The Journal of Immunology, 1998, 160: 253-258.
Copyright © 1998 by The American Association of Immunologists
Monoclonal Anti-Cardiolipin Antibodies from New Zealand Black x New Zealand White F1 Mice React to Thrombomodulin1
Kazuhiko Haruta*,
,
Shigeto Kobayashi2,,
Sachiko Hirose
,
Aiko Horiai,
Mutsuko Ohyanagi,
Mitsuhiko Tanaka,
Terunaga Kawano,
Toshikazu Shirai,
Yoshinari Takasaki and
Hiroshi Hashimoto
*
Research Laboratory, Zenyaku Kogyo Co. Ltd., and Departments of
Rheumatology and Internal Medicine and
Pathology, Juntendo University School of Medicine, Tokyo, Japan
 |
Abstract
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The reactivity with and affinity for thrombomodulin (TM) of
monoclonal anti-cardiolipin Abs (MoaCL), derived from a New Zealand
Black x New Zealand White F1 (NZB/W
F1) mouse, were studied to investigate the pathogenicity of
anti-cardiolipin Abs (aCL). Four of eighteen MoaCL were found to
react with rabbit TM when examined using ELISA. These four MoaCL also
reacted with synthetic peptide that included the epidermal growth
factor-like domain of human TM, a binding site for thrombin. The
reaction with TM of these four MoaCL was inhibited by bovine thrombin.
When the affinity for TM of the MoaCL was determined, the dissociation
constants (Kd) ranged from 4.8 x
10-9 to 4.7 x 10-8 M. By contrast,
examination of the affinity for cardiolipin (CL) gave values from
8.3 x 10-6 to 7.4 x 10-5 M. Thus,
these MoaCL reacted to TM with a higher affinity than to CL. Moreover,
these MoaCL also bound to TM on HUVEC and down-regulated the expression
level of TM on the surface of HUVEC due to internalization of TM. The
binding of thrombin to TM is known to initiate rapid protein C
activation, and complexes of activated protein C and protein S show
anticoagulatory activity. Thus, the present studies suggest that
certain pathogenic aCL cross-react with TM and induce down-regulation
of TM on endothelial cells, followed by induction of thrombosis.
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Introduction
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It
has been reported that
anti-phospholipid Abs (aPL)3 are closely associated
with thrombosis, thrombocytopenia, and intrauterine fetal death in
patients with systemic lupus erythematosus (SLE) as well as
antiphospholipid syndrome (APS) (1). However, anti-cardiolipin Abs
(aCL) have also been detected in sera from subjects who seldom have
thrombosis, such as those with juvenile rheumatoid arthritis, AIDS, or
syphilis, and even in sera from healthy individuals (2, 3, 4). A
relatively new method for determination of aCL revealed that aCL in the
presence of ß2-glycoprotein I (ß2GPI)
has additional pathogenic implications (5, 6). Moreover, Matsuura et
al. reported that aCL recognized ß2GPI, but not
cardiolipin (CL) (7). However, the mechanism of the pathogenicity of
aCL is not well understood.
It was postulated that aCL affects the thrombin-thrombomodulin (TM)
complex and inhibits the activation of protein C (8, 9, 10). TM is an
integral glycoprotein present on the surface of endothelial cells and
serves as a potent receptor of thrombin. When thrombin binds to TM, the
former loses procoagulatory and platelet-stimulating actions, then the
thrombin-TM complex activates the zymogen, protein C (11, 12).
Activated protein C together with protein S degrade the active blood
coagulant cofactors, factor V and factor VIII, and stimulate
anticoagulatory activity (13). Thus, TM plays an important role in
preventing coagulation on blood vessel walls. Since some monoclonal aCL
(MoaCL) exhibit polyreactivity (14, 15), it seems important to
determine whether TM is one of the cross-reactive Ags responsible for
the development of thrombosis in APS. In the present studies, we
established MoaCL from a 6-mo-old NZB/W F1 mouse to
determine whether aCL could react with TM.
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Materials and Methods
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Reagents
mAbs against human TM (3H1 and 9H12) were gifts from Fuji
Chemical Industries Ltd. (Toyama, Japan). Goat polyclonal Abs against
rabbit TM and against human TM were purchased from American
Diagnostica, Inc. (Greenwich, CT), the TM preparation from rabbit lung
was obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan), a
synthetic EGF-5 peptide that includes the thrombin binding site of
human TM (residues Glu408 to Glu426 of human TM
corresponding to the fifth epidermal growth factor-like domain) (16)
was purchased from Peptide Institute, Inc. (Osaka, Japan), CL was
obtained from Avanti-Polar Lipids Co. Ltd. (Alabaster, AL), bovine
thrombin was purchased from Sankyo Co. Ltd. (Tokyo, Japan), human
IgG-Fc was obtained from Cappel (Durham, NC), and ß2GPI
was purchased from Yamasa Corp. (Chiba, Japan).
