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Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4103-4111
A Point Mutation Thr799Met on the  2 Integrin
Leads to the Formation of New Human Platelet Alloantigen
Sita and Affects Collagen-Induced Aggregation
By
Sentot Santoso,
Julia Amrhein,
Heiko A. Hofmann,
Ulrich J.H. Sachs,
Matthias M. Walka,
Hartmut Kroll, and
Volker Kiefel
From the Institute for Clinical Immunology and Transfusion Medicine,
Justus Liebig University Giessen, Giessen, Germany; the Department of
Neonatology, Virchow Hospital, Humboldt-University, Berlin, Germany;
and the Institute for Clinical Immunology and Transfusion Medicine,
University of Leipzig, Leipzig, Germany.
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ABSTRACT |
A new platelet-specific alloantigen, termed Sita, was
identified in a severe case of neonatal alloimmune thrombocytopenia. The Sita alloantigen is of low frequency (1/400) in the
German population. Immunochemical studies demonstrated that the
Sita epitopes reside on platelet glycoprotein (GP) Ia.
Nucleotide sequence analysis of GPIa cDNA derived from
Sita-positive platelets showed
C2531 T2531 point mutation, resulting in Thr799Met dimorphism. Analysis of genomic DNA from 22 Sita-negative normal individuals showed that the
Thr799 is encoded by ACG2532 (90.9%) or
ACA2532 (9.1%). To establish a DNA typing technique, we
elucidated the organization of the GPIa gene adjacent to the
polymorphic bases. The introns (421 bp and 1.2 kb) encompass a 142-bp
exon with the 2 polymorphic bases 2531 and 2532. Polymerase chain
reaction-restriction fragment length polymorphism analysis on DNA derived from 100 donors using the restriction enzyme Mae III showed that the Met799 form of GPIa is restricted to
Sita (+) phenotype. Analysis of stable Chinese hamster
ovary transfectants expressing allele-specific recombinant
forms of GPIa showed that anti-Sita exclusively reacted
with the Glu505Met799, but not with the
Glu505Thr799 and the
Lys505Thr799 isoforms. In contrast,
anti-Bra (HPA-5b) only recognized the
Lys505Thr799 form, whereas anti-Brb
(HPA-5a) reacted with both Glu505Thr799 and
Glu505Met799 isoforms. These results
demonstrated that the Met799 is responsible for formation
of the Sita alloantigenic determinants, whereas amino acid
505 (Lys or Glu) specifically controls the expression of
Bra and Brb epitopes, respectively. Platelet
aggregation responses of Sita (+) individuals were
diminished in response to collagen, indicating that the
Thr799Met mutation affects the function of the GPIa/IIa complex.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
IMMUNIZATION AGAINST human platelet
antigens (HPA) is the central event in the pathophysiology of neonatal
alloimmune thrombocytopenia (NAIT) and in posttransfusion purpura (PTP)
and is also of importance in some patients refractory to platelet transfusions.1
Currently, 5 HPA systems, HPA-1 (PlA, Zw), -2 (Ko, Sib), -3 (Bak, Lek), -4 (Pen, Yuk), and -5 (Br, Zav), are officially recognized. In addition, a number of low frequency alloantigens, HPA-6bW
(Caa, Tua), -7bW (Moa), -8bW
(Sra), -9bW (Maxa), -10bW (Laa),
Groa, and Iya, have also been reported.
Meanwhile, the alloantigenic determinants of all of these HPAs could be
localized on platelet membrane glycoprotein (GP) Ia,
GPIb , GPIb , GPIIb, and GPIIIa. All of
these have been found to result from point mutations in the encoding genes, which lead to single amino acid substitutions.2-13
GPIIb/IIIa, also known as the integrin
 IIb 3,14 which serves as
fibrinogen receptor on platelets, carries the majority of these HPAs.
Another integrin, 21 (GPIa/IIa), which
mediates platelet adhesion to both fibrillar and nonfibrillar
collagen,15,16 is known to bear the alloantigenic
determinant of the HPA-5.17 Recently, we could demonstrate
that the HPA-5a, -5b phenotypes are associated with a single base
G1648A substitution of the GPIa gene leading to an
aminoacid Glu505Lys dimorphism.6
Alloimmunization against HPA-5 has been associated with
NAIT,18 PTP,19 platelet transfusion
refractoriness,20 and thrombocytopenia due to passively transferred platelet antibodies after transfusion of blood
products21 or bone marrow transplantation.22
In addition, the important functional role of GPIa in primary
hemostasis is stressed by the observation of congenital and acquired
bleeding tendency in patients with deficient GPIa and with
autoantibodies. In vitro studies showed impaired reactivity of
platelets with collagen in all these cases.23-26
Current evidence suggests that adhesion and activation of platelets by
collagen are mediated through distinct receptors. Activation of
platelets by collagen is believed to occur via a 2-site, 2-step model
in which GPIa/IIa provides an initial interaction (adhesion) that
brings a second site in the collagen molecule into the activitory receptor GPVI.27
Recently, Kunicki et al28 were able to identify clusters of
linked silent polymorphisms within the coding sequence of the 2 gene that which are responsible for the variation in
21 receptor density on the platelet
surface. Three allelic differences could now be identified by 4 dimorphisms at positions 807, 837, 873, and 1648. Allele 1 (T807/T837/A873/G1648)
is associated with increased levels of
21, whereas allele 2 (C807/T837/G873/G1648)
and allele 3 (C807/C837/G873/A1648)
are associated with lower levels of
21.29 Carriers of the allele
1 express high levels of GPIa/IIa, whereas individuals who carry
alleles 2 and 3 exhibit lower expression of the platelet integrin.
