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Blood, 1 March 2002, Vol. 99, No. 5, pp. 1833-1839
TRANSPLANTATION
HPA-1a phenotype-genotype discrepancy reveals a naturally
occurring Arg93Gln substitution in the platelet 3 integrin that
disrupts the HPA-1a epitope
Nicholas A. Watkins,
Elisabeth Schaffner-Reckinger,
David L. Allen,
Graham J. Howkins,
Nicolaas H. C. Brons,
Graham A. Smith,
Paul Metcalfe,
Michael F. Murphy,
Nelly Kieffer, and
Willem H. Ouwehand
From the Department of Haematology, Division of
Transfusion Medicine, University of Cambridge, United Kingdom; the
Laboratoire Franco-Luxembourgeois de Recherche
Biomédicale, (CNRS/CRP-Santé), Luxembourg, Grand-Duchy
of Luxembourg and National Blood Service East Anglia Centre, Cambridge
and Oxford Centre, Oxford, United Kingdom; and the Division of
Haematology, National Institute for Biological Standards and Control,
Potters Bar, United Kingdom.
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Abstract |
A single nucleotide polymorphism (SNP) at position 196 in the 3
integrin causes a Leu33Pro substitution in the mature protein. Alloimmunization against the 3Leu33 form (human platelet antigen [HPA]-1a, PlA1, Zwa) in patients
who are 3Pro33 homozygous (HPA-1b1b,
PlA2A2, Zwbb) causes neonatal
alloimmune thrombocytopenia, posttransfusion purpura, or refractoriness
to platelet transfusion. Studies with recombinant proteins have
demonstrated that amino acids 1 to 66 and 288 to 490 of the 3
integrin contribute to HPA-1a epitope formation. In determining the
HPA-1a status of more than 6000 donors, we identified a donor with an
HPA-1aweak phenotype and an HPA-1a1b genotype. The
platelets from this donor had normal levels of surface IIb 3 but
reacted only weakly with monoclonal and polyclonal anti-HPA-1a by
whole blood enzyme-linked immunosorbent assay (ELISA), flow cytometry,
and sandwich ELISA. We reasoned that an alteration in the primary
nucleotide sequence of the 3Leu33 allele of this donor was
disrupting the HPA-1a epitope. In agreement with this hypothesis,
sequencing platelet RNA-derived IIb and 3 cDNA identified a novel
G/A SNP at position 376 of the 3 integrin that encodes for an
Arg93Gln replacement in the 3Leu33 allele. Coexpression of the
3Leu33Gln93 encoding cDNA in Chinese hamster ovary cells with human
IIb cDNA showed that the surface-expressed IIb 3 reacted
normally with 3 integrin-specific monoclonal antibodies but only
weakly with monoclonal anti-HPA-1a. Our results show that an Arg93Gln
mutation in the 3Leu33 encoding allele disrupts the HPA-1a epitope,
suggesting that Arg93 contributes to the formation of the HPA-1a B-cell epitope.
(Blood. 2002;99:1833-1839)
© 2002 by The American Society of Hematology.
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Introduction |
The 3 integrin subunit (glycoprotein [GP]
IIIa, CD41) forms a heterodimeric complex with the IIb integrin
subunit (GPIIb) on the surface of platelets ( IIb 3, GPIIb/IIIa,
CD61/41) and functions as a major fibrinogen receptor. Activation of
IIb 3 occurs through so-called inside-out signaling that follows
the binding of platelet receptors to components of the subendothelial cell matrix (eg, the binding of 2 1 and
GPVI to collagen) or soluble ligands (eg, adenosine diphosphate and
thrombin). The activated conformation of IIb 3 binds fibrinogen,
fibronectin, and vitronectin and has a pivotal role in clot formation
after blood vessel damage.1,2
The gene encoding 3 integrin has several single nucleotide
polymorphisms (SNPs) that result in single amino acid substitutions of
immunologic, and possibly functional, consequence.3
Platelet alloantigen systems encoded by SNPs in the 3 integrin gene
are of clinical relevance. The C196T SNP, encoding for a Leu33Pro substitution, is the most immunogenic human platelet alloantigen (HPA)
system.4 In 3Leu33-negative (ie, 3Pro33 homozygous), HLA-DRB3*0101-positive persons, exposure to the 3Leu33 form is highly immunogenic and alloimmunization causes neonatal alloimmune thrombocytopenia, posttransfusion purpura, and platelet
refractoriness.3,5-7 Alloimmunization against 3Leu33
occurs in 1 in 365 pregnant women, and 3Leu33-specific
maternal alloantibodies (anti-HPA-1a) cause severe thrombocytopenia in
1 in 1100 neonates.8,9 In such cases, the treatment of
choice is 3Leu33-negative donor platelets, the provision of which
requires the phenotyping of large numbers of donors.5,10
We have previously reported on a recombinant human immunoglobulin
(Ig)G1 specific for 3Leu33, which can be used for large-scale donor
phenotyping.11-13 In the process of phenotyping more than
6000 donors using this assay, we identified one donor with a
3Leu33weak phenotype but a heterozygous genotype. Here
we describe the molecular basis of this unique phenotype, suggesting
that Arg93 of the 3 integrin contributes to the formation of the
HPA-1a B-cell epitope.
