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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2555-2561
Posttranslational Processing of the Thrombopoietin Receptor Is
Impaired in Polycythemia Vera
By
Alison R. Moliterno and
Jerry L. Spivak
From the Division of Hematology, Department of Medicine, Johns
Hopkins University School of Medicine, Baltimore, MD.
 |
ABSTRACT |
Recently, we demonstrated a marked reduction in the expression of
the thrombopoietin receptor, Mpl, in polycythemia vera (PV) platelets
and megakaryocytes using an antiserum against the Mpl extracellular
domain. To further examine this abnormality, we raised an antibody to
the Mpl C-terminus. Immunologic analysis of PV platelets with this
antiserum confirmed the reduction in Mpl expression. However, the
C-terminal antiserum detected 2 forms of Mpl in PV platelets in
contrast to normal platelets, in which a single form of Mpl was
detected by both the extracellular domain and C-terminal antisera.
Two-dimensional gel electrophoresis studies with isoelectric focusing
in the first dimension identified normal platelet Mpl as an 85 to 92 kD
protein with an isoelectric point (pI) of 5.5. PV platelets contained
an additional 80 to 82 kD Mpl Mpl isoform with a pI of
6.5. Analysis of Mpl expressed by the human megakaryocytic cell line,
Dami, showed 2 isoforms similar to those found in PV platelets
suggesting a precursor-product relationship. Digestion of Dami cell and
normal platelet lysates with neuraminidase converted the more acidic
Mpl isoform to the more basic one, indicating that the 2 isoforms
differed with respect to posttranslational glycosylation. Futhermore,
in contrast to normal platelet Mpl, PV platelet Mpl was susceptible to
endoglycosidase H digestion, indicating defective Mpl processing by PV
megakaryocytes. The glycosylation defect was specific for Mpl, as 2 other platelet membrane glycoproteins, glycoprotein IIb and multimerin,
were processed normally. Importantly, the extent of the PV platelet Mpl
glycosylation defect correlated with disease duration and extramedullary hematopoiesis.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
POLYCYTHEMIA VERA (PV) is a clonal
disorder of unknown etiology, involving a multipotent hematopoietic
progenitor cell that causes the accumulation of phenotypically normal
red blood cells, white blood cells, and platelets and
their precursors in the absence of a known physiologic
stimulus.1 Although trilineage hyperplasia is the hallmark
of PV, erythrocytosis is its most prominent clinical manifestation and,
as a consequence, most investigations of PV have focused on
erythropoiesis and in particular, on the erythropoietin receptor. To
date, however, structural studies of the erythropoietin receptor gene,
its protein expression, and ligand binding have been normal in
PV.2-4 Most recently, PV erythroid progenitor cells
cultured in vitro have been shown to be resistant to apoptosis in the
absence of erythropoietin, a behavior that may be related to the
hypersensitivity of these cells to other growth factors such as
insulin-like growth factor-1 (IGF-1), interleu- kin-3 (IL-3), stem
cell factor (SCF), and granulocyte-macrophage colony-stimulating factor
(GM-CSF).5-9
Because neither acquired nor congenital abnormalities of the
erythropoietin receptor can be implicated in the pathogenesis of PV and
because the disorder involves all of the major hematopoietic cell
lineages, if a receptor abnormality is involved, that receptor must
initially be both expressed and active in primitive multipotent hematopoietic progenitor cells. The thrombopoietin receptor, Mpl, is
such a candidate receptor.10 For example, thrombopoietin (TPO) or Mpl knock-out mice have reduced numbers of multilineage and
committed hematopoietic progenitor cells, while TPO augments the
proliferation of primitive hematopoietic stem cells in
vitro.11,12 Importantly, forced expression studies of
either TPO or Mpl recapitulate many clinical aspects of PV:
overexpression of TPO in mice causes myelofibrosis and extramedullary
hematopoiesis, while ectopic expression of Mpl induces a fatal
erythroblastosis.13,14 Furthermore, a truncated Mpl gene is
the oncogenic protein of the myeloproliferative leukemia virus (MPLV),
which immortalizes hematopoietic progenitor cells in vitro and causes
polycythemia when administered to mice in vivo.15,16
Finally, TPO has been observed to prevent apoptosis of erythroid
progenitor cells deprived of erythropoietin in vitro.17
Circulating platelets express a functional TPO receptor. To our
surprise, in contrast to normal platelets, TPO failed to stimulate PV
platelet protein tyrosine phosphorylation, a defect specific for PV and
a related blood disorder, idiopathic myelofibrosis (IMF). Impaired TPO
signaling in PV platelets was associated with a reduction in platelet
and megakaryocyte Mpl expression as determined by immunologic analysis
using an antiserum raised against the Mpl extracytoplasmic
domain.18 The diminished function and expression of a
hematopoietic cytokine receptor in patients with a disorder characterized by growth factor hypersensitivity and excessive production of hematopoietic cells was an unique and unexpected finding.
