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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4084-4092
Ex Vivo Cultured Megakaryocytes Express Functional Glycoprotein
IIb-IIIa Receptors and Are Capable of Adenovirus-Mediated Transgene
Expression
By
Nauder Faraday,
Jeffrey J. Rade,
David C. Johns,
Gopal Khetawat,
Stephen J. Noga,
John F. DiPersio,
Ying Jin,
Janet L. Nichol,
Jeff
S. Haug, and
Paul F. Bray
From the Department of Anesthesiology and Critical Care Medicine, the
Divisions of Cardiology and Hematology, the Department of Medicine, and
the Department of Oncology, Johns Hopkins University School of
Medicine, Baltimore, MD; the Division of BMT and Cell Biology,
Washington University School of Medicine, St Louis, MO; and Amgen, Inc,
Thousand Oaks, CA.
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ABSTRACT |
Investigation of the molecular basis of megakaryocyte (MK) and
platelet biology has been limited by an inadequate source of genetically manipulable cells exhibiting physiologic MK and platelet functions. We hypothesized that ex vivo cultured MKs would exhibit agonist inducible glycoprotein (GP) IIb-IIIa activation characteristic of blood platelets and that these cultured MKs would be capable of
transgene expression. Microscopic and flow cytometric analyses confirmed that human hematopoietic stem cells cultured in the presence
of pegylated recombinant human MK growth and development factor
(PEG-rHuMGDF) differentiated into morphologic and phenotypic MKs over 2 weeks. Cultured MKs expressed functional GPIIb-IIIa receptors as
assessed by agonist inducible soluble fibrinogen and PAC1 binding. The
specificity and kinetics of fibrinogen binding to MK GPIIb-IIIa
receptors were similar to those described for blood platelets. The
reversibility and internalization of ligands bound to MK GPIIb-IIIa
also shared similarities with those observed in platelets. Cultured MKs
were transduced with an adenoviral vector encoding green fluorescence
protein (GFP) or  -galactosidase (-gal). Efficiency of gene
transfer increased with increasing multiplicities of infection and
incubation time, with 45% of MKs expressing GFP 72 hours after viral
infection. Transduced MKs remained capable of agonist induced
GPIIb-IIIa activation. Thus, ex vivo cultured MKs (1) express agonist
responsive GPIIb-IIIa receptors, (2) are capable of expressing
transgenes, and (3) may prove useful for investigation of the molecular
basis of MK differentiation and GPIIb-IIIa function.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
INVESTIGATION OF megakaryocyte (MK) and
platelet biology has been limited by an inadequate source of
genetically manipulable cells capable of physiologic MK and platelet
functions. Megakaryocytes comprise only 0.02% to 0.05% of human bone
marrow,1 and the quantity of human marrow available for MK
purification is insufficient for investigative purposes. As a result,
the processes involved in MK and platelet development are incompletely
understood. Although the abundance and accessibility of peripheral
blood platelets has promoted an understanding of mature platelet
physiology, information regarding many crucial aspects of platelet
physiology remains unclear. One such area of incomplete knowledge is
the process of activation-dependent high-affinity binding of soluble
fibrinogen to platelet glycoprotein (GP) IIb-IIIa receptors (integrin
 IIb3). Although numerous assays have been
developed to characterize the function of this important adhesive
receptor,2-4 information about the molecular signals
involved in its activation is incomplete. The anucleate nature of
platelets has contributed to this slow advance of knowledge, because
molecular modification of mature platelets is not possible.
Immortalized megakaryocytic5,6 and
GPIIb-IIIa-transfected3 cell lines have limited use,
because none demonstrates physiologic, agonist-inducible, inside-out
exposure of soluble fibrinogen binding sites characteristic of
GPIIb-IIIa activation on platelets.
To circumvent these problems, many investigators have developed in
vitro MK culture systems using various growth factors to induce
megakaryopoiesis from hematopoietic precursors.7-9 The cloning and expression of the c-Mpl ligand and its identification as
megakaryocyte growth and development factor have been major advances in
this endeavor.10-12 A number of investigators have demonstrated that stem/progenitor (CD34+) cells cultured in
the presence of the c-Mpl ligand will differentiate into morphologic
and antigenic MKs.9,13,14 We now show that ex
vivo-generated MKs express functional GPIIb-IIIa receptors and are
capable of expressing exogenously introduced transgenes.
