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BRIEF REPORT
From the Division of Transfusion Medicine, University
of Cambridge and National Blood Service East Anglia Centre, United
Kingdom; National Institute for Biological Standards and Control,
Potters Bar; Queen Elizabeth NHS Trust Hospital, King's Lynn, United
Kingdom; Central Laboratory of the Netherlands Red Cross Blood
Transfusion Service, Amsterdam, The Netherlands; and Thrombosis
Research Section, Department of Medicine, Baylor College of Medicine,
Houston, TX.
Autoimmune thrombocytopenia is generally caused by autoantibodies
against glycoprotein (GP) IIb-IIIa or GPIb-IX and occasionally against
GPIa-IIa or GPV. By investigating 38 rheumatoid arthritis (RA) patients
on gold therapy, 10 with profound thrombocytopenia and 28 nonthrombocytopenic controls, we showed that in all 10 patients with
thrombocytopenia, the platelet autoantibodies preferentially targeted
GPV but the presence of gold was not required for their reactivity.
Elevated levels of platelet-associated IgG (PAIgG) were observed in 8 of the 10 patients in whom the tests were performed. In 5 patients with
sufficient autologous platelets, the GPV specificity of PAIgG was
confirmed. Tests with GPV transfectants revealed that the antibodies
reacted with GPV independent of GPIb Autoimmune thrombocytopenia (AITP) in patients with
rheumatoid arthritis (RA) on gold therapy is a rare, but severe,
event.1 Platelet autoantibodies generally target
glycoprotein (GP) IIb-IIIa or GPIb-IX,2-4 but
GPIa-IIa5-7 and GPV8,9 may also be targeted. GPV is a leucine-rich repeat domain protein, present in 1 to 2 stoichiometry with GPIb Platelets and sera from 38 RA patients treated with gold were
examined for platelet autoantibodies. Of these 38, 10 had severe AITP;
for 7 patients acute-phase platelet counts were available and the
remaining 3 samples were from our AITP archive sample bank. The other
28 patients were controls without thrombocytopenia. In 8 of the
patients with thrombocytopenia, autologous platelets were available for
a direct immunofluorescence platelet-associated IgG (PAIgG) test and in
5 for a specificity investigation by direct monoclonal antibody
immobilization of platelet antigens (MAIPA) assay.13-15
Indirect MAIPA tests using the patient's sera were performed in all 10 patients. In one archive case (patient 10), autoantibodies eluted from
the patient's own platelets using ether16-18 were
also tested.
Because it was likely that the patients' sera contained gold at the
time of testing, the requirement of gold for antibody reactivity was
assessed using protein G-purified IgG. Furthermore, possible
augmentation of antibody binding by gold was investigated through the
addition of sodium aurothiomalate as a source of gold at physiological
concentrations19 during the MAIPA assay.
For direct immunofluorescence, the patients' platelets were washed
using phosphate-buffered saline containing 0.01 M EDTA and 0.25%
bovine serum albumin. Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-human IgG (1 µg per 5 × 106 platelets)
was then added, and after 30 minutes at 20°C the platelets were
washed again and examined for bound FITC by flow cytometry. The
patients' own platelets were also used for the direct MAIPA as
follows. After washing, platelets were incubated with these GP-specific
monoclonal antibodies (mAbs), used as either culture supernatants (1 in
10 dilution) or ascites (1 in 1000 dilution): RFGP56 for GPIIb-IIIa,
CLB-MB45 for GPIb-IX, CLB-SW16 for GPV, and CLB-10G11 for GPIa-IIa.
Following further washes, the platelets were solubilized and the
lysates were added to microplate wells coated with goat anti-mouse Ig
to capture the mAb-GP complexes. Bound human antibodies were then
revealed with goat anti-human IgG. For the indirect MAIPA, sera were
tested against platelets of known human platelet antigen (HPA)
genotype. Results from all assays were compared with those obtained
with platelets or sera from healthy blood donors tested in parallel.
Purified IgG fractions were prepared from 5 patients' sera using MAb
Trap GII kits (Amersham Pharmacia Biotech, Bucks, England), according
to the manufacturer's instructions. The sera and IgG fractions were
retested in MAIPA in the absence and presence of gold at final
concentrations of 0.1 to 100 µg/mL.
