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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2366-2373
Quinine-Dependent Antibodies Bind a Restricted Set of Epitopes on the
Glycoprotein Ib-IX Complex: Characterization of the Epitopes
By
Janette K. Burgess,
Jose A. Lopez,
Michael C. Berndt,
Ian Dawes,
Colin N. Chesterman, and
Beng H. Chong
From the University of New South Wales, Centre for Thrombosis and
Vascular Research, Department of Haematology, Prince of Wales Hospital,
Sydney, Australia; Baylor College of Medicine, Houston, TX; Baker
Medical Research Institute, Melbourne, Australia; and the School of
Biochemistry and Molecular Genetics, University of NSW, Sydney,
Australia.
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ABSTRACT |
Severe immune thrombocytopenia is an idiosyncratic complication of
quinine therapy. Although in most cases the responsible antibody is
directed against platelet membrane glycoprotein (GP) Ib-IX, specificity
for GPIIb-IIIa or both epitopes has also been reported. The objective
of this study was to characterize the binding site of GPIb-IX-specific
quinine-dependent antibodies. Antibody binding to Chinese hamster ovary
cells or mouse L cells stably transfected with various combinations of
the three genes (Ib , Ib , or IX) that encode this complex was
detected using flow cytometry, monoclonal antibody-specific
immobilization of platelet antigens assay, and differential adsorption
studies. IgG in sera from 15 patients with quinine-induced
thrombocytopenia binding to the cells, in the presence of quinine,
showed three distinct patterns. Group 1 sera contained at least two
antibody populations, one which binds to GPIb and another which
recognizes GPIX. Group 2 sera contained an antibody which binds drug
dependently to GPIX, and Group 3 sera contained an antibody which
recognizes a quinine-dependent epitope on GPIb. Thus, the
quinine-dependent antibodies fall into two distinct populations that
bind to GPIb and GPIX independently. Using proteases which cleave
GPIb at specific sites, we have shown that the GPIb-specific
antibody binds to an 11-amino acid (283 to 293) region. Peptide
inhibition studies provide confirmatory evidence that this region
contains the epitope for the GPIb-specific quinine-dependent
antibody.
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INTRODUCTION |
DRUG-INDUCED thrombocytopenia is a common
hematologic problem in clinical practice.1 Drugs frequently
implicated include quinine and its optical isomer, quinidine. In
quinine-/quinidine-induced thrombocytopenia, drug-dependent binding of
antibodies to platelets leads to increased platelet clearance by the
reticuloendothelial system and results in severe thrombocytopenia of
acute onset. In the majority of cases the antibody is directed against
platelet membrane glycoprotein (GP) Ib-IX complex,2,3
although antibodies with specificity against GPIIb-IIIa have also been
reported.4,5
GPIb is a major sialoglycoprotein on the platelet cell surface with
approximately 25,000 copies per platelet.6 GPIb is composed
of two subunits, the subunit of 143 kD which is
disulphide bonded to a smaller subunit of 24 kD. GPIb can be
cleaved by a Ca2+-dependent protease resulting in a
single-chain, water-soluble glycoprotein of 120 kD (glycocalicin) and a
small membrane-bound fragment. GPIb is noncovalently linked to GPIX,
which has a molecular mass of 20 kD. The three glycoproteins have
leucine-rich motifs and they exist as a heterodimeric complex in the
platelet membrane.7,8 GPIb-IX is associated noncovalently
with GPV, a glycoprotein of 82 kD, which also has leucine-rich
motifs.9 The components of GPIb-IX are all encoded by
separate genes.10
Kunicki et al11 first showed that GPIb-IX was involved in
quinine-induced thrombocytopenia when they reported that the
quinine-/quinidine-dependent antibodies failed to react with platelets
from an individual with Bernard-Soulier Syndrome. We provided the first
direct evidence that the GPIb-IX complex was the platelet autoantigen
for the antibody in quinine-induced thrombocytopenia when we showed the drug-dependent immunoprecipitation of GPIb and GPIX using a patient's serum.12 We subsequently showed2 that the
portion of GPIb-IX that remains associated with the platelet membrane
after removal of glycocalcin by proteases and the NH2
terminal of the GPIb are the components of the complex that is
involved with antibody binding. The precise antibody binding site(s) on
GPIb-IX has not been reported.
In this study we have shown that antibodies from patients with
quinine-induced thrombocytopenia react with only a limited number of
epitopes within the GPIb-IX complex. We identified only two binding
sites for the quinine-dependent antibodies. One site is on GPIX and the
other on GPIb, which we have narrowed to a stretch of 11 amino
acids, encompassing residues 283 to 293.
