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Evaluating systemic lupus erythematosus disease activity using molecular markers of hemostasis.

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ARTHRITIS & RHEUMATISM
Vol. 39, No. 2, February 1%. pp 287-291
Q 19%, American College of Rheumatology
287
EVALUATING SYSTEMIC LUPUS ERYTHEMATOSUS DISEASE
ACTIVITY USING MOLECULAR MARKERS OF HEMOSTASIS
MASAYUKI INOH, MICHIAKI TOKUDA, HIROY UKI KIUCHI,
NORIYUKI KURATA, and JIRO TAKAHARA
Objective. To determine the usefulness of measuring sensitive markers of the coagulation-fibrinolysis
system (i.e., thrombin-antithrombin 111 complex
[TAT], D dimer fragments [DD], and plasmin-q
plasmin inhibitor complex [PIC]) for evaluating disease
activity in patients with systemic lupus erythematosus
(SLE).
Methods. We studied 57 SLE patients. Plasma
concentrations of DD were measured by latex agglutination using monoclonal antibodies; TAT and PIC were
determined by sandwich enzyme-linked immunosorbent
assay. Disease activity was determined by using the SLE
Disease Activity Index (SLEDAI).
Results. Levels of TAT, DD, and PIC were higher
in SLE patients than in healthy controls ( P < 0.05).
Levels of TAT and DD showed good correlations with
SLEDAI scores (for TAT r = 0.66, P < 0.001; for DD
r = 0.50, P < 0.001). Elevated levels of TAT, DD, and
PIC were decreased following treatment.
Conclusion. These results strongly suggest that
measurement of molecular markers of hemostasis is useful
for evaluating disease activity in patients with SLE.
Systemic lupus erythematosus (SLE) is an inflammatory disease that affects many organs including
the skin, joints, and kidneys. The morbid condition of
SLE is generally known t o consist mainly of angiitis
induced by immune complexes (1). Precipitation of
immune complexes causes vascular endothelial disorders both directly and indirectly (2), probably resulting
in a coagulation-fibrinolytic abnormality (3).
Recently developed molecular markers, such as
-
Masayuki Inoh, MD. Michiaki Tokuda, MD, Hiroyuki
Kiuchi, MT, Noriyuki Kurata, MD, Jiro Takahara, MD: Kagawa
Medical School, Kagawa, Japan.
Address reprint requests to Masayuki Inoh, MD, First
Department of Internal Medicine, Kagawa Medical School, 1750- 1
Ikenobe, Miki-cho, Kita-gun. Kagawa, 761-07 Japan.
Submitted for publication June 16, 1995; accepted in revised form August 19, 1995.
thrombin-antithrombin 111 complex (TAT) (4), crosslinked fibrin degradation fragment D dimer (DD) (3,
and plasmin-q-plasmin inhibitor complex (PIC) (6),
can reflect more minute changes in the coagulationfibrinolysis system than other parameters, such as
prothrombin time (FT), activated partial thromboplastin time (APTT), and fibrin or fibrinogen degradation
product (FDP). We therefore measured these new
parameters in SLE patients to investigate their correlation with the disease activity.
PATIENTS AND METHODS
Patients. This study included 57 female patients with
SLE (mean ? SD age 30.61 2 9.89 years) who met the 1982
classification criteria proposed by the American College of
Rheumatology (formerly, the American Rheumatism Association) (7). The patients had been treated as outpatients and
inpatients at Kagawa Medical College Hospital between
June 1 and October 31, 1994.
The study excluded patients who were taking oral
contraceptives (8), were positive for antibody to cardiolipin
and lupus anticoagulants (%I l ) , had diabetes mellitus (12),
had hyperlipemia (13) (serum cholesterol >230 mg/dl), and
had renal insufficiency (14) (creatinine clearance 4 0 ml/
minute). It is well known that coagulation-fibrinolytic abnormalities already exist in these patients.