To further examine the defective expression of PV platelet Mpl, we
raised an antiserum to the C-terminal domain of Mpl. We now report both
quantitative and qualitative PV platelet Mpl abnormalities. In contrast
to normal platelet Mpl, PV platelet Mpl is incompletely glycosylated.
Furthermore, the degree of impairment of Mpl glycosylation correlated
with disease duration and the extent of extramedullary hematopoiesis.
This is the first report of a qualitative defect in a growth factor
receptor in a myeloproliferative disorder.
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MATERIALS AND METHODS |
Antisera.
Rabbit polyclonal antiglycoprotein IIb was kindly supplied by Dr Paul
Bray (Johns Hopkins University School of Medicine, Baltimore, MD). An
affinity-purified, polyclonal rabbit IgG antibody to the soluble
extracellular domain of human Mpl was a gift from the Kirin Brewery
(Maebashi, Gunma, Japan). Monoclonal antimultimerin antiserum was
provided by Dr Catherine Hayward (McMaster University, Ontario,
Canada). Polyclonal rabbit antiserum was raised to the C-terminus of
human Mpl after immunization with a KLH-linked peptide of the terminal
14 amino acids of full-length human Mpl (ANHSYLPLSYWQQP).
Subjects.
The study protocol was approved by our Joint Committee on Clinical
Investigation and informed consent was obtained from each patient. The
diagnosis of PV was based on the criteria of the Polycythemia Vera
Study Group and included an elevated red blood cell mass. The diagnosis
of the other chronic myeloproliferative disorders was based on standard
clinical criteria.19
Reagents.
N-glycosidase F, neuraminidase, and endoglycosidase H were purchased
from Boehringer-Manheim (Indianapolis, IN). All other reagents were
purchased from Sigma (St Louis, MO).
Dami cell culture.
The human megakaryocytic cell line, Dami, was supplied by Dr Paul F. Bray. Dami cells were grown in Iscove's modified Dulbecco's medium
containing 10% fetal bovine serum. Dami cell lysis buffer consisted of
10% glycerol, 1% Triton x-100, 20 mmol/L Tris pH 7.4, 75 mmol/L NaCl,
0.5 mmol/L EDTA, 50 mmol/L NaF, 1 mmol/L Na Vanadate, 1 mmol/L
phenylmethyl sulfonyl fluoride (PMSF), 20 ng/mL leupeptin, 20 ng/mL
pepstatin, and 20 ng/mL aprotinin.
Platelet preparation.
Whole blood was drawn from healthy volunteers and patients after
informed consent. Platelet-rich plasma, obtained after centrifugation at 12,000 rpm for 12 minutes, was centrifuged for 15 minutes at 2,000 rpm to form a platelet pellet. The platelets were washed 2 times with
phosphate-buffered saline supplemented with 0.6% sodium citrate and
0.1% bovine serum albumin and then lysed in the Dami cell lysis
buffer. Protein concentrations of the platelet lysates were obtained
with the Pierce protein assay kit (Rockford, IL).
