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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 1999-2004
Differential Effects of Anti- 2-Glycoprotein I and Antiprothrombin
Antibodies on the Anticoagulant Activity of Activated Protein C
By
Monica Galli,
Luisa Ruggeri, and
Tiziano Barbui
From the Department of Haematology, Ospedali Riuniti, Bergamo, Italy.
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ABSTRACT |
Antiprothrombin and anti- 2-glycoprotein I (2-GPI) antibodies
belong to the family of antiphospholipid (APL) antibodies and represent
the phospholipid-dependent inhibitors of coagulation. They may be
distinguished by analyzing the coagulation profiles generated by the
comparison of the ratios of two coagulation tests, the Kaolin Clotting
Time (KCT) and the dilute Russell's Viper Venom Time (dRVVT), commonly
adopted for their diagnosis. The KCT profile is caused by
antiprothrombin antibodies, whereas anti-2-GPI antibodies are
responsible for the dRVVT coagulation profile. The presence of aPL
antibodies is frequently associated with acquired resistance to
activated Protein C (APC-R), but limited information is
available regarding the role of the different antibodies in its
development. We studied the time-course of activated Factor V (FVa)
generation and inactivation in the plasma of 42 patients with
well-defined phospholipid-dependent inhibitors of coagulation: 24 displayed the dRVVT coagulation profile, whereas the other 18 cases
showed the KCT profile. In normal pooled plasma, the peak
values of FVa (mean ± standard deviation, [SD]: 16.307 ± 4.372 U/mL) were reached in 4 to 5 minutes and an almost complete inactivation (0.088 ± 0.123 U/mL) was obtained within 20 minutes. At
this time point, values of residual FVa exceeding 2 SD the mean of
controls (0.344 U/mL) were considered abnormal. Patients belonging to
the KCT coagulation profile group reached the maximal amount of FVa in
plasma (22.740 ± 7.693 U/mL, P = not
significant v controls) within 4 to 5 minutes; at
20 minutes, the residual amount of FVa in plasma ranged from 0 to 1.09 U/mL (0.293 ± 0.298; P = .027), but it was found
abnormal in only six of the 18 cases. The time-course of FVa in plasma
of patients belonging to the dRVVT coagulation profile group differed
from that of normal controls in that the peak values (10.955 ± 5.092 U/mL) were reached at 10 minutes and the amount of residual FVa at 20 minutes ranged from 0.320 to 14.450 U/ml (2.544 ± 3.580 U/mL;
P = .0191 v normal controls and P = .0114 v KCT group patients). Twenty of the 24 patients belonging to
the dRVVT profile group had an abnormal inactivation of FVa
( 2 = 0.001 v KCT group patients).
History of venous thrombosis was experienced by 15 patients: an
abnormal rate of FVa inactivation was found in 11 of them (73%) versus
15 of the 27 cases without thrombosis (56%) (x2
= 0.2556). The effect of affinity-purified IgG
phospholipid-dependent inhibitors of coagulation on the time-course of
FVa generation and inactivation in normal plasma was also investigated.
Anti-2-GPI, but not antiprothrombin antibodies, hampered the
inactivation of FVa by endogenous APC, thus reproducing the behavior of
the original plasmas. This effect was strictly 2-GPI-dependent. In conclusion, our findings confirm that anti-2-GPI antibodies
identify patients with phospholipid-dependent inhibitors of coagulation at increased risk of thrombosis and suggest acquired APC-R as a
possible explanation of the pathogenesis of the thromboembolic events.
