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Prepublished online as a Blood First Edition Paper on May 24, 2002; DOI 10.1182/blood-2002-03-0997.
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Blood, 15 September 2002, Vol. 100, No. 6, pp. 2102-2107
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Amelioration of the macrothrombocytopenia associated with
the murine Bernard-Soulier syndrome
Taisuke Kanaji,
Susan Russell, and
Jerry Ware
From the Roon Center for Arteriosclerosis and
Thrombosis, Division of Experimental Hemostasis and Thrombosis,
Department of Molecular and Experimental Medicine, The Scripps Research
Institute, La Jolla, CA.
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Abstract |
An absent platelet glycoprotein (GP) Ib-IX receptor results in the
Bernard-Soulier syndrome and is characterized by severe bleeding and
the laboratory presentation of macrothrombocytopenia. Although the
macrothrombocytopenic phenotype is directly linked to an absent GP
Ib-IX complex, the disrupted molecular mechanisms that produce the
macrothrombocytopenia are unknown. We have utilized a mouse model of
the Bernard-Soulier syndrome to engineer platelets expressing an
 -subunit of GP Ib (GP Ib) in which most of the extracytoplasmic
sequence has been replaced by an isolated domain of the -subunit of
the human interleukin-4 receptor (IL-4R). The IL-4R/GP Ib
fusion is membrane expressed in Chinese hamster ovary (CHO) cells, and
its expression is facilitated by the presence of human GP IX and the
 -subunit of GP Ib. Transgenic animals expressing a chimeric receptor
were generated and bred into the murine Bernard-Soulier
syndrome-producing animals devoid of mouse GP Ib but expressing the
IL-4R/GP Ib fusion sequence. The characterization of these mice
revealed a 2-fold increase in circulating platelet count and a 50%
reduction in platelet size when compared with platelets from the mouse
model of the Bernard-Soulier syndrome. Immunoprecipitation confirmed
that the IL-4R/GP Ib subunit interacts with filamin-1 and
14-3-3 , known binding proteins to the GP Ib cytoplasmic tail.
Mice expressing the chimeric receptor retain a severe bleeding
phenotype, confirming a critical role for the GP Ib extracytoplasmic
domain in hemostasis. These results provide in vivo insights into the
structural elements of the GP Ib subunit that contribute to normal
megakaryocyte maturation and thrombopoiesis.
(Blood. 2002;100:2102-2107)
© 2002 by The American Society of Hematology.
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Introduction |
Giant platelet disorders are a heterogeneous group
of hematopoietic defects with structurally abnormal platelets in
peripheral blood smears.1,2 Many of these disorders are
hereditary and present with a reduced platelet count, leading to their
classification as hereditary macrothrombocytopenias. Representative of
the group are the Bernard-Soulier syndrome, Epstein syndrome,
May-Hegglin anomaly, Fechtner syndrome, and X-linked
macrothrombocytopenia.2 The genetic basis for each of
these syndromes is different, yet the members of the group are unified
in their presentation of macrothrombocytopenia. In each case, the
molecular pathology leading to the macrothrombocytopenia has remained
obscure but is assumed to be linked to abnormal megakaryocyte
maturation and platelet release.
