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Vol. 269, No. 11. Issue of "arch 18. pp. 8439-8444. 1994
Prinltzd in U.S.A.
BI,,1.,K;1<'.iI.
CHB\ll'iTKI
8 1994 by The American Society for Biochemistry and Molecular Biology. Inc.
A Point Mutation Leads to an Unpaired Cysteine Residue and a
Molecular Weight Polymorphism of a Functional Platelet
p3 Integrin Subunit
THE Sr" ALLOANTIGEN SYSTEM OF GPIIIa"'
(Received for publication, August 10, 1993, and in revised form, November 21, 1993)
Sentot SantosotP, RainerKalbSn, Hartmut KrollS, Matthias WalkaS, Volker Kiefelt,
Christian Mueller-Eckhardt8, and PeterJ. Newmanil**
From the $Institute for Clinical Immunology and Pansfusion Medicine, Justus Liebig Uniuersity, 0-35392 Giessen,
Germany, the IIBlood Research Institute, The Blood Center of Southeastern Wisconsin, and the ""Departments of Cellular
Biology and Pharmacology, The Medical College of Wisconsin, Milwaukee, Wisconsin 53233
form the major platelet fibrinogen receptor, GPIIbLIIa (integrin aIIbP3) on human platelets ( l ) ,or with a , to form the
vitronectin receptor, a&, which is more widely distributed.
Mature GPIIIa protein consists of 762 amino acids, including
six potential N-glycosylation sites in the extracellular domain
that have been shown to be post-translationally modified with
high-mannose carbohydrate residues (2-4). GPIIIa also contains 56 cysteine residues in highly conserved locations within
the extracellular domainof the molecule, all of which are normally disulfide-linked. Thirty-one of these cysteine residues
comprise a cysteine-rich, protease-resistant core of t h e molecule and are clustered between residues 433 and 655 into four
tandemly repeated segmentsof about 40 amino acids each (4).
Another 7 cysteines are concentrated within the first 50 aminoterminal amino acids (4, 5 ) . The cysteine pairing pattern of
GPIIIa has been largely established by Calvete et al. (6), who
showedthatanumber
of longrangedisulfidebondsform
within the molecule, bringing together
at least two linearly
distant polypeptide segments. Overall, these cysteine residues
likely serve an important role in preserving the overall threedimensional structure of the integrin complex,as perturbation
or absence of 1 or more of these residues has been shown to
severely affect stability and/or ligand binding function (7).
In addition to its role in mediating
cell adhesive interactions,
GPIIIa is the most polymorphic
of the integrin subunits, with six
currently recognized alleles known to exist in the human gene
pool. Differences in these allelic isoforms have been shown to be
responsible for eliciting an alloimmune response leading to
platelet destruction in two clinically significant pathologic syndromes, post transfusion purpura and neonatal alloimmune
thrombocytopenia (for recent reviews, see Refs. 5,8,9). The adIntegrins constitute a large family
of cell surface aP het- vent of platelet mRNAPCR technology (10) has made it possible
erodimers that are widely distributed on many cells and are
to elucidate the molecular basis for a numberof these platelet
involved in cell-matrix orcell-cell interactions. Human platelet membrane glycoprotein polymorphisms. To date, all of these
glycoprotein IIIa (GPIIIa)' is the common/3 subunit of the P3 have been found to result from single nucleotide, and
consesubfamily of integrins and associates with either
cqlb (GPIIb) to quently single amino acid substitutions (11-171, which are in
turn thought to subtly affect the conformation of the protein,
* This work was supported in part by Grant HL-44612 (to P. J. N.) leading to expression of t h e offending antigenic determinant.
Recently, we described a new low frequency platelet alloanfrom the National Institutes of Health. The costs of publication of this
article were defrayed in part by the payment of page charges. This tigen on GPIIIa, termed Sra, that was responsible for
a case of
article must therefore be hereby marked "aduertisement"in accordance neonatal alloimmune thrombocytopenia (18). Since that time
with 18 U.S.C. Section 1734 solely to indicate this fact.
B To whom correspondence should be addressed: Institute for Clinical several other private platelet alloantigens (CA, Va, Mol t h a t
reside on GPIIIa have also been reported (16, 19-21). In this
Immunology and Transfusion Medicine, Justus LiebigUniversity,
D-35392 GiessedLanghansstrasse 7, Germany.
study, we have further characterized the biochemical and mo1Portions of this work constitute a doctoral thesis.
lecular properties that underlie the polymorphism of human
' The abbreviations used are: GPIIIa, glycoprotein IIIa; PCR, polymerase chain reaction; NEM, N-ethylmaleimide; PBS, phosphate-buff- platelet GPIIIa that is responsible for the immunogenicity of
ered saline; BSA, bovine serum albumin; mAb, monoclonal antibody; t h e Sr" alloantigen, as well as their effects on expression and
PAGE, polyacrylamide gel electrophoresis; bp, base pair(s).
function of this unique integrin isoform.