Furthermore, Kritzig et al29 could demonstrate that the
density of GPIa/IIa on the platelet surface correlates with the rate of
platelet attachment in whole blood to type I collagen. A clinical
impact of these findings was demonstrated by the association of the
GPIa C807T gene polymorphism with nonfatal myocardial
infarction and stroke in younger individuals.30,31
In this report, we describe another case of alloimmunization against a
new genetic variant of GPIa that was responsible for a case of severe
NAIT, and we describe the biochemical, molecular, biological, and
functional properties of platelets carrying this antigen.
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CASE REPORT |
A healthy 19-year-old white mother (Sit) gave birth to her first child
after a normal pregnancy. The male child (2,150 g) was delivered at
term with an Apgar score of 8. At birth, a severe thrombocytopenia was
observed (31 × 109/L) and the platelet count
decreased continually below measurable value. Clinical and laboratory
examinations showed no other abnormalities. Petechiae were observed at
the age of 4 days and a tentative diagnosis of NAIT was made. Because
maternal platelets were not available, the infant was transfused with
platelets from a healthy donor prepared by platelet apheresis with good
increment.The platelet count increased to 158 × 109/L. In addition, the child was treated with high doses
of intravenous IgG from day 3 to 5 of life and with
prednisolone from day 2 to 10 of life. After a moderate decrease of the
platelet count to 67 × 109/L, the child was
discharged with a normal platelet count on day 18. Ultrasound
examination showed no signs of cerebral hemorrhage.
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MATERIALS AND METHODS |
Blood samples.
Blood samples of the mother, the father, and the child were referred to
us shortly after delivery because of suspected NAIT. Platelets were
isolated from EDTA-anticoagulated blood by differential centrifugation
and stored at 4°C in isotonic saline containing 0.1%
NaN3. Normal donor platelets for the characterization of platelet antibodies were selected from a large number of donors with
known human platelet alloantigens (HPA 1-5) and ABO blood group antigens.
Phenotyping.
Phenotyping of human platelets was performed using the
glycoprotein-specific immunoassay (monoclonal antibody-specific
immobilization of platelet antigens [MAIPA]), as previously
described.32
Antibodies.
Antibodies against Bra and Brb alloantigens
were obtained from mothers of children with NAIT and from
polytransfused patients, respectively.17 Monoclonal
antibody (MoAb) Gi5 and MoAb Gi9 specific for GPIIb/IIIa complex (CD41)
and GPIa (CD49b), respectively, were produced and characterized in our
laboratory.6 MoAb SAM1 directed against the GPIc (CD49e)
subunit and MoAb B1G6 specific for 2 microglobulin of
HLA class I molecule were purchased from Immunotech (Hamburg, Germany).
MoAb FMC 25 against the GPIX (CD42a) subunit of GPIb/IX/V complex was a
generous gift of Dr H. Zola (Adelaide, Australia).
Immunoprecipitation.
Platelets and Chinese hamster ovary (CHO) stable transfectants were
surface-labeled with 5 mmol/L NHS-LC-Biotin (Paesel, Frankfurt, Germany) and were immunoprecipitated as previously
described.9
Isolation of platelet RNA and leukocyte DNA.
Total platelet RNA was isolated from 20 mL EDTA anticoagulated blood of
phenotyped donors by a modification of the guanidium isothiocyanate
method as previously described.4 Platelet RNA was
resuspended in 100 µL diethyl pyrocarbonate (DEPC)-treated H2O and stored at  70°C. Genomic DNA was obtained
from peripheral blood leukocytes derived from 3 mL EDTA anticoagulated
blood after removing platelet-rich plasma. The DNA was extracted by the
salting out procedure as described by Miller et al,33
dissolved in 300 µL TE buffer (10 mmol/L Tris, 1 mmol/L EDTA, pH
8.0), and stored at 4°C.
Primers for GPIa-specific amplification.
To amplify the entire coding region of GPIa by polymerase chain
reaction (PCR), 8 overlapping sets of primers were constructed based on
the published cDNA sequence of GPIa.34 For cDNA
amplification of a region encompassing nucleotides 2304-2858, forward
primer P48 (2212-GAAAGGTGCC TGCAGAAG-2229), reverse primer P40
(2858-TCAGCATCATACAGGAGAGG-2839), and nested primer P18
(2304-CTCTTTGGATTTGCGTGTGGACATC-2328) were used. In addition, primer
pair P61 (2414-GTGGTGAGGATGGACTTTGC-2433), P42
(2572-CAATTCCAGTGTTGTATGCAC-2552) and primer pair P6 (2551-AGTGCATA CAACACTGGAAT-2570), P7 (2699-TTTAAAGCAGGGTAGCCTAC-2680) were used to
amplify GPIa gene from genomic DNA.
PCR amplification of cDNA.
