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*
Division of Rheumatology, Department of Medicine, University of California, Los Angeles, CA 90095; and Departments of
Medicine and
Pathology, University of California at San Diego, La Jolla, CA 92093
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2 glycoprotein I and prothrombin (PT). Recently,
anti-PT Ab (aPT) were found to be associated with thrombosis by
some investigators, although this is not confirmed by others.
Considering that aPT are heterogeneous in patients and that PT is
converted into thrombin, we hypothesize that certain aPT in patients
may bind to thrombin, and that some of such anti-thrombin Ab may
interfere with thrombin-antithrombin (AT) interaction and thus reduce
the AT inactivation of thrombin. To test this hypothesis, we searched
for anti-thrombin Ab in APS patients and then studied those found for
their effects on the AT inactivation of thrombin. The results revealed
that most, but not all, aPT-positive patient plasma samples contained
anti-thrombin Ab. To study the functional significance of these Ab, we
identified six patient-derived mAb that bound to both PT and thrombin.
Of these mAb, three could reduce the AT inactivation of thrombin,
whereas others had minimal effect. These findings indicate that some
aPT in patients react with thrombin, and that some of such
anti-thrombin Ab could inhibit feedback regulation of thrombin. Because
the latter anti-thrombin Ab are likely to promote clotting, it will be
important to develop specific assays for such Ab and study their roles
in thrombosis in APS patients. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Accumulated studies show that aPL represent a heterogeneous group
of immunologically and functionally distinct Ab that recognize various
PL, PL-binding plasma proteins, and/or PL-protein complexes. The
involved plasma proteins include plasma protein
2 glycoprotein-1
(2GPI), prothrombin (PT), protein C, and
protein S (9, 10, 11, 12, 13, 14). To date, the Ab against
2GPI and its complexes with cardiolipin
probably account for most of the positive findings on tests for aCL in
APS (15, 16), whereas Ab against PT and
2GPI are responsible for the majority of the
LAC activity (11, 17, 18, 19).
Recently, increasing attention has been paid to anti-PT Ab (aPT) and their roles in thrombosis in APS patients (18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29). The prevalence of aPT in patients varies among different studies, ranging from 30 to 60% in APS patients when tested by ELISA using immobilized human PT on activated polyvinyl chloride plates (20, 23, 25). However, aPT were found to be associated with thrombosis (21, 25, 28), although this is not confirmed by other investigators (23). These conflicting data may reflect the heterogeneity of aPT present in individual patient sera and different sets of these autoantibodies in clinically diverse patient populations in different studies.
To understand the functional and pathogenic property of aPT, Rao and coworkers (11, 18) affinity purified IgG aPT and found that the purified Ab bound to immobilized phosphatidylserine in the presence of Ca2+ and PT. These results suggested that IgG aPT cross-linked PT molecules, and thus increased the valence of interactions between PT and phosphatidylserine. Subsequently, those investigators showed that IgG purified from a LAC-positive plasma sample (designated LAC IgG; from a patient with hypoprothrombinemia) enhanced the binding of PT to HUVEC and increased conversion of PT to thrombin on the surface of HUVEC (22).
Thrombin is a key effector enzyme in the coagulation cascade. It converts fibrinogen to fibrin, leading to the formation of fibrin clots. It also feedback amplifies the cascade by activating factors V and VIII, which in turn, enhance conversion of PT to thrombin (30). Therefore, once thrombin is generated in vivo, it is tightly regulated by antithrombin (AT) that binds to thrombin in the presence of heparin-like glycosaminoglycans on the endothelial cell (EC) surface and inactivates the enzyme irreversibly (30, 31, 32). Considering that thrombin is derived from the zymogen PT, it is conceivable that some aPT may bind to thrombin at a site where thrombin interacts with AT, and therefore inhibit AT inactivation of thrombin. In this study, we report the detection of Ab against thrombin in APS patients and the inhibitory effects of three patient-derived IgG monoclonal anti-thrombin Ab on the AT inactivation of thrombin. These findings define a novel anti-thrombin autoantibody in APS and they show that such Ab may interfere with negative feedback regulation of thrombin in circulation, and thus contribute to thrombosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasma samples were obtained from 13 patients and 5 normal controls at University of California, Medical Center (Los Angeles, CA) and University of California Medical Center (San Diego, CA). All 13 APS patients satisfy the Sapporo classification criteria for definite APS (8). The diagnosis of APS was confirmed by a medical record review.