Recently, we described a low frequency platelet alloantigen on human platelet membrane glycoprotein
(GP) IIIa, termedSr", that was involved in neonatal alloimmune thrombocytopenia. To identify the molecular
nature of the Sr" alloantigen, we analyzed the nucleotide sequence of polymerase chain reaction-amplified
GPIIIa mRNA, andfounda
CZoo4+Tsubstitutionin
Arg636
seven Sr" positive individuals which results in an
+ Cys polymorphism within the cysteine-rich region of
GPIIIa. Analysis of allele-specific recombinant forms of
GPIIIa that differed only at amino acid residue 636
showed that anti-Sr" alloantibodies reacted with the
but
not
the
recombinant form of GPIIIa.
Interestingly, underreducing
conditions, the
form of GPIIIa migrated with slightly
a
increased apparent molecular weight compared with the
form.
Following treatment with Endoglycosidase H, both allelic forms of GPIIIa exhibited the same mobility, however the Sr" epitope was lost. Sr" positive platelets express thesame number of GPIIb-IIIa complexes on their
surface as wild-type Sra negative platelets, and also aggregate normally in responseto a varietyof platelet agonists. Based upon these results, we conclude that 1)
GPIIIa residue 636 specifically controls the formation
and expression of the Sr" alloantigenic determinant,
and 2) an unpaired cysteine residue alters theN-linked
glycosylation pattern of the extracellular domain of
GPIIIa, but affects neither the degreeof surface expression nor the adhesive function of the GPIIb-IIIa complex.
8439
This is an Open Access article under the CC BY license.
8440
The Sr" Alloantigen
System
of
GPIIIa
97 "C for 5 min, 10 pl of Taq polymerase dilution (2.5 units, Boehringer
Mannheim, Germany) was added a t 85 "C and "hot-start" PCR was
Serological Studies-Typing of human platelets for the presence of
performed in a DNA thermal cycler (Biometra, Gottingen, Germany).
P1" and Sr" alloantigens was performed using the
glycoprotein-specific
Amplification proceeded for 30 cycles, with denaturation for 1.5 min at
immunoassay, MAIPA, usinganti-PIA1-andanti-Sr"-specificalloan94 "C, annealing of primers for 1.5 mina t 50 "C, and extensionfor 3 min
tisera and the monoclonal antibody Gi5 directed against GPIIb/IIIa a s
at 72 "C.
capture antibody (22). The platelet-specific alloantibodies
PIA1and Sr;'
Isolation and Amplification of Genomic DNA-Genomic DNA was
wereobtained from mothers of childrenwithneonatalalloimmune
isolated from peripheral blood leukocytes using the proteinaseisalting
thrombocytopenia, a s previously described (18).Monoclonal antibodies
out procedure of Miller et al. (27). A 335-bp regionof the GPIIIa gene
(mAbs) Gi5 and Gi22 directed against GPIIb/IIIa and GPIb,, respecencompassing the Sr polymorphic nucleotide was amplified using the
tively, were produced in our laboratory (23, 24).
PCR primer pair shown inTable I. PCR was performed using 1-2 pg of
Labeling ofPlatelets-Platelets were surface labeled with
5 mM NHSDNA and 0.5 p~ of each primer using 2.5 units of Taq polymerase in
LC-Biotin (Paesel, Frankfurt, Germany) as previously described (25). PCR buffer in a totalvolume of 100 pl as described above. Thirty cycles
For thiol-specific labeling, washed platelets were radiolabeled withtriof 1.5 minat 96"C, 1.5min a t 57 "C, and 3 min at 72 "C were performed.
tiated N-ethylmaleimide (NEM) as described by Kalomiris and Coller
Analysis of PCR Products-Five pl of PCR-amplified products were
(26)with minor modifications. Briefly, aliquots of 5 x loRwashed plate- analyzed on 1.4% agarose gel containing ethidium bromide (Dianova,
lets in PBS buffer, pH 7.4, containing 10 mM EDTA and 10 mM benzaHamburg,Germany).Selectedamplified
cDNAs werepurified
by
midine hydrochloride (PBSIEB)were incubated with 30pCi of tritiated
Geneclean (Dianova, Hamburg, Germany) andsubcloned into the plasNEM (55 Ci/mM, New England Nuclear,Bad Homburg, Germany)for 60
mid vector pGEM-5Zf (Promega Biotech, Madison, WI). Plasmidsfrom
min a t 37 "C. After the reactions were completed, platelet suspensions
positive clones were sequenced by the dideoxy termination method uswere diluted 10-fold with PBS/EB containing 0.296 BSA (PBS/EB/BSA) ing a Sequenase 2.0 (United States Biochemical, Bad Homburg, Gerand incubated at 37 "C for 1 h. The platelets were pelleted, washed
many) as recommended by the manufacturer. Computer analyses
of
twice, and resuspended in 100 pl of PBSIEBIBSA. To identify proteins
protein and nucleic acid sequences were performed using the program
containing disulfide bonds, plateletswere pretreated with 1 mM dithioPC/GENE (Intelligenetics Inc andGenofit SA) on an IBM PC-compatthreitol for 60 min at 37 "C prior t o labeling.