One hundred microliters of platelet RNA were heated to 68°C for 10 minutes and quickly cooled on ice water. The first-strand cDNA was
synthesized using 10 µmol/L oligo dT, 40 U RNAse inhibitor (Boehringer Mannheim, Mannheim, Germany), 2 mmol/L of each dNTP (Pharmacia, Freiburg, Germany), 500 U Moloney's murine leukemia virus
(MMuLV) reverse transcriptase, and 5× enzyme buffer
(GIBCO BRL, Eggenstein, Germany) for 40 minutes at 45°C in a total
volume of 30 µL and was stopped by chilling to 0°C. Aliquots of
20 µL of cDNA were preheated to 90°C for 5 minutes and were
chilled to 0°C. After digestion with 2 U of RNAseH enzyme (GIBCO
BRL) at 37°C for 20 minutes, cDNA was stored at 20°C.
Five microliters of cDNA were diluted with 10× PCR buffer, 0.3 µmol/L of each primer (P48, P40), 175 µmol/L dNTP, and 2.5 U Taq
Gold polymerase (Perkin Elmer, Norwalk, CT) in a total
volume of 50 µL. Amplification was performed on DNA thermal cycler
480 (Applied Biosystem, Weiterstadt, Germany) for 20 cycles. Each cycle
consisted of denaturation at 93°C for 1 minute, annealing at
53°C for 1 minute, and extension at 72°C for 2 minutes. In the
final cycle, the samples were kept at a temperature of 72°C for 10 minutes and then chilled to 4°C. Two-microliter aliquots of PCR
products were reamplified for 30 cycles using nested primer P18 and
reverse primer P40 under identical PCR conditions. PCR products were
analyzed on 1.6% SeaKemGTG agarose gel (Biozym, Hamel, Germany)
containing ethidium bromide.
PCR amplification of genomic DNA.
Two hundred nanograms of genomic DNA were amplified using primer pairs
P61, P42 and P6, P7 as described above. Thirty-two cycles of
denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, and extension at 72°C for 2 minutes were performed.
Sequence analysis of PCR products.
Amplified DNA was purified on SeaKemGTG gel (Biozym) by QIAquick
(Qiagen, Düsseldorf, Germany). Purified DNA was trimmed using
Klenow DNA polymerase (Biolabs, Schwalbach, Germany) and were subcloned
into the EcoRV cloning site of the plasmid vector pGEM-5Zf
(Promega Biotech, Madison, WI) and then transformed into the DH5
high efficiency competent Escherichia coli (GIBCO BRL). Plasmid
DNA was prepared for nucleotide sequencing analysis by QIAprep
(Qiagen). The inserts from 8 positive clones were sequenced using
Taq-FS Dye-Terminator Cycle Sequencing Kit according to the
manufacturer's protocol and were analyzed on ABI PRISM Genetic Analyzer 310 (Applied Biosystem). SP6 and T7 primers that hybridize to
vector sequence and flank the cloned insert were used for sequencing.
Direct sequencing of PCR fragment.
The DNA fragment of interest was gel purified from 50 µL PCR reaction
and directly sequenced as described above. PCR primers were used as
sequencing primers.
Genotyping of Sita and HPA-5 alloantigens by restriction
fragment length polymorphism (RFLP).
For genotyping of Sita alloantigen, 3 to 5 µg genomic DNA
was amplified using forward primer P61 (see above) and an intronic reverse primer P83 5'-TACCGGTAGGGAGAATGATGC-2602-3 under the
following PCR conditions: 30 cycles at 94°C for 1 minute, 58°C
for 1 minute, and 72°C for 2 minutes, followed by final extension
at 72°C for 10 minutes. Aliquots of 3 µL of PCR products were
digested in a thermal cycler with 2 U Mae III endonuclease
(Boehringer Mannheim) for 6 hours at 55°C, respectively.
Restriction fragments were analyzed on 1.6% agarose gel using Tris
borate buffer system (Biozyme). Genotyping for HPA-5 was performed by
RFLP using Mnl I endonuclease as previously
described.35
Genotyping of GPIa C807T dimorphism by sequence-specific
PCR (PCR-SSP).
Genotyping of the C807T dimorphism was performed using
PCR-SSP as recently described.30
Generation of allele-specific GPIa cDNA constructs.
A full-length human GPIa cDNA (A1648AG/AC2531A)
in the mammalian expression vector pMPSV,36
which encodes Lys505Thr799 GPIa isoform, was kindly provided by Dr E. Klein (Department of Dermatology, University of Würzburg, Würzburg, Germany). An
allele-specific recombinant form
G1648AG/AC2531A encoding the
Glu505Thr799 GPIa isoform was produced by
cartridge mutagenesis. A 1,435-bp cDNA fragment spanning nucleotides
1486 to 2921 of platelet GPIa mRNA derived from a Brb
homozygous donor was amplified by PCR.6 After digestion
with Bgl II and BstEII endonucleases (Biolabs), the
1,374-bp fragments containing the polymorphic base G1648
were then shuttled into the pMPSV expression vector containing the GPIa
insert (A1648AG/AC2531A), which had been
digested with the same enzymes. To exclude contamination of undigested
plasmid, the pMPSV expression vector was linearized with Swa I
endonuclease (Biolabs) before ligation. After subcloning in E
coli, the resulting plasmid constructs were amplified in PCR (bases
1486-1900) and were screened for A1648G dimorphism by RFLP
analysis using Mnl I endonuclease. Specific mutations CATG at positions 2531 and 2532 were introduced into the
Lys505Thr799 and
Glu505Thr799 constructs by site-directed
mutagenesis using QuickChange Mutagenesis Kit (Strategene, Heidelberg, Germany).