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Patients, materials, and methods |
Donor samples
More than 6000 EDTA-anticoagulated whole blood donor samples
were 3Leu33 phenotyped by enzyme-linked immunosorbent assay (ELISA)
with our recombinant antibody CAMTRAN-007 as described previously.11 A single donor (Donor A) with a
3Leu33weak phenotype and a heterozygous genotype was
identified. Genomic DNA samples from healthy apheresis donors were from
the National Blood Service donor DNA repository. Informed consent was
obtained for all samples.
Antibodies
Mouse monoclonal antibodies (mAbs) specific for platelet
integrins and glycoproteins were obtained as follows. Anti- IIb 3 integrin clones RFGP56 and NIBSC-85/661 have been reported in detail
elsewhere14,15; anti- 3 integrin clone Y2/51 from DAKO (Cambridge, United Kingdom); anti- 3 integrin mAb P37 was a kind gift
from Dr J. Gonzalez-Rodriguez (Instituto de Quimica Fisica, Madrid, Spain). Recombinant human IgG1 anti- 3Leu33 clone
CAMTRAN-00711,13 and mouse mAb clone 9E10 specific for the
c-myc tag were provided by The International Blood
Group Reference Laboratory (Bristol, United Kingdom). Recombinant human
IgG1 anti- 3Leu33 clones 19-7 and 23-15 were a kind gift from Dr
Louis Thiobault (Héma-Québec, Canada).16 Human
polyclonal sera were from the National Blood Service serum archives and
were obtained from patients previously referred for investigation of
neonatal alloimmune thrombocytopenia.
3Leu33 typing
Whole blood phenotyping using the recombinant human IgG1
anti- 3Leu33 CAMTRAN-007 was performed as described
previously.11 Results were interpreted as 3Leu33
negative if the optical density (O.D.) was less than 0.2 and as
positive if the O.D. was more than 1.2. Any O.D. between these values
was considered indeterminate, and repeat testing was performed.
Polymerase chain reaction with sequence-specific primers
(PCR-SSP) was performed according to the method of Cavanagh et
al.17
Monoclonal antibody immobilization of platelet antigens
The binding of human polyclonal anti- 3Leu33 and
anti- 3Pro33 was studied using monoclonal antibody immobilization of
platelet antigens (MAIPA) with platelets from healthy donors and Donor A.18,19 MAIPA was performed using platelet-rich plasma
obtained from citrate-anticoagulated donor blood samples and the mAb
NIBSC-85/661 to specifically capture IIb 3 from lysed platelets.
Bound human IgG was revealed with an alkaline-phosphatase-labeled goat
anti-human IgG (Jackson Immunoresearch, West Grove, PA) using
Sigma-104 phosphatase substrate. O.D. was read on an ELISA plate reader
(Tecan Spectra) at 405 nm. Sera from nontransfused group AB male blood
donors were used as negative controls.
Platelet immunofluorescence test
Binding of antibodies to platelets was detected using the
platelet immunofluorescence test.20 Stained platelets
(10 000) were analyzed on a Coulter XL running System II software
(Beckman-Coulter, High Wycombe, United Kingdom). Binding of human and
murine antibodies was detected using fluorescein isothiocyanate
(FITC)-labeled rabbit anti-human IgG (DAKO) and rabbit anti-mouse
IgG (DAKO), respectively. Whole blood HPA-1a phenotyping was performed
with FITC-labeled CAMTRAN-007 as described
previously.13
cDNA amplification and sequencing
Total platelet RNA was prepared from 109 platelets
using 1 mL RNA STAT-60 following the manufacturer's protocol (AMS
Biotechnology, Witney, United Kingdom). Isolated RNA was resuspended in
100 µL diethyl-pyrocarbonate (DEPC)-treated water and used as a
template for cDNA synthesis, as follows. Random hexamers (3 µg) and
20 µL platelet RNA were incubated at 70°C for 10 minutes and then immediately transferred to ice. Forty units SuperRT reverse
transcriptase, 80 U RNAsin, 1 mM each dNTP, and DEPC-treated water to
give a total volume of 50 µL were added, and the mixture was
incubated at 42°C for 40 minutes. Resultant cDNA was used as a
template for PCR amplification of both IIb and 3 integrins.
