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Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 205-211
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Threonine-145/Methionine-145 variants of baculovirus
produced recombinant ligand binding domain of GPIb express HPA-2
epitopes and show equal binding of von Willebrand factor
Chester Q. Li,
Stephen F. Garner,
Julian Davies,
Peter A. Smethurst,
Mark R. Wardell, and
Willem H. Ouwehand
From the Department of Hematology, University of Cambridge, National
Blood Service East Anglia, Cambridge, UK; National Institute for
Biological Standards and Control, Potters Bar, UK; and the Department
of Biochemistry and Molecular Biophysics, Washington University School
of Medicine, St. Louis, MO.
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Abstract |
Glycoprotein (GP) Ib is the functionally dominant subunit
of the platelet GPIb-IX-V receptor complex, with the von
Willebrand factor (vWF) binding site residing on the
amino-terminus. A threonine for methionine-145 replacement of GPIb
is associated with the human platelet antigen (HPA)-2
system. To study the structural and functional consequences of this
mutation, both forms of GPIb were expressed as calmodulin fusion
proteins in insect cells. Both recombinant proteins were
recognized by their respective alloantibodies, independent of
glycosylation or intactness of disulfide bonds, and gave similar
results to platelet-derived GPIb in antibody detection assays.
Resonant mirror studies showed that vWF binding was not affected by the
HPA-2 mutation; however, vWF binding was partially inhibited by IgG
HPA-2 antibodies. Our data are compatible with an involvement of the
leucine-rich repeat domain of GPIb in vWF binding and
indicate that recombinant GPIb may be used to detect HPA-2
antibodies. (Blood. 2000;95:205-211)
© 2000 by The American Society of Hematology.
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Introduction |
The platelet glycoprotein (GP)Ib-IX-V complex plays an
essential role in the maintenance of normal hemostasis by mediating initial adhesion of platelets to the vessel wall via von Willebrand factor (vWF). Initial studies mapped the binding sites for vWF and
 -thrombin to the amino-terminal part of GPIb, and more recent data indicate the involvement of the leucine-rich repeat region (LRR)
in vWF binding.1,2 In vivo vWF binding of the GPIb-IX-V complex requires both immobilization of vWF in the subendothelium and
the presence of high shear forces.3 In vitro binding can be
initiated by both ristocetin, a peptide antibiotic, and botrocetin, a
snake venom protein.1
In earlier studies, we and others obtained evidence that the di-allelic
human platelet antigen (HPA)-2 system is localized on the amino
terminal 45 kDa fragment of GPIb and that the alloantigens co-segregate with a cytosine to thymine mutation at position 434 (the
codon for residue 145 in the fifth LRR) of the GPIb
gene.4-6 This strongly suggests that the alloantigens are
based on the Thr/Met-145 exchange arising from this mutation. The two
alleles, HPA-2a and HPA-2b, have respective frequencies in the
Caucasian population of approximately 0.926 and 0.074.7-9
Exposure to nonself forms of the HPA-2 antigens via either pregnancy or
transfusion may lead to alloantibody formation that can cause neonatal
alloimmune thrombocytopenia, refractoriness to platelet transfusions,
and posttransfusion purpura.10
To date there is only limited proof that the HPA-2 B cell epitopes are
based solely on this single amino acid substitution, as studies have
been based on use of a single example of anti-HPA-2b.6 Furthermore, the roles of glycosylation and disulfide bonds in the
formation of the epitopes remain to be determined. Similarly, many of
the studies on vWF binding to GPIb have been performed with GPIb
in its native stoichiometry on the platelet membrane in the context of
GPI , GPIX, and GPV.
Interestingly, HPA-2 antibodies abolish ristocetin-mediated
agglutination of platelets.4 Why HPA-2 antibodies inhibit
is not well understood, because residue 145 is located in the fifth LRR
of GPIb and is distant from the proposed vWF binding site (residues
252-287).11 Obvious explanations might be the presence of a
second vWF binding site, or alternatively the antibody-mediated inhibition is based on allosteric hindrance.
In the current study, both forms of a truncated GPIb (residues
His1-Val289) with either a Thr or Met at position 145 were expressed in
a baculovirus/insect cell system as calmodulin (CaM) fusion proteins.
The use of CaM as a tag for proteins has been reported
previously.12 The fusion proteins were used in studies designed to answer the above questions on the molecular nature of the
HPA-2 alloantigens, the effect of the mutation on vWF binding, and the
effect on the inhibition of vWF binding by HPA-2 antibodies.
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Materials and methods |
Platelets, human HPA-2 antibodies, and monoclonal antibodies
HPA-2a2a and -2b2b platelets from donors genotyped by
PCR-SSP13 were supplied by the Platelet Immunology
Laboratory of the National Blood Service (NBS), East Anglia Centre,
Cambridge, UK. Negative control sera were obtained from a nontransfused
group AB male blood donors. Sera containing anti-HPA-2a and -2b were kind gifts from Professor Shibata (Tokyo University, Tokyo, Japan), Mr.