Of the 13 patients, there were 8 primary APS (62%) and 5 secondary APS (38%). Three with secondary APS had SLE, one had SLE and Sjogren’s syndrome, and the fifth had undifferentiated connective tissue disease. All were positive for LAC and 10 were positive for aCL. For clinical manifestations, all had thrombosis (5 arterial and 10 venous), and two of the eight females had two or more unexplained fetal losses. The ethnicity and gender for primary APS were six Caucasians and two Hispanics, and six females and two males. All of the secondary APS were Caucasians with a gender ratio of two females/three males. The average age (in years) at diagnosis for primary APS was 43.3 (range of 17–63) and that for secondary APS was 26.8 (range of 12–41).
Five healthy donors were recruited as normal controls and were designated N1 to N5. Their ethnicity was two Asians and three Caucasians, and gender was three females and two males. Their average age at the time of donation of their sample was 33.6 years (range of 21–53).
ELISA for Ab against PT and thrombin
The ELISA for aPT was done as described previously
(33). Briefly, high-binding ELISA plates (Costar,
Cambridge, MA) were coated with 10 µg/ml of human PT (Enzyme Research
Laboratories, South Bend, IN) in TBS (0.05 M Tris-HCl and 0.15 M NaCl,
pH 7.5). After incubating overnight at 4°C, plates were blocked with
TBS containing 0.3% gelatin. Then test plasma samples (1/100 dilution)
or mAb (1.0 µg/ml) in TBS/0.1% gelatin were distributed to wells in
duplicate and were incubated for 1.5 h at room temperature. The
IS6 monoclonal IgG aPT was used as a positive control and as a
reference aPT in all assays (33). After washing with TBS,
bound human IgG and total Ig (denoted as Igs) were detected,
respectively, with HRP-conjugated goat anti-human IgG (
-chain
specific; BioSource International, Camarillo, CA), anti-human Igs
(all isotypes; Jackson ImmunoResearch Laboratories, West Grove, PA),
and peroxidase substrate tetramethylbenzidine (Kirkegaard &
Perry Laboratories, Gaithersburg, MD). The ELISA for anti-thrombin Ab
was done similarly except that plates were coated with 10 µg/ml of
human 
-thrombin (Hematologic Technologies, Essex Junction, VT). Of
note, the IS6 monoclonal aPT was found to react with thrombin during
the initial study of anti-thrombin Ab (see Results) and thus
was used as a reference Ab in all subsequent anti-thrombin Ab ELISA.
The results were expressed in abstract units (AU) with 1 AU equivalent
to the OD of the IS6 mAb at 2 µg/ml.
A competitive inhibition assay was used to study the binding properties of selected mAb to PT and thrombin. Briefly, each mAb (1.5 µg/ml) was preincubated for 1.5 h with various concentrations of either PT or thrombin. Then, the mixture was distributed to the PT- or thrombin-coated wells in duplicate. After incubation, bound IgG was measured. The inhibition data of each mAb were used to calculate its relative Kd toward PT and thrombin (34).
Functional assay for thrombin activity and the AT inactivation of thrombin
The effects of thrombin-reactive mAb on thrombin activity were
studied by mixing 25 µl of human -thrombin (80 nM) separately with
25 µl of a test mAb (128 µg/ml), normal human IgG, or an isotype
control monoclonal IgG3 for 1 h at room temperature. Then, to each
reaction mixture was added 200 µl of the thrombin chromogenic
substrate S-2238
(H-D-Phe-Pip-Arg-p-nitroanilide, 150 µM;
Chromogenix, Molndal, Sweden). After 1 min, generation of
p-nitroaniline was monitored by measuring OD at 405 nm. The
activity of thrombin was determined as the rate of hydrolysis of S-2238
from the linear range of absorbance at 405 nm with time.