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MATERIALS AND METHODS |
Human subjects.
Leukopheresis units were purchased from the Johns Hopkins Hemapheresis
Center (Baltimore, MD). Processed bone marrow was obtained from the
Johns Hopkins Oncology Center and the Division of Bone Marrow
Transplantation of Washington University (St Louis, MO). This research
was approved by the Johns Hopkins investigational review board. All
donors provided informed consent.
Antibodies and reagents.
Biotinylated CD34 antibody was obtained from the Johns Hopkins Oncology
Center. Phycoerythrin-labeled GPIb (PE-GPIb) antibody and fluorescein
isothiocyanate-labeled GPIIb (FITC-GPIIb) antibody were obtained from
Immunotech (Marseille, France). FITC-labeled PAC1 (FITC-PAC1) antibody
and fluorescently labeled IgG isotype controls were obtained from
Becton Dickinson (San Jose, CA). 10E5 antibody was the generous gift of
Barry Coller (Mount Sinai Hospital, New York, NY). LM609 antibody was
the generous gift of David Cheresh (Scripps Research Institute, La
Jolla, CA). Fibrinogen (Sigma Chemical Co, St Louis, MO) was labeled
with FITC (Pierce, Rockford, IL) as previously described.15
Propridium iodide (PI) and ethydium bromide were from Boehringer
Mannheim (Indianapolis, IN). Adenosine diphosphate (ADP), thrombin
receptor activating peptide (TRAP), and arg-gly-asp-ser (RGDS) peptide
were obtained from Sigma. Cyclic KGD heptapeptide (Integrilin) was the
generous gift of Pascal Goldschmidt-Clermont (Columbus, OH).
Enrichment of CD34+ cells from leukopheresis units
or bone marrow.
To obtain stem cells from leukopheresis units, low-density mononuclear
cells were first separated by centrifugation over Histopaque 1077 (Sigma) as per the manufacturer's suggested protocol. Mononuclear cells were washed twice in 1% bovine serum albumin (BSA) in
phosphate-buffered saline (PBS). Cells were incubated with biotinylated
CD34 antibody for 30 minutes and then washed free of excess antibody.
CD34+ cells were isolated from the mononuclear fraction by
passage over an avidin column (CellPro Inc, Bothell, WA) using the
manufacturer's suggested protocol. In a second protocol, bone marrow
for homologous transfusion was obtained and processed by our clinical
oncology department (Johns Hopkins University, Baltimore, MD).
Low-density mononuclear cells were separated from marrow by automated
elutriation centrifugation. The mononuclear fraction was labeled with
biotinylated CD34 antibody and passed over an automated avidin column.
The CD34-depleted fraction, which contains 10% to 40% of the total CD34+ population, was obtained for further CD34 cell
isolation. (The CD34+ fraction was retained by the oncology
service for clinical use.) This CD34-depleted fraction was washed with
1% BSA/PBS and relabeled with biotinylated CD34 antibody. Excess
antibody was washed free and cells were passed over an avidin column as
described above for the leukopheresis product. In both preparations,
CD34+ cells were recovered from the avidin column and
resuspended in media for culture. Yields from the 2 separation
techniques were as follows: leukopheresis: 0.8 to 1.2 × 106 CD34+ cells from 1 × 109
mononuclear cells; CD34-depleted bone marrow fraction: 3 to 7 × 106 CD34+ cells from 1 × 109
mononuclear cells.
Induction of megakaryopoiesis in liquid culture.