The Bernard-Soulier syndrome is one of the best-characterized syndromes
among those presenting with macrothrombocytopenia.3 The
disorder is due to mutations within any of the 3 subunits constituting
the membrane receptor, glycoprotein (GP) Ib-IX.4 Most
commonly, mutations in the GP Ib-IX complex will prevent translocation
of the receptor to the megakaryocyte surface, resulting in the
Bernard-Soulier syndrome. The bleeding associated with the
Bernard-Soulier syndrome is more severe than the macrothrombocytopenia would predict and reflects the critical role for the GP Ib-IX receptor
in platelet adhesion during hemostasis.4 Indeed, the role
of the GP Ib-IX complex in hemostasis is well established, but the link
between an absent GP Ib-IX complex and the generation of a
macrothrombocytopenia is less obvious. Speculation on the molecular
basis has been fueled by work characterizing an interaction between GP
Ib and proteins of the platelet membrane cytoskeleton, such as
filamin-1.5-7 However, an equally plausible explanation might be a GP Ib link to signal transduction proteins, such as 14-3-3, which would be absent in the Bernard-Soulier syndrome and
would potentially, via unknown mechanisms, lead to abnormal megakaryocytopoiesis or thrombopoiesis.8,9 Moreover, a
role for GP Ib extracytoplasmic sequences in platelet formation
cannot be excluded, because anti-GP Ib antibodies can inhibit
proplatelet formation in vitro.10
We have recently described a murine model of the human Bernard-Soulier
syndrome where the mice exhibit all of the salient features of the
human syndrome, including bleeding and
macrothrombocytopenia.11 Moreover, the mice display an
abnormal megakaryocyte maturation, which may lead to the development of
the macrothrombocytopenia.11,12 In the current study we
test the hypothesis that the cytoplasmic domain of the -subunit of
GP Ib is necessary for normal megakaryocytopoiesis and platelet
release. We present the characterization of transgenically engineered
mouse platelets expressing a fusion protein composed of an
extracytoplasmic domain of the human interleukin-4 receptor -subunit
(IL-4R) fused to the transmembrane and cytoplasmic tail of
human GP Ib. The results demonstrate that the fusion protein is
efficiently surface expressed and stabilized by GP Ib and GP IX
subunits. A chimeric receptor was expressed in the murine model of the
Bernard-Soulier syndrome, a model missing the endogenous murine GP
Ib subunit. In vivo, the chimeric complex results in a 2-fold
increase in platelet count and in an approximate 50% reduction in
platelet size when compared with platelets devoid of mouse GP Ib.
The results provide insights into the structural elements of the GP
Ib-IX complex that contribute to normal platelet function, and
implications for the role of the GP Ib-IX complex in normal
thrombopoiesis are discussed.
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Materials and methods |
Generation of IL-4R/GP Ib receptor constructs
DNA constructs encoding the ectodomain of the human IL-4
receptor (residues 1-198 of the mature -subunit of the
receptor13) and residues 473 to 610 of the human GP Ib
mature subunit were generated.14 Specifically, a
HindIII/BamHI restriction fragment of the
IL-4R cDNA was generated in a polymerase chain reaction (PCR) using the complete IL-4R cDNA provided by Dr Kenji
Izuhara (Saga Medical School, Japan). PCR primers for IL-4R
generated a 5' product containing a HindIII restriction site
(italics), a consensus translation initiation site (underlined), and an
initiating Met codon (bold) (forward primer,
5'-GTAAGCTTGCCACCATGGGGTGGCTTTGC-3'). A reverse primer generated 3 Gly codons immediately 3' to the IL-4R
Asn198 codon followed by a BamHI restriction site (reverse primer, 5'-CTGGATCCACCGCCGTTGTGCCACTTGGTGC-3'). A second PCR
generated a BamHI/EcoRI restriction fragment
containing the coding sequence of the mature GP Ib subunit from
residues Ser474 to Leu610 (forward primer,
5'-GGTGGATCCAGAAATGACCCTTTTCTC-3'; reverse primer,
5'-GCGAATTCAGAGGCTGTGGCC-3'). The PCR products were cloned
and their DNA sequence confirmed. The cloned PCR products were ligated
together using BamHI cohesive ends producing in a 5' to 3'
direction the coding sequence for the extracytoplasmic domain of the
IL4-R, 3 Gly residues, and the carboxyl terminus of the GP Ib
subunit. Again, the intact coding sequence was confirmed by DNA
sequence analysis.
Once assembled, the IL-4R/GP Ib coding sequence was cloned as a
HindIII/EcoRI restriction fragment into the
eukaryotic expression vector, pcDNAzeo3.1+ (Invitrogen, Carlsbad, CA).
This construct was used for transfection into heterologous cells. For
the generation of transgenic animals, a 3-kb GP Ib promoter cassette
was cloned as a HindIII fragment immediately 5' to the
coding sequence. The generation and use of the GP Ib promoter
cassette has been previously described.11,15
Microinjection of the transgenic construct into pronuclei was performed
by The Scripps Research Institute transgenic core facility.
