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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 5 (March 1), 2000:
pp. 1849-1855
TRANSFUSIONMEDICINE
Single amino acid substitution in human platelet glycoprotein
Ib is responsible for the formation of the platelet-specific
alloantigen Iya
Ulrich J. H. Sachs,
Volker Kiefel,
Micaela Böhringer,
Vahid Afshar-Kharghan,
Hartmut Kroll, and
Sentot Santoso
From the Institute for Clinical Immunology and Transfusion Medicine,
Justus Liebig-University, Giessen, Germany; the Department of
Transfusion Medicine, University of Rostock, Rostock, Germany; and
Baylor College of Medicine and Veterans Affairs Medical Center,
Houston, TX.
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Abstract |
We recently described a new low-frequency platelet alloantigen on
the human platelet glycoprotein (GP) Ib-IX complex, termed Iya, which was implicated in a severe case of neonatal
alloimmune thrombocytopenia. Immunoprecipitation studies with
trypsin-treated platelets indicated that the Iya
alloantigenic determinants are formed by the membrane-associated remnant moiety of GP Ib (GP Ibr) together with GP
Ib and GP IX. To elucidate the molecular basis underlying the
Iya alloantigen, we amplified
GPIbr, GPIb, and
GPIX genes by polymerase chain reaction (PCR).
Nucleotide-sequence analysis of these 3 genes showed a G to A
transition at position 141 on GPIb gene in a subject
positive for Iya. This transition resulted in a
Gly15Glu dimorphism on the N-terminal domain of
GPIb. This finding was confirmed by genotyping analysis of 6 Iya-positive subjects by restriction fragment length
polymorphism (RFLP) studies using NarI endonuclease.
In 300 randomly selected healthy blood donors, one
Iya-positive individual was found. Phenotypes determined by
monoclonal antibody-specific immobilization of platelet antigens assay
and genotypes determined by RFLP were identical in this population. Analysis of Iya-positive platelets showed that the point
mutation affected neither the degree of surface expression nor the
function of the GP Ib-GP Ib-IX complex on the platelet surface.
Transient expression of the GP Ib-IX complex in CHO cells using
wild-type GP Ib (Gly15) or mutant GP Ib
(Glu15) allowed us to demonstrate that this single amino
acid substitution is sufficient to induce Iya epitope(s).
(Blood. 2000;95:1849-1855)
© 2000 by The American Society of Hematology.
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Introduction |
Human platelet glycoproteins (GP) are carriers of
alloantigenic determinants that can elicit an alloimmune response
leading to platelet destruction, such as occurs in neonatal alloimmune thrombocytopenia (NAIT), posttransfusion purpura, and platelet transfusion refractoriness.1 Four GP subunits (GP Ia, GP
Ib , GP IIb, and GP IIIa) on the platelet surface are known to be
polymorphic and immunogenic in humans.2,3 Two allelic
variants have been found for GP Ia and GP Ib, bearing human platelet
alloantigens (HPA) 5a/5b (Brb/Bra) and 2a/2b
(Kob/Koa), respectively.4,5 GP IIb
exists in 3 allelic variants carrying HPA-3a/3b
(Baka/Bakb) and HPA-9bW
(Maxa).6,7 GP IIIa is the most polymorphic
molecule. Ten allelic variants encoding GP IIIa have been found in the
human gene pool so far, 9 of which are immunogenic as carriers of
HPA-1a (PlA1) and HPA-4a (Yukb or
Pena), HPA-1b (PlA2), HPA-4b (Yuka
or Penb), HPA-6bW (Caa), HPA-7bW
(Moa), HPA-8bW (Sra), HPA-10bW
(Laa), HPA-11bW (Groa), and
Oea alloantigenic
determinants.8-15 Most of these alloantigens result from
point mutations in wild-type DNA that produce single amino acid
substitutions and lead to the expression of the offending alloantigenic
determinants. The Oea variant is an exception because it
results from a deletion of a codon of the mutated GP IIIa
(PlA2) isoform.15
We recently described a new low-frequency platelet alloantigen on the
GP Ib-IX complex, termed Iya, that was responsible for a
case of severe NAIT.16 GP Ib-IX complex is a receptor for
both von Willebrand factor and thrombin and plays an essential role in
adhesion of platelets to the subendothelium. GP Ib is a heterodimer
consisting of a large chain (molecular weight [MW], 143 kilodaltons [kd]) and a smaller disulfide-linked chain (MW, 27 kd). In the platelet membrane, GP Ib forms a noncovalent complex with
GP IX (MW, 22 kd).17 GP Ib is also weakly associated with
GP V (MW, 82 kd) in a noncovalent manner.18 All 4 GP are members of the leucine-rich GP family containing a variable
number of leucine repeats.19-22 GP Ib, GP Ib, GP IX,
and GP V are known to be derived from distinct genes, with the entire
open reading frame of the mature protein located within a single
exon.23-26 The GPIb gene is located on
chromosome 17,27 whereas the GPIb gene is on
chromosome 22.24 The GPIX and GPV genes are
on distinct sites of the long arm of chromosome 3, on band q21 and band
q29, respectively.28
We here report the first molecular variant of GP Ib responsible for
the formation of a clinically important alloantibody in NAIT. Because
this new alloantigen was found in 1 of 300 healthy blood donors tested,
it may be involved in other cases of alloimmune thrombocytopenia.