CD34+ cells isolated from leukopheresis or bone marrow were
grown in StemPro Serum Free Media (Life Technologies, Gaithersberg, MD)
reconstituted with nutrient supplement and 2 mmol/L L-glutamine as per
the manufacturer's guidelines (formulation of this product is
proprietary). Cells were plated in 1-mL aliquots on 24-well culture
plates at an initial cell density of 2.5 to 5.0 × 105
cells/mL. Pegylated recombinant human MK growth and development factor
(PEG-rHuMGDF) was generously provided by Amgen, Inc (Thousand Oaks, CA)
and was added to initial cell cultures at a final concentration of 50 ng/mL. Cells were maintained in a humidified incubator with 5%
CO2 at 37°C. Cells were inspected on a daily basis and
harvested for experiments on days 7 to 14 of culture.
Identification of cultured MKs by morphology, membrane phenotype,
and ploidy.
Morphologic examination of cultured cells was performed on days 7 to
14. Cytospins of cultured cells were stained by modified Wright-Giemsa
method and examined by light microscopy. MKs were characterized by
membrane phenotype and ploidy using 2-color flow cytometry as
previously described with modifications.8,16 Cultured cells
were harvested on days 7 to 14 and diluted in culture media to 2.5 × 105 cells/mL. Cells were incubated with FITC-GPIIb
antibody and/or PE-GPIb antibody for 30 minutes at 22°C. In ploidy
experiments, cells were permeablized with Triton X-100 (0.02%) and
incubated with PI (50 µg/mL) and RNAse (50 µg/mL) in hypotonic
sodium citrate (0.1%). Cytometric analysis was performed on a FACScan
flow cytometer (Becton Dickinson) equipped with a 15-mW argon laser set
with excitation wavelength of 488 nm. Typically, 3 × 105 CD34+ cells yielded a heterogeneous
population of 1 × 106 total cells by day 14 of
culture. Preliminary experiments demonstrated maximal MK
differentiation by days 12 to 14 of culture, with 75% of the total
cell population demonstrating cell surface expression of GPIb and/or
GPIIb. PI staining of cultured cells showed a ploidy pattern consistent
with in vitro megakaryocytic differentiation (50%, 38%, 8%, and 2%
of GPIIb-positive cells being 2N, 4N, 8N, and 16N, respectively). The
rate and magnitude of MK differentiation observed in our culture system
were similar to those reported by others.8-10,14
Functional studies of MK GPIIb-IIIa receptors.
Cultured cells were harvested for physiologic studies of GPIIb-IIIa
function on days 7 to 14. Cells, diluted to 2.5 × 105/mL, were incubated with PE-GPIb antibody and FITC-PAC1
antibody or FITC-labeled fibrinogen (FITC-FGN) in the presence and
absence of TRAP or ADP at 22°C. Preliminary studies demonstrated
that the number of cultured cells capable of agonist-induced GPIIb-IIIa activation reached maximal levels between 10 and 14 days of culture. Therefore, all subsequent experiments assessing GPIIb-IIIa activation were performed during this interval. The specificity of fibrinogen binding was determined by incubating samples, in the presence and
absence of TRAP, with a series of blocking antibodies and peptides. The
kinetics of MK GPIIb-IIIa activation were determined by varying the
concentration of agonist, activation interval, and fibrinogen
concentration. Reversibility of FITC-FGN binding was determined by the
addition of ethylenediaminetetraacetic acid (EDTA; final concentration,
10 mmol/L) to samples after 5 or 20 minutes of agonist stimulation. To
determine if surface-bound FITC-FGN and FITC-PAC1 became internalized,
ethidium bromide (final concentration, 50 µg/mL), a cell-impermeable
quencher of FITC fluorescence,17,18 was added to samples
after 5 or 20 minutes of TRAP stimulation. Samples were diluted with
ice-cold PBS immediately before ethidium bromide and incubated on ice
for 5 minutes before cytometric analysis. In preliminary studies on
fixed TRAP-activated platelets, ethidium bromide quenched FITC-FGN
fluorescence with an efficiency of 76%. Samples were examined using
flow cytometry and 2-color analysis performed as described below (see
analysis of cytometric data).
Analysis of cytometric data.