This is the first study to define the quinine-dependent antibody
binding sites on GPIb-IX in such detail. This is the first report that
has been able to localize the binding site of one of these antibodies
to a region of 11 amino acids on GPIb.
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MATERIALS AND METHODS |
Materials.
Bovine serum albumin (BSA), quinine hydrochloride, phenylmethylsulfonyl
fluoride (PMSF), EDTA, disodium salt, bacitracin, benzamidine,
dithiothreitol (DTT), dimethylsulfoxide, iodoacetamide, propidium
iodide, and 2,2 -azinobis 3-ethylbenzthiazolinesulfonic acid
(ABTS) were purchased from Sigma (St Louis, MO);
3,3,5,5-tetramethylbenzidine dihydrochloride from
Kirkegaard & Perry (Gaithersburg, MD); hydrogen peroxide (30% wt/vol),
Triton X-100, and Tween-20 from BDH (Poole, Dorset, UK);
sulfosuccinimidobiotin from Pierce (Rockford, IL); polyscreen poly
vinylidene difluoride (PVDF) transfer membrane and Western blot
chemiluminesence reagents from DuPont (Boston, MA); and sheep
anti-mouse IgG-coated dyna beads from Dynal (Oslo, Norway). All
chemicals were of analytical reagent grade.
Antibodies.
The goat anti-mouse Ig and the horseradish peroxidase (HRP)-conjugated
goat anti-human Ig (Jackson, West Grove, PA), rabbit anti-mouse
HRP-conjugated antibody (Dako, Carpentaria, CA), and the streptavidin
HRP-conjugated antibody (Amersham, Bucks, UK) were
purchased as indicated.
All monoclonal antibodies (MoAbs) were of the IgG class. MOPC21 (Becton
Dickinson, San Jose, CA), a murine IgG1 myeloma protein, was used as a control Ig, and SZ2 was purchased from Immunotech (Marseille, France). Gi27 was a generous gift from Dr S. Santoso (Giessen, Germany). AK2, AK3, and FMC25, which are all directed against epitopes on various parts of the human GPIb-IX complex, were
raised and characterized in one of our laboratories (M.C.B.) and the
preparation of these antibodies has been previously
described.8,13
Drug-dependent antibodies.
Sera or plasma from 15 patients (6 men and 9 women; ages 18 years 10 months to 76 years 0 months; mean, 53 years 10 months) with
quinine-induced thrombocytopenia were used in this study. The samples
were selected at random from patients presenting at this hospital and
other hospitals from which samples were forwarded to our laboratory for
testing. These patients developed thrombocytopenia when they were
receiving quinine and their thrombocytopenia resolved after the
cessation of the drug. Other causes of thrombocytopenia were clinically
excluded. The diagnosis of quinine-induced thrombocytopenia was
confirmed by a positive platelet antibody test.
Cell lines.
Chinese hamster ovary (CHO) cells and mouse L (tk )
cells were obtained from American Type Tissue Collection (Rockville,
MD) and maintained as recommended by the supplier.
Synthetic peptides.
The peptide specific for amino acids 283-293 of GPIb
(H-DTEGDKVRATR-NH2), the nonspecific peptide that had the
same amino acid sequence in reverse (H-RTARVKDGETD-NH2),
and the unrelated nonspecific peptide
(H-PTLGDEGDTDLYDYY-NH2) were manufactured by Chiron
Technologies (Clayton, Vic, Australia).
Transfection of CHO and L cells with GPIb-IX genes.
CHO DUK (DHFR) cells and mouse L
(tk) cells were stably transfected with cDNA
encoding the GPIb-IX subunits in various combinations as previously
described.14 The cloning of the cDNAs for GPIb, GPIb,
and GPIX has been reported previously.15-17 The three cDNAs (each containing the entire coding sequence and the
3-untranslated region) were cloned separately into the
eukaryotic expression vector pDX (a kind gift from Dr K. Berkner,
Seattle, WA) in which transcription is driven by the adenovirus major
late promoter and the SV40 enhancer. The CHO cells were transfected
with the following combinations of GPIb-IX subunit cDNAs: CHO GPIb + GPIb + GPIX, GPIb + GPIb, GPIb + GPIX, and GPIb + GPIX.
The L cells were transfected with GPIb + GPIb and GPIb + GPIX.
Expression of the GPIb-IX subunits in the cell lines was substantiated
by Northern blot analysis to detect mRNA and by flow cytometry and enzyme-linked immunosorbent assay (ELISA) to ensure expression of the
subunits on the cell surface.