In 4 patients with thrombocytopenia there were sufficient sera for a
sandwich enzyme-linked immunosorbent assay (ELISA) with a mouse
fibroblast L-cell line20 stably expressing human GPV. A
3.5-kilobase (kb) DNA fragment encoding GPV was cloned into the ZEM229R
expression vector and transfected as described
previously.21 The assay was based on the indirect MAIPA,
with the L cells incubated with the patients' sera and GPV-specific
mAb before solubilization and subsequent capture of the mAb-GP
complexes. Use of these transfectants enabled further definition of
antibody specificity because, unlike platelets, GPV in these cells is
expressed without the associated GPIb Increased PAIgG levels were detected in all 8 AITP patients
tested, with results between 1.9 and 22.8 times higher than in the
negative controls (Table 1). Direct MAIPA
in 5 patients tested showed almost exclusive GPV reactivity, with the
ELISA OD values clearly greater than those obtained with controls
(Table 1). Reactivity with capture mAbs against other GPs was negative
in all but one patient (patient 7) in whom weak reactivity with
GPIIb-IIIa, Ib-IX, and Ia-IIa was seen. The ELISA OD values with these
additional GPs were all 0.36 or lower, which is considerably weaker
than the value of greater than 3.5 with GPV (Table 1). The most likely explanation for this weak additional reactivity is incomplete membrane
solubilization, but the presence of additional autoantibodies cannot be
excluded. By indirect MAIPA, GPV antibodies were detected in the eluate
from patient 10 and in sera of all but 2 patients (patients 1 and 10).
Serum reactivity was weaker than reactivity in the direct tests, which
is typical of blood cell autoimmunity when autoantibodies are
predominantly cell-bound. Antibodies to GPIb-IX were not seen. The
average OD for the 10 sera was 0.07 (SD = 0.06), and the negative
control sera gave an average OD of 0.07 (SD = 0.07). Similarly,
reactivity was not seen with GPIIb-IIIa or with GPIa-IIa.
GPV-specific antibodies were detectable in purified IgG from all 5 patients tested (OD values 0.34 or greater; Table
2). Addition of gold to the sera and
purified IgG at concentrations ranging from 0.1 to 100 µg/mL did not
enhance reactivity (Table 2). These results indicate that gold does not
act as a hapten. It therefore appears to break tolerance for self by
inducing autoantibody formation, as is found with That gold is not required for autoantibody binding agrees with earlier studies using eluted autoantibodies.23 However, our specificity results are not in agreement with a previous suggestion that autoantibodies in gold-induced AITP are against GPIIb-IIIa.23 This latter conclusion was inferred from the lack of reactivity with platelets from patients with Glanzmann thrombasthenia rather than from positive test results. To investigate whether the epitopes recognized by autoantibodies are
uniquely expressed on GPV, sera from patients 6, 7, 8, and 9 were
tested using ELISA with fibroblasts expressing human GPV. All sera gave
positive results, and OD values were 0.31, 0.27, 0.25, and 0.37, respectively, compared with a value of 0.03 with the negative control.
This reactivity demonstrates that the epitopes are confined to GPV and
are not dependent on the presence of GPIb All but one of the 28 negative control RA patients without thrombocytopenia (platelet counts 153 × 109/L or higher; range, 153-582 × 109/L; mean, 285 × 109/L) had a negative direct PAIgG test. The single positive patient had a platelet count of 329 × 109/L, and the PAIgG result was 2.9 times that of the negative control. There was insufficient sample for further investigation of the PAIgG, but indirect testing showed a weak alloantibody of undetermined specificity. In summary, we obtained significant evidence that in gold-associated AITP, autoantibodies recognize GPV. Cloning of these autoantibodies is required to determine whether the loss of tolerance is for single or multiple epitopes and whether the same epitopes are targeted in the different clinical groups. Such recombinant mAbs would also be ideal tools for investigating the functional effects of GPV-specific antibodies because it has recently been suggested that they may affect collagen-induced platelet aggregation.24
We thank Professor A. E. G. Kr von dem Borne and Dr L. Porcelijn for their gifts of the CLB mAbs and for referring patient 10, respectively. We also thank Dr Peter Smethurst for the IgG purification, the staff at the Platelet Immunology Reference Laboratory at the National Blood Service East Anglia Centre for their expertise in serology, and Dr John Williams for reviewing the rheumatoid arthritis cases.
Submitted September 21, 2001; accepted February 19, 2002.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Stephen F. Garner, National Blood Service East Anglia Centre and Division of Transfusion Medicine, University of Cambridge, Long Rd, Cambridge, CB2 2PT, United Kingdom; e-mail: sfg21{at}cam.ac.uk.
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© 2002 by The American Society of Hematology.
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  R. H. Aster and D. W. Bougie Drug-Induced Immune Thrombocytopenia N. Engl. J. Med., August 9, 2007; 357(6): 580 - 587. [Full Text] [PDF]
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| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
, GPIb
, or GPIX.
Autoantibodies recognizing GPV were not seen in the 28 nonthrombocytopenic control RA patients. Thus, GPV seems to be targeted
in gold-induced autoimmune thrombocytopenia.
(Blood. 2002;100:344-346)