C. Hurd, and Mrs. C. Quader (NBS, East Anglia, and NBS, Sheffield, UK),
and Dr. L. Porcelijn (CLB, Amsterdam, The Netherlands). All sera were
stored at  20°C until required for use.
The GPIb (CD42b)-specific monoclonal antibody (mAb) MB45 was a gift
from Professor A. E. G. Kr von dem Borne (CLB).
Construction of plasmids
To facilitate protein purification and subsequent immobilization on
a resonant mirror sample cuvette, both forms of GPIb were expressed
as CaM fusion proteins in a baculovirus/insect cell system. A
complementary DNA fragment coding for the signal peptide and mature
protein residues His1-Val289 of GPIb was obtained by polymerase
chain reaction from the plasmid pDX,14
which contained the full-length GPIb gene. The sense and anti-sense oligonucleotide primers used for cloning were (1)
5'-ACCTCGAGATGCCCTCCTCCTCTTGCTG-3'(contains a
XhoI site); and (2)
5'-GCGGCCGCTGTATGGCTTTGGTGGGGAA-3'12 (contains a NotI site). The
complementary DNA fragment amplified using these primers was digested
with the restriction enzymes XhoI and NotI and ligated
into the vector pND162 that contained the CaM gene (kindly provided by
Dr. D. Neri, Medical Research Council Laboratory of Molecular Biology,
Cambridge, UK).12 The resulting plasmid was then digested
with XhoI and EcoRI to release the fusion gene encoding
a GPIb fragment in frame with the CaM gene. The fusion construct was
ligated into the baculovirus expression vector pAcSG2 and its sequence
confirmed. A construct with a 145-Met codon was generated by
mutagenesis using the QuickChange method15 (Stratagene, La
Jolla, CA) and the mutation was verified by sequencing.
Expression, purification, and characterization of recombinant
GPIb/CaM
Cell culture.
Sf9 and High Five insect cells were routinely propagated in Sf-900 II
SFM and Express Five SFM media, respectively, as adherent cell
monolayers in 25 cm2 tissue-culture flasks or as suspension
cultures in spinner flasks at 27°C.
The Sf9 and High Five cells were either seeded at a density of
1 × 106 cells/mL in spinner flasks and grown to
2 × 106 cells/mL with constant stirring at 100 rpm,
or seeded for adherence in flasks at a density of
1.2 × 105 cells/cm2 for Sf9 cells and
5 × 104 cells/cm2 for High Five cells
and grown to 80% confluency.
Expression.
Recombinant baculovirus transfer vectors containing either fusion
construct were transfected into Sf9 cells according to the manufacturer's protocol (PharMingen, San Diego, CA). Briefly, 0.5 µg
of linearized BaculoGold virus DNA and 2.5 µg recombinant baculovirus
transfer vector were mixed for 5 minutes in a microcentrifuge tube at
room temperature and then 1 mL of transfection buffer was added. A
60-mm2 Falcon tissue culture dish was seeded with
2 × 106 Sf9 cells; after 20 minutes cells were
ready for transfection, and medium was replaced with 1 mL
Grace's medium and the above DNA mixture. The cells were incubated for
4 hours at 27°C, and the medium was then replaced with fresh
medium. After 5 days of culturing at 27°C, the medium
was collected for plaque purification or further virus amplification.
High Five cells were cultured in 100 mL spinner flasks with 50 mL cell
culture medium at constant stirring (100 rpm) and infected with high
titre recombinant virus stock at 10 20 multiplicities
of infection. The infected High Five cells were grown for 72 hours,
and, following centrifugation, the medium was collected for protein
purification. To produce nonglycosylated GPIb/CaM protein, cells
were cultured in the presence of tunicamycin at 10 µg/mL.
Purification of CaM tagged GPIb.
Fifty mL of culture medium was applied to a 5 mL column of W-7 agarose
4B resin (Sigma, Poole, UK) that was equilibrated in TBSC buffer (50 mM
Tris, 150 mM NaCl, and 1 mM CaCl2, pH 7.4) with a flow rate
of 25 mL/h. Nonspecifically bound material was removed by washing with
high salt buffer (50 mM Tris, 600 mM NaCl, and 1 mM CaCl2,
pH 7.4). Bound GPIb/CaM fusion protein was eluted with 20 mM EGTA in
TBS buffer (50 mM Tris, 150 mM NaCl, pH 7.4). Eluted protein solution
was concentrated by ultrafiltration (Amicon, Beverly, MA), and protein
concentration was determined by the method of Lowry.16
Gel electrophoresis and immunoblotting.
Purified recombinant proteins (containing Thr-145 or Met-145) were
separated on 10% minigels for 45 minutes according to the method of
Laemmli,17 but substituting
2-amino-2-methyl-1,3-propanediol for Tris base. To determine the
importance of the disulfide bridges for the HPA-2 epitopes, recombinant
proteins were treated with reducing reagents before electrophoresis.