ible computer.
Immunoprecipitation Analysis-Labeled
plateletsortransfected
Allele-specific Oligonucleotide Hybridization Analysis-Ten p1 of amCOS-7 cells were lysed in 50 mM Tris, 150 mM NaCl, 1% Triton X-100,
plified DNA was applied to nylon membranes ( N Y 13N, Schleicher &
pH 7.4 (TB), containing 2mM phenylmethylsulfonyl fluoridefor 30 min
Schuell, Dassel, Germany) and hybridized at 54 "C with 17-mer oligoat 4 "C. After centrifugation (16,000x g, 15 min, 4 "C)cell lysates were nucleotide probes having specificity for either the SP or Srh sequence,
precleared twice with normal IgG and 50.p1of protein A-Sepharose
differing only in the central nucleotide (shown in Table I). The oligobeads (Pharmacia, Freiburg, Germany)for 30 min. Precleared lysates
nucleotide probes were end-labeled with digoxigenin-ll-2',3'-dideoxywere incubated with human sera
or mAb overnight at 4 "C on a Nutator uridine-triphosphate (DIG-11-ddUTP, Boehringer
Mannheim,
Gerand precipitated with 50pl of protein A-Sepharose beadscoupled with
many) aspreviously described (28) and were
immunologically detected
rabbit anti-human or anti-mouse IgG (Dako, Hamburg, Germany).Afusing alkaline phosphatase-conjugated polyclonal sheep anti-digoxigeter five washings with TB, immunoprecipitated complexes were eluted
nin Fab fragment antibody and
AMPPDTM(Serva, Heidelberg, Gerby boiling for 10 min in SDS buffer. In some instances, the immune
many) as a chemiluminescent substrate. Positive reactions were visucomplexes were treated withEndoglycosidase H before elution from the
alized by exposing the nylon membrane t o Kodak X-Omatt X-ray film
beads (see below). Immunoprecipitates were analyzed on 7.5% SDS(Rochester, N Y ) .
PAGE under reduced conditions. Gels containing biotinylated proteins
Construction and Expression ofAllele-specific Recombinant Formsof
were transferred to nitrocellulose by immunoblotting and visualized
GPZIZa-Allele-specific recombinant formsof GPIIIa were created using
using streptavidin-horseradish peroxidase and chemiluminescent subcartridge mutagenesis. A full-length GPIIIacDNA, the internal EcoRI
strate as recommended by the manufacturer (ECL Western Blotting
restriction site of which had been removed by site-directed mutagenesis
Detection System; Amersham Buchler, Braunschweig, Germany). Gels
(kindly provided by Dr. Gilbert C. White 11, University of North Carocontaining radiolabeled proteins were dried and fluorographed or aulina, Chapel Hill, NC) was used as a host
vector for construction of
allelic GPIIIa isoforms. Plasmids (pGEM-BZf,Promega Biotech, Maditoradiographed on Kodak X-Omatt film using an intensifying screen
son, WI) from two clones containing GPIIIa nucleotides 1666-2237 of
(Cronex Hi-Plus; DuPont, Frankfurt, Germany). "GMethylated rainor a C at position 2004 were
platelet GPIIIa, and having either a T
bow proteinmixture(Amersham
Buchler, Braunschweig,Germany)
digested with MluI and AflII (New England Biolabs, Bad Schwalbach,
was run asmolecular weight markers in parallel.