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INTRODUCTION |
ANTIPHOSPHOLIPID (APL) antibodies
represent a large group of autoantibodies that comprise, among the
others, two phospholipid-dependent inhibitors of coagulation:
anti-2-glycoprotein I (2-GPI)1-4 and antiprothrombin
antibodies.5 Their nature may be identified on the basis of
the distinctive coagulation profiles generated by the comparison of the
ratios of two in-house assays, the Kaolin Clotting Time (KCT) and the
diluted Russell's Viper Venom Time (dRVVT), thus avoiding the
purification and characterization of the antibodies.6
The clinical relevance of APL antibodies resides in their association
with arterial and venous thrombosis, recurrent miscarriages, and
thrombocytopenia, which defines the so-called Antiphospholipid Syndrome
(APS).7 Venous thrombosis is particularly frequent, as it
occurs in about 20% of the patients.7 APL antibodies have
been suggested to be involved in the development of the thromboembolic events and several hypotheses have been put forward. Among them, the
interference of APL antibodies with the anticoagulant activity of the
Protein C pathway has been studied by several investigators, who showed
that APL antibodies inhibit the inactivation of activated Factor V
(FVa) by activated Protein C (APC) on a phospholipid surface.8-12 The term "acquired" resistance to APC
(APC-R) has been recently coined to identify this
condition,13 which could explain, at least in part, the
increased risk of venous thromboembolism of patients with APL
antibodies. At present, it is unclear whether the "acquired"
APC-R is a common property of all APL antibodies or pertains to a few
antibody subsets. Therefore, in the present study, we have investigated
the time-course of FVa generation and subsequent inactivation by
endogenous APC in the plasma of a large group of patients with
well-characterized phospholipid-dependent inhibitors of coagulation.
The effect of affinity-purified anti-2-GPI and antiprothrombin
antibodies on the time-course of FVa generation and inactivation in
normal plasma was also studied.
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MATERIALS AND METHODS |
Patients.
Forty-two patients with APL antibodies were included in the present
study. They were 8 men and 34 women, aged 19 to 76 years (median, 41 years). Only patients with persistently positive antibodies were
enrolled. Patients with APL antibodies developed during or after an
infectious disease were excluded. APL antibodies were associated with
an underlying disease in 22 cases: systemic lupus erythematosus
(SLE)/SLE-like diseases (n = 9), other autoimmune diseases (n = 6;
vasculitis, autoimmune hemolytic anemia, rheumatoid arthritis, primary
adrenocortical deficiency and two cases of Basedow's disease), other
diseases (n = 7; thrombotic thrombocytopenic purpura, two cases of
non-Hodgkin's lymphoma, two cases of chronic liver disease, and two
cases of epilepsy). At the time of the study, seven patients were
thrombocytopenic (platelet count <150 × 109/L).
Eleven patients (32%) experienced at least two spontaneous, sequential
miscarriages. APL antibodies were idiopathic in the other 20 patients.
Clinical history was positive for thrombotic events in 21 patients
(50%): 15 of them experienced at least one deep vein thrombosis
and/or pulmonary embolism; peripheral arterial thrombosis,
ischemic stroke and/or transient ischemic attacks were
diagnosed in six cases. Diagnostic methods used for detecting thrombosis were ultrasonography or venography for deep vein thrombosis, radionuclide lung scanning or angiography for pulmonary embolism, arteriography for peripheral arterial occlusions, computerized tomography scan, magnetic resonance imaging, or angiography for cerebral thrombosis. The diagnosis of cerebral transient ischemic attack required a focal neurologic deficit resolved within 24 hours.
Thirteen patients were on oral anticoagulation at the time of the
study. Primary APS was diagnosed in 17 patients. Twenty normal,
apparently healthy, subjects represented the control group.
Diagnosis of the phospholipid-dependent inhibitors of coagulation.
Venous blood was collected in plastic tubes containing one tenth volume
of 3.8% sodium citrate and centrifuged at 2,500g for 20 minutes to obtain platelet-poor plasma. Plasma was divided in small
aliquots and stored at  70°C until use. The revised criteria proposed by the Scientific Standardization Committee
Subcommittee for Standardization of Lupus Anticoagulants were used for
the diagnosis of the phospholipid-dependent inhibitors of
coagulation.14 At the time of the study, all patients
satisfied these criteria. Each test was performed in duplicate on a
manual coagulometer and the mean values of the two determinations were
used.15 Ratios were calculated by dividing the coagulation
time of patients' plasma by that of normal pooled plasma.
Coagulation profiles were generated for each patient's
plasma6: when the ratio of the KCT exceeded that of the
dRVVT, the patient was allocated to the KCT coagulation profile group.