CHO cells with inducible GP Ib and GP IX cDNAs
Chinese hamster ovary (CHO) cells were maintained in 5%
CO2 and grown in Dulbecco modified Eagle medium
(DMEM) supplemented with 10% fetal calf serum, 0.5 mM
nonessential amino acids, and 2 mM L-glutamine (Whittaker
Bioproducts, Walkersville, MD). Transfection of CHO cells was performed
using liposomes (TransFast, Promega, Madison, WI). A CHO cell line
expressing the tetracycline-controlled transactivator (CHO-AA8,
Tet-Off) was purchased from BD Biosciences Clontech (Palo
Alto, CA). The cDNAs for GP Ib and GP IX16,17 were
cloned into the tetracycline-OFF expression vector, pTRE (Clontech),
and transfected into CHO-AA8 cells along with an additional selection
plasmid, pTK/Hyg, which facilitated the selection of stable
transformants with hygromycin (800 µg/mL, Invitrogen). Incubation of
the selected CHO cells in the presence of tetracycline (200 ng/mL)
repressed gene expression, and the removal of tetracycline induced both
gene products. Stable CHO cells expressing both human GP Ib and GP
IX were subsequently transfected with a plasmid encoding the
IL-4R/GP Ib subunit and selected in the presence of Zeocin (800 µg/mL, Invitrogen). Stable transfectants expressing the fusion
protein were identified using a fluorescein isothiocyanate (FITC)-labeled anti-IL-4R antibody in flow cytometry.
Immunologic reagents and protein analysis
A monoclonal antibody recognizing the human IL-4 receptor was
purchased from R&D Systems (cat. no. MAB230; Minneapolis, MN). The
antibody was directly labeled with FITC according to an established protocol.18 Anti-GP Ib antibodies were generated by
immunizing rabbits with a peptide corresponding to the carboxy-terminal
14 residues of GP Ib. An anti-GP IX monoclonal antibody has been previously described.19 A monoclonal antibody labeled with
FITC and recognizing the mouse integrin IIb subunit was
obtained from Pharmingen (La Jolla, CA). A purified rabbit polyclonal
anti-14-3-3 antiserum was purchased from Santa Cruz Technologies
(catalog no. sc-1019; Santa Cruz, CA). A rabbit antimouse filamin-1
antisera was kindly provided by Drs Hoffmeister and Stossel (Brigham
and Women's Hospital, Boston, MA).
Immunoprecipitation experiments were performed with washed platelets
(3.4 × 108 platelets) resuspended in 250 µL modified
Tyrode buffer (137 mmol NaCl, 2.7 mM KCl, 2.8 mmol dextrose, 0.4 mmol
NaH2PO4, 5 mM HEPES
[N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid] [pH 7.4]) and
lysed with an equal volume of solubilization buffer (2% Triton X-100,
0.1 mol Tris [tris(hydroxymethyl)aminomethane], 0.01 mol EGTA
[ethyleneglycoltetraacetic acid], 0.15 mol NaCl, and 2 mmol Pefabloc
SC [Boehringer Mannheim, Indianapolis, IN] [pH 7.4]). The
mixture was kept on ice for 45 minutes and centrifuged (13 000g, 10 minutes) to remove the insoluble material. The
lysates (500 µL) were mixed with 100 µL (50% vol/vol) protein A
beads (IPA-300, Repligen, Cambridge, MA) and 10 µg of the indicated antibody for 90 minutes. The beads were then washed 4 times in an equal
volume of modified Tyrode buffer and solubilization buffer. Bound
proteins were eluted by boiling in sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample
buffer and analyzed by SDS-PAGE and Western blotting. Immunoreactive
proteins were visualized using a chemiluminescence kit (Amersham
Pharmacia Biotech, Piscataway, NJ) and Kodak BIOMAX MR film.
Screening of transgenic mice
Mouse blood was obtained from a periorbital puncture from
anesthetized animals. Blood was collected with heparin-coated capillary tubes and transferred to a tube containing acid-citrate-dextrose (NIH
formula A) anticoagulant. Flow cytometric analysis on whole blood was
determined using an FITCanti-IL-4R monoclonal
antibody. Transgenic animals expressing the IL-4R/GP Ib fusion
protein were bred to mice lacking mouse GP Ib alleles, the mouse
model of the human Bernard-Soulier syndrome. Using a breeding strategy
as previously described,11 animals deficient in both
murine GP Ib alleles but expressing the IL-4R/GP Ib subunit
were identified after 2 generations of breeding. Briefly, GP
Ib / mice were bred to mice containing the human
transgene (mGP Ib+/+,
IL-4R/IbTg). Southern blot analysis of the
offspring confirmed the presence of heterozygous murine GP Ib
alleles, whereas immunologic screening using an
FITCanti-IL-4R monoclonal antibody identified mice also
expressing the human transgene product (mGP Ib+/,
IL-4R/IbTg). Mice containing heterozygous murine GP
Ib alleles and a functional human transgene were again crossed with
GP Ib-deficient mice ([mGP Ib+/,
IL-4R/IbTg] × [mGP Ib/]), and the
resultant offspring were genotyped and immunologically screened. This analysis identified mice with a murine GP
Ib/ locus and a functional human transgene (mGP
Ib/, IL-4R/IbTg). For results
comparing mGP Ib-deficient platelets to mGP Ib-deficient platelets expressing the IL-4R/GP Ib transgene, littermates were
compared from crosses of mGP Ib/ × mGP
Ib/, IL-4R/IbTg, following their
genotypic classification.