The gels were either stained with Coomassie Blue or transferred to
nitrocellulose filters for Western blots. For immunoblotting, the
membranes were blocked with 0.01% Tween-20 in phosphate buffered
saline (PBS) for 30 minutes and then reacted overnight at
4°C with a 1 in 10 dilution of anti-HPA-2a and -2b sera, with
continuous shaking. After extensive washing with blocking buffer, the
membranes were incubated with a 1 in 1000 dilution of
peroxidase-labeled anti-human IgM (for anti-HPA-2a) or IgG (for
anti-HPA-2b) for 2 hours followed by extensive washing. Binding of
antibody to the recombinant proteins was detected by the ECL method
(Amersham, Arlington Height, IL).
Detection of HPA-2-specific antibodies
Enzyme-linked immunoabsorbent assay (ELISA).
A buffer containing 10 mM Tris, 145 mM NaCl, pH 7.4, 0.5%
Nonidet P40, 0.05% Tween 20, 0.5 mM CaCl2, and 0.2%
bovine serum albumin (BSA) was used for all wash steps and for diluting
reagents, except where stated otherwise. Human sera were diluted 1 in
20 with the use of an ELISA specimen diluent (Abbott Laboratories, Abbott Park, IL). Microplate wells were coated with goat
anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc, West Grove,
PA) diluted in sodium carbonate buffer, pH 9.6 by incubating overnight at 4°C. The plates were then washed twice and subsequently blocked by incubating with 3% BSA for 60 minutes at 37°C, followed by five
washings. The CD42b mAb MB45 (1 in 1000 dilution of ascites) was added
to the microplate wells and incubated for 60 minutes at 22°C,
followed by five washes. The Thr- and Met-145 forms of recombinant
GPIb (2.5 µg/mL) were added to the wells and incubated for 30 minutes. After five more washes, the diluted human sera were added and
incubated for 30 minutes. Following five more washes, bound human
antibody was detected with an alkaline phosphatase conjugated goat
anti-human IgG or IgM reagent (Jackson) with the use of p-Nitrophenyl
phosphate substrate in Tris buffer, and optical density was measured in
an ELISA reader (MR5000) at 405 nm.
Monoclonal antibody-specific immobilization of platelet antigens
(MAIPA) assay.
The MAIPA was performed as previously described.18,19
Briefly, platelets from either HPA-2a2a or -2b2b donors were incubated with sera, washed, and then incubated with mAb MB45. The platelets were
washed again, solubilized, and centrifuged, and the clear lysates added
to microplate wells that had been previously coated with goat
anti-mouse IgG to capture the mAb-GPIb/IX complex. Human IgG bound to
the complex was detected with alkaline phosphatase conjugated goat
anti-human IgG (Jackson). The optical density was measured in an ELISA
reader (MR5000) at 405 nm.
Resonant mirror technology.
An IAsys Auto+ resonant mirror biosensor (Labsytems Affinity Sensors,
Cambridge, UK) was also used to study the reactivity of HPA-2
antibodies and vWF binding to both forms of the recombinant GPIb/CaM
fusion protein in the presence and absence of ristocetin (see below).
vWF binding studies
The 23-mer CaM-binding oligopeptide (CAAARWKKAFIAVSAANRFKKIS)
cross-linked to BSA20,21 was coupled to the
carboxymethylated dextran surface of the sample cuvette in 10 mM sodium
acetate, pH 5, according to the manufacturer's instructions. This
coupling allowed capture of the CaM-tagged GPIb in the
presence of calcium, with an insignificant off rate. For subsequent
assays, the recombinant GPIb/CaM fusion protein was removed from the
cuvette surface by the addition of 10 mM EDTA. All experiments were
performed in the presence of Tris buffered saline (50 mM Tris-HCl, 150 mM NaCl) containing 20 mM CaCl2 at 25°C. The factor
VIII concentrate Alphanate (Alpha Therapeutic Corporation, Los Angeles,
CA) was used as a source of vWF and diluted according to the
manufacturer's instructions to give a final vWF concentration of 5 µg/mL.
Binding of vWF to GPIb.
Following capture of Thr-145 GPIb vWF (5 µg/mL) and ristocetin
(1.5 mg/mL; Sigma Aldrich Company Ltd, UK) were added to the cuvette,
and binding of vWF was observed. Thr-145 GPIb and vWF were then
removed by washing of the cuvette with a buffer containing 10 mM EDTA,
and the experiment repeated with Met-145 GPIb.
Inhibition of vWF binding to GPIb by affinity
purified anti-HPA-2b.
Microtitre plate wells were coated with 2.0 µg/mL of BSA-CaM binding
peptide in 100 µL of binding buffer 100 mM NaCl,50 mM Tris-HCl, pH
7.4 and incubated overnight at 4°C. The plates were then blocked
with 3% BSA for 2 hours at room temperature. After blocking, 1.0 µg/mL of Met-145 GPIb/CaM fusion protein was added to the wells in
a 100-µL volume of binding buffer and incubated overnight at 4°C.