Transient transfections of L cells with GPIX cDNA were also
performed by liposome-mediated DNA transfer using a commercial kit
(GIBCO-BRL, Gaithersburg, MD). Twelve µL of liposome suspension and 4 µg of GPIX cDNA were separately mixed in 300 µL of serum-free Dulbecco's modified Eagle's medium (DMEM) nutrient mixture F-12 (DMEM/F-12) (Trace, Sydney, Australia). The two
suspensions were then combined, mixed gently, and incubated for 30 minutes at room temperature. The mixture was diluted in 1.8 mL of
serum-free DMEM/F-12 and added to the cells that had been washed once
with the same medium. The cells were exposed to the DNA/liposome
mixture for 7 hours under standard culture conditions
(37°C, 5% CO2), then 1.6 mL of DMEM/F-12
containing fetal bovine serum (GIBCO-BRL) was added to a final
concentration of 10% (vol/vol). The medium was changed 24 hours after
the start of the transfection. After 48 hours the cells were detached
from the culture dishes with 0.53 mmol/L EDTA/phosphate-buffered saline
(PBS) and evaluated by flow cytometry for surface expression of GPIX.
Flow cytometry.
Flow cytometry was performed on a FACStar Plus cytometer (Becton
Dickinson) fitted with a 100-mW air-cooled argon ion laser using the
488-nm green line for fluorescence excitation. The cell emission
spectra were collected on FL1 (green) using a band pass filter 530 DF
30. Dead cells were excluded using propidium iodide on FL3 using a 700 LP filter. Before analysis the cells were detached from the culture
flasks with 0.53 mmol/L EDTA/PBS and resuspended in 1% BSA/PBS. The
cells (at a concentration of 2 × 105 per tube) were
blocked in 2% BSA/PBS for 10 minutes at room temperature, washed once,
pelleted by centrifugation at 2,000g, and resuspended before
incubation with MoAb (10 µg/mL) or patient serum (1 in 200 dilution)
for 10 minutes at room temperature in the presence or absence of
quinine (0.3 mmol/L). In experiments performed in the presence of
quinine, the working buffer contained the drug beyond this stage at a
concentration of 0.3 mmol/L. The cells were then washed three times in
1% BSA/PBS before being incubated for 10 minutes at room temperature
with the relevant fluorescein isothiocyanate (FITC)-conjugated
secondary antibody (goat anti-mouse IgG at 1 in 200 dilution or goat
anti-human IgG at 1 in 100 dilution). The cells were washed four times
before being resuspended in 1% BSA/PBS and were kept in the dark until
analysis.
In some experiments the cells were incubated with a proteolytic enzyme
before blocking with 2% BSA/PBS. The cells were resuspended in 1%
BSA/PBS at 3.5 × 106 cells/mL. The proteolytic
enzymes mocarhagin and trypsin (porcine 10× stock, specific
activity 1:250; GIBCO-BRL) were added separately to 300 µL of cells
at a final concentration of 50 µg/mL. The mocarhagin18 was purified in our laboratory. The cells were incubated at 22°C for 10 minutes before the reactions were stopped by the addition of 2 mmoL of PMSF. The cells were divided into six tubes and blocked with
2% BSA/PBS. The labeling of these cells was then continued as
described above.
ELISA.
Cells were allowed to grow to confluence before being washed with 1%
BSA/PBS and detached from the tissue culture vessel. After resuspension
in 1% BSA/PBS the cells were seeded at 5 × 104 cells
per well in a 96-well microtiter plate (Corning, Cambridge, MA). The
plate was spun at 200g for 10 minutes at 4°C before the supernatant was removed and the cells were allowed to dry overnight at
37°C. The plate was stored at  20°C, with desiccant, in
an airtight container until analysis.
After defrosting, the plates were washed twice with 0.05%
Tween-20/PBS. The wells were blocked with 200 µL of 2% BSA/PBS for 60 minutes at 37°C and washed three times. The appropriate MoAbs, at a final concentration of 2 µg/mL in 1% BSA/PBS, were added and
incubated for 90 minutes at 37°C. The plates were washed four times
before the rabbit anti-mouse HRP-conjugated secondary antibody (diluted
1 in 1,000 in 1% BSA/0.05% Tween-20/PBS) was added and incubated for
30 minutes at 37°C. The plate was then washed five times and the
ABTS substrate solution was left to develop for 30 minutes in the dark
at room temperature. The reaction was stopped with 3% (wt/vol) oxalic
acid and the absorbance read on a Titertek photometer (Labsystems,
Helsinki, Finland) at 414 nm.
MoAb-specific immobilization of platelet antigens (MAIPA) assay.