The effects of monoclonal anti-thrombin Ab on the AT inactivation of
thrombin were studied in a functional assay for the thrombin activity
in the presence of AT and heparin, according to Bock et al.
(35) with minor modifications. In particular, human AT
(Enzyme Research Laboratories) was used at a concentration that was at
least 10-fold higher than that of human -thrombin, and experiments
were conducted in 50 mM HEPES, 125 mM NaCl, 1 mM EDTA, and 0.1%
polyethylene glycol 8000, pH 7.4, at 25°C in microtiter plates. The
assay was initiated by incubating 25 µl of thrombin (80 nM)
separately with 25 µl of a test mAb (128 µg/ml), normal human IgG,
or the isotype control monoclonal IgG3 in duplicate for 1 h at
room temperature. Then, to each reaction mixture was added 50 µl of
AT (400 nM or the indicated concentrations) in the buffer containing
heparin, resulting in a final heparin concentration of 0.1 USP unit/ml
(U/ml) or the indicated concentrations. Subsequently, 200 µl of the
chromogenic substrate S-2238 was added, and OD at 405 nm was measured
at 1 min unless stated otherwise. The final concentrations of thrombin,
AT, and IgG were 6.7 nM, 67 nM, and 10.7 µg/ml, respectively. The
percentage of thrombin inactivation by AT was calculated as (1 -
(the residual thrombin activity with AT)/(the initial thrombin activity
without AT)) x 100%.
For the comparative analysis of AT inactivation of human -thrombin
and -thrombin (Hematologic Technologies), the experiments were done
similarly as the above, except that either one of the two above
thrombin variants was used in each functional assay of AT inactivation
of thrombin.
Statistical analysis
The mean AU plus 3 SD of the five normal controls was used as the cutoff, and the plasma samples with AU values consistently higher than the cutoff in two separate experiments were considered positive. Differences in the test Ab-induced inhibition of thrombin activity or the thrombin inactivation by AT were analyzed using paired ANOVA followed by the Bonferroni multiple comparison test. Values of p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test the hypothesis that some aPT in patients may bind to
thrombin, we developed an ELISA for anti-thrombin Ab and used the assay
to analyze plasma samples from 13 APS patients (designated P1 to P13)
and 5 healthy normal controls (designated N1 to N5); multiple plasma
samples from 3 patients were available and were all analyzed. All
samples were analyzed at the 1/100 dilution. The results showed that
using mean plus 3 SD of the normal controls as the cutoff,
anti-thrombin Ab were detected in 10 of 13 patients, and IgG
anti-thrombin Ab were detected in 3 of 13 patients (Fig. 1
, A and B).
Patient P1 had the highest titer of anti-thrombin Ab, but had no
detectable IgG anti-thrombin Ab. In contrast, patient P4 had the
highest titer of IgG anti-thrombin Ab, but only a medium titer of
anti-thrombin Ab. Of the three patients with multiple plasma samples,
their anti-thrombin Ab titers fluctuated over time, which paralleled
the previously reported fluctuation of aCL titers in APS patients.
Specifically, only one of three samples from patient P1 had
anti-thrombin Ab, and only one of three samples from patient P6 had IgG
anti-thrombin Ab.
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, C and D, aPT were found in 10 of 13 patients and
IgG aPT was found in 5 of 13 patients. Except for the P2c plasma
sample, there was a good quantitative correlation between aPT and IgG
aPT titers; noticeably, patient P5 had abundant aPT and IgG aPT. Of the
three patients with multiple plasma samples, their aPT and IgG aPT
titers fluctuated over time, similar to the above fluctuation in
anti-thrombin Ab titers. When the presence of aPT and anti-thrombin Ab in patient samples were compared, 11 of 13 aPT-positive patient plasma samples had anti-thrombin Ab, and three of the seven IgG aPT-positive samples had IgG anti-thrombin Ab. These data suggest that many aPT (including the IgG isotype) may also react with thrombin, or that two different Ab for each Ag often occur together, similar to autoantibodies against Sjogren syndrome Ag A and B in SLE (1). In contrast, sample P1b had high titers of anti-thrombin Ab, but no aPT, indicating that some anti-thrombin Ab in APS patients do not react with PT and that anti-thrombin Ab are heterogeneous in patients.