For PCR amplification, 2-nucleotides (underlined) mismatched forward
primer
5'-GTTAACATTTTCAGTAATGCTGAAAAATAAAAGGG-3' and reverse primer
5'-CCCTTTTATTTTTCAGCATTACTGAAAATGTTAACC-3'
from base 2513 to 2548 of GPIa cDNA were constructed. After
denaturation for 30 seconds at 95°C, aliquots of 20 ng plasmid were
amplified for 12 cycles (denaturation for 30 seconds at 95°C,
annealing for 60 seconds at 55°C, and extension for 17 minutes at
68°C). PCR products were digested with Dpn I endonuclease
for 1 hour at 37°C and transformed into DH5 high efficiency
competent E coli. Plasmid DNA from positive clones were
amplified by PCR using primers P18 and P40. PCR products were subjected
to RFLP analysis with Mae III as described above. For
subsequent transfection studies, all GPIa allele-specific constructs
were validated by nucleotide sequence analysis.
Stable expression of allele-specific constructs in CHO cells.
CHO (American Type Tissue Collection, Rockville, MD) cells were grown
in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS;
Seromed, Berlin, Germany), 1% sodium pyruvate, 1% glutamine, and 1%
penicilline/streptomycine (GIBCO BRL; complete medium) and were
transfected with allele-specific GPIa constructs by the use of the
reagent Lipofectin (GIBCO BRL). In brief, 6 µg of the GPIa construct
was mixed with 25 µL Lipofectin in 2 mL OptiMEM Medium (GIBCO BRL)
and then added to a subconfluent 10-cm plate of CHO cells for 12 hours.
For antibiotic selection, cotransfection with 6 µg RSV neo plasmid
(Invitrogen, Leek, Netherlands) containing Neomycin resistent gene was
performed. Nine milliliters of complete medium was added and the
incubation was continued for 48 hours. After splitting, CHO
transfectant was then selected with Genicitin (G418; final
concentration, 1 mg/mL; GIBCO BRL) for approximately 2 weeks. Positive
clones expressing GPIa/IIa were enriched by adhesion of 3 × 106 stable transfectant onto 3.6-cm petri dishes coated
with collagen type I (5 µg/mL of 0.1 mol/L NaHCO3). After
subcloning, the surface GPIa/IIa expression was analyzed by flow
cytometry (see below). Stable cell lines were grown in complete medium
supplemented with 200 µg/mL of G418.
Flow cytometry.
Stable transfectants were harvested with trypsin-EDTA (GIBCO BRL),
washed in phosphate-buffered saline (PBS; GIBCO BRL), and fixed with
1% paraformaldehyde for 3 minutes. After three washes, 450 µL
(106/µL) of cell suspensions were incubated with 50 µL
MoAb of Gi14 (20 µg/mL) or 20 to 30 µL human sera
(anti-Bra, anti-Brb, and anti-Sita)
for 30 minutes at room temperature. Labeled cells were then washed
twice, stained with 500 µL fluorescein isothiocyanate-conjugated rabbit antimouse IgG or antihuman IgG (dilution 1:40; Dako, Hamburg, Germany), and analyzed by flow cytometry (Ortho Diagnostic,
Neckargemünd, Germany).
Adhesion of platelets to immobilized collagen.
Adhesion of platelets to immobilized collagen was investigated as
recently described.37 Platelets were isolated from
ACD-anticoagulated blood by differential centrifugation and washed with
AMPL buffer (140 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L
MgCl2, 10 mmol/L glucose, 10 mmol/L sodium pyruvate, 5 mmol/L sodium malate, 10 mmol/L HEPES, and 0.3 mmol/L albumin, pH 7.4)
containing 0.02 U/mL apyrase and 5 U/mL hirudin (Paesel & Lorey,
Frankfurt, Germany). Aliquots of 1 × 109 washed
platelets in PBS (137 mmol/L NaCl, 2 mmol/L KCl, 8 mmol/L Na2HPO4, and 1 mmol/L
KH2PO4, pH 7.4) were labeled with 16 µL 75 mmol/L NHS-LC-Biotin (Paesel) at room temperature for 15 minutes. Labeled platelets were washed twice with 5 mL AMPL buffer and resuspended in PBS at a final concentration of 3 × 108/mL. For adhesion, microtiter wells were coated
overnight with collagen type I, III, or V (50 µg/mL; Sigma, Dreieich,
Germany) or bovine serum albumin (BSA; 10 mg/mL; Sigma), washed 3 times with 200 µL PBS, and blocked with 200 µL 1% BSA in PBS for 1 hour at 37°C. Aliquots of 100 µL of biotinylated platelets were added in triplicate to wells coated either with BSA or collagen and were
permitted to adhere at 37°C for 1 hour. To evaluate the functional effect of anti-Sita alloantibodies, platelets were mixed
with either 40 µL affinity-purified AB-sera or of
anti-Sita for 30 minutes in a 5% CO2
atmosphere. Nonadherent cells were removed by gentle absorption onto
absorptive pads and by washing of the wells 3 times with 150 µL PBS.