Amplification reactions were performed in a total volume of 50 µL
containing 200 µM each dNTP, 1.5 mM MgCl2, 15 pmol each
primer, 5 U Taq polymerase, and 5 µL cDNA. The mixture was incubated
at 95°C for 5 minutes, and then 30 cycles consisting of 95°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute were performed.
Four and 5 overlapping fragments spanning the complete open-reading
frames (ORFs) of IIb and 3 integrins were amplified, respectively.
Amplified DNA was purified from agarose gels using the QIAquick gel
extraction kit (Qiagen, Crawley, United Kingdom) and was directly
sequenced using the Thermosequenase dye terminator cycle sequencing kit
(Amersham Life Science, Cleveland, OH). Sequences obtained were
compared to published IIb and 3 integrin sequences (accession
numbers J02764 and M20311, respectively).21,22 The 3
integrin PCR product obtained with primers 5'-GGCGGACGAGATGCGAGC-3' and
5'-GCATCTCGGTTCCGTGACAC-3' containing the SNPs C196T and G376A was
cloned into the TA vector according to the manufacturer's protocol
(Invitrogen BV, Groningen, The Netherlands). Recombinant clones were
sequenced to confirm the presence of the observed SNPs.
Construction of the mutant 3 integrin cDNA
For generation of the pcDNA 3.1( )Zeo 3Leu33Gln93 construct,
a 500-bp XbaI/KpnI wild-type (WT; Leu33Arg93)
fragment was replaced with the fragment encoding Gln93. The
3Pro33Arg93 construct was generated by site-directed mutagenesis of
the 3Leu33Arg93 construct using the Altered Sites in vitro
mutagenesis kit and the mismatched primer
5'-TGGTGCTCTGATGAAGCTTTGCCTCCGGGCTCA-3' according to the manufacturer's instructions (Promega, Southampton, United Kingdom). The above primer also introduces a silent mutation encoding a HindIII restriction site (underlined) that allows the rapid
identification of recombinant mutant clones. The full-length 3
integrin cDNA thus obtained (Pro33Arg93) was excised from the pAlter
phagemid and cloned into the pBJ1 mammalian cell expression vector. All constructs were verified by nucleotide sequencing before transfection.
Transfection and selection of stable cell clones
Plasmids for transfection were mixed with 40 µg LipofectAMINE
(Life Technologies, Merelbeke, Belgium) in a final volume of 200 µL
Iscoves modified Dulbecco medium (IMDM). The mixture was added to
either nontransfected Chinese hamster ovary (CHO) cells or cells that
had been pretransfected with human IIb integrin cDNA and grown to
60% confluence in 100-mm tissue culture plates. Twenty-four hours
after transfection, fetal calf serum was added to the culture medium;
48 hours after transfection, the medium was replaced with selective
medium (IMDM containing 10% fetal calf serum and 0.8 mg/mL zeocin
[Invitrogen]). Positive transfectants were analyzed with the
anti- 3 integrin mAb P37 for cell surface expression of the
recombinant human 3 integrin, associated with either the endogenous
hamster v or with human IIb integrins. Stable transfectants were
subcloned by limiting dilution and controlled for cell surface
expression of human 3 integrin.
RT-PCR and cDNA sequencing of Chinese hamster ovary
transfectants
Total RNA was isolated from 5 × 106 transfected
cells according to the method of Chomczynski and Sacchi.23
First-strand cDNA synthesis from 2 µg total RNA was directed with
oligo(dT) primer using an RNA-PCR kit (Perkin Elmer). The coding
sequence, corresponding to the mutated 3 integrin region, was
amplified using 3-specific primers, and products were analyzed by
agarose gel electrophoresis and directly sequenced using the
fmol DNA sequencing kit (Promega).
Western blot analysis
Platelets or cultured CHO cells were washed and lysed in 300 µL lysis buffer (150 mM NaCl, 20 mM Tris, pH 8, 1 mM
CaCl2, 1 mM MgCl2, 1% Triton X-100, 10 µg/mL
leupeptin, 10 µg/mL pepstatin A, 50 µM AEBSF). Lysates were cleared
by centrifugation at 12 000 rpm for 10 minutes at 4°C, and the
protein concentration was determined using the BCA protein assay
(Pierce, Rockford, IL). Fifty micrograms total cell lysate was then
resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and was transferred onto a nitrocellulose membrane. The
membrane was blocked for 1 hour in blocking buffer (TBS containing
0.1% [vol/vol] Tween and 5% [wt/vol] nonfat dry milk) and was
incubated overnight with primary antibody diluted in blocking buffer.