Identification of monoclonal aPT and anti-thrombin Ab
Because anti-thrombin Ab are heterogeneous in patients, it
would be important to obtain monoclonal anti-thrombin Ab and use such
mAb to study the functional significance of anti-thrombin Ab in APS.
Accordingly, we searched for patient-derived monoclonal aPT and
anti-thrombin Ab. Previously, we found that the IS6 monoclonal aPT
cross-reacts with cardiolipin (33), and that the P11
monoclonal aPT F(ab')2 (isolated by panning a
phage display Ab library on PT) reacts with both PT and
2GPI, with relative
Kd values of 3.2 x
10-6 M for PT vs 1.6 x
10-6 M for 2GPI
(36). These data raised the possibility that some of our
monoclonal aCL/anti-2GPI Ab might
reciprocally cross-react with PT. Therefore, we screened seven
patient-derived monoclonal IgG aCL (37) for binding to PT;
IS1 and IS2 are IgG1, and the other mAb are IgG3. The results showed
that five of seven mAb reacted with PT (Fig. 2A). Moreover, the aPT
activities of CL1, CL15, and IS3 were even better than that of the IS6
monoclonal IgG aPT (33) at the same concentration.
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B, all six aPT (CL1, CL15,
CL24, IS3, IS4, and IS6) bind to thrombin. Interestingly, their
relative binding pattern to thrombin is similar to their binding
to PT. Effects of anti-thrombin mAb on thrombin activity and thrombin inactivation by AT
To study the effects of anti-thrombin mAb on thrombin
activity, thrombin was incubated separately with test mAb for 1 h,
and then the thrombin chromogenic substrate activity was assessed. As
can be seen in Fig. 3, CL15 and IS3
slightly reduced thrombin activity, whereas the other four
anti-thrombin mAb did not affect thrombin activity.
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Under these conditions, AT inactivated 88% of thrombin activity
(data not shown), and the degrees of thrombin inactivation by AT were
not changed by the presence of either polyclonal human IgG or a
monoclonal human IgG3 isotype control (Fig. 4A). In contrast, CL24 and
CL15 reduced the degrees of thrombin inactivation to 61% and 71%,
respectively. CL1 showed a small but significant inhibitory effect on
AT inactivation of thrombin, reducing thrombin inactivation to 78%.
The remaining three other monoclonal anti-thrombin Ab did not affect AT
inactivation of thrombin.
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B, accumulated substrate conversion in the presence of CL24
increases dramatically over those in the absence of CL24 in 5
min.
Because the plasma concentration of AT is 2 µM
(38) and the above experiments were done with the final
concentration of AT at 67 nM, we studied the effects of CL24 on
thrombin inactivation in the presence of AT from 67 to 533 nM, due to
the prohibitory cost of AT. The results showed that the CL24-mediated
reduction in AT inactivation of thrombin remained constant over the
tested range of AT concentrations (Fig. 4C). Subsequently,
we studied the effects of CL24 and CL15 over a range of heparin
concentrations that had been used by other investigators
(39). The results show that CL24 and CL15 significantly
reduced AT inactivation of thrombin in the presence of heparin
concentrations from 0.025 to 0.2 U/ml (final concentrations in the
thrombin and AT mixture), and the Ab-mediated reduction disappeared
when heparin concentration reached 0.4 U/ml (Fig. 4D). A
maximal reduction in the thrombin inactivation by AT occurred at 0.05
U/ml of heparin, reducing the inactivation of thrombin from 76% in the
presence of the control IgG3 to 30% in the presence of CL24 (Fig. 4D). In contrast, IS6 did not significantly affect the AT
inactivation of thrombin.
The binding properties of three chosen mAb to PT and thrombin
Based on the above data, three representative mAb were
chosen for analysis by competitive and cross inhibition. These were
CL24 (which inhibits AT inactivation of thrombin), IS3 (which inhibits
thrombin per se), and CL15 (which inhibits both thrombin per se and the
AT inactivation of thrombin). The results showed that soluble thrombin
is more effective than PT in inhibiting all three mAb binding to either
PT or thrombin (Fig. 5). Importantly, PT
could only inhibit binding of all tested mAb to the immobilized PT but
not thrombin. These results demonstrate that these three mAb are more
specific for thrombin than PT. Based on the thrombin inhibition data
for binding to thrombin, the relative Kd
values of these Ab to thrombin were calculated to be 7.5 x
10-6, 1.7 x 10-6,
and 7.4 x 10-6 M for CL15, CL24, and IS3,
respectively.