Phenotypic characterizations of mice
Mouse tail bleeding times were determined prior to genotyping
analysis by removing 1 to 3 mm of distal mouse tail and immediately immersing the tail in 37°C isotonic saline. A complete cessation of
bleeding was defined as the bleeding time. Bleeding time measurements exceeding 10 minutes were stopped by cauterization of the tail. Circulating blood counts were determined using manuals methods (Unopette, Becton Dickinson, Franklin Lakes, NJ) and an automated cell
counter (Baker, Allentown, PA). The flow cytometric analysis data were
collected, and a determination of the geometric mean for peaks was
determined using the software FCSPress (available at
http://www.fcspress.com/).
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Results |
In vitro characterization of an interleukin-4 receptor/GP Ib
fusion protein
We have previously described a murine model of the human
Bernard-Soulier syndrome.11 These mice mimic the human
syndrome with a severe bleeding phenotype and the presence of large
platelets in peripheral blood smears. The bone marrow of these animals
displays abnormal megakaryocyte maturation, which presumably results in an atypical platelet release.11,12 To address the
structural requirements within the GP Ib subunit contributing to
thrombopoiesis, we generated a DNA construct encoding the -subunit
of the human interleukin-4 receptor (IL-4R)20 fused to
the COOH terminus of human GP Ib (Figure
1). The choice of using the human GP
Ib subunit, as opposed to mouse GP Ib, is based on our previous demonstration that the complete human GP Ib sequence rescues the mouse Bernard-Soulier syndrome phenotype.11
The expressed polypeptide would predictably be composed of the
extracytoplasmic domain of the IL-4R fused to 3 tandem Gly residues
followed by the transmembrane and cytoplasmic sequences of GP Ib. A
short portion of the GP Ib extractyoplasmic domain was included (13 residues) containing the tandem Cys484/Cys485 of GP Ib that normally disulfide-link to GP Ib just beyond the transmembrane domain (Figure 1).
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| Figure 1.
Schematic representation of an interleukin-4R/GP
Ib-IX receptor.
The platelet GP Ib-IX receptor complex is composed of the
disulfide-linked - and -subunits of GP Ib and noncovalently
associated GP IX. A coding sequence was generated replacing most of the
GP Ib extracytoplasmic sequence (residues 1-472) with the
extracytoplasmic domain (residues 1-198) of the interleukin-4 receptor
chain (IL-4R). Studies are presented characterizing the
phenotypic consequences of the IL-4R/GP Ib subunit in
heterologous cells and in a murine model of the Bernard-Soulier
syndrome.
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The IL-4R/GP Ib construct was transfected into CHO cells
expressing human GP Ib and GP IX. Stable CHO cell lines were
established expressing all 3 gene products in the same cell:
IL-4R/GP Ib, GP Ib, and GP IX. Constitutive expression of the
IL4-R/GP Ib construct was driven by a cytomegalovirus
(CMV) promoter. Expression of both GP Ib and GP IX was
inducible due to the presence of a tetracycline-inducible promoter
(Tet-Off, see "Materials and methods"). As shown in Figure
2 for a representative cell line, the
induction of GP Ib and GP IX caused an approximate 10-fold increase
in the surface expression of the IL-4R/GP Ib subunit. This result
was corroborated by Western blot analysis of CHO cell lysates using an
anti-IL-4R antibody. Upon induction of GP Ib and GP IX subunits, a
detectable IL-4R antigen was observed (Figure 3). In the absence of GP Ib and GP IX,
no IL-4R antigen was detected (Figure 3). In the same induced samples,
an anti-GP Ib polyclonal antibody recognized a high molecular weight
protein of similar mobility to the IL-4R/GP Ib polypeptide that
disappears upon reduction and becomes a single immunoreactive species
of about 22 kDa, as predicted for the reduced GP Ib subunit of the
GP Ib-IX complex.4 Thus, expression of the IL-4R/GP
Ib subunit is stabilized by induction of GP Ib and GP IX,
and the fusion protein associates with GP Ib in a manner mimicking
the intact human GP Ib subunit.