After several washes, the membrane was incubated for 1 hour with
horseradish peroxidase-conjugated sheep anti-mouse IgG diluted in
blocking buffer (Amersham Pharmacia Biotech, Roosendaal, The
Netherlands). Membranes were then washed in TBS, and bound antibody was
visualized using enhanced chemiluminescence according to the
manufacturer's instructions (Pierce).
Immunofluorescence and flow cytometric analysis of CHO
transfectants
Flow cytometry was used to detect antibody binding to
transfected CHO cells. Briefly, selected transfectants were detached from culture plates with EDTA buffer and were washed twice in incubation buffer (137 mM NaCl, 5 mM KCl, 50 mM HEPES, 1 mg/mL glucose,
pH 7.4). Transfected cells (5 × 105) were incubated on
ice for 1 hour with directly labeled antibodies. Cells were then washed
once, resuspended in incubation buffer, and analyzed on an Epics XL
flow cytometer (Beckman-Coulter). Phycoerythrin-labeled anti-human
CD61 (PharMingen, San Diego, CA) was used to determine total 3
expression, whereas expression of the HPA-1a epitope was determined by
staining with FITC-labeled CAMTRAN-007.
Taqman-based genotyping for the Gln93-encoding allele
Genomic DNA samples were genotyped for the WT Arg93 and
novel Gln93-encoding alleles using the primers
5'-TCAAGTCAGTCCCCAGAGGATT-3' and 5'-AGGTCTCTCCCCGCAAAGAG-3' with the
FAM-labeled WT probe 5'-TCCGGCTCCGGCCAGGTAG-3' and the
VIC-labeled Gln93-specific probe
5'-CTCCGGCTCCAGCCAGGTAGG-3'. The polymorphic nucleotide is
highlighted in bold. Amplification reactions were performed with 900 nM
each primer and 50 nM each probe at an annealing temperature of 64°C.
Allelic discrimination was subsequently determined by a post-PCR plate
read using a Perkin Elmer 7700 (Applied Biosystems, Warrington,
United Kingdom).
 |
Results |
Donor screening
One hundred 3Leu33-negative blood donors and one donor
(Donor A) with a repeatedly indeterminate phenotype were identified after the automated phenotyping of 6311 donor samples using whole blood
phenotyping ELISA (Figure 1). Of the 100 3Leu33-negative donors, 54 were anti-cytomegalovirus negative and
therefore were eligible for enrollment on the 3Leu33
(HPA-1a)-negative therapeutic platelet panel. Genomic DNA was obtained
from these 54 donors and from Donor A. Genotypes of these 55 samples
were determined using PCR-SSP; 54 were homozygous for the
3Pro33-encoding allele (data not shown), but Donor A genotyped as
3Leu33Pro33 heterozygous by PCR-SSP (Figure
2). This heterozygous genotype was
confirmed by Taqman-based genotyping and direct sequencing of 3
integrin cDNA (data not shown).

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| Figure 1.
Whole blood 3Leu33 phenotyping results for donor
Donor A.
Whole blood samples were phenotyped for the presence of 3Leu33
as described. Samples obtained from Donor A repeatedly gave an
HPA-1aweak phenotype. Control samples of 2 3Pro33
homozygous (HPA-1b1b) and one each of 3Leu33Pro33 heterozygous
(HPA-1a1b) and 3Leu33 homozygous (HPA-1a1a) were included in
each assay.
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| Figure 2.
Genotype of Donor A by PCR-SSP.
Genomic DNA samples from Donor A, his mother (Donor C), and his son
(Donor B) were HPA-1 genotyped by PCR-SSP. The 3 control
samples (HPA-1a1a, 1a1b, and 1b1b) show the expected amplicons of 90 bp
for HPA-1 (filled arrows) and control amplicons obtained with primers
specific for human growth hormone (429 bp; open arrows). The PCR-SSP
genotypes of the test samples are HPA-1a1b, HPA-1a1a, and HPA-1a1b for
Donor A, Donor C, and Donor B, respectively.