In the opposite case, the patient was allocated to the dRVVT
coagulation profile. We recently showed that the former profile is
associated with the presence of antiprothrombin antibodies, whereas the
latter one is caused by the presence of anti-2-GPI
antibodies.6 Both the KCT and the dRVVT were performed by
"in-house" techniques.
Measurement of anti-2-GPI
antibodies. IgG and IgM anti-2-GPI antibodies
were measured essentially according to the enzyme-linked immunosorbent
assay (ELISA) procedure described by Loizou et al16 for
anticardiolipin (aCL) antibodies. IgG and IgM were
expressed as G-antiphospholipid (GPL) and M-antiphospholipid
(MPL) units according to Harris et al,17 1 U
being equivalent to 1 µg of affinity-purified aCL antibodies/mL of
sample. Values exceeding 15 GPL or MPL U were considered abnormal.
IgG and IgM anti-2-GPI antibodies were also evaluated
for their binging to 2-GPI in solid phase.18 Results
were expressed as absorbance at 405 nm. Values exceeding 2 standard
deviation (SD) the mean of controls (ie, 283 and 121 m-optical density
[mOD] for IgG and IgM anti-2-GPI antibodies,
respectively) were considered abnormal.
Human 2-GPI, purified according to Polz,19 was a kind
gift of Dr E.M. Bevers (Dept. of Biochemistry, Cardiovascular Research Institute, Maastricht University, The Netherlands).
Detection of antiprothrombin antibodies.
The binding of IgG and IgM antibodies to human prothrombin in solid
phase and complexed to phosphatidylserine via calcium ions was
evaluated by ELISA, as previously described.20
Purification of IgGs.
Total IgG fractions were affinity-isolated from patients' and normal
pooled plasmas over Protein A-sepharose CL-4B (Pharmacia Fine, Uppsala,
Sweden). Affinity-purified IgG fractions containing high
levels of anti-2-GPI antibodies were prepared as previously described.2 The protein content of the preparations was
assayed according to Sedmak and Grossberg.21
Evaluation of anticoagulant activity of IgG immunoglobulins.
The anticoagulant activity of IgG was evaluated by dRVVT both in
normal human plasma and in 2-GPI-depleted plasma (prepared according to Galli et al20) as previously
described.20 IgG were evaluated also by diluted activated
partial thromboplastin time (aPTT) as follows: 50 µL of
plasma were mixed with 25 µL IgG and 25 µL Thrombofax (Ortho,
Raritan, NJ; 1:10 in Tris-buffered saline [TBS]). After
a 3-minute incubation at 37°C, 50 µL 25 mmol/L CaCl2 was added to
the mixture and the time to form the clot recorded. Each test was
performed in duplicate and the results expressed as the mean of the two
values. In the coagulation tests, total IgG preparations were used at
the concentration of 5 mg/mL and anti-2-GPI antibodies at the
concentration of 0.5 mg/mL.
Assessment of FVa generation and inactivation in plasma.
The generation and inactivation of FVa was analyzed in defibrinogenated
plasma essentially according to Marciniak and Romond.9 The
effect of total IgG and aCL antibodies was evaluated in a similar way,
on mixing 50 µL of normal pooled plasma with 25 µL of IgG instead
of TBS. Total IgG preparations were used at the concentration of 5 mg/mL and anti-2-GPI antibodies at the concentration of 0.5 mg/mL.
Some experiments were similarly performed using 2-GPI-depleted
human plasma.
Assessment of the R506Q (Factor V Leiden) mutation.
Genomic DNA was prepared from citrated blood by standard procedures.
Mutant Factor V was detected by amplification of the Factor V gene by
polymerase chain reaction (PCR) and digestion of the fragment with
Mnl1.22 None of the patients with APL antibodies enrolled
in the study were found to be carriers of the mutation. The plasmas of
six carriers (heterozygous, n = 5; homozygous, n = 1) were selected for
studying the time-course of FVa generation and inactivation.
Measurement of Protein C and Protein S.
Antigenic levels of Protein C and Protein S were measured by
Asseraplate (Stago, Asnieres, France), according to the manufacturer's instructions. Their function was assessed by Protein C Reagent (Istituto Behring, Scoppito, Italy) and by Protein S
Clotting-Test (Stago), according to the manufacturer's instructions.