Bound platelets were detected by the addition of 100 µL alkaline
phosphatase-labeled streptavidin (500 µg/mL, dilution 1:1,500;
Dianova) and incubation at 37°C for 1.5 hours. After a further 3 washes with PBS and one wash with diethanolamine buffer, pH 9.8, 100 µL of p-nitro-phenylphosphate (1 mg/mL; Sigma) was added. The
reaction was stopped after 5 minutes at room temperature by the
addition of 100 µL of 1.0 mol/L NaOH and was read in an enzyme-linked
immunosorbent assay (ELISA) plate reader (OD405, Titertex
Multiskan; Pharmacia).
Platelet aggregation.
Platelet-rich plasma (PRP) of ACD anticoagulated blood from
Sita-phenotyped individuals was ajusted to 3 × 105 platelets/µL by dilution with autologous plasma. To
aliquots of 180 µL PRP, 20 µL of various dilutions of ADP (5, 10, and 25 µmol/L; Sigma) or collagen (2.5, 5, and 10 µg/mL; Nycomed,
Munich, Germany) were added and the change in the optical density was monitored using APACT aggregometer (Labor Timer, Ahrensburg, Germany) with continuous stirring at 37°C. In some instances, PRP was
incubated with anti-Sita alloantibodies as described above.
After stimulation with 10 µg/mL collagen, platelet aggregation was
recorded as described above.
| |
RESULTS |
Serologic identification and family studies.
When serum from the mother Sit was tested against autologous, paternal,
or donor panel platelets using a platelet adhesion immunofluorescence
test, a positive reaction was only observed with paternal platelets
(data not shown). An IgG antibody from serum Sita reacted
strongly with GPIa/IIa immobilized by MoAb Gi9 from paternal platelets
only, but not with epitopes on GPIc/IIa, GPIIb/IIIa, GIb/IX, and HLA
class I (Fig 1). Platelets from 11 typed
panel donors, including those of Bra and Brb
homozygous individuals, did not react either. These results indicate that the Sita serum recognizes a new low-frequency platelet
alloantigen residing on GPIa/IIa complex. Because the Sita
alloantigenic determinants could be immobilized by MoAb directed against GPIa/IIa only, but not by MoAb specific for GPIc/IIa complex, Sita epitopes are probably localized on the GPIa subunit.
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| Fig 1.
The reactivities of anti-Sita with paternal
platelets in MAIPA assay using MoAbs FMC25 (anti-GPIb/IX), Gi9
(anti-GPIa/IIa), SAM-1 (anti-GPIc/IIa), Gi5 (anti-GPIIb/IIIa), and B1G6
(anti-2m) as capture antibodies.
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In a population of 400 unselected donors, GPIa/IIa of 1 individual
(Dre) carried the Sita antigen.
Figure 2 shows the pedigrees of the family
Dre and the index family Sit. Phenotyping analysis of both families
demonstrated that Sita antigen is inherited as an autosomal
dominant trait. In addition, all Sita (+) individuals were
also phenotyped for Bra and Brb
(Table 1). Three Brb homozygous
Sita (+) individuals (B.II.2, II.4, and III.3) were found
(Fig 2B) in family Dre, indicating that Sita alloantigen is
inherited with the Brb allele.
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| Fig 2.
The pedigrees of the index family Sit (A) and family Dre
(B). Solid symbols represent Sita (+), open symbols
represent Sita () individuals. The child with NAIT is
indicated. The Bra and Brb phenotypes of
Sita-phenotyped individuals are shown.
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Table 1.
The  2 cDNA Sequences (Bases 807, 1648, 2531, and 2532) of the Family Sit (A) and Family Dre (B) and Their
Br and Sit Phenotypes
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Immunochemical investigations.
To confirm the localization of Sita alloantigen on
GPIa/IIa, immunoprecipitation analysis of biotin-labeled glycoproteins
derived from Sita (+) and Sita ()
Bra/Brb heterozygous individuals was performed.
As shown in Fig 3, anti-Sita
(lanes 1 and 4) only precipitated GPIa/IIa complex from
Sita (+) platelets, but not from Sita ()
platelets. In the control experiments, anti-Bra (lanes 2 and 5) and anti-Brb (lanes 3 and 6) precipitated GPIa/IIa
complex from both Bra/Brb heterozygous
individuals.
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| Fig 3.
Immunoprecipitation analysis of biotin surface-labeled
platelets derived from a Sita () and a Sita
(+) individual with anti-Sita (lanes 1 and 4),
anti-Bra (lanes 2 and 5), and anti-Brb (lanes 3 and 6). Immunoprecipitates were separated on 7.5% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreduced
conditions, transferred onto nitrocellulose membrane, and visualized
using streptavidin-horseradish peroxidase and chemiluminescent
substrate.
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Genetic analysis.
To analyze the nucleotide sequence encoding for GPIa, platelet mRNA was
sequentially amplified by reverse transcription using 8 sets of
primers, subcloned, and sequenced. Nucleotide sequence analysis of the
554 bp encompassing nucleotides 2304-2858 derived from a
Sita (+) individual showed 2-base mutations
C2531A2532
 T2531G2532 (Fig 4) in 10 of 12 subcloned examined,
consistent with the hypothesis that all Sita (+)
individuals found to date are heterozygous for this character. These
mutations predicted Thr799
(AC2531A2532) in the Sita ()
and Met799 (AT2531G2532) in the
Sita (+) phenotype. Analysis of the other 7 regions of GPIa
mRNA showed no other nonconservative nucleotide differences.