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Characterization of surface expression of IIb 3 and 3Leu33
epitope on Donor A's platelets
The cell surface level of IIb 3 was estimated by flow
cytometry using saturating concentrations of mAb Y2/51 and a commercial phenotyping kit (ADIAflo; American Diagnostica, Greenwich, CT). Reactivity with mAb Y2/51, which recognizes a linear 3 epitope, was
comparable to that obtained with control platelets indicating normal
levels of 3 on Donor A's platelets (Table
1). The level of IIb 3 expression
was within the normal range of the ADIAflo phenotyping kit (data
not shown).
Expression of the 3Leu33 (HPA-1a) epitope on Donor A's platelets
was studied in detail by flow cytometry using 3 recombinant human IgG1
3Leu33 antibodies (CAMTRAN-007, 19-7, and 23-15).13,16 Median fluorescence intensity obtained with these mAbs (Figure 3A) was significantly reduced in
comparison to that observed with platelets from control 3Leu33Pro33
heterozygous donors. A reduced reactivity of Donor A's platelets with
FITC-labeled CAMTRAN-007 was also observed in the whole blood, flow
cytometry-based phenotyping assay (Figure 3B). The reduced reactivity
of Donor A's platelets with anti- 3Leu33 was also observed with
polyclonal antisera in MAIPA, suggesting that the epitope recognized by
polyclonal and monoclonal anti- 3Leu33 is disrupted (Figure
4). Normal reactivity was observed with 2 polyclonal 3Pro33-specific antisera with Donor A's platelets when
compared to those obtained with a control heterozygous donor,
indicating normal expression of the 3Pro33 epitope on Donor
A's platelets.

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| Figure 3.
Reactivity of platelets from Donor A with monoclonal
anti- 3Leu33 in flow cytometry.
(A) Washed platelets from Donor A and control donors were
stained with 3 human 3Leu33-specific mAbs (CAMTRAN-007, 19-7, and
25-13) in a platelet immunofluorescence test. All 3 mAbs show
significantly reduced binding to the platelets from Donor A compared to
the heterozygous control. (B) Whole blood 3Leu33 phenotyping was
performed using FITC-labeled CAMTRAN-007 as described. Reactivity for
Donor A is reduced compared to the 3Leu33 homozygous and
heterozygous controls. Reactivity for Donor C is reduced to the
level observed with a heterozygote. Median fluorescence intensity (MFI)
is presented in each case.
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| Figure 4.
3Leu33 phenotyping by MAIPA with polyclonal antisera.
MAIPA was performed using platelets from Donor A and control donors
with 3 3Leu33-specific antisera (CP, MC, RF) and 2 3Pro33
antisera (CH, DW). Sera from 2 nontransfused group AB male blood donors
(AB1 and AB2) were used as negative controls. Donor A shows normal
reactivity with both 3Pro33 antisera but strongly reduced reactivity
with all 3 3Leu33 antisera (HPA-1aweak).
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IIb and 3 cDNA sequence analysis
Nine cDNA fragments encoding the complete ORFs of IIb and 3
integrins were amplified from RNA extracted from Donor A's platelets (data not shown). Sequencing of PCR products revealed a single G376A
SNP resulting in a 3Arg93Gln substitution, for which Donor A was
heterozygous (Figure 5). The presence of
the G376A SNP was confirmed by reverse transcription (RT)-PCR using 2 separate platelet RNA preparations (data not shown) and by sequencing
the PCR product after cloning it into the TA vector. Moreover, both
clones with the 376A nucleotide, encoding Gln93, also encoded Leu at
position 33.

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| Figure 5.
A G376A polymorphism was identified in the Leu33
encoding 3 integrin allele from Donor A.
The complete ORFs of both IIb and 3 integrins were
sequenced from cDNA obtained from the platelets of Donor A. Sequencing identified a single, novel polymorphism in the 3 cDNA
with adenine or guanine at position 376, for which Donor A is
heterozygous. This SNP, indicated by the arrow, results in the
replacement of arginine with glutamine at position 93 in the
3Leu33 allele.
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Expression of the recombinant mutant 3 integrin subunits in
Chinese hamster ovary cells
After stable transfection of the mutant 3Leu33Gln93 encoding
cDNA into CHO cells, 2 cell lines were produced expressing
3Leu33Gln93 complexed with either hamster v or human IIb
integrins, Cam11 and Cam12, respectively. The presence of the correct
3 integrin (Arg93 or Gln93) in transfected cell lines was confirmed
by RT-PCR and direct sequencing of the amplified cDNA fragment (data
not shown). Analysis of the expression of the recombinant 3 integrin subunits by Western blot with mAb P37 showed that the
Leu33Gln93-encoding 3 integrin was expressed in both Cam11 and Cam12
clones. In addition, Western blotting showed that 3Leu33Gln93
migrated with an identical electrophoretic mobility to recombinant WT
3Leu33Arg93 and native, platelet-derived 3 integrin (Figure
6A). Cell surface expression of the
Leu33Gln93 mutant 3 integrin was confirmed in both cell lines by
staining with mAb P37 (Figure 6B).