Germany). The resulting 249-bp fragment encoding the polymorphic
Analysis of N-Linked Glycosylation-One-hundred-pl aliquots of bibase was gel-purified and ligated
to full-length GPIIIaA3luescript cDNA
otinylated platelet detergent lysates were incubated with
50 milliunits
which had been digested withthe same enzymes. After subcloning, the
of Endoglycosidase H (Boehringer Mannheim, Germany) overnight at
resulting plasmid construct inserts were removed with EcoRI, flushed
37 "C and immunoprecipitated with human sera, and analyzed
by SDSPAGE as described above. For comparison, washed immunoprecipitates using Klenow DNA polymerase, and finally subcloned into the EcoRV
site of the mammalian expression
vector pcDNA N E 0 (ITC, Heidelberg,
were divided into two 50-pl aliquots and resuspended 50
in pl of acetate
for subsequent transfection were
buffer (0.15M sodium acetate,0.1 M phenylmethylsulfonyl fluoride,0.170 Germany). Purified plasmids used
SDS, pH 5.5) in the absence or presenceof 15 milliunits of Endoglyco- validated by nucleotide sequence analysis.
COS cells were transfectedwithboth
allele-specific recombinant
sidase H. After an overnight incubation a t 37 "C, the reaction was
forms of GPIIIa using the DEAE-dextran method, as
previously destopped by adding an equal volume of two times SDS buffer.
COS cells were coIsolation and Amplification of Platelet RNA-Human platelet mRNA scribed (29). For surface expression experiments,
transfected with full-length GPIIb/pcDNANEO (kindly
provided by Dr.
was isolated from EDTA-anticoagulated blood as previously described
Ronggang Wang, Blood Research Institute, TheBlood Center of South(12).Twenty-nine-p1 aliquots of platelet mRNA were heated to 68 "C for
10 min and quickly
cooled on icewater before reverse transcription. The eastern Wisconsin, Milwaukee, WI). Recombinant proteins synthesized
by the transfected COS cells were metabolically labeled with ["SlmeGPIIIa-specific PCR primers used in these studies
were constructed
based on the published numbering systemof Fitzgerald et al. (4). Prim- thionine and immunoprecipitated as described above.
Determination of Antibody-binding Siteson Platelets-Quantitation
ers 1 and 2 (GPIIIa bases 5'46-72-3' and 5'-698-674-3') were used t o
of anti-PIA1- and anti-&-"-binding sites on washed platelets was peramplify a 642-bp segment of GPIIIa cDNA encompassing nucleotides
formed using unlabeled human alloantibodies in a competitive enzyme56-698, while primers 3 and 4 (GPIIIa bases 5'-1666-1695-3' and 5'linked immunoassay (30). Aliquotsof 2.5 x lo7 washed typed platelets
2237-2208-3') were used for amplification of GPIIIa 16662237 (571
were incubated with increasing amount of alloantisera or normal hubp). First strand cDNA synthesis was carried out in the presence
of
either 0.5 p~ of antisense primer 2 or primer 4, 500 p~ of each dNTP man sera (20-400 pl) at37 "C for 45 min. The sensitized platelets were
washed four times in isotonic saline with 0.2% BSA and then resus(Pharmacia,Freiburg,Germany),
40 units of RNasin(Boehringer
pended in PBS buffer containing 2% BSA. Platelet-bound IgG was deMannheim, Germany), 500 units of cloned Moloney murine leukemia
termined in various
cell dilutions (25,000,5,000, and
1.000 platelets/pl).
virus reverse transcriptase, and
five timesreactionbuffer(GIBCO,
Platelet Aggregation-Platelet-rich plasma was obtainedby differenEggenstein, Germany) in a totalvolume of 50 pl. cDNA synthesis was
whole blood derived from S F
carried out at 37 "C for 60 min and was stopped by chilling t o 0 "C. tial centrifugation ofACD-anticoagulated
cDNA was diluted with 25 p1 of four times buffer (50 mM KCl, 0.04% typed donors and control healthy subjects. Platelet aggregation was
induced by severaldifferentagonists,including
1 PM ADP, 5 pg/ml
gelatin) and mixed with 0.5 p~ sense primer (1 or 3) and 0.25 p~
collagen, and 1.5 mg/ml ristocetin using standard procedures.
antisense primer (2 or 4) in a total volume of 90 pl. After heating a t
MATERIALSANDMETHODS
The SP Alloantigen System of GPIIIa
8441
codes for cysteine a t amino acid 636 of the mature GPIIIa
polypeptide chain.The presence of anadditional cysteine
within thecysteine-rich region of GPIIIa islikely to be responsible for the formation of the Sr" epitope.
Biochemical Properties of Recombinant GPIIIa Allelic
Cys polyForms-To examine directly whether the Arg""
morphism actually controls the formation of the SP antigenic
determinant, we constructedan allele-specific recombinant
form of GPIIIa that differs only by the presence of T a t nucle_ -c otide 2004 and analyzed theCys""" protein product in a mamPIA' alloantibody
malian cell transfectionsystem.Whereas
bound equally well to either the
Arg6"" or CYS"~"
isoforms of the
GPIIIa molecule (Fig. 3B, compare lanes 2 and 3 with lanes 4
and 5),anti-Sr" reacted only with the Cysfisfiform (Fig. 3A).