The MAIPA assay was performed as previously described with minor
modifications.19 Briefly, the cells detached from the
culture flasks were centrifuged in test tubes at 2,000g for 2 minutes to give a total of 1.5 to 2 × 106
cells per tube. The cells were resuspended in 100 µL PBS/0.2% EDTA
buffer with 0.3% BSA/1% FCS (PEBF buffer) and added to 50 µL of
patient serum, with or without 5 µL of quinine (final concentration, 0.5 mmol/L), and incubated at 37°C for 30 minutes. The cells were washed once with PEBF buffer. Quinine (0.3 mmol/L) was included in the
washing buffer throughout the procedure if the cells were incubated
with the drug together with the patient serum at the beginning of the
experiment. Twenty µL of MoAb (final concentration, 10 mg/mL) was
added and incubated at 37°C for 30 minutes. The cells were pelleted
and washed three times with PEBF buffer. The cells were lysed by
resuspension in 100 µL 0.01 mol/L Tris-buffered saline (TBS)
containing 0.5% Triton X-100 and 0.05% Tween-20 ± 0.3 mmol/L
quinine, pH 7.4. The cell lysate was centrifuged at 12,000g for
30 minutes at 4°C. Seventy µL of the supernatant was diluted in
180 µL of TBS buffer. The diluted supernatant (100 µL) was added in
duplicate to the wells of a microtiter plate (Immunotech) that had been
coated with 100 µL of affinity-purified goat anti-mouse IgG (1/500 in
0.05 mol/L sodium carbonate buffer) by overnight incubation at 4°C,
washed, and blocked with 250 µL per well of TBS buffer for 60 minutes
at 4°C. The microtiter plate was incubated for 90 minutes at
4°C and washed four times with TBS buffer. Anti-human IgG HRP
conjugate (100 µL diluted 1:10,000 in TBS buffer) was added to each
well and incubated for 120 minutes at 4°C. After six washes, 100 µL of substrate solution (0.42 mmol/L 3,3,5,5-tetramethylbenzidine dihydrochloride in 100 mmol/L sodium acetate/citric acid, pH 6.0, with 1.3 mmol/L hydrogen
peroxide) was added. The color reaction was stopped by the addition of
50 µL 2 mol/L sulfuric acid after 15 minutes and read at the dual wavelengths of 450 and 492 nm in a Titertek photometer (Labsystems). The positive control for the assay was normal pooled platelets at a
concentration of 20 × 106 plus 50 µL patient serum ± 6.5 µL 7.5 mmol/L quinine (final concentration 0.5 mmol/L).
Biotin labeling of GPIb-IX subunits.
The cells were incubated with 70 µmol/L sulfosuccinimidobiotin for 30 minutes at room temperature in a capped tube, with constant rotation.
The free biotin was quenched with a twofold molar excess of glycine for
10 minutes at room temperature, before being washed once. MoAb (AK2,
AK3, or SZ2; final concentration 10 µg/mL) was then incubated with
the cells for 30 minutes at 4°C, before the cells were washed three
times. The cells were lysed, in the presence of protease inhibitors
(PMSF 200 mmol/L, bacitracin 10 mg/mL, and benzamidine 200 mmol/L ) by
resuspension in 100 µL 0.01 mol/L TBS containing 0.5% Triton X-100,
0.05% Tween-20, and incubation at 4°C for 30 minutes. The cells
were then centrifuged at 12,000g for 30 minutes at 4°C. The
supernatant (70 µL) was diluted in 430 µL PBS and 1 × 107 goat anti-mouse IgG coated dyna beads were added. After
incubation at 4°C for 45 minutes, the dyna beads were washed three
times using the MPC-E-1 magnet before they were resuspended in 2 × Laemmli buffer and stored at 80°C until sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.
Proteolytic digestion of GPIb.
Cell surfaces labeled with biotin as described above were subjected to
proteolytic digestion. Mocarhagin or trypsin was added to 300 µL of
cells (3.5 × 106 cells/mL) at a final concentration
of 50 µg/mL. The cells were incubated at 22°C for 10 minutes
before the reactions were stopped by the addition of 200 mmol/L PMSF.
The cells were washed once and the cells and supernatant were
transferred to separate tubes. AK3, AK2, or SZ2 were added to the cells
and the supernatant samples at a final concentration of 10 µg/mL.
After incubation for 30 minutes at 4°C the cells were washed and
lysed as described above. The cell lysates and supernatant samples were
added to dyna beads (monoclonal sheep anti-mouse IgG) for 45 minutes at
4°C. The dyna beads were separated from the supernatant using the
Dynal MPC-E-1 magnet and washed extensively. The samples were stored at
80°C until SDS-PAGE analysis.
SDS-PAGE and detection of proteins.