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| Figure 6.
Expression of recombinant 3 integrins in CHO cells.
(A) Western blot analysis of recombinant 3 integrin expression in
CHO cells. Cell lysates of transfected CHO cells were prepared, and
protein concentration was determined as described in "Materials and
methods." Equal amounts of protein from 3-transfected CHO cells
(50 µg) were resolved by 8% SDS-PAGE under nonreducing conditions,
transferred onto nitrocellulose, and immunoblotted with a mAb to human
3 (P37). Platelet lysate (5 µg protein) was run in parallel as a
positive control. Clone A10, CHO- IIb 3Leu33Arg93; clone CAM12,
CHO- IIb 3Leu33Gln93; clone A13,
CHO- vhamster 3Leu33Arg93; clone CAM11,
CHO- vhamster 3Leu33Gln93; clone E05,
CHO- vhamster 3Pro33Arg93. (B) FACS analysis of CHO
cells in suspension after indirect immunofluorescence labeling with the
anti- 3 integrin mAb P37. Negative control cells (bold solid line),
CAM11 (solid line), CAM12, A13, E05 (dotted lines), A10 (dashed
line).
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Reactivity of the 3Leu33-specific mAb CAMTRAN-007 with CHO
transfectants expressing 3Leu33Arg93 or 3Leu33Gln93
To study the expression of the 3Leu33 (HPA-1a) epitope on the
Leu33Gln93-encoding recombinant 3 integrin expressed in CHO cells,
we performed flow cytometry using FITC-conjugated CAMTRAN-007. In
these studies, the relative binding of CAMTRAN-007 to the
3Leu33Gln93 mutant was reduced to 60% of that observed with the WT
(Leu33Arg93) 3 integrin, indicating that the Arg93Gln mutation has a
modifying effect on the HPA-1a epitope (Figure
7). Interestingly, the reduction in
reactivity of CAMTRAN-007 was independent of the association of the
3 integrin with either human IIb or hamster v integrins (Figure 7). CAMTRAN-007 did not react with the E05 cell line that expresses 3Pro33Arg93 (HPA-1b), confirming that the mAb is
allospecific (Figures 6B, 7).

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| Figure 7.
The Arg93Gln mutation does impair the 3Leu33-specific
mAb CAMTRAN-007 binding to 3 integrins expressed in CHO cells.
Adherent CHO cells were detached with EDTA buffer, washed, and directly
labeled with the anti-CD61-PE or CAMTRAN-007-FITC for 30 minutes on
ice. Cells were washed and analyzed by flow cytometry. To control for
the variations in 3 expression between the different cell clones,
CAMTRAN-007 binding was normalized to the total 3 integrin
expression determined using a 3-integrin-specific mAb
(anti-CD61-PE). This ratio, CAMTRAN-007:CD61, was then expressed as a
percentage of the ratio obtained for the CHO cell clone A10 with a
CAMTRAN-007 dilution of 1:50 (100%). Data are representative of 4 different experiments. Clones as per Figure 6, plus clone A06,
CHO- v 3Leu33Arg93. - - - indicates A10; ×, A13; , A06;
___, E05; , Cam12; and , Cam11.
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Genomic analysis
A clear differentiation between the WT and mutant alleles was
obtained by Taqman-based 3G376A SNP genotyping (data not shown). Typing of 300 genomic DNA samples from random donors did not identify additional examples of the Gln93-encoding allele. However, typing of
Donor A's immediate family members showed the presence of the Gln93
allele in his mother (Donor C; Figure 8).
The Taqman Gln93-positive genotype of Donor C was confirmed by direct
sequencing of genomic 3 integrin DNA (data not shown).

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| Figure 8.
Pedigree of Donor A's family showing members positive
for the Gln93-encoding allele.
Genomic DNA samples were obtained from members of Donor A's immediate
family and were genotyped for the presence of the Gln93 encoding 3
integrin allele. A complete HPA genotype was also obtained by PCR-SSP
(data not shown). The mutant 3Gln93 integrin allele was found in the
mother (Donor C) and in the proband. Black symbols represent
3Gln93-positive members, open symbols represent 3Gln93-negative
members, and the arrow indicates the proband (Donor A). Samples were
not available from members represented by broken symbols.