"
. n
These results demonstrate that aminoacid 636 is directly in"
- .
-.volved in the expression of the Sr epitopes. Interestingly, the
c"
recombinant
isoform (lane 4 ) migrated slightlyslowerin
T G C-A
SDS-PAGE than the recombinant wild-type Argfi3"form (lane
FIG.1. DNA sequence analysis of amplified GPIIIa cDNA de- 3 ) , even under reducing conditions. Identical results were obrived from an Sr"positive individual. The 571-bpPCR product was tained usingtwo independently constructedclones (lanes 2 and
subcloned into the plasmid vector pGEM-5Zf and sequenced on both 5 ).
to the SP6 and T7 RNA polystrands using primers corresponding
It is possible that the presence of a n additional cysteine
merase promoter sequences. The single base substitution
of a T for a C
residue in theS P allelic form of GPIIIa could have altered the
a t base 2004 is indicated with urrows.
conformation of the molecule such that the glycosylation pattern would also be affected. To examine whether the altered
RESULTS
mobility of the Cys6"" isoform was uniqueonly to therecombiAmplification a n d Analysis of the NH2-and COOH-terminal nant COS-7 cell product, or might be intrinsically present in
Regions of GPIIIa Platelet mRNA-Previous immunochemical Sf' positive versus SF' negative platelets,biotin surface-labeled
studies have shown that anti-SPalloantibodies react with the platelets were subjected to immunoprecipitation analysis. As
68-kDa fragment of GPIIIa generated by chymotryptic treat- shown in Fig. 4, GPIIIa molecules derived from the plateletsof
ment of intact platelets (18). Since this fragment has been an Sr" positive (heterozygous) individual migratedas a doublet
shown to lack residues 121-348 of the large disulfide-bonded (lane 1), corresponding to the wild-type (lower band) and
Sr" (upper band) allelic forms. As predicted, only the
loop formed by disulfide bondingof Cys5 with Cys4"" (6,31-33),
we were able to predict that the remaining region formed by upperGPIIIabandwasreactivewithanti-SPalloantisera
amino acid residues 1-120 and 349-762 (encompassing nucle- (lane 2 ) , confirming the results obtainedabove using recombiotides 99-470 and 1143-2384, respectively) of GPIIIa is likely nant isoforms produced in COS cells (Fig. 3). GPIIIa derived
to carry theSr" epitope. Thus, we amplified this region as two from wild-type Sr" negative platelets (lane3 ), was presentas a
separate segmentsof 642 (nucleotides 56-698) and 571(nucle- single band, as expected, and was not reactive with anti-Sr"
otides 1666-2237) base pairs. Nucleotide sequence analysis of antibodies (lane4 ) .After deglycosylation with Endoglycosidase
the resulting571-bp COOH-terminalfragment derived from an H, however, both GPIIIa allelic forms migrated with the same
Sr" positive individual revealed a single C -> T nucleotide sub- mobility (lane 5),indicating that the molecular size polymorstitution at base 2004 (Fig. 1)in five of seven subclones exam- phism observed in the S P positive allele of GPIIIa is due toa
ined, consistent with the fact that all Sr" positive individuals variable degree of N-linked glycosylation, most likely the presexamined to date have
been serologically heterozygous for this ence of an additional single
high mannose moiety. In contrast to
low frequency allelicform of GPIIIa. In contrast,all clones from continued reactivity with anti-PIA' alloantibody (lane 5, and
an Sr" negative individual encoded a C a t this position (not Ref. 34), however, deglycosylated GPIIIa failed to react with
shown). No other nucleotide differences between Sr" positive anti-SF alloantibodies (lane 6).These data suggest that the
Sra antigenic determinant is,
at least in part, dependent
on the
versus negative individuals werefound.
Correlation of C2004 T Polymorphism with Sr Allotype-In
presence of one or more of the high-mannose carbohydrate
resiorder to determine whether the C +) T polymorphism seen in dues that are known to comprise approximately 15% of the
the one Sr" positive individual was associated with Sr pheno- molecular mass of the GPIIIa molecule. Whether or not the
as a result of the Arg"""
type, genomic DNA from six Sr" positive and 10 different Sr" additional carbohydrate residue added
negative individuals (four family members and six unrelated -'Cys amino acid substitution forms part of the alloantibodydonors) were amplified using PCR to yield a 335-bp product combining site remains tobe determined.