Samples for SDS-PAGE analysis were treated with 2 µL 0.5 mol/L DTT
and boiled for 5 minutes. Free DTT was quenched by the addition of 4 µL 0.5 mol/L iodoacetamide. SDS-PAGE was performed according to the
method of Laemmli20 using 4% to 15% Tris-glycine gradient
gels (Bio-Rad, Hercules, CA). After completion of electrophoresis the
proteins were transferred to PVDF membrane. Membranes were blocked
overnight in 5% (wt/vol) skim milk (Diploma, Bonlac Foods Ltd, Melbourne, Australia) and the following morning washed 5 times in
PBS/0.05% Tween-20. Streptavidin HRP diluted 1 in 2,000 in 2%
BSA/PBS/0.05% Tween-20 was incubated with the membrane for 60 minutes
with rocking. After five washes the presence of a signal was detected
using the Western blot chemiluminescence reagents according to the
manufacturer's instructions.
Peptide inhibition of quinine-dependent antibody binding.
To 10 µL of patient serum (diluted 1 in 200) was added a specific
peptide (H-DTEGDKVRATR-NH2) or either a reverse amino acid sequence nonspecific peptide (H-RTARVKDGETD-NH2) or an
unrelated nonspecific peptide (H-PTLGDEGDTSLYDYY-NH2) at a
final concentration of 200 µmol/L in the presence of quinine (0.3 mmol/L). After incubation at room temperature for 10 minutes this
mixture was added to L cells that had been blocked with 2% BSA.
The cells were then prepared for flow cytometry as previously
described. The optimal concentration of peptide for inhibition was
determined using a dose-response curve for the specific peptide.
Statistical analysis.
Statistical analysis was performed using the one-tailed Mann-Whitney U
test.
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RESULTS |
Quinine-dependent antibodies binding to GPIb-IX.
Flow cytometry and MAIPA assays were used to test the binding of sera
from 15 patients with quinine-induced thrombocytopenia to mouse L cells
that had been stably transfected with either the human GPIb and GPIX
components of GPIb-IX (L IX cells) or the human GPIb and GPIb
components (L cells). Binding was not observed in the absence of
quinine. In the presence of quinine three distinct binding patterns
were observed (Table 1). Group 1 (sera from
6 patients) bound to both cell types. Groups 2 (7 patients) and 3 (2 patients) bound only to L IX cells and L cells, respectively.
Several group 2 sera were assayed for their ability to bind to L
cells before and after transfection with GPIX cDNA. Binding was
observed neither in the absence nor in the presence of quinine before
transfection of the L cells with GPIX cDNA but was observed in
the presence of quinine after transfection with the GPIX cDNA (Fig 1).
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| Fig 1.
Reactivity of sera from group 2 with L cells
before and after transfection with GPIX cDNA. L cells were
transfected with GPIX cDNA using the GIBCO-BRL lipofectAMINE kit as
described in Materials and Methods. The cells were labeled with primary
antibody (either anti-GPIX MoAb FMC25 [A and B], or patient sera from
group 2 plus quinine [C and D]) followed by a FITC-conjugated
secondary antibody and examined by flow cytometry. L cells did
not react with anti-GPIX MoAb before transfection with GPIX cDNA (A)
but bound anti-GPIX MoAb after GPIX cDNA transfection (B). Patient sera
from group 2 did not react with L cells before transfection with
GPIX cDNA (C) but were able to bind to L cells expressing GPIX
on their surfaces after transfection with GPIX cDNA (D). The graphs are
representative of the results observed with three different patients'
sera. The solid peak in each graph represents the negative control.
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Patient sera from all three groups were examined for their ability to
bind to CHO cells expressing GPIb alone on the surface, in the
presence of quinine. (CHO cells were initially transfected with
GPIb and GPIb but the cells rapidly lost expression of GPIb on
repeated cell passage.) Sera from groups 1 and 3 bound this cell type
but the sera from group 2 did not bind (results not shown).
Altogether, these data indicate that (1) group 1 sera contain at least
two antibody populations, one that binds to GPIb, and another that
recognizes either GPIb or GPIX; (2) group 2 sera contain an antibody
that binds drug dependently to GPIX; and (3) group 3 sera contain an
antibody that recognizes a quinine-dependent epitope on GPIb.
Competitive MAIPA.
Group 1 sera were further analyzed using competitive MAIPA. As shown
previously the antibody binding was only observed in the presence of
quinine.
The MAIPA assay is an antigen-capture ELISA which uses a MoAb to
capture GPIb-IX and the human drug-dependent antibody then binds to the
GP complex at a site distant from the MoAb-binding site. If the human
antibody and the murine MoAb-binding sites coincide or are in close
proximity to each other, the MoAb cross-blocks the human antibody and a
negative result is obtained. Group 1 serum showed quinine-dependent
antibody binding to CHO cells when the non-cross-blocking MoAb,
AK2, was used to capture the antigen. Similarly, strong
quinine-dependent antibody binding to CHO IX cells occurred when the
non-cross-blocking MoAb, FMC25, captured the GPIb-GPIX complex.