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| Fig 4.
Nucleotide sequence analyses of amplified GPIa cDNA
derived from 2 Sita (+) individuals (A.I.1 and A.II.1;
Fig 2). PCR product encompassing nucleotides 2304-2854 was subcloned
into the plasmid vector pGEM-5Zf and sequenced on both strands using
primers corresponding to the SP6 and T7 RNA polymerase promotor
sequences. The base exchanges of the wild-type (WT) CA or CG to the
mutant TG at positions 2531 and 2532 (arrows) result in a
Thr799 (ACA or ACG) Met799 (ATG)
substitution.
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Genotyping analysis of Sita.
To establish genomic DNA typing, we first elucidated the exon-intron
boundaries surrounding the Sit polymorphic base by PCR. When PCR with
genomic DNA was performed using exonic primer pairs P61, P42 and P6,
P7, fragments of 580 bp and nearly 1,400 bp were obtained. Nucleotide
sequence analysis of both PCR products identified 421-bp and 1.2-kb
introns (Fig 5) with conserved donor and
acceptor splice junctions, which are classified as phase 2 and 0, respectively. These results demonstrate that these 2 introns enclose a
142-bp (bases 2478-2619) exon encoding 47 amino acids. Based on these intron sequences, reverse primer P83 downstream of the 142-bp exon was
constructed. After 35 cycles of amplification using primer P61 as
forward primer, the expected 630-bp product was obtained from genomic
DNA derived from peripheral blood lymphocytes
(Fig 6). The PCR product was digested with
Mae III endonuclease, which cleaves 5'-GTAAC-3'
(wild-type) but not 5'-GTAAT-3' sequences (mutant).
Sita-negative individuals could be clearly distinguished
from Sita heterozygous individuals by the absence of the
uncut 630-bp band. The results of genotyping by PCR-RFLP of 400 Sita-phenotyped donors were consistent with the serological
phenotyping.
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| Fig 5.
PCR strategy for the elucidation of the GPIa gene
surrounding the polymorphic base at position 2531 (arrow). Genomic DNA
was amplified by PCR using primer pairs P61, P42 and P6, P7. PCR
products were sequenced for the determination of exon-intron
boundaries. The polymorphic exon 20 (bases 2478-2619) encoding the
amino acids Glu782-Pro828 is flanked by 2 introns (421 and 1,200 bp) of phases 2 and 0 (italic). For genotyping
analysis of the polymorphic bases 2531 and 2532 (bold), a 630-bp
fragment was amplified using primer pair P61, P83.
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| Fig 6.
Restriction map of the 630-bp PCR products (top). The
arrow indicates the position of the restriction site for Mae
III endonuclease. The length of the restriction fragments is shown
above. RFLP analysis of PCR-amplified genomic DNA derived from
peripheral blood cells (PBL) of Sita-phenotyped
individuals. DNA fragments were analyzed on 1.6% agarose gel stained
with ethidium bromide (bottom). The undigested 630-bp PCR product is
shown in lane 1. Lanes 2 and 3 represent the analysis of Mae
III-digested PCR product derived from DNA of a Sita (+)
heterozygous individual and a Sita () individual,
respectively. Lane 4, DNA size standards (pBR328 DNA.BglI + pBR328
DNA.HinfI).
|
|
Amino acid 799 of 2 integrin is encoded by different
codons.
Because different triplets theoretically could code the wild-type
Thr799 (ACA, ACC, ACG, and ACT), we analyzed the members of
the families Sit and Dre (Table 1) by direct nucleotide sequencing. Two
different codons, ACA and ACG (Fig 4), both of which encode the
wild-type Thr799 form of GPIa, were found among
Sita () homozygous and Sita (+)
heterozygous individuals (Table 1). To determine the distribution of
the codon usage for Thr799, we analyzed 22 unselected blood donors by DNA sequencing. Nineteen individuals presented with codons
ACG/ACG, 2 with ACG/ACA, and 1 with ACA/ACA. Our results suggest that
the codon usage for Thr799 is 90.9% ACG and 9.1% ACA.
Other conceivable codons (ACC and ACT) were not found in our population
so far. It is of note that the Thr799 ACA allele is
uniquely recognized by the restriction enzyme TspRI and
therefore represents a potentially useful genetic marker for RFLP (data not shown).
Thus, the mutation C2531T in Sita (+)
individuals most probably occurred in the high-frequeny codon ACG (Thr)
to ATG (Met) due to a 1-point mutational event.
Linkage association between C2531T dimorphism with
A1648G, C807T and A2532G
polymorphisms.
To analyze the linkage associations between C2531T with
A2532G, A1648G, and C807T
polymorphisms, we genotyped all members of the families Sit and Dre
for A1648G, C807T, and A2532T
dimorphisms by PCR-RFLP, PCR-SSP, and direct nucleotide sequencing
analysis, respectively. Their genotypes are summarized in Table 1.
In accordance with our previous phenotyping results by MAIPA assay, we
could confirm that the Sita allele segregated together with
the Brb allele. Four Sita (+) individuals
(A.II.1, B.II.2, B.II.4, and B.III.3) are homozygous for
the G1648 allele.