(not shown). Allele-specific oligonucleotide typing was then
perEffect of a Free Thiol Group on GPIIIa Expression a n d Funcformed using 17-base probescontaining the putative
genotype- tion in SF Positive Platelets-In order to verify biochemically
specific nucleotide in the middle (Table I, right). As shown in the presence of a n additional, unpaired cysteineresiduein
the top panel of Fig. 2, the putative SF-specific probe contain- GPIIIa derived from Sr" positive individuals, intact platelets
ing a T inthe middle hybridizedwith thePCR products derived were incubated with tritiated NEM to derivatizeaccessible free
thiol groups, solubilized in Triton X-100, and then immunoprefrom all six Sr" positiveheterozygous individuals, but was
monoclonal antibody,
wild- cipitated with the anti-GPIIIa murine
negative with 10 SF negative individuals. In contrast, the
type "Srb"-specific probe, containing a C in themiddle, hybrid- Gi5. Since the P-subunit of the GPIb complex has previously
ized with both Sr" heterozygous and Sr" negative individuals. been shown to contain a free sulfhydryl group (26), aliquotsof
weresubjected to immunoprecipitation
Based upon these results, we conclude that the observed base thesesamelysates
anti-GPIbp-specific monoclonal antibody
change at nucleotide 2004 segregates directly with the pheno- analysisusingthe
typic presence of the Sr" alloantigen. Importantly, the C -> T Gi22, which served to validate the specificity of the labeling
CGT codon for arginine intoa TGT that reaction. As shown in Fig. 5 , GPIb, was visualized by autoramutation changes a --j
"
"
"-9
"
Q
The Sf' Alloantigen
System
of
GPIIIa
8443
TABLE
I1
Quantitation of surface GPIIIa on SF positive and negative platelets
Anti-PIA1or anti-Sr;' allooantibodies were added in increasing concentrationst o 2.5 x lo7 washed human platelets and incubatedfor 45 min a t
37 "C. Platelets were then washed inPBS-BSA, and bound IgG was determined using a competitive enzyme linked immunoassay, as previously
described (30).
Moleculesiplatelet ( n = 3 )
Donor
Phenotype
1
2
3
Anti-PI"'
Anti-Sr'
Normal serum
50,800 ? 5,000
48,300 ? 6,500
27,300 ? 3,500
25,700 ? 4,300
1,650 ? 230
ND"
1,600 ? 200
666 ? 200
1,400 ? 800
" ND, not determined.
aggregation, plateletsfrom a Sr" positive individual were compared with Sr" negative (control) platelets in standard platelet
aggregation assay. Fig. 6 shows the platelet aggregation responses obtained using PRP from 2 Sr" positive individuals
compared with a single Sr" negative healthy donor. As shown,
M ADP resulted ina completely normalaggreaddition of
gation response in platelets derived from both Sr" positive individuals. Similar findings were obtained using collagen (5 pgl
ml) or ristocetin (1.5 mg/ml) as agonists (not shown). These
results suggest that neither theexpression nor function of Sr"
positive platelets are adversely affected by the presence of an
unpaired cysteine residue on the cell surface.
ADP
+
DISCUSSION
Although the importance of integrins in mediating cell-cell
and cell-extracellular matrix interactions is well recognized,
the fact that a number of integrin a- and P-subunits may be
encoded by multiple allelic forms is not well appreciated.
GPIIIa ( P 3 ) is the most polymorphic integrin subunit in man,
and is most frequently responsible for eliciting an alloimmune
response leading tothrombocytopenia and perhaps more widespread damage within the vasculature. To date, six different
genetic variants of the GPIIIamolecule have been identified at
the serological and molecular biological level, and these are
summarized inTable 111. The PIA1allelic form is by far themost
common, with a gene frequency of nearly 85% within the Caucasian population and differs from the second most common
form of P3, PIA2,by a single Leu33 + Pro amino acid substitution. Other alleles of GPIIIa, including the Sr" isoform of this
study, are much less frequently represented, butalso appear to
have arisen by point mutations of the PIA' ancestral allele.
The Sr" alloantigen was originally described serologically in
a case of alloimmune thrombocytopenia, and subsequent immunochemical studies localized the Sr" antigen to the 68-kDa
membrane-bound protease-resistant core of GPIIIa (18),either
on the amino-terminal 121 amino acid residues or on the carboxyl-terminal segment bounded by residues 349-692. By sequencing cDNA derived from PCR-amplified platelet mRNA
(lo), we have localized the polymorphism underlying the Sr"
polymorphism to a C2004+ T substitution, which in turn results in an Arg ~> Cys polymorphism at amino acid 636, just
proximal to the membranefrom the cysteine-rich region of the
molecule. Using mammalian transfection techniques, we were
cysteine residue at
able t o demonstrate that the additional
position 636 of GPIIIa controls the formation of the Sr" alloantigenic epitope. Other three-dimensional structural featuresof
GPIIIa appear tobe required as well, however, since both disulfide bond reduction (18) as well as deglycosylation (Fig. 4)
abolish the presentation of the Sr" epitope.