SLE disease activity was scored using the SLE
Disease Activity Index (SLEDAI) (15).
Methods. TAT and PIC were determined by sandwich
enzyme-linked immunosorbent assay (Enzygnost-TAT and
Enzygnost-PAP micro; Behringwerke AG, Marburg, Germany); DD was determined by latex agglutination (LPIA-DD
dimer; Jatron, Kanda, Tokyo, Japan). APTT was determined by a modification of Langdell and coworkers' method
(16); Sysmex APTT (Sysmex, Akasaka, Tokyo, Japan)
served as partial thromboplastin. PT was determined by the
1-stage method of Quick et a1 (17); Sysmex PT served as
tissue thromboplastin. FDP was determined by latex agglutination (LPIA-FDP; Jatron).
Twenty healthy adults (mean 2 SD age 30.5 2 5.07
years) served as controls. The normal TAT level was 0.95 2
0.863 ng/ml, the DD level was 0.22 2 0.146 ng/ml, the PIC
level was 256.15 119.084 &ml, the AFTT was 30.9 5 0.7
*
288
INOH ET AL
*
t
30:
5:
1400
25-
16001
1200
4-
.*
m
1800
6-
35-
t
n
m
.
SLE
Control
SLE
Control
SLE
Control
Figure 1. Distribution of the 3 hemostatic parameters, thrombin-antithrombin 111 complex
(TAT), D dimer fragments (DD), and plasmin-a,-plasmin inhibitor complex (PIC), in patients
with systemic lupus erythematosus (SLE) and in healthy control subjects. Bars show the mean.
* = P < 0.005; t
*
=
P < 0.05.
seconds, the F
T was 10.8 1.2 seconds, and the FDP level
was 3.1 -c 0.7 &ml (all mean SD). The mean + 2SD was
regarded as the upper limit of normal for the individual
indices; all levels above that were regarded as abnormal.
Statistical analysis. We used the statistical software
package StatView 54.02. The chi-square test was used to
compare differences in proportions. The paired r-test was
used for the comparison of 2 related groups, and the unpaired t-test for analysis of independent groups. Spearman's
*
rank correlation coefficient was used to determine the correlation between 2 variables. Results are expressed as the
mean 2 SD. Differences (2-sided) at P < 0.05 were regarded
as significant.
RESULTS
Plasma levels of TAT, DD, and PIC in the SLE
patients were significantly higher than those in the
351
30
5'
25
4-
.
p20-
W
m
400
0 4
SLEDAI score (points)
8 16 19 23
SLEDAI score (points)
0
0
4
8
16 19 23
SLEDAI score (points)
Figure 2. Correlation between hemostatic parameters, thrombin-antithrombin I11 complex (TAT), D dimer fragments (DD), and plasmin-a,-plasmin inhibitor complex (PIC),
and the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score.
289
MOLECULAR MARKERS OF HEMOSTASIS IN SLE
Table 1.
scores*
Levels of hemostatic markers in all SLE patients, categorized according to SLEDAI
S L E patients
_______~_______
Variable
TAT,ng/ml
DD, ng/ml
PIC, &ml
APTT, seconds
PT, seconds
FDP. d m l
0.95 t
0.22 2
256.15 2
30.9 2
10.8 k
3.1 ?
~~
Group 1, SLEDAI <10
(n = 40)
Healthy controls
(n = 20)
Group 2, SLEDAI 210
(n = 17)
*
2.77 1.2951
0.74 2 0.8519
351.97 rf: 137.4399:
31.1 ? 1.9#
11.1 2 0.8#
3.4 2 1.0#
0.863
0.146
119.084
2.6
1.2
0.7
11.77 2 8.57tt
3.06 ? 5.1197
516.85 2 449.31§(1
30.8 2 2.3#
10.9 2 0.9#
3.3 f 1.1#
* Values are the mean ? SD. S L E = systemic lupus erythematosus; SLEDAI = SLE Disease Activity
Index; TAT = thrombin-antithrombin Ill complex; DD = cross-linked fibrin degradation fragment D
dimer; PIC = plasmin-a,-plasmin inhibitor complex; APTT = activated partial thromboplastin time;
PT = prothrombin time; FDP = fibrin or fibrinogen degradation product.
t P < 0.001 versus healthy controls.