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Discussion |
The 3 integrin is associated with IIb integrin in a
noncovalent and cation-dependent manner on the platelet surface, where it binds fibrinogen, fibronectin, and vitronectin and mediates the
aggregation of platelets and subsequent thrombus formation. The genes
encoding IIb and 3 integrins are polymorphic, and 8 SNPs are at
the basis of HPA alloantigens.3,6 The bi-allelic HPA-1
system, which is based on a Leu33Pro polymorphism in 3 integrin, is
clinically the most significant HPA system.9
The exact molecular nature of the HPA-1 epitope has been studied in
some detail. Site-directed mutagenesis studies have confirmed that
amino acid 33 of the 3 integrin is essential for the formation of
the HPA-1 epitopes.24 Additional studies using recombinant 3 integrin fragments suggest that the HPA-1 epitope is expressed within the N-terminal 66 amino acids.25,26 However, the
reactivity of HPA-1a antisera with recombinant fragments was variable
and only involved investigations with a small number of samples. More recently, the human HPA-1a epitope has been introduced into mouse 3
integrin by substituting human amino acids into the mouse sequence. The
reactivity of anti- 3Leu33 with 42 amino acid recombinant fragments
(residues 9 to 50) demonstrated that amino acids 30, 32, and 39, in
addition to 33, are critical for allo-antibody binding.27
Further studies have investigated the role of disulfide bonds and
noncontiguous sequences in the formation of the HPA-1a epitope. Alanine
replacement experiments with 3 integrin, designed to investigate the
role of the various disulfide bonds in HPA-1a epitope formation,
suggested 2 types (type 1 and type 2) of anti-HPA-1a that were split
by their difference in reactivity with the Cys435Ala 3
isoform.28 It is assumed that Cys435 forms a disulfide
bond with Cys5 linking the presumed cloverleaf-like, HPA-1a epitope, to
the Cys-rich 3 core.29 Both type 1 and type 2 anti-HPA-1a required an intact, conformationally native IIb 3
because the replacement of Cys for Ala at N-terminal positions 5, 23, 26, and 38 abrogated reactivity.28 Inhibition of
anti-HPA-1a binding by the mouse mAb LK-4 also demonstrates a split in
allo-antibody reactivity.30,31 However, the 2-epitope
model, proposed on the basis of these experiments, remains in dispute
because most antisera were from patients with posttransfusion purpura
that are known to contain IIb 3 autoantibodies in addition to the HPA-1 alloantibodies.32 There is ample evidence that
HPA-1a antibodies do not bind 3 integrin-derived oligopeptides
spanning the Leu33Pro33 polymorphism,27,33 findings that
are in agreement with those obtained with chimeric 3
molecules.34 Studies with the latter suggested that
sequences flanking the Cys435 position that encompassed amino acids
288-490 were important in epitope formation and that these sequences
were brought into proximity with the Cys26-Cys38 loop by long-range
disulfide bonds, such as the Cys5-Cys435 bond.34
Here we report on a unique donor with a normal level of platelet 3
integrin, as indicated by the reactivity of the mAb Y2/51 (Table 1),
but with a severely reduced reactivity with monoclonal and polyclonal
anti- 3Leu33 (anti-HPA-1a; Figures 1, 3, 4). The 3 3Leu33-specific monoclonals show more than 80% reduction in binding
to Donor A platelets compared with the heterozygous control (Figure
3A), a level of reduction also seen with polyclonal anti- 3Leu33 in
the MAIPA assay (Figure 4). A previously reported similar discrepancy between HPA-1 phenotype and genotype was attributed to the donor identified as a carrier of Glanzmann thrombasthenia.35
However, several lines of evidence suggest this was not the case with
Donor A. First, he does not carry a silent 3Leu33 allele, as judged from the 3Leu33 mRNA level and from sequencing data (Figure 5). Second, the platelet membrane 3 integrin copy number was normal (Table 1). A significant reduction in mAb Y2/51 reactivity would have
been observed in the case of a silent 3 allele (eg, in carriers of
type 1 Glanzmann thrombasthenia). Finally, sequencing 3 and IIb
integrin cDNA identified a novel G/A SNP at position 376 of the 3
cDNA that encodes for an Arg93Gln substitution linked with the
3Leu33 allele (Figure 5). Expression of the mutant 3Leu33Gln93 integrin cDNA in CHO cells with hamster v and human IIb integrins confirmed that the Arg93Gln mutation was responsible for the reduced reactivity with anti- 3Leu33. Reactivity with 3 integrin-specific mAbs demonstrated that the Leu33Gln93-encoded 3 integrin was expressed at the cell surface and was of the correct size (Figure 6A-B). However, CAMTRAN-007 (anti- 3Leu33) showed a 40% reduction in
binding to 3Leu33Gln93 relative to 3Leu33Arg93 (Figure 7). That a
greater reduction of anti- 3Leu33 binding was seen with the platelets
of Donor A and his mother compared with the CHO transfectants was
attributed to the homozygous nature of the transfectants in contrast to
the heterozygous platelets, to differences in glycosylation, or both.