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As a first step to define the epitope recognized by CL24, we
comparatively analyzed the effects of CL24 on -thrombin and
-thrombin, a proteolytic variant of -thrombin with impaired
exosite I. The exosites are patches of positively charged amino acid
residues on the thrombin surface that interact with the thrombin
substrate, inhibitor, and modifier (i.e., thrombomodulin; Ref.
40). As can be seen in Fig. 6, AT alone inactivated 67–69% of
-thrombin activity in the presence of either polyclonal human IgG or
a monoclonal human IgG3 isotype control. Under the same conditions,
CL24 reduced the degree of -thrombin inactivation to 40%,
equivalent to a 42% reduction (based on the 69% for the monoclonal
human IgG3 isotype control) in AT inactivation of thrombin.
Importantly, this CL24-induced reduction on AT inactivation of
-thrombin is similar to the 42% reduction of AT inactivation of the
-thrombin by CL24 (decreasing from 88% AT inactivation of
-thrombin to 51%). The results suggest that CL24 is unlikely to
react with thrombin at its exosite I and/or the surrounding
regions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A and B), and several patient-derived IgG mAb
bound to thrombin (Fig. 2B). Importantly, of the six
monoclonal anti-thrombin mAb, CL15 and IS3 inhibited thrombin activity
per se (Fig. 3), whereas CL1, CL15, and CL24 significantly reduced the
AT inactivation of thrombin in the presence of heparin (Fig. 4A), and CL24 at 10.7 µg/ml could inhibit AT inactivation
of thrombin in the presence of AT at 533 nM (Fig. 4C). Of
note, the human plasma concentrations of AT and IgG are 2 µM and 10
mg/ml, respectively (38). Moreover, although the reduction
of AT inactivation was only moderate, the effect of such an Ab on
cumulative thrombin cleavage products increases rapidly over time (Fig. 4B). In addition, the binding of three functionally
representative mAb (i.e., CL15, CL24, and IS3) to PT and thrombin was
inhibited more effectively by soluble thrombin than by PT (Fig. 5),
indicating that these three mAb are more specific for thrombin than PT.
Combined, these data show that some anti-thrombin Ab in APS patients
(like CL24 at 
0.1% of the plasma IgG concentration) could interfere
with the AT inactivation of thrombin, thus allowing for prolonged
coagulation in blood once clotting is initiated in patients carrying
such autoantibodies. Obviously, prolonged and unchecked thrombin
activity is most likely to promote and/or sustain thrombosis.
Fig. 4D shows that CL24-mediated inhibition of AT
inactivation of thrombin was decreased by increasing concentrations of
heparin. As noted earlier, the addition of heparin is to approximate
the in vivo inactivation of thrombin by AT, which often binds
toanticoagulantly active heparan sulfate proteoglycans on the
vascular endothelium (31, 41). During the in vitro
heparin-dependent AT inactivation of thrombin, heparin binds
simultaneously to thrombin and AT, and thus bridges thrombin and AT to
form a trimolecular complex of heparin-thrombin-AT, leading to a
several thousand-fold increase in the rate of AT
inactivation of thrombin (42). In this context, the above
data suggest that CL24 may bind to thrombin’s heparin binding site
(the exosite II) and thus interfere with the binding of heparin to
thrombin, and this effect is overcome by increased concentrations of
heparin, which could compete for the same binding sites. This
contention is consistent with the finding that CL24 also inhibited the
AT inactivation of -thrombin, which contains the intact exosite II
but impaired exosite I (Fig. 6).