Generation of MoaCL
MoaCL were obtained by hybridizing the myeloma cell line
P3-X63-Ag8.653 with spleen cells from a 6-mo-old female NZB/W
F1 mouse, as described previously (17). Binding
activities of culture supernatants to CL were screened by ELISA.
Namely, 50 µl of CL solubilized in ethanol (10 µg/ml) were
immobilized in each well of a microtiter plate (Immulon 2, Dynatech
Laboratories, Inc., Chantilly, VA) by evaporation of the ethanol. After
blocking the nonspecific binding sites by incubation with 1% BSA, the
plate was washed with 0.05% Tween-20 in PBS (PBS/Tween-20) and
incubated with culture supernatant. The plate was washed with
PBS/Tween-20 followed by incubation with 50 µl of appropriately
diluted biotinylated Ab against mouse IgG. The preparation was then
washed with PBS/Tween-20 followed by incubation with 50 µl of
avidin-conjugated horseradish peroxidase. Each incubation was performed
for 1 h at 37°C. After washing with PBS/Tween-20, 200 µl of
0.4 mg/ml substrate (o-phenylenediamine
dihydrochloride) diluted in 0.1 M citrate/0.2 M phosphate buffer, pH
4.2, with 0.012% H2O2 was added, and the
reaction was stopped by adding 50 µl of 2.5 N
H2SO4 to each well after 5 min. Reactivity (OD)
was determined using a MicroPlate Reader (model 450, Bio-Rad, Hercules,
CA) at 490 nm. Positive clones were subcloned at least twice by
limiting dilution, and 18 MoaCL clones were obtained.
Determination of isotypes of MoaCL
The isotypes of MoaCL were determined using Mouse Mono Ab-ID EIA
Kits (Zymed Laboratories, San Francisco, CA).
Reactivities of MoaCL to TM, EGF-5 peptide, and IgG-Fc
Reactivities of MoaCL with TM, EGF-5 peptide, and IgG-Fc were
determined using the ELISA procedures described above, except for
immobilization of Ags, which was performed by incubation of each Ag (50
µl/well) in wells of a microtiter plates for 1 h at 37°C.
Inhibition of reactivity with TM by thrombin
To determine whether MoaCL react with the thrombin binding site
on TM, an inhibition assay was performed. After the TM-coated plate had
been incubated for 1 h with 1% BSA, culture supernatants of MoaCL
or polyclonal Abs against rabbit TM (1 µg/ml) were added in the
presence of various concentrations of bovine thrombin. As a negative
control, BSA was added instead of thrombin. After washing the wells
with PBS/Tween-20, MoaCL or polyclonal Abs against rabbit TM (1
µg/ml) were added. The percentage of TM binding activity was
determined using the following equation: % activity = (OD with
thrombin or BSA/OD without thrombin or BSA) x 100.
Effects of ß2GPI on binding of MoaCL to CL
In experiments to examine the effects of
ß2GPI on binding of MoaCL to CL, the reactivity of
protein G-Sepharose column-purified MoaCL (500 ng/ml) to CL was
determined in the presence or the absence of ß2GPI in
PBS, using the ELISA procedure described above.
Competitive inhibition of TM binding activity by TM, EGF-5 peptide,
CL, and ß2GPI
TM binding activities of MoaCL were determined by ELISA in the
presence or the absence of several concentrations of TM, EGF-5 peptide,
CL, ß2GPI, or BSA as competitive inhibitors. In this
experiment, CL was solubilized in PBS by sonication. The percentage of
TM binding activity was calculated as follows: % activity = (OD
with inhibitor/OD without inhibitor) x 100.