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Materials and methods |
Monoclonal antibodies (MAB)
MAB Gi10 and Gi27 against the remnant moiety of GP Ib and GP
Ib, respectively, were raised and characterized in our
laboratory.12,29 MAB SZ2 and AN51 specific for GP
Ib30,31 were purchased from Dianova and Dako (both
Hamburg, Germany). MAB FMC25 against GP IX32 was provided
by H. Zola, Adelaide, Australia.
Phenotyping
Phenotyping of human platelets for the presence of Iya
alloantigenic determinants was performed by using the monoclonal
antibody-specific immobilization of platelet antigens (MAIPA) assay as
described previously.33 MAB FMC 25 was used as capture
antibody.16,32
Immunoprecipitation analysis
Washed platelets from ACD-anticoagulated blood were labeled with
biotin hydrazide (Pierce, Munich, Germany) as described by Fabris et
al, with minor modifications.34 Briefly, 109
platelets in 900 µL of phosphate-buffered saline (PBS)-EDTA (3.72 g/L
of EDTA, 1 µmol/L of leupeptin, 1 mmol/L of phenylmethylsulfonyl fluoride (PMSF), 4 mmol/L of N-ethylmaleimide
in PBS; pH 7.4) were exposed to 100 µL of 12 mmol/L
sodium metaperiodate at 4°C for 10 minutes; 0.6 mol/L of glycerol
was then added. After being washed twice with 500 µL of PBS-EDTA, the
platelets were incubated with 3 mmol/L of biotin hydrazide at room
temperature for 2 hours. Labeled platelets were washed 4 times and
lysed in 1 mL solubilization buffer (25 mmol/L of Tris, 10 mmol/L of
EDTA, 100 mmol/L of sodium chloride (NaCl) containing 1% Triton X-100,
2 mmol/L of PMSF, 1 mmol/L of leupeptin, and 2 mmol/L of
N-ethylmaleimide) for 30 minutes at
4°C.
Aliquots of 109 labeled platelets were digested with 100 µL of trypsin (1 mg/mL; Sigma, Deisenhofen, Germany) for 5 minutes at
37°C. Digestion was stopped by adding 200 µL of soybean trypsin inhibitor (1 mg/mL; Sigma). Trypsin-treated platelets were washed twice
and then solubilized as described above.
After centrifugation (30 minutes at 16 000g at 4°C),
immunoprecipitation was performed as described
previously.12 Immunoprecipitates were separated by using
7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), transferred to nitrocellulose membrane, and visualized with
use of streptavidin-peroxidase and chemiluminescence substrate. Colored
protein MW markers (Rainbow; Amersham, Braunschweig, Germany) were used
as the standard.
Immunoblotting
Aliquots of 109 washed platelets were lysed in 1 mL of
solubilization buffer. After centrifugation (30 minutes at 16 000g
at 4°C), proteins were separated with SDS-PAGE and transferred
to nitrocellulose membrane. Membrane strips were blocked with 1.5% bovine serum albumin (BSA) in PBS and then incubated with an MAB dilution (20 µg/mL) or serum for 30 minutes at room temperature. The
strips were washed twice with Tris buffer (pH 7.4) containing 0.05%
Tween 20 and then incubated with peroxidase-conjugated rabbit antimouse
or antihuman antibodies (1:200 000 dilution; Dianova). After washing,
the recognized protein was visualized by using the enhanced
chemiluminescence substrate system (Amersham).