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| Figure 2.
Surface expression of an interleukin-4R/GP Ib-IX
receptor on the surface of Chinese hamster ovary cells.
A stable CHO cell line containing inducible human GP Ib and GP IX
cDNAs was generated using tetracycline-responsive elements (see
"Materials and methods"). Transfection of this cell line with the
coding sequence for the IL-4R/GP Ib subunit under the control of
a CMV promoter generated a cell line with constitutive expression of
IL-4R/GP Ib and inducible expression of GP Ib and GP IX. Shown
is the fluorescence profile of an anti-IL-4R monoclonal antibody with
repressed GP Ib and GP IX gene expression (Tet-On) and induced GP
Ib and GP IX expression (Tet-Off). An approximate 10-fold increase
in mean fluorescence produced by the IL-4R monoclonal antibody is
generated by the simultaneous expression of GP Ib and GP IX.
Nontransfected CHOs (Neg CHO) are shown for comparison.
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| Figure 3.
Synthesis of the interleukin-4R/GP Ib subunit is
stabilized by the induction of GP Ib and GP IX genes.
Cell lysates of transfected CHO cells induced for the expression of GP
Ib and GP IX (Tet-Off) were analyzed by Western blot to identify the
IL-4R/GP Ib subunit schematically shown in Figure 1. Under
nonreducing conditions an anti-IL-4R monoclonal antibody identified a
dominant immunoreactive species migrating with an apparent molecular
mass of 148 kDa. No IL-4R immunoreactive species was seen in the
absence of induced GP Ib and GP IX expression (Tet-On). Upon
induction (Tet-Off) an anti-GP Ib polyclonal antibody identified a
dominant immunoreactive species seen under nonreducing conditions with
a similar mobility to the major antigen observed with an anti-IL-4R
monoclonal antibody (mAb). Upon reduction a single immunoreactive
species of approximately 22 kDa is seen, consistent with the reduced
single chain molecular weight for the platelet GP Ib
subunit.
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Transgenic expression of an interleukin-4R/GP Ib fusion
protein
A megakaryocytic-specific expression promoter cassette functioning
in vivo to express the human GP Ib subunit in megakaryocytes and
platelets has been described.21 This promoter cassette was inserted 5' to the IL-4R/GP Ib coding sequence and was used to
generate transgenic mice expressing the fusion. Several different founder animals were identified, but 2 lines expressing surface IL-4R antigen were chosen for establishment of mouse colonies. One
transgenic line was designated Tg33 and the other
Tg51. With the establishment of the IL-4R/GP Ib
transgenic lines, we next bred these animals into the mouse model of
the Bernard-Soulier syndrome, a colony lacking endogenous mouse GP
Ib alleles (mGP Ib/). First-generation animals
were identified with heterozygous mouse GP Ib alleles (mGP
Ib+/) and an expressed IL-4R/GP Ib transgene. These
animals were again bred to animals with a mGP Ib/
genotype, and the resultant offspring were screened by Southern blot
analysis to identify mice containing the mGP Ib/
genotype and flow cytometry to identify the product of the transgene. Having eliminated the endogenous mGP Ib alleles, these animals were
used to characterize the phenotypic consequences of expression of the
IL-4R/GP Ib protein in the Bernard-Soulier syndrome model.
As shown in Figure 4A, platelets
from Tg lines 33 and 51 both surface express IL-4R antigen. In
addition, platelets from Tg51 animals surface express more
antigen than platelets from Tg33 animals.