Measurement of affinity
The affinities of MoaCL for CL, TM, EGF-5 peptide, and IgG-Fc
were determined according to the method of Friguet et al. (18). A
constant amount (1 µg/ml) of each MoaCL suspended in PBS/Tween-20 in
5-ml tubes was incubated with serial dilutions of CL, TM, EGF-5
peptide, or IgG-Fc for 2 h at 37°C. CL was sonicated for
preincubation.These samples were then transferred to ELISA wells that
had been precoated with each corresponding Ag, CL at 10 µg/ml, TM at
50 ng/ml, EGF-5 peptide at 5 µg/ml, or IgG-Fc at 50 ng/ml. The
dissociation constant (Kd) of each MoaCL
was calculated from the formula reported by Friguet et al. (18):
A0/(A0 - A) = 1 +
Kd/a0, where A0 is the
OD measured in the absence of Ag, A is the OD measured in the presence
of Ag, and a0 is the concentration of Ag). When
A0/(A0 - A) and 1/a0 are
plotted against one another, the slope yields Kd
for binding of the MoaCL to the Ag (Klotz plots). The affinity is
expressed as 1/Kd.
Flow cytometry
The reactivity of MoaCL to HUVEC was determined using flow
cytometry, as previously described (19). HUVEC (Clonetics Co., San
Diego, CA) was used between the third to ninth passages. Cells in the
flask were removed using a cell scraper and washed with medium 199
containing 5% FCS (washing solution), then incubated with MoaCL for 30
min at 4°C. After washing with washing solution, cells were stained
with phycoerythrin-conjugated goat anti-mouse IgG for 30 min at
4°C and analyzed by flow cytometry using Coulter EPICS Elite
(Hialeah, FL). Mouse IgG2a (myeloma protein UPC10, 1 µg/ml) was used
as the background Ab control for these studies. To determine the
reactivity of MoaCL to TM on the HUVEC, MoaCL was incubated with HUVEC
preincubated with excess amounts of polyclonal Abs against human TM
(100 µg/ml) for 30 min at 4°C, and the reactivity was examined by
flow cytometry, as described above.
Cell-ELISA for detection of TM on cell surface and in cytoplasm of
HUVEC
To determine whether binding of MoaCL to TM modulates the level
of expression of TM on HUVEC, the amount of TM on the cell surface was
measured by cell-ELISA, as previously described (19). Briefly, a
confluent culture of HUVEC in the gelatin-coated 96-well plate (Iwaki
Glass, Tokyo, Japan) was washed with PBS and incubated with MoaCL for
30 min at 37°C. After washing with PBS, HUVEC were fixed by adding
0.2% glutaraldehyde in PBS at room temperature for 20 min, followed by
incubation with 1% BSA for 1 h at 37°C. HUVEC was then
incubated with polyclonal Abs against human TM (1 µg/ml) for 1 h
at 37°C, washed, and incubated with peroxidase-conjugated
anti-goat IgG. To determine the total amounts of TM (on cell
surface and in cytoplasm), the same procedure was performed, except for
treatment of HUVEC with 0.1% Triton X-100 in PBS for 2 min after
fixation.
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Results
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Cross-reactivities of MoaCL with TM, EGF-5 peptide, and
IgG-Fc
Four of 18 MoaCL (CL 19, 31, 34, and 54) reacted with TM and also
the EGF-5 peptide, a synthetic peptide that includes the thrombin
binding site of TM. The maximum reactions of these MoaCL (with the
exception of CL 54) and polyclonal Abs against TM were observed when TM
at 50 ng/ml was used to coat the microtiter plates. No changes in OD
were observed with the other 14 MoaCL, including CL 74, with changes in
the concentration of TM (Fig. 1
). The
reaction of each MoaCL with the EGF-5 peptide depended on concentration
of the peptide (Fig. 2). Two of these
four MoaCL (CL 31 and 34) reacted to IgG-Fc. The reactivities of these
MoaCL with CL, TM, EGF-5 peptide, and IgG-Fc are summarized in Table I. The isotype of all MoaCL reacted with
TM was IgG2a.
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FIGURE 1. Binding of MoaCL to TM. Of 18 MoaCL, CL 19, 31, 34, and 54 reacted with
rabbit TM. Binding to TM was not observed with CL 74. anti-rTM,
polyclonal goat Abs against rabbit TM (1 µg/ml).
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FIGURE 2. Binding of MoaCL with EGF-5 peptide. CL 19, 31, 34, and 54 reacted with
EGF-5 peptide. No reaction was observed with CL 74. 3H1, mAb against
human TM, which reacts with the fifth EGF-like domain of human TM, (1
µg/ml).