They were found normal in all patients who were not receiving oral
anticoagulant treatment at the time of the study.
Statistical analysis.
The 2 test and the Student's t-test for
unpaired data were used: a P value < .05 was considered
statistically significant.
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RESULTS |
Characterization of the coagulation profiles of patients with
phospholipid-dependent inhibitors of coagulation.
Forty-two patients with phospholipid-dependent inhibitors of
coagulation were enrolled in the study. The comparison of their coagulation profiles6 identified 24 patients to belong to
the dRVVT group and the other 18 to the KCT group. The comparison was
performed on the 1:1 mixture of patients' with normal pooled plasma in
the 13 cases under oral anticoagulation.
The main clinical and laboratory data of the patients are reported in
Table 1. The two groups differ with respect
to both the prevalence and the titers of anti-2-GPI antibodies
(measured as aCL antibodies), which were significantly higher in the
dRVVT profile group. When 2-GPI was used as the solid phase antigen, a clear trend towards a higher prevalence of anti-2-GPI antibodies was observed in the dRVVT group, although statistical significance was
not reached. The retrospective analysis of the clinical history of the
patients showed that thromboembolic complications were statistically
associated with the dRVVT profile (P = .0018).
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Table 1.
Clinical and Laboratory Characteristics of 42 Patients
With Phospholipid-Dependent Inhibitors of Coagulation Classified
According to Their Coagulation Profile
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Evaluation of acquired APC-R: time-course of FVa generation and
inactivation in plasma.
First, the time-course of FVa generation and inactivation was evaluated
in normal pooled plasma and in plasma of nine normal controls. The peak
values of FVa were reached within 4 to 5 minutes from calcium addition
(mean ± SD, 16.307 ± 4.372 U/mL) and an almost complete
inactivation by endogenous APC was achieved at 20 minutes (mean ± SD, 0.088 ± 0.123 U/mL) (Fig 1). Levels
of residual FVa in plasma at 20 minutes exceeding 2 SD the mean of controls (ie, 0.334 U/mL) were considered abnormal. In six patients with the R506Q mutation (Factor V Leiden), the maximal amount of FVa
generated in plasma (mean ± SD, 32.537 ± 29.624 U/mL) was approximately twice that of normal subjects (P = .3173). In all cases, the residual amount of FVa at 20 minutes was higher than that of
the control group (range, 0.370 to 14.120 U/mL; mean ± SD, 5.352 ± 5.965 U/mL; P = .004). Therefore, the assay could easily
distinguish carriers of the R506Q mutation, responsible for the APC-R,
from normal individuals.
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| Fig 1.
Time-course of FVa generation and inactivation in the
plasma of normal controls ( ), 18 patients belonging to the KCT
coagulation profile group ( ), and 24 patients belonging to the dRVVT
coagulation profile group ( ). Results are presented as the mean
values of the three groups for each time point; bars represent 1 SD.
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Subsequently, the time-course of FVa generation and inactivation was
measured in the plasma of 42 patients with phospholipid-dependent inhibitors of coagulation. In the 13 cases under warfarin treatment at
the time of the study, this measurement was performed in the 1:1
mixture of patients' with normal pooled plasma. This was based on the
results of experiments performed in plasmas of seven patients under
stable oral anticoagulation for valvular heart prosthesis without APL
antibodies or the R506Q mutation: the increased levels of residual FVa
at 20 minutes measured in the undiluted plasmas (range, 6.500 to 8.140 U/mL; mean ± SD, 7.413 ± 0.605 U/mL, P = .0001 v controls) came down to 0.130 to 0.610 U/mL in the 1:1 mixture
with normal pooled plasma. Therefore, levels of FVa exceeding the upper
value of this range (ie, 0.610 U/mL) were considered abnormal for APL
patients under oral anticoagulation.
In the 42 patients with APL antibodies, the peak values of FVa
generated in plasma (14.173 ± 13.523 U/mL) were similar to those of
normal plasmas. Conversely, abnormal rates of FVa inactivation by
endogenous APC were observed in 26 cases (62%), indicative for the
acquired APC-R. Eleven of them (42%) experienced venous thromboembolic
events versus only four of the 16 patients (25%) with a normal rate of
FVa inactivation. There was a trend towards a correlation between the
acquired APC-R and deep vein thrombosis, which did not reach
statistical significance (P = .2556).