Two-color flow cytometry was used to determine MK phenotype and
GPIIb-IIIa activation in cultured cells. Data from 25,000 to 50,000 events were collected from each sample with scatter data in linear mode
and fluorescent data in logarithmic mode. In all experiments, data were
analyzed using forward and side scatter gates to exclude dead cells and
cell fragments. MKs were defined by GPIb positivity (PE fluorescence)
in combination with their characteristic forward scatter as previously
described.16 FITC fluorescence histograms were generated
from the gated MK population and median fluorescence intensity (MFI) or
percent positivity was used to determine differences in fluorescence
between cell samples. Specific ligand binding was defined as the MFI of agonist-stimulated samples minus the MFI in unstimulated samples. In
some experiments, MFI was converted to fluorochrome equivalents using
standard calibration beads (Flow Cytometry Standards Corp, San Juan,
Puerto Rico) as previously described.15 The fraction of
fibrinogen irreversibly bound was determined by dividing the MFI of
EDTA-treated samples by the MFI of agonist-treated samples in the
absence of EDTA. The fraction of bound fibrinogen that became
internalized was determined by dividing the MFI of ethidium bromide-treated samples by the MFI of activated samples without the
fluorescein quencher and correcting for the quenching efficiency (×0.76).
Adenovirus vector preparation.
Preparation of an adenovirus vector encoding humanized green
fluorescent protein (hGFP) driven by the cytomegalovirus (CMV) promoter
has been described in detail previously.19,20 Briefly, cDNA
for GFP was subcloned from pGreenLantern (Life Technologies, Gaithersberg, MD) into the adenovirus shuttle vector pE1CMV to create
pE1CMVhGFP. The plasmids pE1CMVhGFP and pJM17, which contain the full
human adenovirus serotype 5 genome, were cotransfected into HEK293
cells using LipofectAMINE (Life Technologies). As described previously,
homologous recombination between the shuttle vector and pJM17 replaces
the region of the adenovirus between map units 1.0 and 9.8 with the
expression cassette cDNA. The recombinant virus designated AdCMVhGFP
was expanded in HEK293 cells and the virus was purified by 3 rounds of
cesium chloride banding as previously described.19
A recombinant replication defective adenovirus vector encoding nuclear
targeted -galactosidase (-gal), AdCNLacZ, was generated by
cotransfection of the shuttle vector pAdCNLacZ and full-length adenoviral DNA (serotype 5) into CRE8 cells (both a generous gift from
Dr Stephen Hardy, Somatix Therapy Corp, Alameda, CA) as previously described.21 The pAdCNLacZ shuttle plasmid was constructed
in a 2-step process. First, the Msc I fragment of pAAVRNLacZ (a
generous gift from Dr Robert Kotin, NHLBI, Bethesda, MD) containing the cDNA encoding for nuclear targeted -gal driven by an RSV promoter was cloned into the EcoRV site of pBluescript SK II+
(Stratagene, La Jolla, CA) to generate pBSRNLacZ. In the second step,
pAdCNLacZ was generated by ligating the Sal I-Xba I
fragment of pBSRNLacZ, containing the NLacZ cDNA, into the shuttle
plasmid pAdlox (containing a CMV promoter; also a gift from Dr S. Hardy) digested with Xho I and Xba I. Recombinant virus
was plaque purified and stocks of AdCNLacZ were prepared by propagation
in 293 cells followed by double cesium chloride centrifugation.
Adenoviral transduction of cultured MKs.
Day-11 cultured cells were incubated with AdCMVhGFP, an adenoviral
vector control lacking the sequence for GFP, or viral vehicle control
at varying multiplicities of infection (MOI) for 24 to 72 hours. Cells
were inspected daily for changes in morphology, cell number, and
viability (Trypan blue staining). Expression of GFP was determined by
flow cytometry at 24, 48, and 72 hours after viral incubation. Cell
cultures incubated with virus or vehicle were harvested at the
indicated times and incubated with PE-GPIb antibody for 30 minutes at
22°C before cytometric acquisition of data.
Cultured cells (day 10) were incubated with AdCNLacZ or vehicle control
at varying MOI for 3 to 4 days. Expression of -gal was determined as
previously described.22 Briefly, cell suspensions were
incubated in 2% formaldehyde/0.2% glutaraldehyde for 5 minutes. Cells
were washed with PBS and then incubated in X-gal staining solution
(5-bromo-4-chloro-3-indolyl--D-galacopyranoside) for 3 hours at
37°C. Stained cells were placed on a hemacytometer and transduction
efficiency was estimated by determining the percentage of cells that
appeared blue by light microscopy.