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Inhibition of reactivity of MoaCL with TM by thrombin
The reactivity of one MoaCL (CL 19) with TM deceased when
increasing concentrations of thrombin were used for inhibitor (Fig. 3), even though the reaction with TM of
polyclonal Abs against rabbit TM, which reacted with various epitopes
of TM, was not inhibited by thrombin. The inhibition of reactivity with
TM by thrombin was also observed with CL 31, 34, and 54. However, the
BSA pretreatment did not affect the reactivity of MoaCL with TM (Table II). These findings suggest that these
four MoaCL reacted with thrombin binding sites of TM.
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FIGURE 3. Inhibition of the binding of CL 19 to TM by thrombin. Binding of CL 19
or anti-rTM (polyclonal goat Abs against rabbit TM; 1 µg/ml) to a
TM-coated plate in the presence of the indicated concentration of
bovine thrombin was measured, and the percent binding activity was
determined.
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Effects of ß2GPI on binding of MoaCL to CL
The reactivities of purified MoaCL (CL 19, 31, 34, and 54) with CL
decreased in the presence of a high dose (30 µg/ml) of
ß2GPI compared with reactivities in the absence of
ß2GPI. The reactivity of CL 74, which showed no binding
activity to TM (Table I
), also decreased; however, that of CL 104
increased in the presence of ß2GPI. The serum binding
activity with CL in 8-mo-old NZB/W F1 mice decreased, while
that in 4-mo-old MRL/Mp-lpr/lpr (MRL/l) mice increased in
the presence of ß2GPI (Fig. 4).
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FIGURE 4. Effects of ß2GPI on reactivity of MoaCL with CL.
Binding to CL was determined in the presence of the indicated
concentration of ß2GPI. MoaCL was purified using a
protein G-Sepharose column and used at a concentration of 500 ng/ml.
Pooled sera from 4-mo-old MRL/l or 8-mo-old NZB/W F1 mice
was used at a 1/3200 dilution.
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Competitive inhibition of reactivity of MoaCL with TM
The reactivity of CL 19 with TM decreased when increasing
concentrations of TM and EGF-5 peptide, but not of
ß2GPI or BSA, were added as competitors. The
apparent TM binding activity of CL 19 increased in the presence of high
concentrations of CL (Fig. 5A). This may be due to
the binding of CL to TM in the ELISA system because of the avid binding
capacity of TM to phospholipids such as CL; thus, CL 19 bound both TM
and CL. In contrast, the reactivity of polyclonal Abs against rabbit TM
was inhibited only by TM (Fig. 5B). The same results
were observed with CL 31, 34, and 54 (Table III).
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FIGURE 5. Competitive inhibition of TM binding with various inhibitors. Binding
to TM of CL 19 (A) or anti-rTM
(B; polyclonal goat Abs against rabbit TM; 1 µg/ml)
was determined in the presence of TM, EGF-5 peptide,
ß2GPI, CL, or BSA, and the percent binding activity was
determined.
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Comparison of affinities of each MoaCL for CL, TM, EGF-5 peptide,
and IgG-Fc
The Kd values of each MoaCL for binding to
CL, TM, EGF-5 peptide, and IgG-Fc were determined. As was typical of
other MoaCL, the reactivity of CL 31 to each Ag gradually decreased
when CL 31 was preincubated with increasing concentrations of each Ag.
We obtained a competitive inhibition curve for CL 31 with each CL, TM,
EGF-5 peptide, and IgG-Fc (Fig. 6).
Linear plots (Klotz plots) for determination of the
Kd for the binding of CL 31 to each Ag are shown
in Figure 7. Table IV shows a summary of
Kd values for each MoaCL. The
Kd value revealed high affinities to TM ranging
from 4.8 x 10-9 M (CL 34) to 4.7 x
10-8 M (CL 54). In case of polyclonal anti-TM, the
Kd value was 2.2 x 10-10 M.
Affinities to EGF-5 peptide were lower than those to TM, and the
Kd values ranged from 9.0 x
10-7 M (CL 31) to 6.3 x 10-6 M (CL 19).
These MoaCL had low affinities to CL (Kd
values ranging from 8.3 x 10-6 M (CL34) to 7.4
x 10-5 M (CL 19)) and to IgG-Fc (5.5 x
10-5 M for CL 34 and 1.0 x 10-4 M for
CL 31).
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FIGURE 6. Binding activity of CL 31 preincubated with CL, TM, EGF-5 peptide, or
IgG-Fc to each CL-, TM-, EGF-5 peptide-, or IgG-Fc-coated plate. Based
on these data, the Kd values were calculated as
shown in Figure 7. See details in Materials and
Methods.
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FIGURE 7. Klotz plots for the determination of Kd values
for binding of CL 31 to each Ag. See details in Materials and
Methods.