Figure 2A illustrates the results obtained
with one representative patient's serum; similar results were observed
with three other patients' sera. These data confirm that group 1 serum
contains at least two separate antibodies, one which reacts with
GPIb, and another that reacts with either GPIb or GPIX. When the
experiment was repeated using the anti-GPIX MoAb, SZ1, binding of the
drug-dependent antibody to CHO IX cells was completely inhibited
(Fig 2B), suggesting that the specificity of the second antibody type
in group 1 serum is GPIX rather than GPIb. In contrast, if SZ1 and
CHO IX cells were used in the experiment, we detected a mild
quinine-dependent reaction which was due to the GPIb-specific
antibody in group 1 serum remaining after the GPIX-specific antibody
had been completely cross-blocked by SZ1 (data not shown).
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| Fig 2.
Reactions of group 1 patient sera with CHO cells stably
transfected with cDNAs from various parts of the GPIb-IX complex and
inhibition of quinine-dependent antibody binding by anti-GPIX MoAbs.
MAIPA studies were performed in the presence and absence of quinine
with patient serum and a MoAb as indicated in the figure. (A) The
results of one representative patient's serum binding to
mock-transfected CHO, CHO IX, and CHO cells in the presence of
quinine. Binding was not observed in the absence of quinine (not
shown). In the presence of quinine, binding did not occur with
mock-transfected CHO cells but was observed with CHO IX and CHO cells. (B) The results of one representative patient's serum binding
to CHO IX cells expressing GPIb and GPIX in the presence ( ) or
absence ( ) of quinine and a competitive MoAb (FMC25 or SZ1). No
binding was observed in the absence of quinine. Competition with FMC25
did not inhibit the quinine-dependent antibody binding. Conversely,
competition with SZ1 (specific for GPIX) inhibited binding of the
quinine-dependent antibody.
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Adsorption of drug-dependent antibody subtypes.
Sera from patients in group 1 were absorbed against three different
cell types and the further binding potential of the sera was assessed.
When the sera were absorbed with untransfected L cells no difference in
the binding pattern was observed. Preadsorption of the same sera with L
cells ablated any further binding to the L cells, but the
binding to the L IX cells remained. Conversely, preadsorption of the
patient sera with the L IX cells abrogated further binding to the L
IX cells, but the binding to the L cells was unchanged
(Table 2). These data indicate that group 1 sera which reacted with both L and L IX cells did not contain one antibody (directed against the shared subunit, GPIb) but two
distinct antibodies, one with specificity for GPIb and another with
specificity for GPIX.
Mapping the antibody-binding site on GPIb using selective
enzymatic cleavages.
Three enzymes have recently been shown to cleave GPIb at specific
sites.18 Using sequential cleavage with two of these enzymes the location of the patient antibody-binding site on GPIb was determined (Fig 3A). The degree of
digestion of GPIb, using mocarhagin and trypsin, was determined by
labeling the CHO IX cell surface proteins with biotin before
digestion and extracting the protein components from the cell surfaces
and the supernatants before and after cleavage to be run on SDS-PAGE,
as described in Materials and Methods. Complete digestion of the
GPIb protein was observed with mocarhagin (Fig 3B), with a 70-kD
band immunoprecipitated from the cell lysate by AK3 and a 40-kD band
precipitated by AK2 and SZ2 from the supernatant collected after the
cleavage process. A similar banding pattern was observed after the
digestion with trypsin (results not shown). The GPIX protein was not
affected by these enzymes. GPIb was also unaffected by the
digestions. Flow cytometry was used to analyze the binding of two group
1 serum samples to L cells. The MoAbs AK2, AK3, and SZ2 were used to ensure that specific cleavage had occurred (Fig 3C). After cleavage with mocarhagin, at position 283, AK2 and SZ2 no longer bound,
but AK3 and the quinine-dependent antibody binding was unaffected.
Cleavage with trypsin, at position 293, resulted in loss of both AK2
and SZ2 binding, but the binding site for AK3 remained intact, although
reduced in magnitude. The quinine-dependent antibody binding was
inhibited by cleavage with trypsin. These results indicate that the
quinine-dependent antibody is binding to GPIb between the mocarhagin
and the trypsin cleavage sites, between amino acids 283 and 293 (Fig
3A).
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| Fig 3.