2D-gel electrophoresis.
For 2D-gel electrophoresis analysis, 25 µg of platelet
lysate was applied to the IPGphor system (Amersham, Arlington Heights, IL) and isoelectric focusing was performed according to the
manufacturer's specifications (500 V for 1 hour, 1,000 V for 1 hour
followed by 8,000 V for 2 hours). Second dimension separation was
performed with 10% acrylamide sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE). The gels were electroblotted to
nitrocellulose membranes at 450 mA for 2 hours.
Glycosidase digestion.
A total of 25 µg of Dami cell lysate was incubated with 20 mU
neuraminidase at 37°C for 1 hour. For both N-glycosidase F and endoglycosidase H digestions, 25 µg of platelet lysate were first denatured by boiling in SDS in the presence of a reducing agent, and
then incubated for 18 hours at 30°C in 0.5 mol/L Tris-HCl pH 8.0 (N-glycosidase F, 5 U) or 0.5 mol/L sodium citrate pH 5.5 (endoglycosidase H, 2.5 mU).
Immunoblotting.
Immunoblotting protocols were performed as reported
elsewhere.18 Dilutions for the antisera were as follows:
1:2,500 for the N-terminal antiserum, 1:5,000 for the C-terminal
peptide antiserum, and 1:20,000 for the antiglycoprotein IIb.
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RESULTS |
PV Mpl expression is diminished in PV platelets.
In a previous study, total and surface expression of Mpl appeared
diminished in PV platelets as determined by 3 different assays:
TPO-induced protein tyrosine phosphorylation, flow cytometry, and
immunoblotting using an affinity-purified antiserum against the Mpl
extracytoplasmic domain.18,21 To confirm these results, we
examined the expression of Mpl in normal and PV platelet lysates by
immunoblotting with a polyclonal antiserum raised against the 14 C-terminal amino acids of Mpl (ANHSYLPLSYWQQP). As shown in Fig 1, virtually no PV platelet Mpl was
detected by immunoblotting using the extracytoplasmic domain Mpl
antiserum. By contrast, Mpl was present in the lysates of all PV
patients when the C-terminal Mpl antiserum was used. However, not only
was the quantity of PV platelet Mpl still diminished, but 2 forms of
Mpl were present in PV platelets.
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| Fig 1.
C-terminal antiserum detects Mpl in PV platelets. PV and
control platelet lysates were probed with an antiserum against the
N-terminus and one against the C-terminus of Mpl. The membrane was
reprobed with an antiserum against glycoprotein IIb to control for
equal loading of platelet protein.
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PV platelets express 2 Mpl isoforms.
To further examine the differences in Mpl expression in normal and PV
platelets, we analyzed Mpl in platelet lysates by 2-dimensional gel
electrophoresis using isoelectric focusing in the first dimension and
10% SDS-PAGE in the second followed by immunoblotting with the Mpl
C-terminal antiserum. As shown in Fig 2,
Mpl from control platelets migrated with an apparent molecular mass of
85 to 92 kD and as a series of differently charged species with
isoelectric points spanning a pH range between 5.1 and 5.5, presumably
due to differences in sialation. In contrast, as suggested by
1-dimensional electrophoresis (Fig 1), 2 major Mpl isoforms were
present in PV platelets. One, which we designated the A form, migrated
in the same fashion as normal platelet Mpl, but was reduced in
quantity; the second isoform, designated the B form, migrated with an
apparent molecular mass of 80 to 82 kD and with isoelectric points
ranging from 6.2 to 6.5. In some PV patients, we could detect only the B form (Fig 2, lowest panel).
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| Fig 2.