Determination of GP Ib-IX binding sites
Aliquots of 2 × 107 washed platelets were
incubated with increasing amounts of MAB Gi10 (50-100 µg) at 37°C
for 30 minutes. The sensitized platelets were washed 3 times with 0.2%
BSA in isotonic saline before resuspension in 80 µL of isotonic
saline. Bound MAB were eluted with 40 µL of 100 mmol/L of NaCl (pH
2.2; adjusted with acetic acid) containing 1.5% BSA for 10 minutes at
room temperature. After centrifugation, eluates were neutralized with a
predetermined volume (about 3.3 µL) of 2.5 mmol/L of Tris buffer. A
sandwich enzyme-linked immunoassay (ELISA) using purified normal mouse
IgG (m-IgG) as the standard was used to quantify the number of binding
sites, as reported previously.12 In brief, microtiter wells
were coated overnight with 100 µL of goat antimouse IgG
F(ab')2 (1:1000 dilution in coating buffer; Dianova)
at 4°C. After being washed 3 times with 200 µL of 1% BSA in PBS
(PBS-BSA), wells were blocked with 200 µL of PBS-BSA for 30 minutes
at 4°C and then incubated with either 100 µL of eluates or 100 µL of various dilutions of m-IgG (2500-50 pg) for 1 hour at 37°C.
Afterward, the wells were washed 3 times, and 100 µL of alkaline
phosphatase-labeled goat antimouse IgG Fc (1:1000 dilution, Dianova)
was added. After 1 hour of incubation at 37°C, the wells were
washed 5 times. Finally, 100 µL of p-nitrophenylphosphate
substrate solution (Sigma) was added and the plate was incubated at
room temperature for 30 minutes. The color reaction was stopped by
adding 50 µL of 3 mol/L of sodium hydroxide and was read at 405 nm in
a Titertek photometer (Helsinki, Finland). All samples
were assayed in duplicate.
Platelet function studies
Platelet-rich plasma (PRP) was obtained by centrifugation (200g
for 15 minutes) of ACD-anticoagulated blood collected from Iya-positive and Iya-negative individuals. The
platelet count was adjusted to 3 × 105 per µL by
dilution with autologous plasma. To aliquots of 180 µL of PRP, 20 µL of various dilutions of ristocetin (2.5, 5, 10, and 15 µg/mL)
were added and the change in optical density was monitored by using an
aggregometer, with continuous stirring, at 37°C.
To evaluate the functional effect of anti-Iya antibodies,
aliquots of 180 µL of PRP derived from Iya-positive
individuals were mixed with either 20 µL of isotonic saline, MAB SZ2
(20 µg/mL), or 20 µL eluates of heat-inactivated serum (normal
human serum [NHS] or anti-Iya) and incubated at 37°C
for 30 minutes in an atmosphere supplemented with 5% carbon dioxide
(CO2). After stimulation with ristocetin (15 µg/mL),
platelet aggregation was recorded as described above.
Isolation and amplification of genomic DNA
Genomic DNA was isolated from 10 mL of EDTA-anticoagulated blood
from Iya-phenotyped donors as described
previously.12 Primers used to amplify the
GPIbr, GPIb, and
GPIX genes (Table 1) were
constructed according to published DNA sequences.20,23,25
The coding regions of the GPIb gene encompassing nucleotides
826 to 1965 were amplified in 2 overlapping fragments (bases 826 to
1518 and bases 1192 to 1965) by using primer pairs GP Ib 1-GP Ib
2 and GP Ib 3-GP Ib 4, respectively. Amplification was performed
in a total volume of 50 µL containing 10 µL of genomic DNA (400-600 ng), 0.3 µmol/L of each primer, 200 µmol/L of each dNTP, 1.5 mmol/L
of magnesium chloride (MgCl2), and 1.5 U of Taq
GOLD polymerase on a GeneAmp 9600 DNA thermal cycler (Perkin Elmer,
Weiterstadt, Germany). After heating at 97°C for 5 minutes,
polymerase chain reaction (PCR) was performed under the following
conditions. For amplification of the first region (bases 826-1518),
denaturation was done for 75 seconds at 94°C, annealing for 120 seconds at 52°C, and extension for 180 seconds at 72°C. For the
second region (bases 1192-1965), denaturation was done for 60 seconds
at 95°C, annealing for 90 seconds at 55°C, and extension for
120 seconds at 72°C. Both amplifications proceeded for 36 cycles. In the final cycle, all samples were kept at
72°C for 10 minutes and then chilled to 4°C.
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Table 1.
Sequences and positions of primers used in the
polymerase chain reaction amplification of GPIbr,
GPIb, and GPIX genes
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The entire coding region of GPIb (nucleotides 47-939) was
amplified by using 0.5 µmol/L each of GP Ib 1 and GP Ib 2
primer, 200 µmol/L each of dNTP, 1.5 mmol/L of MgCl2,
10% dimethyl sulfoxide (DMSO), 1.5 U µL of Taq GOLD
polymerase, and 5 µL of 10 × PCR buffer. A cycle consisted of
denaturation at 94°C for 75 seconds, annealing at 48°C for
90 seconds, and primer extension at 72°C for 120 seconds and was
repeated 35 times.