Quantitation of the geometric means for each genetic group is
presented in Table 1. The
phenotypic consequences of transgene expression on platelet size within
the circulating platelet population were examined by flow cytometry
(Figure 4B). The expression of both IL-4R/GP Ib transgenes, 33 and 51, shifts the forward scatter profile to the left as compared with
platelets from mGP Ib/ animals, indicating a
reduction in particle size. However, the higher levels of transgene
expression seen in platelets from Tg51 animals did not
produce an additional correction to the population size (Table 1). The
geometric mean of the forward scatter profile for platelets from
Tg33 animals demonstrated a more than 2-fold decrease in
size compared with platelets from mGP Ib-deficient animals (Table
1). A determination of the number of circulating platelets revealed a
more than 2-fold increase in platelet number as a consequence of
expression of the IL-4R/GP Ib subunit (Table 1). However, the
transgene did not correct the platelet count or platelet size to levels
observed for platelets from wild-type animals. Tail bleeding times were determined for Tg33 animals and were prolonged with more
half of the animals exceeding 10 minutes, indicative of a severe
bleeding phenotype (Figure 5). Normal
tail bleeding times cluster within a 1- to 3-minute range.11 However, tail bleeding times did suggest that the
hemostatic defect in the IL-4R/GP Ib colony is not quite as
severe as in the GP Ib/ colony (Figure 5). This
difference is presumably a direct comparison of the extent of
macrothrombocytopenia between the 2 models and its impact on the tail
bleeding time assay.
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| Figure 4.
Phenotypic consequences of an interleukin-4R/GP Ib
receptor on transgenic mouse platelets.
The IL-4R/GP Ib coding sequence was placed immediately 3' to a
megakaryocytic-specific promoter, and the resultant DNA construct was
injected into mice pronuclei to generate transgenic animals. Two
different transgenic founders, Tg33 and Tg51,
were bred into the murine model of the Bernard-Soulier syndrome. After
2 generations of screening (described in "Materials and methods"),
2 colonies of mice were generated lacking their endogenous murine GP
Ib alleles but still expressing the IL-4R/GP Ib transgene.
(A) Shown is the fluorescence profile of platelets obtained
from Tg33 and Tg51 transgenic lines screened
with an FITCanti-IL-4R mAb. Control platelets are shown
from nontransgenic animals and GP Ib-deficient animals.
(B) Forward light scattering profiles from mouse platelets
display the entire population of platelet sizes. The platelet
population was identified using an antimouse CD41 (integrin
IIb chain) monoclonal antibody. A total of 50 000
recorded events are presented for each genotype. A progressive shift to
the left illustrates decreasing particle (platelet) size. The
quantitation of this analysis from multiple animals is presented in
Table 1.
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Table 1.
Flow cytometry analysis of platelets from mice expressing
an IL-4R/GP Ib fusion in a model of murine Bernard-Soulier
syndrome
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| Figure 5.
Tail bleeding time assays.
Shown are the times obtained for individual GP Ib/
(mouse Bernard-Soulier syndrome) and Tg33
(Tg33; GP Ibnull) animals. Normal mouse
bleeding times range from 1 to 3 minutes.11 Mice lacking
the extracytoplasmic domain of GP Ib have a severe bleeding
phenotype even with an amelioration of the macrothrombocytopenia. These
results support the in vivo relevance of the extracytoplasmic GP Ib
ligand-binding domain in normal hemostasis.
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The cytoplasmic tail of GP Ib has been reported to interact with a
number of different platelet proteins.22 Although the protein-protein interactions have been described, the physiologic relevance of the interactions has been more difficult to establish. Given the ability of the chimeric complex to increase platelet count
and decrease platelet size, we hypothesized that the cytoplasmic tail
has retained the ability to interact with some of these proteins. Therefore, we immunoprecipitated Tg33 platelets with an
IL4-R monoclonal antibody and determined if either of the
well-characterized interaction partners, filamin-1 and the signal
transduction protein 14-3-3, were coimmunoprecipitated. Indeed,
immunoprecipitation with the IL-4R monoclonal antibody coprecipitated
both filamin-1 and 14-3-3 (Figure 6).
Thus, with respect to these 2 interaction partners of the native GP
Ib subunit, the fusion protein is mimicking native GP Ib and
provides a molecular explanation for the amelioration of the
macrothrombocytopenia.
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| Figure 6.
Coimmunoprecipitation of the interleukin-4R/GP Ib
subunit, filamin-1, and 14-3-3.