After washing with buffer, vWF at various concentrations and ristocetin
(1.5 mg/mL) were added to the wells and incubated overnight at 4°C.
vWF binding to GPIb without ristocetin was included as a negative
control. Incubation in the presence of purified IgG (1.0 mg/mL)
prepared from a serum sample containing anti-HPA-2b was used to measure
the inhibitory capacity of anti-HPA-2b on vWF binding, and purified IgG
from an inert serum was included as a negative control. After extensive
washing, bound vWF was detected with a rabbit anti-vWF, 1:1000 that
was incubated for 2 hours at room temperature. After washing with
buffer, the bound rabbit antibodies were detected by horseradish
peroxidase-conjugated pig anti-rabbit antiserum 1:1000 at room
temperature for 1 hour. After five washings with buffer, 100 µL
of peroxidase substrate o-Phenylenediamine buffer was
added to the wells for color development, which was measured at 450 nm
in a multichannel photometer (ThermoMax, Molecular Devices Corporation,
Menlo Park, CA).
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Results |
Binding of antibodies to GPIb/CaM fusion protein
Insect cells infected with recombinant baculovirus expressed high
levels of protein (8.0 mg/L after single-step purification) with the
expected molecular mass of 60 and 56 kDa for glycosylated and
nonglycosylated, amino-terminal 289 residues of GPIb fused with CaM
(Figure 1, lanes 1 and 2, respectively).
The relative molecular masses of the recombinant amino-terminal GPIb
without CaM differed in the presence and absence of tunicamycin,
(Figure 1, lanes 3 and 4, respectively) with a reduction occurring in the presence of tunicamycin. The recombinant GPIb migrated with a
molecular mass 3-4 kDa lower than corresponding platelet-derived fragment (Figure 1, lanes 4 and 5, respectively) because of
differential glycosylation of two N-linked sites in the recombinant
GPIb.
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| Fig 1.
Gel electrophoresis of different forms of glycoprotein
(GP)Ib.
Molecular weight markers (M). Recombinant GPIb/calmodulin (CaM)
(lane 1). Recombinant GPIb/CaM in the presence of tunicamycin (lane
2). Recombinant GPIb in the presence of tunicamycin (lane 3).
Recombinant GPIb (lane 4). Platelet-derived, elastase digested
GPIb (lane 5).
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Binding of HPA-2-specific antibodies to the Thr-145 and Met-145 forms
of the recombinant GPIb/CaM fusion protein at coating concentrations
of 0.625-10 µg/mL was determined by ELISA assay with the use of
single samples of polyclonal IgM anti-HPA-2a and IgG anti-HPA-2b.
Binding was shown to be specific for the expected protein or antibody
combinations, giving dose-response curves reaching a plateau at about
7.5 µg/mL (Figure 2A). As a satisfactory signal-to-noise ratio was obtained at 2.5 µg/mL, this concentration was used for all further assays. Further specific binding of HPA-2 antibodies to their respective forms of recombinant GPIb in ELISA was demonstrated with a panel of anti-HPA-2b (see below). This reactivity was confirmed in resonant mirror studies in which specific antibody binding to the relevant form of GPIb was indicated by an
increased response in arc seconds after washing, when compared with the
response with the irrelevant GPIb. (Figure
3, A to D).
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| Fig 2.
Enzyme-linked immunoabsorbent assay studies of antibody
binding to recombinant glycoprotein (GP)Ib.
(A) Binding of anti-human platelet antigen (HPA)-2a and -2b to Thr-145
and Met-145 forms. (B) Binding of three examples of anti-HPA-2b and a
negative control serum to glycosylated and nonglycosylated (NG)
Met-145.
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| Fig 3.
Binding of human platelet antigen (HPA)-2 antibodies to
both forms of recombinant glycoprotein (GP)Ib measured by resonant
mirror studies.
Addition of recombinant GPIb to biosensor cuvette (1). Washing
with TBS/Ca++ (2). Addition of serum diluted 1 in 10 (3).
Washing with TBS/Ca++(4). Distance between points
y1 and y2 represent specifically bound antibody
remaining after TBS/Ca++ wash, expressed as arc seconds.
(A) Incubation of anti-HPA-2a with Thr-145,
y2 y1 = 83 arc seconds. (B)
Incubation of anti-HPA-2a with Met-145,
y2 y1 = 21 arc seconds. (C)
Incubation of anti-HPA-2b with Thr-145,
y2 y1 = 7 arc seconds. (D)
Incubation of anti-HPA-2b with Met-145,
y2 y1 = 128 arc seconds.
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Glycosylation and disulfide bonds are not critical for the epitopes
recognized by HPA-2 antibodies
Three HPA-2b antisera all gave similar results with glycosylated and
nonglycosylated recombinant GPIb/CaM fusion proteins by ELISA,
suggesting that glycosylation is not relevant for the HPA-2 B cell
epitopes (Figure 2B).