The coding region of GPIX (nucleotides 271-750) was amplified
by using 0.5 µmol/L each of GP IX 1 and GP IX 2 primer, 200 µmol/L
each of dNTP, 1.2 mmol/L of MgCl2, 0.01% gelatin, 1.5 U of
Taq GOLD polymerase, and 5 µL 10 × PCR buffer under
the same PCR conditions used for the GPIb gene except that
the annealing was done at 52°C.
Subcloning and sequencing
All PCR products were purified on 1.5% SeaKem agarose gel (FMC,
Hessisch Oldendorf, Germany) by using Geneclean (Dianova). Purified DNA
was flushed with Klenow DNA polymerase (Biolabs, Schwalbach, Germany)
for blunt-end ligation into the EcoRV site of the pGEM5 plasmid
and then transformed into DH5 high-efficiency competent
Escherichia coli (Gibco BRL, Eggenstein, Germany). Before ligation, the GP Ib PCR product was shortened by digestion with SmaI endonuclease (Biolabs). Recombinant colonies were selected by blue-white screening on indicator plates. For single-strand nucleotide sequencing, plasmid DNA from 8 positive clones of each region was amplified with use of biotinylated forward primer
5'-CGC CAG GGT TTT CCC AGT CAC GAC G-3' and nonbiotinylated
reverse primer 5'-GCT TCC GGC TCG TAT GTT GTG TGG-3' or
vice versa. Biotinylated single-strand DNA was isolated by magnetic
beads (Dynal, Norway), sequenced with SP6 and T7 primers using a
fluorescence DNA-sequencing kit (Perkin Elmer), and then analyzed on
ABI Prism 373 DNA Sequencers (Applied Biosystems, Weiterstadt, Germany).
Genotyping by restriction fragment length polymorphism (RFLP)
Genomic DNA was amplified by using primer pair GP Ib 1-GP Ib 3 as described above, except that "hot start" PCR was saved by
application of Taq GOLD polymerase (1.5 U, Perkin Elmer).
Aliquots of 7 µL of PCR products were subjected to RFLP using 2 U of
NarI endonuclease (Biolabs) and then analyzed on 3.0% NuSieve
agarose gel (Gibco).
Construction of allele-specific GP Ib expression vectors
A full-length complementary DNA (cDNA) encoding wild-type GP
Ib in pDX plasmid was removed with EcoRI (Biolabs) and
ligated into the mammalian pcDNA3.1 Zeo expression vector (Invitrogen, Leek, Holland). Specific mutation G A at position 141 was
induced in the wild-type GP Ib construct by site-directed
mutagenesis with use of a QuickChange Mutagenesis Kit (Strategene,
Heidelberg, Germany). For PCR amplification, single
nucleotide-mismatched sense primer 5'-GGG ACG CTC GTG GAC TGC GAG
CGC CGC GGG CTG ACT TGG-3' and antisense primer 5'-CCA AGT
CAG CCC GCG GCG CTC GCA GTC CAC GAG CGT CCC-3' corresponding to
base 122 to 160 of GP Ib cDNA were constructed. After 12 cycles of
amplification (denaturation for 30 seconds at 95°C, annealing for
60 seconds at 55°C, and extension for 12 minutes at 68°C) in
the presence of 10% DMSO, PCR product was digested with DpnI
and transformed into DH5 high-efficiency competent E coli.
Plasmid DNA from positive clones was amplified by PCR using GP Ib 1 and GP Ib 3 primers, and subjected to RFLP analysis with
NarI as described above. Purified GP Ib allele-specific constructs used for subsequent transfection were validated by nucleotide-sequence analysis.
Cell culture and transfection
CHO cells stably expressing GP Ib and GP IX35 were
transiently transfected by liposome-mediated delivery of plasmid DNA with use of a commercially available kit (Lipofectamine, Gibco BRL,
Grand Island, NY). Cells were grown to approximately 70% confluence on
50-mm2 tissue culture dishes. Twelve microliters of
liposome suspension and 2 µg of plasmid DNA (either pcDNA 3.1 Zeo
vector containing the wild-type or mutated cDNA of GP Ib, or the
vector alone) were separately mixed in 200 µL of serum-free medium.
The 2 suspensions were then combined, mixed gently, and allowed to form
DNA-liposome complexes for 30 minutes at room temperature. The mixture
was diluted in 1.6 mL of serum-free medium and added to the cells, which had been washed twice with the same medium. The cells were exposed to the mixture for 5 hours under standard culture conditions (37°C in 5% CO2), after which 2 mL of medium
containing 10% fetal-calf serum was added. Twenty-four hours later,
the medium was changed.