Immunofluorescence microscopy.
Cell cultures incubated with AdCNLacZ or vehicle for 3 days (day 13 of
culture) were harvested and incubated with PE-GPIb antibody (or PE-IgG
isotype control) and FITC-PAC1 in the presence and absence of 50 µmol/L TRAP for 15 minutes. Samples were fixed in 2%
formaldehyde/0.2% glutaraldehyde for 5 minutes and then cytospun onto
glass slides. Slides were rinsed in PBS and cells were stained in situ
with X-gal solution for 3 hours at 37°C. Slides were viewed by
conventional light and fluorescence microscopy using a Zeiss laser
scanning microscope (LSM). Cells transduced with AdCNLacZ were
identified by blue nuclear staining using conventional light, followed
by immunofluorescent imaging using a krypton-argon excitation laser at
488 nn and emission filters of 505 to 530 nm (FITC) and 570 nm (PE).
Fluorescent images for FITC and PE were obtained separately to ensure
that signal overlap from the 2 fluorochromes could not occur.
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RESULTS |
Cultured MKs possess functional GPIIb-IIIa receptors.
Preliminary studies demonstrated that cultured MKs stimulated with TRAP
bound more FITC-FGN than unstimulated cells. The specificity of agonist
induced soluble fibrinogen binding for GPIIb-IIIa on cultured MKs was
verified by incubating cultured cells in the presence and absence of a
series of GPIIb-IIIa blocking peptides and antibodies
(Fig 1). In each case, KGD peptide, RGDS
peptide, and 10E5 antibody (Fig 1B, C, and D, respectively), which are known to inhibit GPIIb-IIIa-mediated fibrinogen
binding,23-25 blocked the TRAP-induced increase in MK
FITC-FGN binding. In contrast, antibodies that lack specificity for
GPIIb-IIIa, LM609 (block ligand binding to the vitronectin
receptor26) and an irrelevant IgG, had no effect on the
activation-dependent binding of FITC-FGN to cultured MKs (Fig 1E and
F). None of the peptides or antibodies had any effect on the
nonspecific FITC-FGN binding of unstimulated samples. In other control
experiments, no agonist-induced increase in fluorescence was observed
in samples incubated with a nonspecific fluorescent antibody instead of
FITC-FGN (data not shown).
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| Fig 1.
Soluble fibrinogen binding to cultured MKs is specific
for GPIIb-IIIa. In each experiment, cultured cells were incubated with
PE-GPIb antibody and FITC-FGN in the presence (shaded
histograms) and absence (unshaded histograms) of TRAP and with or
without the indicated blocking antibody. FITC fluorescence (FL1)
histograms were generated from the MK population. (A) No blocking
antibody. (B) KGD heptapeptide (Integrilin). (C) RGDS peptide. (D) 10E5
antibody. (E) LM609 antibody. (F) Nonspecific mouse monoclonal IgG.
Data are representative of 3 experiments performed.
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To determine if the kinetics of GPIIb-IIIa activation on MKs were
similar to those described for platelets, we undertook a series of
experiments to determine the following aspects of the receptor-ligand
interaction: (1) the effect of agonist concentration on PAC1 binding,
(2) the effect of duration of activation on PAC1 binding, and (3) the
receptor affinity for fibrinogen. The effect of agonist concentration
on MK GPIIb-IIIa activation was assessed by incubating cultured cells
with PAC1 and increasing concentrations of TRAP or ADP. The results of
these experiments are shown in Fig 2A and
demonstrate a typical dose-response relationship for both agonists,
with maximal PAC1 binding achieved in response to 50 µmol/L TRAP and
20 µmol/L ADP. TRAP activation routinely induced a greater number of
PAC1-positive MKs than was observed with ADP stimulation (Fig 2A,
percentage of PAC1 positivity). The effect of agonist stimulation time
on MK GPIIb-IIIa activation was determined by incubating cultured cells
with PAC1 and 50 µmol/L TRAP for increasing periods of time. These
data demonstrated rapid receptor activation in the presence of TRAP,
with maximal receptor binding occuring by approximately 20 minutes
(data not shown). To determine the dissociation constant
(Kd) of MK GPIIb-IIIa receptors for fibrinogen, cultured
cells were incubated with increasing concentrations of FITC-FGN and 50 µmol/L TRAP for 20 minutes. Figure 2B shows that receptor binding was
saturable and that half-maximal binding occurred at a fibrinogen
concentration of approximately 50 µg/mL (ie, Kd = 1.5 × 10 7 mol/L).