The 42 kDa amino-terminal segment of GPIb contains three disulfide
loops, one amino-terminal to the LRR (Cys-4 to Cys-17) and two located
carboxy-terminal to this region (Cys-209 to Cys-248; Cys-211 to
Cys-264).1 To determine the possible importance of the
disulfide bonds in the expression of the HPA-2 epitopes, the two forms
of recombinant GPIb were analyzed by Western blotting. Both
anti-HPA-2a and anti-HPA-2b were found to only react with 145-Thr and
145-Met, respectively, under the denaturing conditions of the SDS-PAGE
(data not shown). The antibodies were further tested under reducing and
nonreducing conditions and transferred to nitrocellulose in which
binding was then analyzed by Western blotting (Figure
4). The data demonstrated that HPA-2a and
HPA-2b antibodies were able to distinguish their respective
alloantigens on Western blots under both nonreducing and reducing
conditions.
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| Fig 4.
Western blots of Thr-145 and Met-145 forms of recombinant
glycoprotein (GP)Ib under reducing and nonreducing conditions.
(A) Coomassie blue stain of recombinant GPIb under nonreducing
conditions (Thr-145, lane 1, and Met-145, lane 3) or reducing
conditions (Thr-145, lane 2, and Met-145, lane 4). (B) Western blot of
duplicate of panel A with anti-human platelet antigen (HPA)-2a (lanes 1 and 2), and anti-HPA-2b (lanes 3 and 4).
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Comparison between platelet-derived and recombinant GPIb for
HPA-2 antibody detection
The standard technique for the detection and identification of HPA-2
antibodies is the MAIPA assay with the use of HPA-2 typed platelets as
a source of GPIb complexed with GPIb and GPIX. We compared the
MAIPA with our recombinant protein-based antibody detection ELISA with
the use of five HPA-2b antisera. All five reacted strongly with the
Met-145 form of the protein, but not the Thr-145 form, when compared
with the negative control (Figure 5A).
Similar results were obtained by MAIPA, with all five sera reacting
strongly with HPA-2b2b platelets (Figure 5B). Three of the anti-HPA-2b
sera (samples 2, 3, and 4) were titrated to compare sensitivity of the
two assays: sample 2 had a titre of 64 by ELISA and 128 by MAIPA,
whereas samples 3 and 4 had identical titers of 256 and 64, respectively, by both techniques.
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| Fig 5.
Comparison between recombinant and platelet-derived
glycoprotein (GP)Ib for human platelet antigen (HPA)-2 antibody
detection.
(A) An inert negative control serum and five different sera containing
anti-HPA-2b tested against the Thr-145 and Met-145 forms of recombinant
GPIb by enzyme-linked immunoabsorbent assay. (B) The same sera
tested against platelet-derived GPIb by monoclonal antibody-specific
immobilization of platelet antigens.
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Effect of Thr/Met-145 mutation on vWF binding and inhibition by
HPA-2 antibodies
The two forms of recombinant GPIb/CaM fusion protein were used to
investigate possible effects of the Thr/Met-145 mutation on vWF
binding. In the presence of ristocetin, and with equal amounts of
recombinant GPIb immobilized on the biosensor cuvette, vWF was
observed to bind equally to both the Thr-145 and Met-145 forms (Figure
6). No significant uptake of vWF was seen
in the absence of ristocetin.
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| Fig 6.
Demonstration of von Willebrand factor (vWF) binding to
the Thr-145 and Met-145 forms of glycoprotein (GP)Ib with the use of
resonant mirror technology.
Recombinant GPIb-calmodulin was coupled to bovine serum
albumin-peptide immobilized on the carboxymethylated dextran surface
and binding of vWF to both forms of GPIb measured.
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HPA-2 alloantibodies inhibit ristocetin-induced platelet agglutination
but not collagen-induced platelet aggregation.4 The
mechanism of this inhibition is not known. We investigated this finding
further with the use of anti-HPA-2b in an inhibition ELISA. vWF binding to GPIb-145-Met was measured at a single IgG concentration of 1 mg/mL and a range of vWF concentrations from 6.25 to
500 ng/well. Inhibition, which reached a plateau at about 40%, was
observed (Figure 7).
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| Fig 7.
Inhibition of von Willebrand factor (vWF) binding to
recombinant glycoprotein (GP)Ib by IgG anti-human platelet antigen
(HPA)-2b determined by enzyme-linked immunoabsorbent assay.
vWF was allowed to bind to GPIb-Met-145 immobilized on microtitre
plate wells via bovine serum albumin-peptide in the presence of
ristocetin ( ) and IgG anti-HPA-2b ( ). Bound vWF was detected with
a polyclonal antibody. IgG obtained from an inert group AB serum was
used as a negative control ( ). vWF did not bind in the
absence of ristocetin (x).