Two-dimensional gel electrophoresis of PV and control
platelet Mpl. A total of 25 µg of platelet lysate protein was
subjected to isoelectric focusing in the first dimension (horizontal
axis) followed by 10% acrylamide SDS-PAGE in the second dimension
(vertical axis), transferred to a nitrocellulose membrane, and
immunoblotted with the Mpl C-terminal peptide antiserum.
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Mpl isoforms represent differences in sialic acid content.
The extracellular domain of Mpl contains a reduplicated cytokine
binding domain and 4 potential sites for asparagine-linked glycosylation.22 Because the theoretical molecular mass of
Mpl based on its amino acid sequence is 68 kD, we postulated that the
Mpl A and B isoforms differed with respect to their degree of
glycosylation and, in particular, their sialic acid content. To
evaluate this, because platelets do not synthesize protein efficiently,
we first examined Mpl expression in the human megakaryocyte cell line,
Dami. As shown in Fig 3, analysis of Dami
cell Mpl expression by 2-dimensional gel electrophoresis showed 2 Mpl
isoforms with both isoelectric points (pI) and apparent molecular
masses similar to the A and B Mpl isoforms present in normal and PV
platelet lysates, respectively. Furthermore, when Dami cell lysates or normal platelet lysates were digested with neuraminidase before 2-dimensional gel electrophoresis, only the B isoform was observed, suggesting that sialic acid accounts for the differences between the A
and B forms. Additionally, in vitro translation of full-length Mpl cDNA
in a system capable of core, but not complex glycosylation produced a
protein identical in pI and molecular weight as the B form (data not
shown) that was recognized by the Mpl C-terminal antiserum, but not the
Mpl extracytoplasmic domain antiserum, indicating that the latter
antiserum was sensitive to conformation or carbohydrate epitopes.
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| Fig 3.
Mpl isoforms in Dami cell lysates. A total of 20 µg of
Dami cell lysate protein was subjected to 2-dimensional gel
electrophoresis and immunblotting with the Mpl C-terminal peptide
antiserum as described in Fig 2.
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PV platelet Mpl is susceptible to digestion by endoglycosidase H.
The data cited above suggested that PV platelet Mpl was incompletely
glycosylated and, in particular, lacked its full complement of sialic
acid. Protein glycosylation in the endoplasmic reticulum is a
cotranslational process. However, carbohydrate processing to create
complex oligosaccharide side chains takes place in the Golgi apparatus
where incompletely processed proteins can be distinguished by their
sensitivity to digestion with endoglycosidase H.20 Therefore, we examined the sensitivity of Mpl in normal and PV platelet
lysates to endoglycosidases H and F. As shown in
Fig 4, normal platelet Mpl was resistant to
digestion with endoglycosidase H, while a portion of PV platelet Mpl
was not. Both normal and PV platelet Mpl could be digested with
N-glycosidase F, yielding products of identical molecular weight,
indicating similarity of normal and PV platelet Mpl at the polypeptide
level.
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| Fig 4.
Specific and nonspecific endoglycosidase digestion of
control and PV platelet lysates. Platelet lysates from a control and 3 different patients were untreated (lanes 1 to 4), digested with
endoglycosidase H (lanes 5 to 8), or digested with N-glycosidase F
(lanes 9 to 12), and subsequently probed with the Mpl C-terminal
antiserum.
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Expression of 2 other platelet membrane glycoproeins, GPIIb and
multimerin, is normal in PV platelets.
Studies with isolated Dami cell membranes (not shown) also indicated
that the A isoform of Mpl was the predominant Mpl species present in
the plasma membrane, which is consistent with the notion that the B
isoform represents incompletely processed Mpl. To determine if
defective posttranslational processing was specific for PV platelet
Mpl, we examined the expression of 2 other platelet membrane glycoproteins, glycoprotein IIb and multimerin. These glycoproteins were chosen because we were unable to identify by immunoblotting in
platelets expression of the cytokine receptor subunits gp130 and IL-3
beta, which share homology with Mpl. As shown in
Fig 5, based on 2-dimensional gel
electrophoresis, qualitative and quantitative analysis of glycoprotein
IIb was identical in normal and PV platelets. Similar results (not
shown) were obtained with multimerin.