$ P < O.OOO1 versus group 1.
9 P < 0.05 versus healthy controls.
T P < 0.05 versus group 1.
# Not significant versus healthy controls.
*
normal control subjects (5.28 _t 6.130, 1.39 2.935,
and 394.31 & 257.630, respectively, mean +- SD)
(Figure 1). Distribution of the SLEDAI scores ranged
from 0 to 25 points (maximum score 105). When the
correlation between the SLEDAI score and each of
the markers was investigated, there was a good correlation with TAT levels (r = 0.66, P < 0.001) as well as
with DD levels (r = 0.50, P < 0.001) (Figure 2).
Moreover, it was of considerable interest that the
TAT, the PIC, and the DD levels tended to show
marked increases as the SLEDAI exceeded 10 points.
When we grouped the patients according to
SLEDAI scores (group 1 <10 points [n = 401; group 2
210 points [n = 17]), the proportion of patients with
abnormal values for each parameter was considerably
higher in group 2 patients than in group 1 patients: for
TAT, 16 of 17 (94.1%)versus 18 of 40 (45.0%); for DD,
14 of 17 (82.4%) versus 18 of 40 (45.0%); and for PIC,
12 of 17 (70.6%) versus 3 of 40 (7.5%), respectively.
Furthermore, the levels of all indices were more
elevated in group 2 patients than in group 1 patients.
Conversely, the routine markers of the coagulationfibrinolysis system (AFTT, PT, and FDP) did not
show significant correlations with the disease activity
(Table 1).
Comparing the characteristics of patients in
Table 2. Characteristics of patients in groups 1 and 2*
SLEDAI score
Renal
0
4
8
12
16
Immunologic
0
2
4
Age, years
Total cholesterol, mg/dl
Serum creatinine, mddl
Group 1
Group 2
30
0
P
<O.OOOl
10
1
0
0
0
3
4
9
21
19
0
1
<0.001
32.28
?
3
13
10.476
1%.00 rf: 25.775
0.98
rf:
0.089
26.53 rf: 7.706
201.28 rf: 24.195
1.05 ? 0.298
0.467
0.463
0.572
* Systemic Lupus Erythematosus Disease Activity Index (SLEDAIbgenerated weighted index of 9
organ systems; for example, 4 for renal might be urinary casts, hematuria, proteinuria, pyuria, and 2
for immunologic might be low complement, increased DNA binding. Values are the number of patients
or the mean 2 SD.
INOH ET AL
290
1000:
8:
25-
900 :
7:
20 6:
"
=
E5:
PI
,
E
&15C
\
'
V
V
5 :
-z
1\ \
i 600:
.
V
u_
n.
3:
2:
0
500 :
400 :
300:
51:
1
700:
ol
\
84:
10-
800 :
200 :
100'
Before
After
Before
After
Before
After
Figure 3. Effect of treatment on hemostatic parameters, thrombin-antithrombin I11 complex
(TAT), D dimer fragments (DD),
and plasmin-a,-plasmin inhibitor complex (PIC). * = P <
0.005; t = P < 0.05.
groups 1 and 2, there were no significant differences
between the 2 groups in terms of age, serum creatinine
level, or total cholesterol level (Table 2). Of particular
interest was the fact that group 2 patients had a
substantially higher frequency of abnormal urinalysis
results as well as immunologic abnormalities. However, patients with critical organ manifestations in the
central nervous system, with synovitis, and with vasculitis were absent from both groups.