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Discussion |
High-level expression (8 mg/L after purification) of both the Thr-
and Met-145 forms of truncated GPIb (amino acids His1-Val289) fused
to CaM was achieved in insect cells. The CaM tag allowed single-step
purification and reversible calcium dependent binding to the
carboxymethylated dextran surface for resonant mirror studies. Characterization by SDS gel electrophoresis showed proteins of the
expected size (Figure 1), whose migration was decreased on reduction
(Figure 4). Synthesis of the Met-145 form in the presence of
tunicamycin caused a reduction in molecular mass, most likely caused by
the absence of the two N-linked glycan moieties at residues 21 and 159 (Figure 1). The baculoviral protein also had a reduced molecular mass
when compared with the platelet-derived fragment of GPIb obtained
by elastase digestion, indicating different glycosylation in the
recombinant protein. As already mentioned, differences in glycosylation
relate to the two N-linked glycan moieties that have molecular masses
of around 2-3 kDa each, giving a maximum difference of 6 kDa between
glycosylated and nonglycosylated fragments.
The absent or alternative glycosylation does not seem to have a
detrimental effect on the binding of HPA-2 alloantibodies. Indeed, the
observation that these antibodies bind to both the glycosylated and
nonglycosylated recombinant forms of GPIb (Figure 2B) indicates
that glycosylation is not required for the expression of their
respective epitopes and corroborates proper folding of the recombinant
protein. That the glycan moieties are of no significance to the binding
of HPA-2 antibodies is comparable with HPA-1a antibodies, which react
with the E. coli produced amino-terminal 66 amino acids of the
leucine-33 form of GPIIIa (3 integrin
subunit).22 In contrast, binding of HPA-3a23
and HPA-5b24 alloantibodies to the isoleucine-843 form of
the IIb and the lysine-505 form of the 1 integrin,
respectively, is dependent on glycosylation.
Antibody detection by MAIPA and ELISA indicate that a sensitivity can
be achieved with the recombinant antigens comparable to that obtained
with the native platelet derived GPIb. If the nonspecific binding of
IgG to the solid phase can be reduced by modification of the antibody
detection ELISA, the requirement to dilute serum samples 1 in 20 might
be unnecessary, thus avoiding possible loss of sensitivity because of
antibody dilution. Although the recombinant GPIb/CaM fusion proteins
were bound to the solid phase via the CD42b mAb MB45 to enable
comparison with MAIPA, this situation has the potential
problem that human sera with anti-mouse activity25,26 will
give false-positive results. As an alternative, the CaM tag could be
used to bind the recombinant protein to a solid phase. The resonant
mirror results clearly show that rapid, but reversible binding, can be
achieved with specific uptake of HPA-2a and -2b antibodies from
1 : 10 diluted serum samples (Figure 3, panels A to D).
The specific reactivity of the HPA-2 antibodies with their
corresponding Thr-145 or Met-145 form of the protein make it highly unlikely that the reactions were with the CaM fragment, which was
common to both forms of the recombinant GPIb. The apparent minor
cross-reactivity exhibited by some HPA-2b antibodies showing minor
reactivity with the Thr-145 GPIb in ELISA, and similarly anti-HPA-2a
with the Met-145 GPIb in the biosensor studies, is also unlikely to
be because of reactivity with the CaM portion, as it also occurred with
platelet-derived GPIb in the MAIPA assays. It most likely reflects
similar reactivity already described within the HPA-2
system,27 or minor variations in binding that are not
significantly different from variation in negative controls.
By use of a panel of anti-HPA-2b and an anti-HPA-2a in multiple assay
systems, the current study ruled out the possibility that the other
subunits of the GPIb-IX-V complex are associated with or required for
the formation of both the HPA-2a and HPA-2b B cell epitopes and showed
that the epitopes are not dependent on disulfide bond formation (Figure
4). The latter is in contrast with the results of experiments with the
Thr-145 and Met-145 forms of a 302-residue GPIb fragment expressed
in mammalian cells, which showed that a single example of the antibody
only reacted with its respective antigen by dot blot using nonreduced
recombinant protein.6 This discrepancy is most likely a
reflection of the different immunoassays used for
antibody detection, but it may also be a feature of the single-antibody
sample used in the study.
Previous work with HPA-2a and -2b platelets did not produce evidence
that the Thr/Met-145 polymorphism has an effect on ristocetin-mediated platelet agglutination.7 However, a small effect of the
Thr/Mer mutation on the binding kinetics of vWF to
GPIb might have been masked in the aggregation assay. The
recombinant forms of GPIb were, therefore, used to investigate the
possible effects of the mutation in finer detail. The resonant mirror
studies of vWF binding to CaM immobilized GPIb confirmed that the
binding kinetics to both forms of GPIb are identical (Figure 6).