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| Fig 2.
Binding kinetics of activated GPIIb-IIIa receptors on
cultured MKs. Cultured cells were incubated with PE-GPIb antibody and
FITC-PAC1 or FITC-FGN in the presence and absence of TRAP or ADP. MFI
was determined from MK fluorescence histograms. The percentage of
PAC1-positive MKs is shown in parentheses. Fluorochrome equivalents
were determined from the MFI as described in Materials and Methods. (A)
Effect of agonist concentration on PAC1 binding. Data are
representative of 2 experiments performed. (B) Fibrinogen binding
kinetics. Arrows indicate fibrinogen concentration at which binding was
half maximal (Kd). Data are the mean ± SEM of 3 experiments.
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Agonist-induced binding of fibrinogen to GPIIb-IIIa receptors on
platelets has been reported to be reversible, particularly during the
early stages of activation.27,28 To determine the reversibility of this receptor-ligand interaction on MKs, EDTA was
added to cultured cells that had been incubated with FITC-FGN in the
presence and absence of TRAP or ADP for 5 or 20 minutes. Approximately
half of MK FITC-FGN binding was reversed after 5 minutes of TRAP
activation, but binding was nearly irreversible by 20 minutes
(Table 1). Similar results were observed
when cells were activated with ADP. It has been reported that
fibrinogen bound to platelet GPIIb-IIIa receptors becomes rapidly
internalized after agonist stimulation.28 To determine if
internalization of surface-bound fibrinogen follows MK activation,
ethidium bromide was used to quench fluorescence from surface-bound
FITC-FGN on TRAP-activated MKs. These experiments showed that 19% ± 6% and 33% ± 3% of surface-bound FITC-FGN was internalized
by activated MKs at 5 and 20 minutes, respectively (Table 1). A similar
reduction in FITC-PAC1 fluorescence was also detected in
TRAP-stimulated MKs treated with ethidium bromide (data not shown). In
parallel experiments on washed platelets, 37% ± 3% and 46% ± 12% of FITC-FGN was internalized by TRAP-activated platelets
at 5 and 20 minutes, respectively (n = 3).
Successful transduction of cultured MKs with adenoviral vectors.
Cells were incubated with varying MOI of AdCMVhGFP, control virus, or
vehicle on day 11 of culture and were harvested for cytometric analysis
24 to 72 hours later. MK expression of GFP was detected after 24 hours
of viral incubation and increased over the next 48 hours
(Fig 3). The efficiency of transduction increased with increasing MOI and incubation time
(Table 2). There was a 10% reduction in
cell viability for the highest MOI at 48 and 72 hours as determined by
Trypan blue staining and scatter profile analyses. Incubation of
cultured cells with AdCNLacZ also resulted in successful transduction
with low cell toxicity (Fig 4). Transduction efficiency at 4 days, as assessed by visualization of
X-gal staining, was 9%, 18%, 22%, and 28% for MOI of 50, 100, 200, and 500, respectively.
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| Fig 3.
GFP expression in cultured MKs after adenoviral
transduction. CD34+ cells were cultured with PEG-rHuMGDF
for 11 days before incubation with AdCMVhGFP (MOI of 200), viral
control (MOI of 200), or vehicle control. Cells were harvested after 72 hours of viral infection and incubated with PE-GPIb antibody. FITC
fluorescence (FL1) histograms were generated from the MK population for
samples incubated with AdCMVhGFP (shaded), viral control (dotted line,
unshaded), and vehicle control (solid line, unshaded). M1 is 1%, 2%,
and 46% positivity, respectively, for vehicle control, viral control,
and AdCMVhGFP. Data are representative of 2 experiments performed.