Interestingly, the
(Sr") allelic form of GPIIIa was
found t o have a higher apparent molecular weight than the
wild-type Arg636 form. This molecular weight polymorphism
appears to be attributable to differential glycosylation of the
altered conformer of GPIIIa induced by the presence of an
1 rnin
FIG.6. ADP
M ) inducedplateletaggregation of two different SP
positive individuals (A and B ) compared with a normal Sra negative
healthy donor (C).
unpaired cysteine residue, since deglycosylation, but not disulfide bond reduction, allowed the Sr" form of GPIIIa to co-migrate on SDS-PAGE with the wild-type allele. At the present
time, wedo not know whether the glycosylation differences
observed are due to altered trimming
of the high-mannose carbohydrate side chains, or to the presence of additional carbohydrate moieties to normally cryptic, unmodified sites.
Following incubation with [3H1NEM, label became incorporated into GPIIIa from Sr" positive platelets, confirming at a
biochemical level the presence of a free sulfhydryl groupin this
rare allelic form of GPIIIa.Parallelexperimentsusing
Sr"
negative platelets, however, revealed no incorporation of
[3H]NEM into platelet GPIIIa,confirming the structural analysis of Calvete et al. (6). The finding of a naturally occurring
isoform of GPIIIa having 57 cysteine residues is remarkable,
since both the number and theposition of the 56 cysteine residues areabsolutely conserved in the integrin P-subunit family
ranging from Drosophila to man (36, 37). One might have expected the presence of an additional, unpaired
cysteine to have
a deleterious effect on either expression of this integrin P-subunit or on platelet function, resembling the effects of similar
mutations that have been shown to be responsible for the congenital platelet functional disorder, Glanzmann thrombasthenia (5,7,38).
However, not only was expressionof the Sr" allelic
form of GPIIIa quantitatively normal, Sr" positive platelets
aggregated to the same extent as
wild-type Sr" negative platelets. Moreover, individuals carrying theSr" allele also show no
obvious hemostatic, immunologic, or vascular abnormalities,
suggesting that theadhesive functions of cells expressing the
vitronectin receptor a& are also unaffected. Examination of
the effects on expression and function of other molecular variations and alloantigenicforms of platelet surfacereceptors
should continue to provide insights into the structural features
of these molecules that influence biosynthesis, trafficking, and
ligand binding capacity.
The SF Alloantigen
System
8444
of
GPIIIa
TABLE111
The six currently recognized forms of the integrin p3 subunit
Allelic form
Gene
frequency"
serologic
designation
0.85
0.15
<0.01
<0.01
<0.01
PlA'
P F
<0.01
a
REFERENCES
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
C f i
SP
In the Caucasian population. Gene frequencies differ in African and Asian populations.
Acknowledgments-We gratefully acknowledge the excellent technical assistance of Micaela Bohringer. We are also grateful toDavid Wilcox for the technical advice in performing the expression studies and to
Jarnilshah Roudy for assistance with~lele-specific O~igonuc~eotide
H~.
bridization
analysis.
1.
2.
3.
4.
Mo
Kieffer, N., and Phillips, D. R. (1990) Annu. Reu. Cell. Biol. 6, 329-357
McEver, R. P., Baenziger, J. U., and Majerus, P. W. (1982) Blood 59, 80-85
Tsuji, T., and Osawa, T. (1986) J . Biochem. (Tokyo) 100, 1387-1398
Fitzgerald, L. A., Steiner, B., Rall, S. C., Jr.,Lo, S. S.,and Phillips, D. R. (1987)
J. Biol. Chem. 262,3936-3939
Newman, P. J. (1991) Thromb. Haemostasis 66, 111-118
Calvete, J. J., Henschen, A., and Gonzalez-Rodriguez, J. (1991) Biochem. J.
274,63-71
Newman, P. J., Seligsohn, U., Lyman, S., and Coller, B. S. (1991) Proc. Natl.