The time-course of FVa generation and inactivation was analyzed
according to the coagulation profiles of the 42 patients (Table 1 and
Fig 1). The prevalence of the acquired APC-R was significantly higher
in patients belonging to the dRVVT profile group (83%) compared with
those of the KCT profile group (33%) (2 = 0.001). The
level of FVa residual at 20 minutes measured in the plasma of patients
belonging to the dRVVT group (range, 0.320 to 14.450 U/mL; mean ± SD, 2.544 ± 3.580 U/mL) was signficantly higher than that of both
the control group (P = .0191) and that of patients belonging to
the KCT coagulation profile group (range, 0.0 to 1.090 U/mL; mean ± SD, 0.293 ± 0.298 U/mL) (P = .0114). When
compared with controls, the KCT group patients also had an abnormal
inactivation of FVa (P = .027), even though the levels of
residual FVa at 20 minutes of the six pathologic cases ranged from 0.43 to only 1.090 U/mL. No correlation between anti-2-GPI antibody
titers and the levels of residual FVa in plasma was found.
Effect of anti-2-GPI and antiprothrombin antibodies on the
time-course of FVa generation and inactivation in normal and in
2-GPI-depleted plasma.
IgG preparations were isolated from the plasma of eight patients with
phospholipid-dependent inhibitors of coagulation. They were three IgG
affinity-purified by means of cardiolipin-containing liposomes
(Table 2, numbers 1-3) and five total IgG
fractions (Table 2, numbers 4-8). Their behavior in ELISA assays for
anti-2-GPI and antiprothrombin antibodies, as well as their effect
on the dRVVT and diluted aPTT performed in normal pooled plasma and in 2-GPI-deficient plasma is presented in Table 2. The first four IgG
preparations had high titers of anti-2-GPI and antiprothrombin antibodies measured by ELISA and were able to inhibit the dRVVT and the
dilute aPTT performed in normal plasma, but not in 2-GPI-deficient plasma. Therefore, they were classified as anti-2-GPI antibody preparations (formerly defined aCL-type A antibodies).6 IgG preparations number 5-8 had low titers of anti-2-GPI antibodies, high content of antiprothrombin antibodies, and displayed anticoagulant activity in both a dRVVT and dilute aPTT system, irrespective of the
presence or absence of 2-GPI in plasma. Therefore, they were
classified as antiprothrombin antibodies.6
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Table 2.
Characterization of the Immunologic and Anticoagulant
Properties of Purified Anti-2-GPI and Antiprothrombin IgG
Antibodies
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The effect of the eight IgG fractions on the time-course of FVa
generation and inactivation was studied in normal pooled plasma (Fig 2): anti-2-GPI antibodies
(Table 3, preparations number 1-4), but not
antiprothrombin antibodies (Table 3, preparations 5-8), were able to
hamper the inactivation of FVa by endogenous APC. Thus, the different
phospholipid-dependent inhibitors of coagulation reproduced the pattern
displayed by the original plasmas. The acquired APC-R induced by
anti-2-GPI antibodies was clearly dependent on the IgG
concentration (data not shown). It was also strictly
2-GPI-dependent, as the four IgG preparations containing anti-2-GPI antibodies failed to cause acquired APC-R in
2-GPI-depleted plasma (Table 3).
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| Fig 2.
Effect of isolated anti-2-GPI IgG antibodies () (n
= 4) and antiprothrombin antibodies () (n = 4) on the time
course of FVa generation and inactivation in normal pooled plasma. IgG
from normal pooled plasma served as control IgG (). Results are
presented as the mean values for each time point; bars represent 1 SD.