Flow cytometry
Forty-eight hours after transfection, cells were detached from the
dishes with 0.54 mmol/L of EDTA and washed twice with PBS. Cells were
fixed in 1% paraformaldehyde and washed twice. A total of 400 000
cells were counted by hemacytometer, resuspended in 200 µL of PBS,
and incubated with 50 µL of serum (either anti-Iya serum
or NHS) for 30 minutes at 37°C. The cells were washed twice with
PBS and resuspended in 200 µL of PBS. They were incubated with 40 µL of a 1:40-diluted fluorescein isothiocyanate-conjugated rabbit
antihuman antibody (Dianova) for 30 minutes at 37°C. To remove
unbound antibody, cells were washed twice. A total of 10 000 cells
from each transfection were analyzed with an Ortho Cytoron Absolute
flow cytometer (Ortho Diagnostic Systems, Raritan, NJ).
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Results |
Immunochemical characterization
To characterize the Iya antigen, immunoprecipitation
studies with surface-labeled biotinylated platelets were performed
(Figure 1). When anti-Iya
immunoprecipitate was electrophoresed under reducing conditions, 3 bands GPIb, GPIb, and GP IXcould be detected, with apparent MW
of 145 kd, 27 kd, and 22 kd, respectively (Figure 1, left panel, lane
1). In the control experiments, these bands could not be precipitated
from Iya-negative platelets (Figure 1, lane 2).
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| Fig 1.
Immunoprecipitation analysis of the GPIb-Ib-IX
complex of Iya-phenotyped platelets.
Platelets from Iya-positive (lane 1) and
Iya-negative (lane 2) individuals were surface labeled with
biotin, lysed, and immunoprecipitated with anti-Iya (left
panel) and monoclonal antibody (MAB) Gi27 (right panel).
Immunoprecipitates were analyzed with 7.5% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under
reducing conditions, transferred to nitrocellulose membrane, and
visualized by using a streptavidin-horseradish
peroxidase-chemiluminescence substrate system.
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To analyze the surface expression of the GP Ib-IX complex in
Iya-positive platelets, we compared the levels of GP Ib-IX
precipitates from Iya-positive and Iya-negative
platelets by immunoprecipitation (Figure 1, right panel). When MAB Gi27
directed against GP Ib was used, similar amounts of GP Ib, GP
Ib, and GP IX were precipitated from both platelet phenotypes. In
addition, in both platelet types, GP Ib, GP Ib, and GP IX
subunits migrated with similar mobility. Similar results were obtained
with MAB SZ2 directed against the GP Ib subunit and with MAB FMC25
directed against the GP IX subunit (data not shown). These observations
indicate that normal amounts of GP Ib-IX complex are expressed on the
surface of Iya-positive platelets and that the
Iya antigen is not associated with an MW polymorphism.
To further localize the epitope recognized by anti-Iya
antibodies, we took advantage of the fact that trypsin cuts off the amino-terminal part of GP Ib, leaving GP Ibr as the
remnant moiety, which is associated with GP Ib and GP IX on the
platelet surface.36 After trypsin treatment,
anti-Iya antibodies (Figure 2,
lane 4) still precipitated the GP Ibr-Ib complex (MW,
65 kd) and GP IX subunit (MW 22, kd) under nonreducing conditions, as
did MAB Gi10 and Gi27 (Figure 2, lanes 2 and 3). In contrast, MAB SZ2
(Figure 2, lane 1), directed against the glycocalicin moiety, did not
precipitate any platelet proteins.
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| Fig 2.
Immunoprecipitation analysis of the GPIb-Ib-IX
complex of Iya-phenotyped platelets after trypsin
treatment.
Aliquots of 109 biotinylated platelets from an
Iya-positive individual were treated with trypsin, washed,
lysed, and immunoprecipitated with MAB SZ2 (lane 1), MAB Gi10 (lane 2),
MAB Gi27 (lane 3), and anti-Iya antibodies (lane 4).
Immunoprecipitates were analyzed with 7.5% SDS-PAGE under nonreducing
conditions.
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In immunoblotting analysis, no reactivity of anti-Iya
antibodies with any platelet proteins was detectable (Figure
3, lane 1) under either nonreducing or
reducing conditions. In the control experiments, MAB SZ2 (Figure 3,
lane 2) reacted with GP Ib (both and subunits) under
nonreducing conditions. Under nonreducing conditions, MAB Gi27 (Figure
3, lane 3) showed binding to GP Ib and to its proteolytic fragment, GP
Ibr. Under reducing conditions, MAB SZ2 recognized the GP Ib subunit
and MAB Gi27 recognized the GP Ib subunit.
