgG is 10 mg/ml and that IgG anti-FXa antibodies in a patient with APS account for 1% of the possible
total IgG, the concentration of antibodies used in this
assay was ⬃30% of the possible total IgG anti-FXa
antibodies. Therefore, we studied the concentrationdependent effects of anti-FXa antibodies on AT inactivation of FXa. The mAb, polyclonal mouse IgG, and
mouse monoclonal IgG1 isotype were analyzed in a
graded series of 2-fold lower concentrations (from 3.13
␮g/ml up to 50 ␮g/ml). The results showed that at a
lower concentration of 12.5 ␮g/ml (resulting in an IgG:
FXa molar ratio of 67:1), M2-11 and M4-0 reduced the
degree of FXa inactivation to 26% and 74%, respectively
(Figure 6B). Of note, increasing the concentration of
M2-11 almost completely inhibited the anticoagulant
function of AT on FXa (Figure 6B).
DISCUSSION
Figure 6. Reduction of antithrombin III (AT) inactivation of activated FXa with M2-11 and M4-0, 2 of the IgG aCL mAb. A, FXa was
preincubated separately with the test mAb (each at a concentration of
33.3 ␮g/ml), polyclonal mouse IgG, or monoclonal mouse IgG1 isotype
control (mIgG1), and AT was then added to each reaction mixture (in
the presence of heparin), which was followed immediately by the
addition of the FXa chromogenic substrate S-2765. Bars show the
mean and SEM percentage of FXa inactivation by AT (n ⫽ 2
experiments). ⴱⴱ ⫽ P ⬍ 0.01 versus buffer alone (as the untreated
control). B, The concentration dependence of the inhibition of AT
inactivation of FXa by the test mAb was assessed. The experiment was
performed in a manner similar to that described in A except that a
series of graded concentrations of mAb (ranging from 0 to 50 ␮g/ml)
was used. Bars show the mean ⫾ SEM percentage of inactivation of
FXa by AT in the presence of heparin (n ⫽ 2 experiments). See Figure
3 for other definitions.
polyclonal mouse IgG control or monoclonal mouse
IgG1 isotype control (Figure 6A). In contrast, the mAb
M2-11 and M4-0 reduced the degree of FXa inactivation
Previously, we 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 (Figure 1).
Moreover, the antibodies produced in the plasminimmunized MRL/MpJ mice reacted with human ␤2GPI
(Figure 2B), and the IgG antiplasmin antibody levels
were highly correlated with those of IgG aCL and IgG
anti-␤2GPI (Figures 2C and D), 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-␤2GPI activity
might be due to ␤2GPI contamination in plasmin, we
used Western blotting analysis with 40 ␮g plasmin; the
results (available from the corresponding author upon
request) indicated that there was no detectable ␤2GPI
contamination.
Taken together, these results suggest that aCL
are tightly regulated in normal mice with a normal
3116
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 ␤2GPI (Figure 3C), and some of the monoclonal
IgG aCL cross-reacted with other human serine proteases, namely, thrombin and FXa (Figures 3D and E).
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 adjuvant 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%
(Figure 4). Interestingly, the strength of binding of the
mAb to plasmin (Figure 3A) did not directly correlate
with the profiles of plasmin inhibition (Figure 4), 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% (Figure 5), and 2
mAb, M2-11 and M4-0, reduced the inactivation of FXa
by AT to 3% and 38%, respectively (Figure 6). 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 procoagulant 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 (9,10,14). Furthermore, these 5 plasminreactive aCL were observed to bind to plasmin with
relative Kd values in the range of 10⫺7 (14), which are
EDE ET AL
30–100-fold higher than the affinities of known IgG aCL
toward ␤2GPI, 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 (28,29).
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 (30,31). 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-␤2GPI antibodies, and APS. Future studies are
warranted to test the above-mentioned hypothesis.
Alternatively, we recently reported that some
aPL in APS patients recognize conformational epitopes
shared by ␤2GPI and the homologous enzymatic domains of several serine proteases, including plasmin (33).
Specifically, 2 patient-derived IgG anti-␤2GPI mAb bound
to thrombin and plasmin, and 1 antithrombin mAb reacted
with ␤2GPI. In addition, the binding of a cross-reactive
mAb to ␤2GPI was inhibited by ␣-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-␤2GPI 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.
ACKNOWLEDGMENT
We thank Dr. Brian Skaggs for teaching Dr. M. Wu
and providing reagents for the Western blot analyses.
PLASMIN INDUCTION OF POTENTIALLY PROTHROMBOTIC IgG aCL IN MRL/MpJ MICE
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Ede had full access to all of the
data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Study conception and design. Ede, Hwang, Tsao, P. P. Chen.
Acquisition of data. Ede, Hwang, C.-C. Wu, M. Wu, Yang, Lin, Chien,
P.-C. Chen, McCurdy, P. P. Chen.
Analysis and interpretation of data. Ede, Hwang, C.-C. Wu, M. Wu,
Lin, P.-C. Chen, Tsao, P. P. Chen.
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