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| Fig 5.
Two-dimensional electrophoresis analysis of glycoprotein
IIb in control and PV platelets. Platelet lysates from a control (upper
panels) and a PV patient (lower panels) were analyzed by 2-dimensional
gel electrophoresis and immunoblotted with the Mpl C-terminal antiserum
(left) or antiserum to glycoprotein IIb (right).
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Expression of the PV platelet Mpl B isoform correlates with disease
progression.
To understand the significance of the Mpl B isoform in PV platelets, we
performed 2-dimensional gel electrophoresis and immunoblotting using
the C-terminal Mpl antiserum on platelet lysates from 24 PV patients
who fulfilled the diagnostic criteria of the Polycythemia Vera Study
Group.23 These studies yielded the following results: first, as we previously reported using the Mpl extracytoplasmic domain
antiserum and as shown in Fig 1 for 3 of the patients, all 24 patients
had reduced amounts of platelet Mpl. Second, 4 patterns of PV platelet
Mpl expression emerged: the presence of the A isoform alone (but
diminished in total amount compared with normal), equal quantities of
the A and B isoforms, a lesser quantity of A compared with B, or B
only. Third, as shown in Table 1, the 4 isoform patterns segregated according to disease duration and degree of
splenomegaly, which is a measure of extramedullary hematopoiesis.24 Thus, in the patients with a relatively
short disease duration from the time of diagnosis, only the A isoform was present. In the patients with either extended disease duration and/or the development of clinically evident splenomegaly, the B
isoform was predominant.
Study of 3 PV patients over time supported the contention that Mpl
isoform expression was a function of disease progression. As
shown in Fig 6 for a representative
patient, by 1-and 2-dimensional gel electrophoresis and immunoblotting,
there was a reduction of platelet Mpl expression and an evolution from
primarily the A isoform at diagnosis to emergence of the B isoform 20 months after diagnosis, with phlebotomy as the only therapy.
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| Fig 6.
Mpl isoform evolution in a single patient. (A)
Immunoblots after 1-dimensional electrophoresis of patient platelet
lysate obtained at diagnosis in May 1997 and again in January 1999 were
probed with the Mpl C-terminal antiserum. The immunoblot was reprobed
with the glycoprotein IIb antiserum to verify equal protein loading.
(B) Two-dimensional gel electrophoresis of the platelet lysates shown
in (A) were probed with the C-terminal antiserum.
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The observed pattern of Mpl isoform expression was independent of white
blood cell or platelet count, concurrent therapy, previous therapy,
splenectomy, or iron status. As controls for this study, we examined 5 patients with essential thrombocytosis, 5 with secondary
erythrocytosis, 1 with sideroblastic anemia, 1 with chronic myelogenous
leukemia (CML), 1 with thrombocytosis due to iron deficiency, and 6 patients with hemochromatosis undergoing regular phlebotomy. All 19 of
these patients had normal Mpl isoform expression as assessed by
2-dimensional gel electrophoresis (data not shown).