However, studies of coronary heart disease and cerebral vascular
disease have found an increased frequency of the HPA-2b allele,
suggesting that the mutation does have a functional
effect.28,29 These conflicting observations may reflect
differences between in vitro ristocetin-induced vWF binding and binding
in vivo. Alternatively, the HPA-2 polymorphism could be genetically
linked to two other polymorphisms that influence GPIb structure
(variable number of tandem repeats polymorphism)30,31 and
expression level (Kozak polymorphism)32 and have potential effect on thrombotic disease.
In conclusion, our studies demonstrate that the two forms of
recombinant GPIb/CaM fusion protein are useful for the detection of
HPA-2 antibodies and that disulfide bond formation and glycosylation are not important for formation of the B cell epitopes. At the molecular level, there is no evidence that the Thr-/Met-145 mutation alone affects vWF binding. That HPA-2 antibodies inhibit vWF binding is
suggestive of an involvement of the LRR in vWF binding, as it is
unlikely that the inhibition is explained by allosteric hindrance.
| |
Acknowledgments |
We gratefully acknowledge the continuous support of the Apheresis
Clinic and Platelet Immunology Reference Laboratory at the National
Blood Service East Anglia Centre during this study and thank Dr N. A. Watkins for helpful discussions during preparation of the manuscript.
The generous donation of monoclonal antibodies and HPA-2 antibodies by
Prof von dem Borne, Mr C. Hurd, Dr L. Porcelijn, Mrs C. Quader, and
Prof Shibata is greatly acknowledged.
| |
Footnotes |
Submitted March 30, 1999; accepted September 8, 1999.
J. Davies was supported by grant G9410995 from the Medical Research
Council, UK; C. Q. Li and S. F. Garner were supported by a grant from
the English National Blood Service.
Reprints: Willem H. Ouwehand, Division of Transfusion Medicine,
Department of Hematology, University of Cambridge, National Blood
Service East Anglia, Long Road, Cambridge, CB2 2PT, UK; e-mail:
who1000{at}cam.ac.uk.
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.
| |
References |
1.
Lopez JA.
The platelet glycoprotein Ib-IX complex.
Blood Coagul Fibrinolysis.
1994;5:97-119[Medline]
[Order article via Infotrieve].
2.
Berndt MC, Shen Y, Romo GA, Kenny DA, Lopez JA, Andrews RK.
Identification of monoclonal antibody epitopes and the ristocetin-dependent binding site on the -chain of the platelet GPIb-IX-V complex using canine-human chimaeras [abstract].
Blood.
1998;92:703a.
3.
Savage B, Saldivar E, Ruggeri ZM.
Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor.
Cell.
1996;84:289-297[Medline]
[Order article via Infotrieve].
4.
Kuijpers RW, Ouwehand WH, Bleeker PM, Christie D, von dem Borne AE.
Localization of the platelet-specific HPA-2 (Ko) alloantigens on the N-terminal globular fragment of platelet glycoprotein Ib.
Blood.
1992;79:283-288[Abstract/Free Full Text].
5.
Kuijpers RW, Faber NM, Cuypers HT, Ouwehand WH, von dem Borne AE.
NH2-terminal globular domain of human platelet glycoprotein Ib has a methionine145/threonine145 amino acid polymorphism, which is associated with the HPA-2 (Ko) alloantigens.
J Clin Invest.
1992;89:381-384.
6.
Murata M, Furihata K, Ishida F, Russell SR, Ware J, Ruggeri ZM.
Genetic and structural characterization of an amino acid dimorphism in glycoprotein Ib involved in platelet transfusion refractoriness.
Blood.
1992;79:3086-3090[Abstract/Free Full Text].
7.
Mazzucato M, Pradella P, de Angelis V, Steffan A, de Marco L.
Frequency and functional relevance of genetic threonine145/methionine145 dimorphism in platelet glycoprotein Ib in an Italian population.
Transfusion.
1996;36:891-894[Medline]
[Order article via Infotrieve].
8.
Simsek S, Faber NM, Bleeker PM, Vlekke AB, Huiskes E, Goldschmeding R, et al.
Determination of human platelet antigen frequencies in the Dutch population by immunophenotyping and DNA (allele-specific restriction enzyme) analysis.
Blood.
1993;81:835-840[Abstract/Free Full Text].
9.
Steffensen R, Kaczan E, Varming K, Jersild C.
Frequency of platelet-specific alloantigens in a Danish population.
Tissue Antigens.
1996;48:93-96[Medline]
[Order article via Infotrieve].
10.
von dem Borne AE, Ouwehand WH.
Immunology of platelet disorders. In:
Caen JP, ed.
Platelet disorders. Bailliere Tindall: London.; 1989:749-781.
11.
Katagiri Y, Hayashi Y, Yamamoto K, Tanoue K, Kosaki G, Yamazaki H.
Localization of von Willebrand factor and thrombin-interactive domains on human platelet glycoprotein Ib.
Thromb Haemost.
1990;63:122-126[Medline]
[Order article via Infotrieve].
12.
Neri D, De Lalla C, Petrul H, Neri P, Winter G.
Calmodulin as a versatile tag for antibody fragments.
Biotechnology.