Acad. Sei. U. S. A. 88, 3160-3164
Aster, R. H. (1989) in Platelet Zmmunobiology (Kunicki, T. J.,and George, J . N.,
eds) pp. 3 8 7 4 3 5 , Lippincott, Philadelphia
Mueller-Eckhardt, C., Kiefel, V., and Santoso, S. (1990) Dansfus. Med. Reu. 4,
98-109
Newman, P. J.,Gorski, J., White, G. C., Gidwitz, S., Cretney, C. J., and Aster,
R. H. (1988) J. Clin. Invest. 82, 739-743
Newman, P. J., Derbes, R. S., andAster, R. H. (1989) J. Clin. Inuest. 83,
1778-1781
Lyman, S., Aster, R. H., Visentin, G. P., and Newman, P. J . (1990) Blood 75,
2343-2348
Wang, R., Furihata, K., McFarland, J . G., Friedman, K., Aster, R. H., and
Newman, P. J. (1992) J. Clin. Inuest. 90, 2038-2043
Kuijpers, R. W. A. M., Faber, N.M., Cuypers, H. T. M., Ouwehand, W. H., and
Vondemborne, A. E. G. K. (1992) J. Clin. Invest. 89, 381-384
Santoso, S., Kalb, R., Walka, M., Kiefel, V., Mueller-Eckhardt, C., and Newman, P. J. (1993) J. Clin. Inuest. 92, 2427-2432
Wang, R., McFarland, J. G., Kekomaki, R., and Newman,
P. J . (1993)Blood 82,
3386-3391
Wang, L., Juji, T., Shibata, Y., Kuwata, S., and Tokunaga, K. (1991) Proc. Jpn.
Acad. 67, 102-196
18. Kroll, H., Kiefel, V., Santoso, S., and Mueller-Eckhardt. C. (1990) Blood 76,
2296-2302
19. Kekomaki, R., Raivio, p., and Kero, p. (1992) nuns. Med. 2,27-33
20. Kekomaki, R., Jouhikainen, T., Ollikainen, J., Westman, P., and L a w M.
(1993) Br J. Haematol. 83, 306-310
21.
A. Kuijpers,
W.
R.
M., Simsek, S., Faber, N. M., Goldschmeding, R., van
Wermerkerken. R. K. V.. and Von dem Borne. A. E. G. K. (1993) Blaad 81.
70-76
22. Kiefel, V., Santoso, S., Weisheit, M., and Mueller-Eckhardt. C. (1987) Blood 70,
1722-1726
23. Santoso, S., Kiefel, V., and Mueller-Eckhardt. C. (1989) Br J . Haematol. 72,
191-198
24. Kiefel, V., Santoso, S., Kaufmann, E., and Mueller-Eckhardt, C. (1991) Br J.
Haematol. 79,256-262
25. Smith, J.W., Hayward, C. P. M., Warkentin, T.E., Horsewood, P., and Kelton,
J . G. (1993) J. Immunol. Methods 158, 77-85
26. Kalomiris, E. L., and Coller, B. S. (1985) Biochemistry 24, 5430-5436
27. Miller, S. A,, Dykes, D. D., and Polesky, H. F, (1988) Nucleic Acids Res. 16,
1215-1216
28. McFarland, J. G., Aster, R. H., Bussel, J. B., Gianopoulos, J. G., Derbes, R. S.,
and Newman, P. J. (1991) Blood 7 8 , 2 2 7 6 2 2 8 2
29. Goldberger, A., Kolodziej, M., Poncz, M., Bennett, J. S., and Newman, P.J.
(1991) Blood 78,681-687
30. Kiefel, V., and Mueller-Eckhardt, C. (1990) in Laboratory Methods i n Zmmunology (Zola, H., ed) pp. 241-248, CRC Press, Boca Raton, FL
31. Niewiarowski, S., Norton, K. J., Eckardt, A,, Lukasiewicz, H., Holt, J. C., and
Kornecki, E. (1989) Biachim. Biophys. Acta 983,91-99
32. Beer, J., and Coller, B. S. (1989) J. Biol. Chem. 264, 17564-17573
33. Kouns, W. C., Newman, P. J., Puckett, K. J., Miller, A. A,, Wall, C. D., Fox, C.
F., Seyer, J . M., and Jennings, L. K. (1991) Blood 78,3215-3223
34. Newman, P. J., Martin, L. S., Knipp, M. A,, and Kahn, R. A. (1985) Mol.
Immunol. 22, 719-729
35. Newman, P. J., Allen, R. W., Kahn, R. A,, and Kunicki, T. J. (1985) Blaad 65,
227-232
36. Bogaert, T., Brown, N., and Wilcox, M. (1987) Cell 51,929-940
37. DeSimone, D. W., and Hynes, R. 0. (1988) J. Biol. Chem. 263,5333-5340
38. Burk, C. D., Newman, P. J., Lyman, S., Gill, J., Coller, B. S., and Poncz, M.
(1991) J. Clin. Znuest. 87, 270-276
39. Zimrin, A. B., Gidwitz, S., Schwartz, E., Bennett,
J. S., White, G. C., and Poncz,
M. (1990) J. Biol. Chem. 265,8590-8595