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| Fig 4.
-gal expression in cultured cells after
adenoviral transduction. CD34+ cells were cultured with
PEG-rHuMGDF for 10 days before incubation with AdCNLacZ (MOI of 200).
-gal expression was visualized by light microscopy after cell
fixation and X-gal staining as described in Materials and Methods.
Photomicrograph of adenovirus infected cells (400×). Note the blue
nuclear staining of successfully transduced cells. No blue coloration
was detectable in any cell from samples that were similarly stained but
were incubated with virus control.
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To determine if transduced MKs retained the capacity for
agonist-induced GPIIb-IIIa activation, cells infected with AdCNLacZ were analyzed by microscopy for simultaneous expression of -gal, GPIb, and TRAP-induced PAC1 binding. Results of these experiments are
depicted in Fig 5.
Although cells on each slide were heterogeneous, nuclear X-gal staining
could be easily identified in transduced cells. (Blue X-gal stained
nuclei appear black in black and white light transmission photograph.)
As expected, the majority of cells were megakaryocytic by GPIb
positivity at this point in culture (Fig 5F), and TRAP stimulation
induced high-level PAC1 binding (Fig 5G). Successful transduction of a
morphologic and phenotypic MK can be observed in Fig 5E through H. The
arrow indicates an MK characterized by its bi-lobed X-gal-stained
nucleus, strong PE-GPIb fluorescence, and bright FITC-PAC1 fluorescence
after TRAP activation. Thus, exogenous gene transfer to cultured MKs was accomplished using adenoviral vectors and transduced MKs retained their characteristic morphologic and phenotypic features.
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| Fig 5.
Transduced MKs retain typical MK morphology and
phenotype. PEG-rHuMGDF-cultured cells that were incubated with
AdCNLacZ or control were harvested for simultaneous analysis of -gal
expression and GPIIb-IIIa activation. Cells were incubated with PE-GPIb
antibody (or PE-IgG control) and FITC-PAC1 in the presence or absence
of TRAP before fixation and in situ X-gal staining. In each set of 4 photomicrographs, the first panel shows images using light transmission
microscopy, the second and third show the same field using fluorescence
microscopy, and the fourth shows a computer generated overlay of the
first 3 images. (A through D) Virus control, PE-IgG control, FITC-PAC1,
unstimulated for light transmission, PE fluorescence, FITC
fluorescence, and overlay, respectively. (E through H) AdCNLacZ,
PE-GPIb, FITC-PAC1, TRAP for light transmission, PE fluorescence, FITC
fluorescence, and overlay, respectively. Note that in these black and
white light transmission images X-gal-stained nuclei appear as black
instead of blue. The arrow indicates a successfully transduced
morphologic and phenotypic MK. All photomicrographs are 400×.
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DISCUSSION |
Most studies of MK and platelet biology have investigated separately
the processes of MK differentiation and platelet physiology. Although
the influence of MK development on platelet function is recognized, the
ability to link these 2 processes has been limited by an inadequate
source of genetically manipulable cells capable of physiologic platelet
responses. This study demonstrates that ex vivo-cultured MKs are
capable of (1) agonist-induced GPIIb-IIIa activation characteristic of
blood platelets and (2) expression of an exogenous gene product after
adenoviral transduction and that (3) transduced MKs retain physiologic
GPIIb-IIIa activation. These data suggest that ex vivo-cultured MKs
represent a genetically manipulable substrate suitable for
investigation of GPIIb-IIIa function.