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| Fig 3.
Immunoblotting studies.
Aliquots of 109 washed platelets from an
Iya-positive individual were lysed, and proteins were
separated by using 7.5% SDS-PAGE under nonreducing (n.r.) and reducing
(r.) conditions. After proteins were transferred to a nitrocellulose
membrane, membrane strips were incubated with anti-Iya
antiserum (lane 1), MAB SZ2 (lane 2), and MAB Gi27 (lane 3). Antibody
binding was detected by using corresponding peroxidase-conjugated
antibodies and chemiluminescence substrate.
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Amplification and analysis of GPIb, GPIb, and
GPIX genes
Because anti-Iya antibodies bind to trypsin-treated
platelets, we predicted that the region formed by amino acid residues
450 to 610 (nucleotides 1440-1920) of GPIb, together with
GPIb and GPIX, would carry the Iya
epitope(s). To analyze this region, we amplified the GPIb
gene in 2 overlapping fragments (nucleotides 826-1518 and 1192-1965) and the entire coding regions of the GPIb (nucleotides
47-939) and GPIX genes (nucleotides 271-750). PCR products of
GPIb, GPIb, and GPIX genes from an
Iya-positive individual migrated with the same
electrophoretic mobility as PCR products derived from an
Iya-negative individual (data not shown). All PCR products
were subcloned, and 6 independent clones from each fragment were
subjected to nucleotide-sequence analysis.
Nucleotide-sequence analysis of GPIb and GPIX
fragments from an Iya-positive individual and an
Iya-negative individual did not show any differences in
nucleotides (data not shown). However, nucleotide-sequence analysis of
the 517-base-pair (bp) fragment of GPIb encoding
nucleotides 47-563 from an Iya-positive individual revealed
a single G to A substitution at base 141 in 3 of 6 subclones examined
(Figure 4). In contrast, all clones from an
Iya-negative individual encoded a G at this position (data
not shown). These results are consistent with the idea that
Iya-positive individuals are usually heterozygous for this
low-frequency antigen. The G to A substitution changes a GGG codon for
glycine to GAG, which encodes for glutamic acid at amino acid 15 of the mature GP Ib.
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| Fig 4.
DNA-sequence analysis of amplified GPIb gene
of an Iya-positive individual.
Polymerase chain reaction (PCR) products of the GPIb gene
encompassing nucleotides 47-563 were subcloned in the plasmid vector
pGEM-5Zf and sequenced on both strands. Nucleotide-sequences of 2 positive clones are shown. (A) The wild-type G in position 141 is
changed to an A (arrow), predicting a glycine to glutamic acid
(GGGGAG) polymorphism at position 15 of the mature
glycoprotein. (B) The sequence is identical to the published wild-type
sequence for GPIb.20
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Correlation of the G141A dimorphism with the
Iya phenotype
Along with the Iya-positive subjects from the index
family,16 one Iya-positive individual (Kr) was
identified among 300 German blood donors phenotyped for the
Iya antigen with the MAIPA assay. The pedigrees of both
families are shown in Figure 5. No case of
NAIT was observed in the Kr family. To determine whether the G to A
substitution at position 141 of the GPIb gene segregates
with the Iya phenotype, we established genomic DNA typing
with a PCR-RFLP technique. The G to A substitution abolishes a cleavage
site for the restriction endonuclease NarI, which cleaves at
5'-GG CGCC-3' but not at 5'-AGCGCC-3'
sequences (Figure 6A). We
used this technique to genotype 3 members of the index family (A.I.1,
A.I.2, and A.II.1 in Figure 5) and all members of the Kr family (Figure
5B and Figure 6B). After amplification of genomic
DNA by using primer pair GP Ib 1 and GP Ib 3, the 332-bp PCR
product was digested with NarI. All Iya-negative
individuals had 140-bp, 96-bp, 58-bp, and 38-bp restriction fragments.
All Iya-heterozygous individuals could be differentiated
from the Iya-negative subjects by the presence of an
additional 178-bp fragment. The results of the genotyping of 300 unrelated individuals correlated with the phenotyping results.
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| Fig 5.
Pedigrees of the index family and the Kr family.
In the Iy (index) family (A), the family member with the index case of
neonatal alloimmune thrombocytopenia (NAIT) is marked with an asterisk
(A.III.2). A third pregnancy was interrupted because of massive
intraventricular bleeding in the fetus (A.III.3). All individuals were
phenotyped with use of the monoclonal antibody-specific immobilization
of platelet antigens assay. Material for genotyping was available only
from individuals A.I.1, A.I.2, and A.II.1. One member of the Kr family
(B) was found in assessing 300 healthy blood donors. All individuals
were phenotyped and genotyped. No case of NAIT was observed in the Kr
family.