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Modulation of cell surface expression of TM on HUVEC by binding of
MoaCL
Figure 8 shows the reactivity of
each MoaCL with HUVEC, examined using flow cytometry. CL 19, 31, 34,
and 54, but not CL 74, reacted with HUVEC, and these activities were
completely inhibited by pretreatment of HUVEC with polyclonal Abs
against human TM, thus indicating that these MoaCL bound cell surface
TM on HUVEC. When HUVEC were preincubated with these MoaCL, the
expression level of TM on the cell surface, detected using polyclonal
Abs against human TM, decreased by 14
36% (Fig. 9). In contrast, there was no change in
the total contents of TM on the cell surface and in the cytoplasm of
HUVEC (data not shown). These results indicate that TM on the cell
surface was internalized as a result of MoaCL binding.
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FIGURE 8. Flow cytometric analysis of reactivities of MoaCL with HUVEC. CL 19,
31, 34, and 54, but not CL 74, reacted with HUVEC. These reactions were
completely inhibited by preincubation of HUVEC with polyclonal Abs
against human TM (100 µg/ml; solid histograms). 9H12, mAb against
human TM which reacts with the third EGF-like domain of human TM;
IgG2a, mouse myeloma protein UPC10.
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FIGURE 9. Effects of MoaCL on the expression of TM on HUVEC. The TM expression
level on the cell surface of HUVEC decreased when HUVEC were
preincubated with CL 19, 31, 34, and 54, but not with CL 74. The
percentage of TM expression was calculated compared with the level
after preincubation with culture medium alone.
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Discussion
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We obtained evidence that our murine MoaCL reacted with rabbit TM
and that these MoaCL were directed to the thrombin binding site on TM,
since the MoaCL reacted with the synthetic form of the EGF-5 peptide.
Furthermore, the reaction with TM was inhibited by bovine thrombin.
Reactions of MoaCL with TM and the EGF-5 peptide were true Ag-antibody
interactions, since they were competitively inhibited by each
respective Ag. Moreover, flow cytometric analysis revealed that these
MoaCL reacted to TM expressed on the surface of HUVEC.
There is controversy as to whether autoantibody against TM is present
in patients with SLE or APS or in the lupus-prone mouse. Gibson et al.
(20) found no autoantibody to TM in patients with SLE. MoaCL from the
MRL/l mouse, which have been reported to show polyreactivity, did not
react with recombinant human TM (15). However, Ruiz-Argulles et al. (8)
reported that the reactivity of aCL with CL in sera from patients with
primary APS was eliminated by preincubation with TM. Furthermore,
Oosting et al. (21) reported that autoantibodies against TM were
transiently present in the sera from two aPL-positive patients with SLE
and in four aPL-negative patients with histories of thrombosis. IgG
fractions of these sera inhibited the activation of protein C and bound
to a recombinant version of the EGF-like portion of TM. These reports
are in agreement with our present findings.
As our MoaCL were polyreactive, we examined the affinity of MoaCL for
CL, TM, EGF-5 peptide, and IgG-Fc by calculating
Kd values. Kd values of
four MoaCL to TM ranged from 4.8 x 10-9 to 4.7
x 10-8 M, and those to EGF-5 peptide ranged from 9.0
x 10-7 to 6.3 x 10-6 M. In contrast,
Kd values to CL ranged from 8.3 x
10-6 to 7.4 x 10-5 M, and those to
IgG-Fc ranged 1.0 x 10-4 for CL 31 and 5.5 x
10-5 M for CL 34. The Kd values to
IgG-Fc were almost of the same magnitude as those of the polyreactive
Abs reported in the literature (22, 23). These results suggest that our
MoaCL bound more strongly to TM than to CL. As for the affinity of
thrombin for TM, the Kd value was reported to be
5 x 10-10 M (24), thereby suggesting a higher
affinity for TM than for our MoaCL. This high affinity was shown to be
due to the secondary binding site of thrombin, glycosaminoglycan linked
to the serine/threonine-rich domain (residues 463–497) on TM (25).