Furthermore, we observed that all Sita (+) individuals were
homozygous for the C807, indicating the linkage association
between the Sita allele with the C807 allele.
Finally, we found that the G1648 haplotype exclusively
segregated with the G2532 allele. All Brb
homozygous family members are homozygous for the G2532
allele. In contrast, the A1648 haplotype most probably
segregates with the A2532 allele, because all
Bra heterozygous individuals were found to be heterozygous
G/A at position 2532.
These observations indicate a linkage disequilibrium between
polymorphisms at positions 807, 1648, 2531, and 2532. Under the consideration of the linkage associations between the polymorphisms at
bases 807/837/873,28 we could now define 4 2
gene alleles by 6 dimorphisms at positions 807/837/873, 1648, 2531, and
2532 (Table 2).
Analysis of recombinant GPIa allelic isoforms.
To examine the involvement of amino acid 799 in the formation of the
Sita antigenic determinants, we transfected allele-specific
expression vectors encoding for the Lys505
Thr799, Glu505Thr799, and
Glu505Met799 GPIa into CHO cells. Stable
transfectants expressing GPIa recombinant proteins were surface-labeled
with biotin and analyzed by immunoprecipitation. As shown in
Fig 7, anti-Sita (lanes 2)
precipitated exclusively the Met799 GPIa isoform, but not
both Thr799 isoforms having either glutamic acid or lysine at position 505. The associated band (110 kD) most probably represents endogenous hamster 1 integrin subunit, which is
coprecipitated with the human GPIa subunit (Mr 150 kD). In the control
experiment, anti-Brb (lanes 1) reacted with
Thr799 and Met799 GPIa isoforms, which have the
amino acid glutamic acid at position 505. In contrast, anti-Bra (lane 3) only recognized the Lys505
GPIa isoform.
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| Fig 7.
Immunoprecipitation analysis of allele-specific
recombinant GPIa isoforms. Recombinant forms of GPIa/IIa complex were
produced in CHO cells transfected either with
Glu505Thr799 (left panel),
Glu50Met799 (middle panel), or
Lys505Thr799 (right panel) form of GPIa. After
surface labeling with biotin, cell lysates were immunoprecipitated with
anti-Brb (lanes 1), anti-Sita (lanes 2), and
anti-Bra (lanes 3). Immunoprecipitates were analyzed on
7.5% SDS-PAGE under nonreduced conditions, transferred onto
nitrocellulose membrane, and visualized using chemiluminescence
substrate.
|
|
These findings demonstrate that amino acid Met799 directly
controls the expression of the Sita epitopes, whereas
residue 505 (lysine or glutamic acid) is responsible for the formation
of Bra and Brb alloantigenic determinants, respectively.
Effect of the Sit Thr799Met dimorphism and
anti-Sita alloantibodies on platelet function.
To determine the possible effects of Thr799Met
mutation on platelet function, we studied platelet
ad- hesion and platelet aggregation of
Sita-phenotyped individuals.
The results of platelet adhesion to immobilized collagen type I of
Sita phenotyped platelets were summarized in
Table 3. The platelet adhesion response to
high collagen (50 µg/mL) and low collagen concentration (1 µg/mL)
of Sita (+) platelets was indistinguishable from
Sita () platelets.
View this table:
[in this window]
[in a new window]
|
Table 3.
Adhesion of Platelets to Collagen Type I in the Absence
(None) and in the Presence of Affinity-Purified IgG From Normal Human
Serum, Anti-Sita, or Murine MoAb Gi9 Specific for
GPIa/IIa
|
|
Furthermore, we analyzed the effect of anti-Sita
alloantibodies on platelet adhesion. In comparison to the control
experiment with MoAb Gi9 specific for a functional epitope of the
GPIa/IIa complex, anti-Sita did not inhibit platelet
adhesion to type I collagen. Similar results were obtained with type
III and V collagens (data not shown).
However, in standard aggregation assay (Fig
8), the platelet aggregation response to low collagen concentration
(2.5 µg/mL) in the Sita (+) individual (B.III.3, see Fig
2) was diminished in comparison to the Sita ()
individual (B.III.2). In contrast, the platelet aggregation of the
Sita (+) individual after stimulation with high
concentration of collagen (10 µg/mL) was indistinguishable from the
Sita () individual. The addition of other agonists
(ADP and ristocetin) to platelets from Sita (+) individuals
resulted in completely normal aggregation response (data not shown).
View larger version (23K):
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| Fig 8.
Platelet aggregation of a Sita (+) donor
(B.III.3; curves 1 and 3) and a Sita () individual
(B.III.2; curves 2 and 4) after stimulation with 2.5 µg/mL collagen
(curves 1 and 2) or 10 µg/mL collagen (curves 3 and 4).
|
|
Similar results were observed with 3 other Sita (+)
individuals (B.II.1, B.II.2, and B.II.4; data not shown).
Because GPIa of both individuals (B.III.3 v B.III.2) only
differs at nucleotide 2531 (T or C), but carries identical nucleotide
sequences at polymorphic positions 807 (homozygous C) and 1648 (homozygous G), our results suggest that only the mutation
ThrMet at position 799 affects the function of GPIa/IIa as a
collagen receptor. The differences in response to collagen thus cannot
be explained by possible differences in GPIa expression density related
to the base C807T polymorphism.