Characterization of the quinine-dependent antibody
binding domain on GPIb. (A) GPIb amino acid sequence
residues 269 to 297. The specific cleavage sites for mocarhagin (a
novel cobra venom metalloproteinase) and trypsin are shown. The MoAbs
AK2, SZ2, and AK3 are specific for GPIb. AK2 binds distal to amino
acid 275, SZ2 between amino acids 276 to 282, and AK3 proximal to amino
acid 294. The quinine-dependent antibody is interacting with GPIb
between amino acids 283 to 293. (B) After labeling the surface proteins
of the CHO IX cells with biotin, the GPIbIX complex was
immunoprecipitated using the MoAb panel. The presence of the GPIb
and GPIX native proteins was shown on the surface of the CHO IX
cells. Lane 1 shows the components immunoprecipitated using AK3, lane 2 with AK2, and lane 3 with SZ2. After cleavage with mocarhagin, AK3
precipitated a 70-kD GPIb component and the intact GPIX protein from
the surface of the cells (lane 4). AK2 (lane 5) and SZ2 (lane 6) were
able to precipitate a 40-kD GPIb component from the supernatant
collected after the cleavage step. (C) Sequential cleavage of GPIb
and checking the ability of the quinine-dependent antibody to continue
binding enabled the definition of the domain to which the
Ib-specific antibody was binding. All MoAbs bound to the surface of
the untreated L cells. AK2 and SZ2 binding were inhibited after
cleavage with mocarhagin. AK3 binding was still present, although at a
lower level after cleavage with trypsin. The quinine-dependent antibody
binding remained after cleavage with mocarhagin but was inhibited after
cleavage with trypsin, indicating that the GPIb-specific
quinine-dependent antibody binding site is located
between amino acids 283 and 293.
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Peptide inhibition of quinine-dependent antibody binding.
A dose-response curve for the specific peptide was obtained ranging
from 2.5 to 500 µmol/L. Optimal inhibition was observed at 200 µmol/L. Incubation of the patient serum with a peptide specific to
the 11 amino acids encompassing the antibody binding site before
exposure to the L cells decreased the level of antibody binding
by 62.0% ± 8.8% (n = 3). In contrast, incubation of the patient
serum with a reverse peptide that was composed of the same amino acid
sequence in reverse resulted in 26.6% ± 5.8% decrease in antibody
binding (n = 3). Incubation with a peptide homologous to an unrelated
region of GPIb resulted in a 15.5% ± 13.4% decrease in
antibody binding. The inhibition with the specific peptide was
significantly greater than the inhibition observed with the unrelated
peptide (P  .05). The observation of the small decrease in
antibody binding in the presence of both unrelated peptides connoted
that this decrease was a nonspecific effect, whereas the effect of the
specific peptide was twofold to threefold stronger.
| |
DISCUSSION |
The aim of this study was to map the binding site(s) of the
quinine-dependent antibodies on GPIb-IX. We have previously
shown2 that the portion of GPIb-IX that remains associated
with the platelet membrane after removal of glycocalcin by proteases
and the NH2 terminal of GPIb are the components of the
complex involved in antibody binding. Cell lines transfected with the
cDNAs of different GPIb-IX subunits were used to further define the
domains to which the quinine-dependent antibodies bind.
Sera from 15 patients who had developed quinine-induced
thrombocytopenia were examined. Three distinct binding patterns with the cells were observed. The sera in group 1 were able to bind to both
the L and the L IX cells, indicating these sera contained at
least two separate antibody populations with binding sites on different
subunits of the GPIb-IX complex. The sera in group 2 were only able to
bind to the L IX cells and sera in group 3 only to the L
cells, indicating that each contained a different antibody type.
The sera in group 1 exhibited drug-dependent binding to more than one
subunit of the GPIb-IX complex. The MAIPA and flow cytometry results
showed that these sera were able to bind to the CHO IX, L ,
CHO IX, L IX, and CHO cells but not to mock-transfected cells
in the presence of quinine. These results indicate that these sera
contain antibodies specific for at least two of the three subunits of
the GPIb-IX complex. The binding to CHO cells expressing GPIb alone
indicates that group 1 sera contain an antibody that reacts
specifically to the GPIb subunit. Because we were unable to generate
CHO or L cells that expressed a single subunit of either GPIb or
GPIX on the surface, alternative experimental approaches were required
to characterize the antibody specificity to other subunits besides
GPIb. Our finding that the anti-GPIX MoAb, SZ1,21 could
completely abolish binding of group 1 sera to CHO IX cells indicates
that these sera contained antibodies specific for GPIX but no antibody
against GPIb. Overall, these data indicate that group 1 sera
contained two antibody populations, one that reacts with GPIX and
another with GPIb. Consistent with our previous
findings,2 we found that the anti-GPIX MoAb, SZ1, strongly
inhibited the binding of group 1 sera to CHO IX cells (>90%
inhibition). Because the quinine-dependent epitope on GPIb and that
on GPIX are not close to each other,2 this MoAb-blocking result indicates that in these sera, the anti-GPIX drug-dependent antibody (rather than the anti-GPIb antibody) is the predominant one.