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| Fig 6.
PCR-restriction fragment length polymorphism analysis of
Iya-phenotyped individuals with use of
NarI.
(A) A 332-base-pair product encompassing nucleotides 47-378 of the
GPIb gene was obtained from genomic DNA by using primers
GPIb 1 and GP Ib 3. The arrows indicate the cleavage sites
recognized by the restriction endonuclease NarI. The
length of the expected digested fragments from Iya-positive
and Iya-negative alleles is also shown. (B) Analysis of
NarI-digested PCR products from genomic DNA of the members of
the Iy family (left panel) and the Kr family (right panel). Lanes are
inscribed according to the pedigrees (Figure 4). Undigested products
from samples obtained from 3 individuals are shown in lanes 1 to 3 (left panel). In lane M, pBr 322 HaeIII DNA fragments are
shown as standards.
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Expression of the wild-type and mutated GP Ib-Ib-IX
complex in CHO cells
To demonstrate that the G to A substitution at nucleotide 141 of
GPIb cDNA is sufficient to induce formation of the epitope recognized by anti-Iya serum, we performed transient
transfection of CHO cells expressing GP Ib and GP IX with eucaryotic
expression vectors carrying wild-type or mutated GP Ib cDNA. Two
days after transfection, surface expression of Iya
epitope(s) was examined by flow cytometry after staining with anti-Iya serum. Sham-transfected CHO cells solely
expressing GP Ib and GP IX failed to bind anti-Iya
antibodies (data not shown). CHO cells expressing the wild-type complex
also did not bind anti-Iya antibodies (Figure
7A, dark curve), whereas
those expressing the mutated GP Ib-IX complex showed antibody binding
(Figure 7B, arrow). NHS was used as a
negative control (Figure 7A and 7B, bright curves). These
results were reproduced in independent transfection experiments in 2 different laboratories.
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| Fig 7.
Detection of expression of Iya epitope on
recombinant GP Ib-Ib-IX complexes by flow cytometry.
CHO cells stably expressing GP Ib and GP IX were transiently
transfected with eucaryotic expression vectors carrying wild-type (A)
or mutated (B) complementary DNA for GPIb. Forty-eight hours after
transfection, expression of the Iya epitope on the cell
surface was determined by using anti-Iya antibodies (dark).
Normal human serum was used as a negative control (bright). The arrow
indicates the subpopulation of cells showing antibody binding.
|
|
Effect of G to A mutation on the expression and function of GP
Ib-IX complex
To determine whether the G to A mutation influences the efficiency
of expression of the GP Ib-IX complex on the platelet surface, binding
isotherms were generated by using MAB Gi10 in a quantitative sandwich ELISA.
In accordance with the findings from our immunoprecipitation analysis,
platelets from Iya-positive individuals bound amounts of
MAB Gi10 (22 753 ± 200 molecules per platelet; n = 3) similar
to the amounts bound by platelets from Iya-negative
individuals (23 000 ± 300 molecules per platelet; n = 3).
To determine the possible effect of the point mutation on the ability
of the GP Ib-IX complex to bind von Willebrand factor, platelets from
an Iya-positive individual were compared with those from 3 Iya-negative individuals in standard platelet aggregation
assays. The ristocetin-induced agglutination of
Iya-positive platelets was indistinguishable from that of
Iya-negative platelets (data not shown). All these
results suggest that neither the expression nor the function of the GP
Ib-IX complex is affected by the Gly15Glu mutation.
When ristocetin-induced agglutination was performed in the presence of
purified anti-Iya antibodies, no inhibition was observed in
platelets from either Iya-positive or
Iya-negative individuals. In the control experiments, MAB
SZ2 (20 µg/mL) completely inhibited the ristocetin-induced
agglutination (data not shown).
| |
Discussion |
We report the characterization of the new platelet-specific
alloantigen, Iya, that was responsible for a severe case of
NAIT. Our immunochemical studies demonstrated that anti-Iya
precipitated GP Ibr, GP Ib, and GP IX from
trypsin-treated platelets. Because we were unable to detect reactivity
of the Iya antibody with either serum or affinity-purified
antibody in immunoblot analysis, we predict that the alloantigenic
determinants of Iya are dependent on protein conformation
sensitive to denaturation by SDS.
To elucidate the molecular basis underlying the Iya
antigen, we amplified GPIbr,
GPIb, and GPIX genes from genomic DNA derived from
Iya-positive and Iya-negative individuals.