Arg93 of the 3 integrin is outside the first 66 amino acids and
amino acids 288-490 that have previously been shown to be involved in
the formation of the HPA-1a epitope.25,26,34 However, replacement of Arg93 with Gln disrupts the binding of anti- 3Leu33 and thus identifies a region of the 3 integrin not previously thought to be involved in HPA-1a epitope formation. That a residue 60 amino acids from the allelic residue has such a dramatic effect on the
HPA-1a B-cell epitope is surprising, and an alternative explanation
could be a major structural change in 3Leu33Gln93. However, several
observations argue against this. First, Donor A's platelets show
normal reactivity with 3-specific mAbs. Second, his platelets show
normal reactivity with polyclonal anti-HPA-3a in MAIPA, confirming
both that the allo-epitope defined by an Ile843Ser substitution in
IIb and that the epitope recognized by the capture monoclonal are
intact (data not shown). Third, the observation that all 3 human mAbs
derived from HPA-1a allo-immunized patients were minimally reactive
with Donor A's platelets strongly suggested that residue 93 provides a
critical contact residue for anti- 3Leu33 binding (Figure 3B). It is
interesting that 2 of the 3 human monoclonal anti-HPA-1a (19-7 and
23-15) react with the N-terminal 66-amino acid fragment of the 3
integrin but that CAMTRAN-007 does not (N.A.W., unpublished
observations, August 2001). We interpret this as evidence that
the former 2 are possibly representative of type 1 HPA-1a antibodies
and that the latter are representative of type 2 antibodies. That all 3 human monoclonal 3Leu33 antibodies did not bind Donor A's platelets
(Figure 3A) suggests that his platelets would not define this split in
antibody types.
Finally, family studies indicated cosegregation of the Leu33 and Gln93
codons (Figure 8). Genotyping of family members showed that the
Leu33Gln93 3 integrin allele was inherited from the mother. The
mother (Donor C) had a 3Leu33 homozygous PCR-SSP genotype (Figure
2). When tested for reactivity with CAMTRAN-007 in whole blood,
however, platelet immunofluorescence gave a signal similar to that of
the HPA-1a1b heterozygous control (Figure 3B). We were unable to
identify any other related or unrelated persons with a 3Gln93 allele
after testing 300 DNA samples from random blood donors (data not
shown). Taken together, these data suggest that we have identified a
private allele unique to this family.
In conclusion, we have identified a rare but informative SNP in the
3 integrin that encodes a glutamine at position 93 instead of the
normal arginine. The presence of Gln93, with Leu33, is coupled with a
strong reduction in the binding of monoclonal and polyclonal 3Leu33
(HPA-1a) allo-antibodies. Amino acid 93 had not previously been thought
to be involved in the formation of the HPA-1 epitope, but our findings
indicate that the conformation of the Leu33Pro33-containing loop
(residues 26 to 38) or that of the Cys-rich core is conformationally
changed by this mutation. If the latter is the correct explanation, it
would support the hypothesis that the HPA-1a epitope is discontinuous
and that residues from several loops (including a loop containing
residue 93) are involved in allo-antibody binding. Continuation of high
throughput HPA-1a phenotyping might identify additional persons with
unique 3Leu33 alleles, allowing a further unraveling of the
molecular structure of the HPA-1a B-cell epitope.
 |
Acknowledgments |
We thank the staffs of the National Blood Service, Oxford and
Cambridge Centres, for collecting and testing samples. We also thank Dr
A. H. Goodall, University of Leicester, and Prof A. E. G. Kr. von dem Borne, Central Laboratory of the Netherlands Red Cross
Blood Transfusion Service, for their kind gifts of monoclonal antibodies.
 |
Footnotes |
Submitted July 3, 2001; accepted October 19, 2001.
N.A.W. is supported by a research grant from DiaMed AG, Switzerland.
E.S.R. and N.H.C.B. are supported by grants from CRP-Santé, Luxembourg.
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: Willem H. Ouwehand, Dept of Haematology, Division
of Transfusion Medicine, University of Cambridge, Cambridge CB2 2PT
United Kingdom; e-mail: who1000{at}cam.ac.uk.
 |
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