The ability of the IL-4R/GP Ib subunit to interact with other
cytoplasmic proteins was investigated by immunoprecipitation. The
immunopurified products were electrophoresed and analyzed by Western
blotting using antibodies that recognize filamin-1 (A) or
14-3-3 (B). Shown is the resulting autoradiograph produced
by the immunoreactive species. Lane 1, normal (wild-type) mouse
platelet lysate (no immunoprecipitation); lane 2, immunoprecipiated
platelet lysate from animals devoid of mouse GP Ib but expressing
the IL-4R transgene (Tg33; GP Ibnull);
lane 3, same lystate as lane 2 but immunoprecipitated with a control
IgG; lane 4, normal (wild-type) mouse platelet lysate
immunoprecipitated with the anti-IL-4R mAb; lane 5, normal human
platelet lysate (no immunoprecipitation).
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Discussion |
An unexplained yet hallmark feature of the Bernard-Soulier
syndrome is macrothrombocytopenia. Numerous studies have characterized the genetic basis for the Bernard-Soulier syndrome, but none have established the molecular basis for the associated
macrothrombocytopenia. The present study was undertaken to identify the
structural features of the GP Ib-IX complex that contribute to normal
platelet biogenesis. Our study focused on the contribution of the
-subunit of the GP Ib-IX complex. The availability of a GP
Ib-deficient mouse facilitated our strategy to produce genetically
engineered platelets with an altered GP Ib subunit. Specifically,
the experiment would determine whether a limited portion of GP Ib
could contribute to the normal process of thrombopoesis.
We chose to express a protein composed of the extracytoplasmic domain
of the human interleukin-4 receptor (IL-4R) fused to the
transmembrane and cytoplasmic tail of GP Ib. The chosen IL-4R fragment has been expressed and crystallized as an isolated
domain,20 supporting the possibility that the fragment
might behave as an isolated domain and, as such, would become a marker
for the expression of the fusion protein. Previous studies had
established that mouse IL-4 is species specific and does not activate
human T cells.23 Thus, the possibility that we might
engineer a new stimulatory effect on circulating mouse platelets with a
human IL-4 receptor seemed unlikely. We also included a limited coding
sequence for the extracytoplasmic domain of GP Ib because this
sequence contains tandem Cys residues that normally disulfide-link to
the GP Ib subunit (Figure 1). To facilitate the independence of each
domain within the polypeptide, a triple glycine repeat was inserted
between the 2 sequences (Figure 1). The expression of the fusion
protein was initially characterized in heterologous cells (Figures 2
and 3). The successful generation of an IL-4R/GP Ib-IX complex on the surface of CHO cells provided preliminary evidence that genetically engineered platelets might express a similar complex.
During the course of our preliminary studies, we demonstrated that the
extracytoplasmic domain of GP Ib is not essential for the surface
expression of a GP Ib-IX complex. Well-documented motifs within each
subunit of the GP Ib-IX complex are the leucine-rich repeats.24 Indeed, the leucine-rich repeats have been
proposed to support protein-protein interactions, leading to
speculation that the leucine repeats within the extracytoplasmic
domains of GP Ib, GP Ib, and GP IX might contribute to surface
assembly of the complex.25 Our results suggest that the
leucine-rich repeats of GP Ib have no major role in assembly of the
complex. We would propose that residues within the transmembrane domain of each subunit or the formation of the GP Ib/GP Ib disulfide bond just distal to the transmembrane domain (Figure 1) are likely to
be key to the assembly and formation of a GP Ib-IX complex.
Relative to our original objective, we demonstrated an amelioration of
the macrothrombocytopenia associated with the Bernard-Soulier syndrome.
However, the animals retained a severe bleeding phenotype similar to
that described for the mouse model of the Bernard-Soulier syndrome.