Preadsorption of the sera from group 1 with L or L IX cells
inhibited binding when the sera was reexposed to the same cell line.
Conversely, on exposure to the other cell line, binding remained. The
inability of the L or L IX cells to completely inhibit
antibody binding after preadsorption of the sera further illustrates
the presence, in the group 1 sera, of two distinct antibody populations
that bind to GPIb and GPIX and the absence of an antibody that binds
to GPIb.
The antibody present in the group 2 sera most probably reacted with the
GPIX subunit on the L IX cells because no binding of these sera was
detected with the L cells. The observation that binding occurred
when the L cells were transfected with GPIX cDNA confirms that
this group of sera contained an antibody that bound specifically to
GPIX.
The sera from group 3 contained an antibody that bound specifically to
GPIb because sera in this group were able to bind to CHO cells
expressing GPIb alone but were unable to bind to CHO or L cells
expressing GPIb and GPIX.
The protease sensitivity of GPIb was used to further define the
binding domain for the GPIb-specific antibody. We have previously defined the specific sites on GPIb where mocarhagin (a cobra venom
metalloproteinase) and trypsin cleave.18 The sequential cleavage of GPIb with these proteases has enabled us to locate the
domain to which the quinine-dependent antibodies are binding. Our
previous work has shown that the binding site for the quinine-dependent antibodies is distal to the trypsin cleavage site at amino acid 293 of
the GPIb protein.2 The binding of the drug-dependent antibody was not affected by cleavage of the GPIb protein with mocarhagin. This study has shown that the binding site is located between the mocarhagin and the trypsin cleavage sites, between amino
acids 283 and 293 on the GPIb protein. The second trypsin-sensitive site on GPIb was also partially cleaved during this study but the
continued presence of the MoAb AK3 binding after this digestion confirmed that complete cleavage at this site did not occur. The cleavage patterns observed by flow cytometry were confirmed by surface-labeling experiments. These data indicated that the
quinine-dependent antibody was able to bind to both the 40-kD fragment
that results from cleavage at amino acid 293 and the 100-kD
glycocalicin fragment that resulted from cleavage close to the surface
of the cell. This result is consistent with that of our previous
study.2
The peptide inhibition studies have shown that the region encompassing
amino acids 283 to 293 of the GPIb protein is essential for the
binding of the quinine-dependent antibody. After incubation of the
patient serum with the specific peptide we observed a 62.0% ± 8.8% drop in the level of antibody binding. The unrelated peptides caused an insignificant decrease in the level of antibody binding. This
inhibition of antibody binding by the peptide specific for amino acids
283 to 293 indicates that the 11-amino acid region represented by this
peptide is involved in the quinine-dependent antibody binding to
GPIb. The incomplete inhibition by the specific peptide possibly
implies the epitope is not entirely linear but involves a degree of
conformational dependency which is not mirrored by the linear peptide.
We have shown that the quinine-dependent antibodies that bind to
GPIb-IX can be defined as two separate populations that bind to
independent domains on GPIX and GPIb, respectively. About 50% of
patients only have the antibody which binds to GPIX alone; only about
10% of them have the antibody which reacts with GPIb alone. About
40% of patients have both antibodies. Detailed characterization of the
binding domain on GPIb has shown that the quinine-dependent antibody
is interacting with the GPIb protein between amino acids 283 and
293. This is the first study to define the quinine-dependent antibody
binding sites on GPIb-IX in such detail. Identification of the binding
domain for the quinine-dependent antibodies may allow more detailed
studies of this and related regions to identify possible genetic
abnormalities that predispose individuals to the development of this
condition.
The amino acid sequence of importance for drug-dependent antibody
binding to platelets in quinine-induced thrombocytopenia may provide an
indication of regions of homology that act as the antigenic epitope on
other glycoproteins in drug-dependent immune reactions. Knowledge
obtained from the analysis of the molecular mechanism of
quinine-induced thrombocytopenia may contribute useful insights to the
pathogenesis of immune drug-induced damage to other tissues.
| |
FOOTNOTES |
Submitted December 8, 1997;
accepted May 21, 1998.
Supported by a grant from the National Health and Medical Research
Council of Australia.
Address reprint requests to Beng H. Chong, MBBS, PhD, Department of
Haematology, Prince of Wales Hospital, Cnr. High & Avoca St, Randwick,
NSW 2031, Australia; e-mail: b.h.chong{at}unsw.edu.au.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr S. Santoso for generously providing the MoAb Gi27 and Dr K. Berkner for the kind gift of the eukaryotic expression vector pDX. We
also thank Sue Evans for assistance with the MAIPA assays and Sasha
Tait for assistance with establishing the ELISA assay and the GPIX cDNA
transient transfection experiments during this study.
| |
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Abstract
Introduction
Abstract