When platelet aggregation by collagen was evaluated in the presence of
purified anti-Sita, no inhibition was found (data not shown).
| |
DISCUSSION |
We report here the identification and characterization of a new
low-frequency platelet alloantigen, Sita, in whites that
was involved in a case of severe NAIT. A study in 400 unrelated
individuals identified another Sita (+) individual, showing
that this low frequency alloantigen is not restricted to a single
family. Studies with immunochemical techniques allowed us to localize
the Sita alloantigenic determinant on GPIa. Examination of
the nucleotide sequence derived from a Sita (+) father
showed a mutation comprising 2 bases CATG at positions 2531 and 2532, respectively, in a heterozygous state in the GPIa mRNA transcripts.
An analysis of the exon-intron structure allowed us to define the
polymorphic site on an exon with a length of 142 bp. A comparison with
other subunits of integrin showed that this exon corresponds to
exon 20 of the x and M
genes.38,39 All 3 exons have identical length (142 bp) and
are flanked by introns with identical phases (2 and 0).
Interestingly, we also discovered an additional silent polymorphism at
base 2532 (G or A) adjacent to the polymorphic base 2531. By direct
nucleotide sequencing of genomic DNA derived from Sita
() individuals, we could define 2 different codons, ACG and ACA,
that encode the wild-type Thr799 form of GPIa.
Because the Sita alloantigen is a low-frequency antigen, it
seems to represent a recent mutational event. According to the current
observations that most polymorphisms responsible for platelet alloantigens are point mutations of the wild-type allele,40 the Sita alloantigen most probably occurred due to the
single base mutation CT at position 2531 of the
high-frequency wild-type allele ACG (90.9%) rather than the
low-frequency ACA allele (9.1%).
Recently, Kritzig et al29 could define 3 allelic
differences by 4 dimorphisms at positions 807, 837, 873, and 1648. Our data indicate that the A2532 allele may be linked to the
C807A1648 allele, whereas the G2532
allele can be associated either with the
C807G1648 or T807G1648
allele. These observations are in accordance with the frequencies of
C807A1648 and A2532 genotypes in
our population (10% v 9.1%). However, analysis of
A1648 and G1648 genotyped individuals among
larger populations is necessary to establish this association. Interestingly, the GPIa cDNA derived from fibroblast cell library also
represented the rare C807A1648A2532
allelic form.34
The mutation C2531T results in a substitution of the polar
uncharged amino acid threonine into a hydrophobic nonpolar residue methionine at position 799 on mature GPIa polypeptide. Stable expression of recombinant allele-specific GPIa in CHO cells led to the
confirmation that the single amino acid substitution
Thr799Met was sufficient to induce Sita epitope
formation recognized by the Sita antibody present in the
mother's serum. As expected from our previous
observations,6 we could demonstrate in this study that the
residue 505 (Lys or Glu) specifically controls the formation of
Bra and Brb alloantigenic determinants, respectively.
Functional studies showed no influence of the Sit-polymorphism on
platelet adhesion to immobilized collagen. In contrast, after
stimulation with a low concentration of collagen, the platelet aggregation response of Sita (+) platelets was reduced as
compared with the response of platelets from Sita () individuals.
Until now, the region comprising the amino acid 799 has so far not been
implicated in the adhesion to collagen, which was mapped to the
I-domain of the GPIa subunit.41,42 Recently, several lines
of evidence suggest that activation of platelets occurs via a 2-site,
2-step model in which the initial interaction occurs via the GPIa/IIa,
allowing binding to GPVI leading to activation.27 It may be
speculated that the mutation ThrMet at position 799 affects
the activation of platelets, possibly through interaction between
GPIa/IIa and GPVI.
Although the aggregation response to collagen in Sita (+)
carriers was diminished, none of these individuals had signs of an altered hemostasis.
In NAIT, alloantibodies against the Bra epitopes on GPIa
often induces moderate thrombocytopenia in affected
children.18 It is interesting to note that NAIT due to
anti-Sita was associated with severe thrombocytopenia. It
is conceivable that the additional functional defect of GPIa/IIa
complex resulted in the pronounced clinical course of this child. An
additional influence of anti-Sita alloantibodies on
platelet adhesion and aggregation by collagen could not be observed.
In conclusion, we have identified that a single point mutation
Thr799Met adjacent to a hot spot in the GPIa gene is
responsible for the formation of a new low-frequency platelet
alloantigen, which we termed Sita. In contrast to other
known platelet alloantigens, this point mutation appears to affect
platelet function.
| |
ACKNOWLEDGMENT |
The authors thank Dr H. Schachinger, who kindly referred this NAIT case
to us. We are grateful to Micaela Boehringer and Monika Kummel for
their technical assistance. Our gratitude is also extended to the
families concerned for their cooperation in this study.
| |
FOOTNOTES |
Submitted April 7, 1999; accepted August 3, 1999.
Supported by Grant DFG Sa480/2-1. This work is part of the PhD theses
of J.A. and U.J.H.S.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Sentot Santoso, PhD, Institute for Clinical
Immunology and Transfusion Medicine, Justus Liebig University Giessen,
Langhansstr. 7, D-35392 Giessen, Germany; e-mail:
Sentot.Santoso{at}immunologie.med.uni-giessen.de.
| |
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ABSTRACT
INTRODUCTION