Thus, it is not known whether the affinity of thrombin for the thrombin
binding site of TM is indeed higher than that of our MoaCL. In fact,
thrombin inhibited the binding of MoaCL to TM only when large amounts
of thrombin were used (Fig. 3). In our preceding studies, we found that
anti-DNA autoantibodies from NZB/W F1 mice showed an
age-associated increase in the number of somatic mutations in IgV
region genes, and that these somatic hypermutations correlated well
with affinity maturation of anti-DNA Abs (17, 26). Based on these
observations, we propose that an age-associated accumulation of somatic
hypermutation in the IgV region genes may lead to the formation of
highly pathogenic autoantibodies in NZB/W F1 mice. In this
study we obtained MoaCL only from a single 6-mo-old NZB/W
F1 mouse. MoaCL with a higher affinity for TM may be
obtained from older mice. Studies are ongoing to clarify relationships
between an age-associated somatic hypermutation in IgV region genes and
the affinity maturation of MoaCL to TM.
Kumada et al. (27) found that i.v. injection of polyclonal Abs against
rat TM accelerated the thrombin-induced thromboembolism in mice.
Moreover, it was demonstrated that the levels of expression of TM on
endothelial cells were decreased by internalization of the TM-thrombin
complex in the presence of anti-TM Abs, resulting in a decrease in
the activation of protein C (28). In the present study, addition of
MoaCL down-regulated the expression level of TM on the cell surface of
HUVEC due to internalization of the TM-MoaCL complex. Thus, it is
suggested that our MoaCL in the circulation directly down-regulate the
expression of TM on endothelial cells, thereby preserving the
procoagulatory action of thrombin.
The mechanism of pathogenicity of aCL is obscure. Vismara et al. (29)
demonstrated that affinity-purified aCL from sera of patients with SLE
reacted with endothelial cells and suggested that these Abs were
responsible for the induction of thrombosis via binding to
phospholipids on these cells. Matsuura et al. (6) reported that the
reactivity with CL of aCL from patients with APS was enhanced in the
presence of ß2GPI. It was also shown that the presence of
ß2GPI in the aCL assay was necessary for quantitation of
thrombosis-related aCL in sera of patients with SLE or APS (5), but not
in the case of thrombosis-unrelated disease, such as syphilis (6).
Moreover, aCL from patients with SLE or lupus-prone mice are directed
against ß2GPI, but not CL, as demonstrated only when
gamma-ray-irradiated microplates are used to coat ß2GPI
(7). ß2GPI has been shown to inhibit the intrinsic blood
coagulation pathway, the ADP-mediated aggregation of platelets, and the
prothrombinase activity of activated platelets (30). Thus, we speculate
that it is the functionally impaired ß2GPI induced by
binding of aCL to ß2GPI that induces thrombosis. However,
there is a report that these anticoagulatory activities of
ß2GPI were enhanced in the presence of aCL (31).
ß2GPI-dependent aCL do not always relate to pathologic
activities (32, 33). In the present studies, binding activities to CL
of our four MoaCL with reactivity to TM did not increase by adding
ß2GPI. It is possible that there might be several
mechanisms by which aCL induce thrombosis because aCL are, in general,
heterogeneous (34). Our present studies suggest that the
cross-reactivity to TM of certain aCL is one of the mechanisms of
aCL-induced thromboembolism. Further studies on the biologic activity
of aCL in relation to reactivity to TM will lead to a better
understanding of the pathogenicity of aCL.
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Acknowledgments
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We thank Dr. David T. Y. Yu, Division of
Rheumatology, Department of Medicine, University of California-Los
Angeles, for kind encouragement, and M. Ohara for helpful comments on
the manuscript.
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Footnotes
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1 This work was supported by grants from the Ministry of Health and Welfare and the Ministry of Education, Science, Sports, and Culture of Japan. 
2 Address correspondence and reprint requests to Dr. Shigeto Kobayashi, Department of Rheumatology and Internal Medicine, Juntendo University School of Medicine, 2–1–1, Hongo, Bunkyo-ku, Tokyo 113, Japan.
3 Abbreviations used in this paper: aPL, antiphospholipid Abs, SLE, systemic lupus erythematosus; APS, antiphospholipid syndrome; aCL, anti-cardiolipin antibodies; ß2GPI, ß2-glycoprotein I; CL, cardiolipin; TM, thrombomodulin; MoaCL, monoclonal anti-cardiolipin antibodies; NZB/W F1, New Zealand Black x New Zealand White F1; EGF, epidermal growth factor.
Received for publication January 27, 1997.
Accepted for publication September 22, 1997.