1995;13:373-377[Medline]
[Order article via Infotrieve].
13.
Cavanagh G, Dunn AN, Chapman CE, Metcalfe P.
HPA genotyping by PCR sequence-specific priming (PCR-SSP): a streamlined method for rapid routine investigations.
Transfus Med.
1997;7:41-45[Medline]
[Order article via Infotrieve].
14.
Lopez JA, Leung B, Reynolds CC, Li CQ, Fox JE.
Efficient plasma membrane expression of a functional platelet glycoprotein Ib-IX complex requires the presence of its three subunits.
J Biol Chem.
1992;267:12,851-12,859[Abstract/Free Full Text].
15.
Papworth C, Braman J, Wright DA.
Site-directed mutagenesis in one day with greater than 80% efficiency.
Strategies.
1996;9:3.
16.
Lowry O, Rosenbrough NJ, Farr AL, Randall RJ.
Protein measurement with Folin phenol reagent.
J Biol Chem.
1951;193:265[Free Full Text].
17.
Laemmli UK.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature.
1970;227:680[Medline]
[Order article via Infotrieve].
18.
Kiefel V, Santoso S, Weisheit M, Mueller Eckhardt C.
Monoclonal antibody-specific immobilization of platelet antigens (MAIPA): a new tool for the identification of platelet-reactive antibodies.
Blood.
1987;70:1722-1726[Abstract/Free Full Text].
19.
Kiefel V.
The MAIPA assay and its applications in immunohaematology.
Transfus Med.
1992;2:181-188[Medline]
[Order article via Infotrieve].
20.
Montigiani S, Neri G, Neri P, Neri D.
Alanine substitutions in calmodulin-binding peptides result in unexpected affinity enhancement.
J Mol Biol.
1996;258:6-13[Medline]
[Order article via Infotrieve].
21.
Bernatowicz MS, Matsueda GR.
Preparation of peptide-protein immunogens using N-succinimidyl bromoacetate as a heterobifunctional crosslinking reagent.
Anal Biochem.
1986;155:95-102[Medline]
[Order article via Infotrieve].
22.
Barron-Casella EA, Kickler TS, Rogers OC, Casella JF.
Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2.
Blood.
1994;84:1157-1163[Abstract/Free Full Text].
23.
Calvette JJ, Muniz-Diaz E.
Localization of an O-glycosylation site in the alpha-subunit of the human platelet integrin GPIIb/IIIa involved in Baka(HPA-3a) alloantigen expression.
Federation of European Biochemical Societies Letters.
1993;328:30-34[Medline]
[Order article via Infotrieve].
24.
Santoso S, Kalb R, Walka M, Kiefel V, Mueller Eckhardt C, Newman PJ.
The human platelet alloantigens Bra and Brb are associated with a single amino acid polymorphism on glycoprotein Ia (integrin subunit 2).
J Clin Invest.
1993;92:2427-2432.
25.
Clofent-Sanchez G, Lucas S, Laroche Traineau J, et al.
Autoantibodies and anti-mouse antibodies in thrombocytopenic patients as assessed by different MAIPA assays.
Br J Haematol.
1996;95:153-160[Medline]
[Order article via Infotrieve].
26.
Clofent-Sanchez G, Laroche Trainean J, Lucas S, et al.
Incidence of anti-mouse antibodies in thrombocytopenic patients with autoimmune disorders.
Hum Antibodies.
1997;8:50-59[Medline]
[Order article via Infotrieve].
27.
Marcelli Barge A, Poirier JC, Dausset J.
Allo-antigens and allo-antibodies of the Ko system, serological and genetic study.
Vox Sang.
1973;24:1-11[Medline]
[Order article via Infotrieve].
28.
Murata M, Matsubara Y, Kawano K, et al.
Coronary artery disease and polymorphisms in a receptor mediating shear stress-dependent platelet activation.
Circulation.
1997;96:3281-3286[Abstract/Free Full Text].
29.
Gonzalez-Conejero R, Lozano M, Rivera J, et al.
Polymorphisms of platelet membrane glycoprotein Ib associated with arterial thrombotic disease.
Blood.
1998;92:2771-2776[Abstract/Free Full Text].
30.
Moroi M, Jung SM, Yoshida N.
Genetic polymorphism of platelet glycoprotein Ib.
Blood.
1984;64:622-629[Abstract/Free Full Text].
31.
Simsek S, Bleeker PM, van der Schoot CE, von dem Borne AE.
Association of a variable number of tandem repeats (VNTR) in glycoprotein Ib and HPA-2 alloantigens.
Thromb Haemost.
1994;72:757-761[Medline]
[Order article via Infotrieve].
32.
Afshar-Kharghan V, Li CQ, Khoshnevis-Asl M, Lopez JA.
Kozak sequence polymorphism of the glycoprotein (GP) Ib gene is a major determinant of the plasma membrane levels of the platelet GP Ib- IX-V complex.
Blood.
1999;94:186191.
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Abstract
Introduction