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DISCUSSION |
Interference of APL antibodies with the anticoagulant activity of the
PC system has been reported by several investigators.8-13 Along with the resistance to APC displayed by plasmas from patients carrying the R506Q mutation (ie, the FV Leiden),22 the term acquired APC-R has been recently coined to define this
property,13 which may, in part, explain the increased
thrombotic risk of patients with APL antibodies. The prevalence of this
acquired APC-R has not yet been clearly established because single
cases or small series of patients have been investigated so far and
also because the type of assay used to evaluate the effect of APL
antibodies on the anticoagulant activity of protein C
(PC) system greatly influences the results. Here, we have
shown impairment of the inactivation of FVa by endogenous APC in
approximately two thirds of a large group of patients
with phospholipid-dependent inhibitors of coagulation. Acquired APC-R
could not be due to the presence of FV Leiden because none of the
APL-positive patients enrolled in this study was found hetero- or
homozygous for the R506Q mutation. The reported prevalence could
neither be an overestimation due to the warfarin treatment received by
a substantial number of cases. In fact, being aware of the effect of
oral anticoagulants on PC function, we evaluated the inactivation FVa
by APC in the 1:1 mixture of warfarin-treated patients' plasma with
normal pooled plasma.
The assay used here to assess the time-course of FVa generation and
inactivation in plasma is a two-stage, PTT-based test, which could
easily discriminate plasmas of carriers of the R506Q mutation from
those of normal controls. The interference of phospholipid-dependent inhibitors of coagulation with this type of assays is a well known in
vitro phenomenon, which makes the results of the majority of the
coagulation tests currently used to evaluate APC-R in APL-positive patients unreliable. The experimental conditions we used to assess the
anticoagulant activity of APC in plasma allowed us to kinetically follow the generation and subsequent inactivation of FVa beyond clot
formation.9 The peak values of FVa measured in our
patients' plasmas and in control plasmas were similar; this makes
unlikely the possibility that the reduced rate of FVa inactivation
observed in the majority of our samples was due to incomplete FV
activation by thrombin. Conversely, as we did not measure the level and
activity of PC inhibitor and  1-antitrypsin in the patients'
plasmas, we cannot exclude a possible influence of these inhibitors on
the anticoagulant activity of APC. However, under conditions very similar to ours, Marciniak and Romond9 showed that the full inactivation of FVa requires the presence in its active form of no more
than 2% of the PC available in plasma. The same investigators reported
impairment or even abolishment of FVa inactivation in all of the 15 plasmas with phospholipid-dependent inhibitors of coagulation analyzed,
due to IgG fractions containing the phospholipid-dependent inhibitors
of coagulation.9 This elegant work was performed before it
became evident that APL antibodies and, in particular, phospholipid-dependent inhibitors of coagulation, are heterogenous and
display different specificities. Therefore, it does not provide information on whether the interference with the anticoagulant activity
of APC is a common property of these inhibitors or it is expressed by
specific subgroups. We approached this problem by analyzing the
time-course of FVa generation and inactivation in plasma according to
the coagulation profiles of the patients: acquired APC resistance was
found to correlate with the dRVVT rather than with the KCT coagulation
profile. We have recently shown that anti-2-GPI antibodies are
responsible for the former coagulation profile, whereas the latter one
is due to the presence of antiprothrombin antibodies.6
Moreover, the retrospective analysis of the clinical history of the
patients showed that the dRVVT profile is statistically associated with
an increased risk of thromboembolic complications.6 Our
present data confirm the relationship between historical thrombosis and
the dRVVT profile and provide a possible pathogenetic explanation of
such an association. In fact, isolated anti-2-GPI antibodies, but
not antiprothrombin antibodies, were able to induce acquired APC-R,
thus reproducing the procoagulant effect observed in the original
plasmas. This effect of anti-2-GPI antibodies was strictly
2-GPI-dependent, as it was abolished in plasma depleted of this
protein. Similar results were recently reported by Matsuda et
al,23 who demonstrated the ability of a rabbit
anti-2-GPI antibody to induce APC-R only when 2-GPI was present
in plasma. 2-GPI has been shown to inhibit the inactivation of FVa
by APC at physiologic concentrations of Protein S and
FVa.24 This effect was proportional to the
amount of 2-GPI present and was more pronounced at low phospholipid concentrations. Based on these data, 2-GPI was suggested to act via
competition with APC for the binding to phospholipids.24 Although they do not clarify the fine details of the
acquired APC-R induced by anti-2-GPI antibodies, these findings and
our present data suggest the possibility that anti-2-GPI antibodies may cause this phenomenon by enhancing the binding of 2-GPI to the
phospholipid surface. Therefore, acquired APC-R would be due to the
same mechanism responsible for the in vitro anticoagulant effect of
anti-2-GPI antibodies. Further experiments will be necessary to
elucidate this point.