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DISCUSSION |
Polycythemia vera is classified as a chronic myeloproliferative
disorder together with IMF, essential thrombocytosis (ET), and CML
because it involves a multipotent hematopoietic progenitor cell with
overproduction of 1 or more of the formed elements of the blood. CML is
unique among the chronic myeloproliferative disorders, as it alone has
an invariant clonal marker, a reciprocal translocation of the distal
portions of chromosomes 9 and 22. The major consequence of this
translocation is a constitutively-active, nonreceptor tyrosine kinase
fusion protein, bcr-abl. Experimentally, expression of bcr-abl in
multipotent hematopoietic progenitor cells in vitro mimics many of the
features of PV progenitor cells cultured in vitro such as growth factor
hypersensitivity and resistance to apoptosis after growth factor
deprivation.25 However, clinically CML differs greatly from
PV, IMF, and ET. First, its duration is measured in years, while their
durations are measured in decades. Second, malignant transformation is
the inevitable consequence of untreated CML, while marrow failure or
acute leukemia are far less common in PV, IMF, and ET, as long as
mutagenic agents are not used in their treatment.19
PV erythroid progenitor cells cultured in vitro are characteristically
hypersensitive to a variety of growth factors and are capable of
surviving in the absence of erythropoietin. PV erythroid progenitor
cells are hypersensitive to IGF-1, an antiapoptotic factor, and the
IGF-1 receptor is constitutively phosphorylated in PV peripheral blood
mononuclear cells.6,26 Because erythroid progenitor cells
expressing a hypofunctional erythropoietin receptor are also
hypersensitive to IGF-1,27 it is interesting to speculate that a similar situation might exist in PV, but involving a receptor more globally expressed than the erythropoietin
receptor.10,12
Mpl was such a candidate given the fact that it can function as an
oncogene by immortalizing multipotent hematopoietic progenitor cells
and rendering them growth factor-independent.16 In contrast to CML platelets, there was no constitutive protein tyrosine
phosphorylation in PV platelets.18,28 Indeed, even after
exposure to TPO, there was no increase in protein tyrosine
phosphorylation in PV platelets, although the machinery for this was
intact. Lack of responsiveness to TPO with respect to tyrosine
phosphorylation and enhancement of adenosine diphosphate
(ADP)-induced platelet aggregation as well as the
inability to cross-link TPO to PV platelets suggested an abnormality in
Mpl.29 Using an antiserum raised against the extracytoplasmic domain of Mpl, we were not only unable to identify surface expression of Mpl in PV platelets by flow cytometry, but total
platelet Mpl was severely diminished as well, as determined by immunoblotting.
Using a C-terminal Mpl antiserum, we now demonstrate that Mpl
expression in PV platelets was not only reduced compared with normal
platelets, but that its posttranslational processing was abnormal as
well. Failure of the Mpl extracellular domain antiserum to recognize
the in vitro translation product of Mpl (which lacks posttranslational
carbohydrate modifications, 4 sites of which are present in the Mpl
extracytoplasmic domain) suggested that this antiserum was conformation
or epitope dependent. Recognition of the Mpl B form in PV platelets by
our C-terminal antiserum, but not the Mpl extracytoplasmic domain
antiserum, further localized the PV Mpl abnormality to the Mpl
extracytoplasmic domain. With respect to specificity, the processing
defect appeared to be limited to Mpl, as 2 other platelet membrane
glycoproteins, glycoprotein IIb and multimerin, were processed
normally, while the erythropoietin and stem cell factor receptors were
demonstrated to be intact by others.2,8 Incomplete Mpl
glycoslyation suggested that its transport from the Golgi apparatus was
impaired and was consonant with our observation of reduced Mpl surface
expression in PV platelets.29 Importantly, this defect was
exaggerated with disease duration from the time of diagnosis and
extent, suggesting that it may be a useful marker of disease activity.
A hematopoietic growth factor receptor abnormality is a natural
candidate for oncogenic transformation due to the effects of these
receptors on cell proliferation, differentiation, and apoptosis. Two
observations illustrate how an acquired abnormality of a growth factor
receptor might participate in human cancer. First, in many cases of the
highly malignant brain tumor, glioblastoma, the epidermal growth factor
receptor (EGFR) gene is amplified and undergoes intragenic deletions
and rearrangements that result in the expression of a receptor protein
lacking a portion of its extracellular domain.30,31 The
mutant EGFR does not bind EGF and its tyrosine kinase domain is
constitutively active. Interestingly, the altered EGFR has been shown
to localize to the endoplasmic reticulum and to have a prolonged
half-life compared with its normal counterpart, a property that may
contribute to aberrant signaling and oncogenesis.32 Second,
the development of acute myelogenous leukemia in patients with severe
congenital neutropenia has been associated with acquired mutations in
the granulocyte colony-stimulating factor receptor
(G-CSFR).33 Recently, mutations of the G-CSFR that truncate
its C-terminal domain have been shown to result in impaired ligand
internalization, defective receptor downmodulation, enhanced cell
proliferation, and impaired differentiation.34,35 Additionally, such mutant receptors have been observed to have a
dominant-negative effect on the function of the normal G-CSFR.