Correlations between changes in the coagulationfibrinolysis system and those in the SLEDAI score
after treatment were also investigated in 11 of 17
patients in group 2. In all patients, steroid pulse
therapy using 1,000 mg of methylprednisolone (or 100
mg of betarnethasone) over 3 consecutive days was
repeated twice at 4-day intervals. It was noteworthy
that each level of TAT, DD, and PIC was significantly
decreased after treatment in all patients except 1 who
showed abnormal values for the individual indices
(Figure 3).
DISCUSSION
In recent years, methods have been developed
for measuring the levels of thrombin and plasmin in
complexed form with their inhibitors. Importantly,
they have been shown to reflect changes in coagulationfibrinolysis more sensitively than the markers routinely
used (APTT, PT, and FDP) (18).
In SLE, the precipitation of immune complexes
has been observed along the vascular endothelium,
and has been thought to cause endothelial damage
both directly and indirectly (19). It seemed natural that
vascular endothelial damage might lead to some turbulence in the coagulation-fibrinolysis system (20).
Based on these assumptions, we determined the level
of TAT in SLE patients as a sensitive marker of the
coagulation system, as well as the levels of DD and
PIC as markers of the fibrinolysis system. In addition,
we investigated correlations between these molecular
markers and SLE disease activity.
The levels of both TAT and DD were found to
be markedly higher in SLE patients than in healthy
adults. This observation was consistent with the
above-mentioned assumption that the coagulationfibrinolysis system might be activated in this disease.
The compelling evidence in its favor was that there
was a good correlation between the level of TAT and
the SLEDAI score. The level of DD also correlated
well with this index. Furthermore, it should also be
noted that levels of TAT, DD, and PIC were substantially higher in all group 2 patients, who had scores of
910 on the SLEDAI, than in group 1 patients, who
had SLEDAI scores of 40.
While the levels of these molecular markers are
influenced to various extents by several factors, such
as the presence of diabetes mellitus, hyperlipemia, or
renal dysfunction (12-14), our study patients clearly
lacked all of these features. Moreover, comparison of
MOLECULAR MARKERS OF HEMOSTASIS IN SLE
characteristics between the 2 patient groups showed
no significant differences in terms of age, cholesterol
level, or creatinine level. Notably, all levels of TAT,
DD, and PIC substantially decreased along with a
decline in the SLEDAI score after corticosteroid therapy in patients in group 2. All these observations
support our assumption that the degree of abnormality
in the coagulation-fibrinolysis system might reflect the
degree of SLE disease activity.
Whereas we could not clarify whether corticosteroids directly influenced the levels of these markers, the absence of a “rebound” increase in these
parameters after the prednisolone dosage was tapered
off might be evidence against this possibility.
It was noteworthy that the frequency of abnormal results on urinalyses as well as the frequency of
immunologic abnormalities was higher in group 2 than
in group I patients. On the other hand, patients in
neither group had critical organ manifestations, including the absence of central nervous system involvement, serositis, and vasculitis with clinically apparent
signs, as defined in the original description of the
SLEDAI. These observations raised the possibility
that lupus nephritis resulting from immunologic
abnormalities might play a pivotal role in causing a
coagulation-fibrinolytic disorder. However, this possibility requires substantiation.
There is as yet no direct evidence for the mechanism of vascular damage and the resultant coagulationfibnnolytic abnormalities seen in SLE. It is possible
that precipitation of immune complexes in the vascular
endothelium may be followed by increased expression
of tissue factor, decreased production of thrombomoddin, and enhanced secretion of plasminogen activation inhibitor 1. These sequential events which take
place on the surface of damaged endothelium may
eventually lead to activation of coagulation and secondary fibrinoiysis. However, more in vitro data are needed
before these questions can be fully answered ( 1 9 ~ 0 ) .
In conclusion, our findings strongly suggest that
the coagulation-fibrinolysis system is perturbed as the
activity of SLE increases. We believe that measuring
these sensitive markers, including TAT, PIC, and DD
levels, might serve as a powerful tool for assessing the
disease activity of SLE.
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