Similar to the in vitro heparin-mediated enhancement of AT inactivation of thrombin, it is thought that in vivo acceleration of AT inactivation of thrombin occurs through the binding of AT and thrombin to the anticoagulantly active endothelial glycosaminoglycans (41). Although the effective activity of anticoagulantly active heparan sulfate proteoglycans on the EC surface is unknown, it can be speculated that in some in vivo circumstances, anti-thrombin Ab similar to CL24 may interfere with the binding of thrombin to these heparan sulfates and may therefore inhibit the formation of thrombin-heparan sulfate-AT complexes and the accelerated AT inactivation of thrombin.
Of the above three likely prothrombotic anti-thrombin Ab, CL15 also
inhibits thrombin activity (Fig. 3). In contrast, IS3 did not reduce AT
inactivation of thrombin, but did inhibit thrombin activity per se
(Fig. 3). The observed inhibitory activity of these two anti-thrombin
mAb was similar to a previously reported monoclonal IgG anti-thrombin
Ab that apparently caused severe bleeding in a patient with monoclonal
gammopathy (43). This latter mAb did not bind to PT, and
thus was similar to the anti-thrombin Ab observed in plasma sample P1b
(Fig. 1).
In light of patient-derived anti-thrombin mAb with different (and
even opposite) functional activities, it would be fruitless to assess
the clinical significance of all anti-thrombin Ab in APS by association
studies of the presence of anti-thrombin Ab to APS, or to study the
functional activities of affinity-purified polyclonal anti-thrombin Ab
from patients. Instead, it will be first necessary to delineate the
thrombin epitopes recognized by various anti-thrombin Ab with different
functional activities such as CL24 (which inhibits AT inactivation of
thrombin), IS3 (which inhibits thrombin activity), and IS4 (which binds
to thrombin but neither inhibits thrombin activity nor interferes with
AT inactivation of thrombin) (Figs. 3 and 4). If anti-thrombin Ab with
different functional activities are found to recognize different
thrombin epitopes, then specific assays for each type of anti-thrombin
Ab may be developed and used to study the roles of CL24-like
anti-thrombin Ab in thrombosis in APS patients, as well as the roles of
IS3-like Ab in Ab-mediated bleeding disorders.
It is intriguing that five of seven mAb generated by screening against
cardiolipin in the presence of bovine serum bind to PT and thrombin. Of
these five mAb, IS3, IS4, CL1, and CL24 also react with
2GPI, the major autoantigen or cofactor for
autoantibodies detected by the conventional aCL ELISA
(37). Viewed as a whole, these data suggest that the
latter four mAb may recognize an epitope shared by PT and
2GPI, and that such an epitope may be
analogous to the one revealed by P11 monoclonal aPT
F(ab')2 isolated by panning a phage display Ab
library on PT (36). However, CL24, IS3, and IS4 differ in
their effects on thrombin activity and AT inactivation of thrombin. In
this context, the combined data may suggest that there is more than one
thrombin epitope that is shared among PT, thrombin, and
2GPI. Clearly, further experimentation to test
these hypotheses is warranted.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kwan-Ki Hwang, Medicine/Rheumatology, 167022, University of California, Los Angeles, CA 90095-1670. E-mail address: hwangkk{at}ucla.edu
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; aCL, anticardiolipin Ab; PL, phospholipid; aPL, anti-PL Ab; APS, antiphospholipid syndrome; PT, prothrombin; aPT, anti-PT Ab; AT, antithrombin; AU, abstract unit; 2GPI, 2 glycoprotein-1; EC, endothelial cell; LAC, lupus anticoagulant; PT, prothrombin.
Received for publication April 20, 2001. Accepted for publication October 12, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-glycoprotein I (apolipoprotein H). Proc. Natl. Acad. Sci. USA 87:4120.2-glycoprotein I. J. Clin. Invest. 90:1100.
2-glycoprotein I in the expression of lupus anticoagulant activity. Blood 83:2878.2-glycoprotein I-dependent antiphospholipid antibodies. Blood 86:617.2-glycoprotein I and antiprothrombin antibodies on the anticoagulant activity of activated protein C. Blood 91:1999.2-glycoprotein-1 and antiprothrombin antigen-binding fragments generated by phage display. J. Immunol. 163:4604.2GP1 antibodies from two patients with the anti-phospholipid syndrome reveals three species of antibodies. Br. J. Haematol. 105:102.[Medline]
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