In contrast with Smirnov et al11 findings, the prevalence
of acquired APC-R reported here cannot be explained by the ability of
antiphosphatidylethanolamine (anti-PE) antibodies to interfere with the
anticoagulant activity of APC by competing with this protein for the
binding to PE. In fact, a survey of IgG anti-PE antibodies performed in
our laboratory showed their presence in only 10% of APL-positive
patients (unpublished observation). Also the possibility that 2-GPI
competes with APC for the binding to PE seems unlikely because we
failed to show binding of purified 2-GPI, either alone or in
combination with anti-2-GPI antibodies, to this phospholipid in
solid phase (data not shown).
Despite the interference of anti-2-GPI antibodies with the
anticoagulant activity of APC, we did not find a correlation between acquired APC-R and the titers anti-2-GPI antibodies measured in
plasma either as aCL antibodies or using 2-GPI as solid phase antigen. Our data are at variance with those recently reported by
Martinuzzo et al,12 who found that acquired APC-R in
APL-positive patients was statistically associated with the presence of
anti-2-GPI antibodies rather than with that of the
phospholipid-dependent inhibitors of coagulation. Once more, these
conflicting results highlight the heterogeneity of the methodology used
for the detection of APL antibodies, whose standardization is far from
being reached. This is underlined also by the low prevalence of
anti-2-GPI antibodies we found in patients belonging to the dRVVT
profile group using the ELISA with 2-GPI in solid phase.
Theoretically, all of them would be expected to have anti-2-GPI
antibodies measured by this system. However, one must keep in mind that
the identification of the different phospholipid-dependent inhibitors
of coagulation by comparing the ratios of the dRVVT and the KCT is
somewhat artificial, and no complete overlap with the ELISA's results
is to be expected. Moreover, the higher prevalence of plasmas reacting
with 2-GPI bound to cardiolipin (88%) than with 2-GPI directly
bound to high-activated polyvinylchloride (PVC) plates
(55%) closely resembles that recently reported for antiprothrombin
antibodies,20 suggesting that the former ELISA system is
more efficient than the latter one in identifying anti-2-GPI
antibodies. Interestingly, the titers of anti-2-GPI antibodies
(measured with cardiolipin in solid phase) were significantly higher in
patients belonging to the dRVVT than to the KCT coagulation profile.
The most likely explanation is that the presence of anti-2-GPI
antibodies, which are responsable for the former coagulation
profile,6 is a necessary condition only in plasmas
displaying the dRVVT profile. Indirectly, this is also supported by the
higher prevalence of anti-2-GPI antibodies in the dRVVT than in the
KCT profile (Table 1).
In conclusion, the present report confirms the association between
thromboembolic events and the anti-2-GPI antibodies and shows their
relationship with the impairment of the anticoagulant activity of the
PC system. The resulting acquired APC-R might represent one of the
possible pathogenetic mechanisms responsible for the thrombotic risk of
this subgroup of APL-positive patients. It is also tempting to
speculate that coagulation tests might appear more efficient than
ELISAs in identifying the anti-2-GPI antibodies that represent a
possible risk factor for thromboembolic events. However, more work is
required to improve the specificity of both coagulation tests and
immunoassays for the detection of APL antibodies with respect to the
clinical events of the antiphospholipid syndrome.
| |
FOOTNOTES |
Submitted June 3, 1997;
accepted October 28, 1997.
Address reprint requests to Monica Galli, MD, PhD, Divisione di
Ematologia, Ospedali Riuniti, L.go Barozzi, 1, 24100 Bergamo, Italy.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We wish to thank Drs E.M. Bevers and J. Rosing (Department of
Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht
University, The Netherlands) for critically reviewing this manuscript.
Dr E.M. Bevers is also acknowledged for kindly giving us
2-GPI-depleted plasma.
| |
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Abstract
Introduction
Abstract