Cytometric studies demonstrated that cultured MKs were capable of
inside-out signal transduction leading to binding of soluble fibrinogen
and PAC1 to activated GPIIb-IIIa receptors. These studies confirm and
extend the findings of previous investigators who have demonstrated
that bone marrow-derived MKs are capable of GPIIb-IIIa-mediated
fibrinogen binding.29,30 However, our data begin to address
the functional similarities and dissimilarities between GPIIb-IIIa
activation on MKs and platelets. Both the agonist concentration and
time course required for maximal GPIIb-IIIa activation on MKs were
similar to those described for receptor activation on
platelets.2,31,32 Furthermore, the affinity of MK
GPIIb-IIIa receptors for fibrinogen was similar to that reported for
platelets.2,15 In the present assay, TRAP was substantially
more effective at activating MK GPIIb-IIIa receptors than ADP, which is
in accord with the relative strengths of thrombin and ADP as platelet
agonists. Although MKs activated with TRAP aggregated in the presence
of fibrinogen, ADP-activated MKs did not (data not shown). Thus, it is
possible that this differential responsiveness to agonist stimulation
may also be due to (1) inadequate numbers of MK ADP receptors, (2)
immaturity of ADP-dependent second messenger signals, and/or (3)
differences between MK and platelet cytoskeletal architecture.
Ligands induced to bind to MK GPIIb-IIIa receptors appear to share
similarities with GPIIb-IIIa-bound ligands on activated platelets.
Fibrinogen binding to MK GPIIb-IIIa receptors was reversible, and the
extent of reversibility was dependent on the duration of cell
activation, as has been described previously for
platelets.27,28 The extent to which fibrinogen binding to
activated MKs was irreversible was similar to that reported for
platelets by some investigators,28 but more than
others.27 Fluorescence quenching experiments determined that a substantial portion of the fibrinogen bound to MKs became internalized within a few minutes of activation, which is similar to
what we observed in platelets. Rapid internalization of
GPIIb-IIIa-bound fibrinogen has been described previously for
activated platelets,28 as has internalization of GPIIb-IIIa
on resting MKs29 and platelets.33 However,
internalization of GPIIb-IIIa-bound fibrinogen can only account for
some of the irreversibly bound ligands on MKs, because the fraction
irreversibly bound was substantially greater than the fraction
internalized. It is possible that cytoskeletal reorganization in
activated MKs contributes to stablization of the GPIIb-IIIa-bound fibrinogen, as has been proposed to occur in activated
platelets.27 Although such a determination is beyond the
scope of this work, our studies suggest that ex vivo-cultured MKs may
be a useful substrate for further characterization of platelet and MK functions.
Viral vectors have been successfully used for exogenous gene transfer
to a number of cell types, including hematopoietic
cells.34,35 Adenoviral vectors have been shown to be
particularly useful for this purpose because of their ability to
transform a variety of postmitotic cells, high multiplicity in culture,
and ability to incorporate large inserts.36 Data from this
study demonstrated that adenoviral vectors were useful for gene
transfer to cultured MKs. Efficient transduction occurred at a variety
of MOI over a 96-hour interval with only a modest reduction in cell
viability. The efficiency, time course, and toxicity we observed with
adenovirus-mediated gene transduction of cultured MKs were very similar
to those recently reported by other investigators.37
Transduction efficiency appeared to be greater with the GFP than with
the -gal vector. The observed difference in transduction efficiency
more likely reflects greater sensitivity of the GFP assay than a true
difference in protein expression; however, we cannot rule out the
latter. The high transduction efficiency of MKs may be related to
high-level v3 expression, which has been
shown to be important for adenoviral infection of a number of cell
types, including MKs.37 Importantly, transduced cells were
shown to retain morphologic and physiologic MK structure and function.
GFP and -gal were chosen as reporter genes because of the ease with
which transformed cells can be identified. However, our data suggest
that targeted gene transduction in cultured MKs is a methodology that
may be useful for elucidating the molecular mechanisms underlying MK
developmental biology and GPIIb-IIIa function.
| |
FOOTNOTES |
Submitted September 11, 1998; accepted August 13, 1999.
Supported by National Institutes of Health Grants No. HL03454 and HL58564.
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 Nauder Faraday, MD, Department of
Anesthesiology/CCM, Johns Hopkins University School of Medicine, 600 N
Wolfe St, Baltimore, MD 21287.
| |
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ABSTRACT
INTRODUCTION