Nucleotide-sequence analysis of the GPIb, GPIb,
and GPIX genes showed a single G to A transition at nucleotide 141 in the GPIb gene, changing glycine to glutamic acid at
residue 15 of the mature GP Ib protein. RFLP analysis using the
restriction endonuclease NarI, which is capable of
discriminating between these 2 alleles, demonstrated that this
nucleotide substitution correlated with the serologic phenotypes of 6 Iya-positive individuals from 2 independent families and
300 Iya-negative unrelated blood donors.
The Gly15Glu dimorphism of the GP Ib protein represents
the only difference between Iya-positive and
Iya-negative individuals. Flow cytometry analysis that used
CHO cells expressing the wild-type (Gly15) or mutated
(Glu15) GP Ib-Ib-IX complex on their surface showed
that this single amino acid substitution is directly responsible for
formation of the Iya alloantigenic determinant(s).
The actual Iya-antibody binding sites are probably
complex-specific, formed by GP Ib and other GP Ib subunits. Which GP
Ib subunitGP Ib, GP IX, or bothis required for the alloantigenic formation remains unclear. However, expression of recombinant mutated
GP Ib in CHO cells confirmed that this single amino acid substitution is sufficient to induce formation of the epitope(s) recognized by anti-Iya serum.
Bernard-Soulier syndrome (BSS) is an extremely rare autosomal recessive
bleeding disorder in which patients have a low platelet count and large
platelets that are unable to adhere to subendothelium. The functional
defect lies in the inability of the platelets to bind von Willebrand
factor.37 In patients with classic BSS, the level of GP
Ib-IX complex is greatly reduced because of abnormalities (mutations
and deletions) in the GPIb-IX genes that lead to a biosynthetic
defect affecting expression, processing, or synthesis of the complex.
In contrast, patients with variant BSS have normal amounts of GP Ib-IX
complex, but the complex is dysfunctional. Recently, BSS variants due
to point mutations in a leucine-rich domain of GP Ib and GP IX have
been identified.38-40 So far, only 2 GP Ib mutations
have been reported to be associated with BSS. The first involved a
giant-platelet syndrome caused by point mutations that led to amino
acid substitutions Tyr to Cys at residue 88 and Ala to Pro at residue
108 of the mature glycoprotein.41 The patient was
heterozygous for these mutations. It was suspected that the Tyr to Cys
transition affected the disulfide linkage between GP
Ib and GP Ib. The second mutation was in a patient with
velocardiofacial syndrome who had a point mutation at position  133 (C133G) and deletion of the other
allele.42
Studies of Iya-positive platelets showed that the
Gly15Glu point mutation of the GP Ib gene does not
impede expression or function of the GP Ib-IX complex. Wright et
al39 observed variable band shifts within the GP Ib
coding region in a few patients with BSS and healthy blood donors by
means of single-stranded conformation polymorphism. However, this
sequence variation did not show any linkage to the BSS phenotype. Our
RFLP analysis of these patients with BSS showed that this polymorphism
is not related to the Gly15Glu polymorphism (data not shown).
So far, all the alloantigenic epitopes responsible for the observed
cases of NAIT, posttransfusional purpura, and refractoriness to
platelet transfusion have been found to be on GP Ia, GP Ib, GP IIb,
or GP IIIa. The elucidation of the Iya
alloantigen represents the first characterization of an immunologically important polymorphism of GP Ib.
| |
Acknowledgments |
We thank Dr M. Vicariot and Dr Y. Giovangrandi of the Centre
Hospitalier et Universitaire de Brest, France, who observed the index
case and provided anti-Iya serum; Dr J. Clemetson, Theodor
Kocher Institute, University of Bern, Switzerland, for helpful
discussions; Dr E. G. D. Tuddenham, Imperial College Medical School,
London, United Kingdom, for providing genomic DNA from patients with
Bernard-Soulier syndrome; and Ms Dagmar Westrup for technical support
in performing nucleotide-sequence analysis at the Institute for
Transfusion Medicine and Immunohematology, German Red Cross Blood Donor
Service, Frankfurt/ Main, Germany.
| |
Footnotes |
Submitted August 13, 1999; accepted October 19, 1999.
Supported by grants from the German Research Foundation (DFG
Sa480/2-1). This work is part of a PhD thesis (UJHS).
Reprints: S. Santoso, Institute for Clinical Immunology and
Transfusion Medicine, Langhansstr 7, D-35392 Giessen, Germany; e-mail:
sentot.santoso{at}immunologie.med.uni-giessen.de.
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.
| |
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Abstract
Introduction