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References
|
|---|
-
Harris, E. N., A. E. Gharavi, M. L. Boey, B. M. Patel, C. G. Mackworth-Young, S. Loizou, G. R. V. Hughes. 1983. Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet 2:1211.[Medline]
-
Caporali, R., A. Ravelli, G. Gennaro, G. Neirotti, C. Montecucco, A. Martini. 1991. Prevalence of anticardiolipin antibodies in juvenile chronic arthritis. Ann. Rheum. Dis. 50:599.[Abstract/Free Full Text]
-
Gharavi, A. E., L. R. Sammaritano, J. Wen, N. Miyawaki, J. H. Morse, M. H. Zarrabi, M. D. Lockshin. 1994. Characteristics of human immunodeficiency virus and chlorpromazine induced anti-phospholipids antibodies: effect of ß2 glycoprotein I on binding to phospholipid. J. Rheumatol. 21:94.[Medline]
-
Cheng, H. M.. 1991. Antiphospholipid antibodies are masked in normal human serum. Immunol. Today 12:96.[Medline]
-
McNeil, H. P., R. J. Simpson, C. N. Chesterman, S. A. Krilis. 1990. Antiphospholipid antibodies are directed against a complex antigen that induces a lipid-binding inhibitor of coagulation: ß2 glycoprotein I (apolipoprotein H). Proc. Natl. Acad. Sci. USA 87:4120.[Abstract/Free Full Text]
-
Matsuura, E., Y. Igarashi, M. Fujimoto, K. Ichikawa, T. Koike. 1990. Anticardiolipin cofactor(s) and differential diagnosis of autoimmune disease. Lancet 336:1778.
-
Matsuura, E., Y. Igarashi, T. Yasuda, D. A. Triplett, T. Koike. 1994. Anticardiolipin antibodies recognize ß2-glycoprotein I structure altered by interacting with an oxygen modified solid phase surface. J. Exp. Med. 179:457.[Abstract/Free Full Text]
-
Ruiz-Arguelles, G. J., A. Ruiz-Arguelles, M. Deleze, D. Alarcon-Segovia. 1989. Acquired protein C deficiency in a patient with primary antiphospholipid syndrome: relationships to reactivity of cardiolipin antibody with thrombomodulin. J. Rheumatol. 16:381.[Medline]
-
Amer, L., W. Kisiel, R. P. Seales, Jr R. C. Williams. 1990. Impairment of protein C anticoagulant pathway in a patient with SLE, anticardiolipin antibody and thrombosis. Thromb. Res. 57:247.[Medline]
-
Freyssinet, J. M., M. L. Wiesel, J. Gauchy, B. Boneu, J. P. Cazenave. 1986. Anti-IgM lupus anticoagulant that neutralizes the enhancing effect of phospholipid on purified endothelial thrombomodulin activity: a mechanism of thrombosis. Thromb. Haemost. 55:309.[Medline]
-
Esmon, C. T.. 1987. The regulation of natural anticoagulant pathway. Science 235:1348.[Abstract/Free Full Text]
-
Dittman, W. A., P. W. Majerus. 1990. Structure and function of thrombomodulin: a natural anticoagulant. Blood 75:329.[Free Full Text]
-
Esmon, C. T., N. L. Esmon, K. W. Harris. 1982. Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation. J. Biol. Chem. 257:7944.[Abstract/Free Full Text]
-
Igarashi, K., M. Umeda, S. Tokita, K. Y. Nam, K. Inoue. 1991. Effective induction of antiphospholipid and anticoagulant antibodies in normal mouse. Thromb. Res. 61:135.[Medline]
-
Ichikawa, K., T. Suzuki, Y. Hashimoto, T. Sumida, H. Tomioka, S. Yoshida, T. Koike. 1992. Monoclonal autoantibodies to cardiolipin derived from SLE mice. Lupus 1:239.[Abstract/Free Full Text]
-
Hayashi, T., M. Zushi, S. Yamamoto, K. Suzuki. 1990. Further localization of binding sites for thrombin and protein C in human thrombomodulin. J. Biol. Chem. 265:20156.[Abstract/Free Full Text]
-
Hirose, S., M. Wakiya, Y. Kawano-Nishi, J. Yi, T. Sanokawa, S. Taki, T. Shimamura, T. Kishimoto, H. Tsurui, H. Nishumura, T. Shirai. 1993. Somatic diversification and affinity mutation of IgM and IgG anti-DNA antibodies in murine lupus. Eur. J. Immunol. 23:2813.[Medline]
-
Friguet, B., F. Allian, L. Djavacli-Ohaninace, M. E. Goldberg. 1984. Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J. Immunol. Methods 77:305.