Together, these results highlight the pleiotropic phenotype produced in
the absence of a GP Ib-IX complex. Although the expression of the
chimeric receptor in the Bernard-Soulier model increases platelet count
and decreases platelet size, it is not possible to definitively state
that the absence of a GP Ib cytoplasmic tail is solely responsible
for generating the macrothrombocytopenia. First, the fusion protein
facilitates the surface expression of the complete complex and, as
such, brings GP Ib and GP IX back to the platelet surface. Do either
of these subunits contribute to the phenotypic corrections observed in
these animals? Certainly, a role for GP IX in this regard seems
unlikely, because it lacks any appreciable cytoplasmic tail and has no
known extracytoplasmic ligand.4 A role for GP Ib in the
process is still possible because both the - and -subunits of GP
Ib may share interaction partners, with the best example being
interactions with dimeric 14-3-3.26,27 A more
definitive answer to this question may come with additional animals
generated using a similar approach where specific mutations are
introduced to block interactions to the GP Ib tail. One set of
mutations must include those residues that interact with
filamin-1.7 Successful generation of these platelets may
also define critical intracellular cross talk mechanisms between the GP
Ib-IX and IIb3 complexes because the
membrane cytoskeleton has been implicated in the GP Ib-IX hemostatic
response.28
The results do illustrate the critical importance for expression of the
GP Ib cytoplasmic tail. However, in the context of the chimeric
complex we did not observe a correction of platelet number or size to
levels observed in normal animals. Indeed, in our previous study,
expression of the complete GP Ib subunit did not correct the
Bernard-Soulier syndrome phenotype to normal levels but did
significantly improve platelet count, circulating platelet size, and
bleeding time.11 One possible explanation is that the
fusion protein is not expressed to high enough levels. However, we
characterized 2 different transgenic lines with differing levels of
surface-expressed IL-4R. An increased level of receptor, such as
that observed on platelets from Tg51 animals, did not
provide an additional correction of the macrothrombocytopenia. Thus, it
appears that expression levels are important but higher levels of the
fusion protein do not produce a further correction of the phenotype.
The partial correction of the macrothrombocytopenia in this model may
also be suggesting that the conformation of the isolated cytoplasmic
tail is somewhat dependent upon the extracytoplasmic domain. Indeed,
conformation changes produced by ligand binding and a change in the
interactions of the cytoplasmic tail with other proteins may be a
central issue in normal GP Ib-IX biology.29 Is there any
evidence linking the structure of the GP Ib extracytoplasmic and
cytoplasmic domains together? Several years ago a variant GP Ib
molecule (type Bolzano) was described containing a missense mutation
within a leucine-rich repeat of the extracytoplasmic domain
(A156V).30 This Bernard-Soulier syndrome variant did not
preclude surface expression of GP Ib (although expression was
reduced as compared with normal platelets) but did produce a giant
platelet phenotype. However, a direct comparison of the type Bolzano
platelet size to other Bernard-Soulier syndrome platelets was not done.
By a strict definition, the transgenic animals characterized in the
current study could be classified as having the Bernard-Soulier syndrome. They have 3 of the hallmark features when compared with platelets from a normal animal: (1) they lack the extracytoplasmic domain of GP Ib; (2) they have a reduced platelet count; and (3)
they have larger-than-normal-sized circulating platelets. Yet, when
compared with the homozygous-deficient animal, they illustrate a less
severe phenotype. Thus, our results highlight an additional level of
heterogeneity that occurs within the designation of a Bernard-Soulier
syndrome, and the type Bolzano variant probably represents variation on
the phenotypic expression of the human syndrome.
The molecular basis for macrothrombocytopenia is likely to be unique to
each of the disorders presenting with the phenotype. However, one
unifying theme will most likely be the disruption or perturbation of
molecular mechanisms controlling platelet morphogenesis and release.
The present study suggests a direct role for the carboxyl terminus of
GP Ib in thrombopoiesis. The utilization of animal models with
defined genetic defects presents an opportunity to define fundamental
aspects of thrombopoiesis and normal platelet function. Indeed, the
cellular environment and cellular characteristics of the megkaryocyte
and platelet make in vivo models an excellent experimental framework to
study both megakaryocyte and platelet biology. Future studies
exploiting these models will define key mechanisms controlling normal
platelet biogenesis and function.
| |
Acknowledgments |
The authors acknowledge the Sam and Rose Stein Charitable Trust for
the establishment of the DNA Core Facility within the Department of
Molecular and Experimental Medicine at The Scripps Research Institute.
The administrative assistance of Ms Pamela Fagan is greatly appreciated.
| |
Footnotes |
Submitted April 1, 2002; accepted May 8, 2002.
Prepublished online as
Blood First Edition Paper, May 24, 2002; DOI
10.1182/blood-2002-03-0997.
Supported by HL50545 from the Heart, Lung, and Blood Institute of the
National Institutes of Health (J.W.) and by the Sumitomo Life Insurance
Welfare Service Foundation (2000) and Uehara Memorial Foundation (2001)
(T.K.).
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Jerry Ware, MEM170, The Scripps Research
Institute, 10550 N Torrey Pines Rd, La Jolla, CA 92037; e-mail:
jware{at}scripps.edu.
| |
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Abstract
Introduction