The observation that an essential multipotent hematopoietic growth
factor receptor was hypofunctional in a chronic myeloproliferative disease whose phenotype is overproduction of hematopoietic cells was
unexpected. An important feature of our observations is that the Mpl
defect was unique to PV and a closely related disorder, IMF. In fact,
so selective was its expression that the Mpl abnormality can be used to
distinguish PV from other forms of erythrocytosis and thus avoid
exposure of patients with a benign disorder to potentially mutagenic therapy.
Our results in ET patients are in direct opposition to the report of
Horikawa et al,36 who found decreased platelet Mpl in some
patients carrying this diagnosis. A difficulty in any study of ET is
the identification of authentic ET patients, due to the lack of
positive identifiers of this elusive disorder. Furthermore, isolated
thrombocytosis may be the first manifestation of PV, IMF, or CML. We
can only state that the patients we classify as ET have been
extensively studied, followed on average for 8 years, and all other
potential causes of an elevated platelet count have been excluded.
Recently, Taskin et al37 also found no abnormalities of the
Mpl gene or impaired responsiveness to thrombopoietin in 9 ET patients.
Finally, it has recently been demonstrated that many patients
considered to have ET actually have a nonclonal disorder, and in some
families with inherited thrombocytosis, an abnormality of the TPO gene
has been demonstrated.38,39
Impairment of Mpl glycosylation is the first molecular defect
identified in PV. Because Mpl expression is impaired in PV
megakaryocytes and there is no evidence that platelets can either
synthesize or metabolize Mpl, PV platelets appear to represent a
"fossil" record of an abnormality in progenitor cell Mpl
expression. Thus, the progressive increase in expression of
underglycosylated Mpl could represent either increasing severity of the
molecular defect or the selection or evolution of clones with a more
severe Mpl defect, possibly providing them with a survival advantage.
To date, we have not identified gross structural abnormalities of the
Mpl gene or its transcripts by Southern or Northern analysis. Although
the mechanism and relevance to the pathogenesis of PV are still
undefined, a unique molecular abnormality, defective Mpl glycosylation,
is now available for use in the diagnosis, staging, and evaluation of
specific therapies in PV patients.
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ACKNOWLEDGMENT |
The excellent technical assistance of Evelyn Connor and Mary Ann Isaacs
is acknowledged with gratitude. We thank W.D. Hankins and Donna
Williams for helpful discussions and our colleagues in the Division of
Hematology, Drs William R. Bell, Paul F. Bray, Chi Van Dang, Lawrence
B. Gardner, and Michael B. Streiff, and the physicians of the Johns
Hopkins Oncology Center for referring patients for this study. We also
acknowledge the unflagging support from the staff of the Johns Hopkins
Hospital Blood Bank. We gratefully acknowledge contributions from the
Myeloproliferative Disorders Support Group.
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FOOTNOTES |
Submitted April 6, 1999; accepted June 14, 1999.
Supported by National Institutes of Health (NIH) Grant No. 58589 from
the National Heart, Lung and Blood Institute (to J.L.S.) and a Doris
Duke Clinical Scientist Award (to A.R.M.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Jerry L. Spivak, MD, Traylor Building, Room
924, 720 Rutland Ave, Baltimore, MD 21205.
| |
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ABSTRACT
INTRODUCTION