-
Sakai, A.. 1996. Inhibition of endothelial cell adhesion molecule expression with SJC 13, an azaindolidine derivative, in vitro. Inflamm. Res. 45:224.[Medline]
-
Gibson, J., M. Nelson, R. Brown, H. Salem, H. Kronenberg. 1992. Autoantibodies to thrombomodulin: development of an enzyme immunoassay and a survey of their frequency in patients with the lupus anticoagulant. Thromb. Haemost. 67:507.[Medline]
-
Oosting, J. D., K. T. Preissner, R. H. W. M. Derksen, P. G. DeGroot. 1993. Autoantibodies directed against the epidermal growth factor-like domains of thrombomodulin inhibit protein C activation in vitro. Br. J. Haematol. 85:761.[Medline]
-
Casali, P., A. L. Notkins. 1989. Proving the human B-cell repertoire with EVB: polyreactive antibodies and CD5+ B lymphocytes. Annu. Rev. Immunol. 7:513.[Medline]
-
Carson, D. A.. 1984. Rheumatoid factor. W. N. Kelly, Jrand E. D. Harris, and S. Ruddy, and C. B. Sledge, eds. Textbook of Rheumatology 155. W. B. Saunders Co., Philadelphia.
-
Esmon, C. T., W. G. Owen. 1981. Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. Proc. Natl. Acad. Sci. USA 78:2249.[Abstract/Free Full Text]
-
Koyama, T., J. F. Parkinson, N. Aoki, N. U. Bang, G. Muller-Berghaus, K. T. Preissner. 1991. Relationship between post-translational glycosylation and anticoagulant function of secretable recombinant mutants of human thrombomodulin. Br. J. Haematol. 78:515.[Medline]
-
Taki, S., S. Hirose, K. Kinoshita, H. Nishimura, T. Shimamura, J. Hamuro, T. Shirai. 1992. Somatically mutated IgG anti-DNA antibody clonally related to germ-line encoded IgM anti-DNA antibody. Eur. J. Immunol. 22:987.[Medline]
-
Kumada, T., W. A. Dittman, P. W. Majerus. 1987. A role for thrombomodulin in the pathogenesis of thrombin-induced thromboembolism in mice. Blood 71:728.[Abstract/Free Full Text]
-
Brisson, C., G. Archipoff, M. Hartmann, D. Hanau, A. Beretz, J. Freyssinet, J. Cazenave. 1992. Autoantibodies to thrombomodulin induce receptor-mediated endocytosis in human saphenous vein endothelial cells. Thromb. Haemost. 68:737.[Medline]
-
Vismara, A., P. L. Meroni, A. Tincani, E. N. Harris, W. Barcellini, A. Brucato, M. Khamashta, G. R. V. Hughes, C. Zanussi, G. Balestrineri. 1988. Relationship between anticardiolipin and antiendothelial cell antibodies in systemic lupus erythematosus. Clin. Exp. Immunol. 74:247.[Medline]
-
Nimpf, J., M. Bevers, P. H. H. Bomans, U. Till, H. Wurm, G. M. Kostner, R. F. A. Zawaal. 1986. Prothrombinase activity of human platelets inhibited by ß2-glycoprotein I. Biochim. Biophys. Acta 884:142.[Medline]
-
Galli, M., E. M. Bevers, P. Comfurius, T. Barbui, R. F. A. Zwaal. 1993. Effect of antiphospholipid antibodies on procoagulant activity of activated platelets and platelet-derived microvesicles. Br. J. Haematol. 83:466.[Medline]
-
Aoki, K., A. B. Dudkiewicz, E. Matsuura, M. Novotny, G. Kaberlein, N. Gleicher. 1995. Clinical significance of ß2-glycoprotein I-dependent anticardiolipin antibodies in the reproductive autoimmune failure syndrome: correlation with conventional antiphospholipid antibody detection systems. J. Obstet. Gynecol. 172:926.
-
Balestrieri, G., A. Tincal, L. Spatola, F. Allegri, E. Prati, R. Cattaneo, G. Valesini, N. D. Papa, P. Meroni. 1995. Anti-beta2-glycoprotein I antibodies: marker of antiphospholipid syndrome?. Lupus 4:122.[Abstract/Free Full Text]
-
Matsuura, E., Y. Igarashi, M. Fujimoto, K. Ichikawa, T. Suzuki, T. Sumida, T. Yasuda, T. Koike. 1992. Heterogeneity of anticardiolipin antibodies defined by the anticardiolipin cofactor. J. Immunol. 148:388.[Abstract]
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Abstract
Introduction
