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New Insights Into the Negative Regulation of Hematopoiesis by Chemokine
Platelet Factor 4 and Related Peptides
By Laurence Lecomte-Raclet, Mònica Alemany, Anabelle Sequeira-Le Grand, Jean Amiral, Gérard Quentin,
Anne Marie Vissac, Jacques P. Caen, and Zhong Chao Han
Platelet factor 4 (PF4) has been recognized as an inhibitor of
myeloid progenitors. However, the mechanism of action of
this chemokine remains poorly understood. The present
study was designed to determine its structure/function
relationship. A series of peptides overlapping the C-terminal
and central regions of PF4 were analyzed in vitro for their
action on murine hematopoietic progenitor growth to assess the minimal sequence length required for activity. The
peptides p17-58 and p34-58 possessed an increased hematopoietic inhibitory activity when compared with PF4, whereas
the shorter peptides p47-58 and p47-70 were equivalent to
the native molecule and the peptide p58-70 was inactive. The
PF4 functional motif DLQ located in 54-56 was required for
the activity of these peptides. The peptide p34-58 impaired
to a similar extent the growth of colony-forming unitmegakaryocyte (CFU-MK) as well as burst-forming uniterythroid (BFU-E) and colony-forming unit–granulocytemacrophage (CFU-GM), whereas PF4 was more active on
CFU-MK. In the experiments using purified murine CD341
marrow cells, statistically significant inhibition induced by
p34-58 was shown at concentrations of 2.2 nmol/L or greater
for progenitors of the three lineages, whereas that induced
by PF4 was seen at 130 nmol/L for CFU-MK and 650 nmol/L
for CFU-GM and BFU-E, indicating that the p34-58 acts
directly on hematopoietic progenitors and its activity is
approximately 60- to 300-fold higher than PF4. The p34-58,
unlike PF4, lacked affinity for heparin and its inhibitory
activity could not be abrogated by the addition of heparin. In
addition, an antibody recognizing p34-58 neutralized the
activity of p34-58 but not whole PF4 molecule. These results
demonstrate that PF4 contains a functional domain in its
central region, which is independent of the heparin binding
properties, and provide evidence for a model of heparindependent and independent pathways of PF4 in inhibiting
hematopoiesis.
r 1998 by The American Society of Hematology.
P
which confers to the molecule a high affinity for heparin and
other sulfated glycans, has been considered to be responsible for
most of the activities of PF4.16 In human erythroleukemia
(HEL) cells, PF4 directly inhibits the growth of HEL cells by
fixation on heparan sulfate proteoglycans on the cell surface.
The binding to cells and the inhibitory effect of PF4 can be
inhibited by addition of exogenous heparin or other glycosaminoglycans (GAG), by treatment with heparinase and
heparitinase as well as the inhibitors of proteoglycan synthesis.17,18 However, PF4 sequence p58-70, which contains the
main heparin-binding domain, does not interfere with megakaryocytopoiesis in vitro and in vivo in mice.19 Furthermore, a
PF4 analogue lacking affinity for heparin has been found to
retain the ability of PF4 to suppress angiogenesis in vivo.20
Recent studies on structure-function relationships of chemokines have shown that the two DLQ motives of PF4 located in
positions 7-9 and 54-56 are necessary for this protein to inhibit
myeloid progenitor proliferation.21 All these observations suggest that functional determinants other than the heparin binding
sequence may also be involved in the mechanism of action of
PF4. To address this issue, a series of peptides overlapping the
C-terminal and central domains of PF4 were tested for inhibitory activity towards hematopoietic progenitor cells. We provide evidence here for a new pathway of hematopoietic
inhibitory action of PF4 through its central functional domain
independent of the heparin binding properties of PF4.
LATELET FACTOR 4 (PF4) is a 7.8-kD protein that is
synthesized by megakaryocytes, stored in a-granules as a
noncovalent tetramer and released from activated platelets.
Each monomer has a conformational flexible N-terminal region
that is anchored by two disulfide bridges to the protein core,
which consists of three antiparallel b-strands and a carboxylterminal a-helix.1-5 PF4 shares 30% to 40% amino acid
homology and general structural identity with the members of
CXC chemokines family, including b-thromboglobulin (bTG)
and its N-terminal cleavage product neutrophil-activating protein-2 (NAP2), interleukin-8 (IL-8), human proto-oncogene
Gro/melanocyte growth-stimulating activity (Gro/MCSA), and
interferon-inducible protein 10 (IP-10). In addition, these
molecules have overlapping and some additive biologic activities in modulating inflammation, hemostasis, hematopoiesis,
cell proliferation, angiogenesis, and glycosaminoglycan activity.6-10
PF4 has been recognized as inhibitor of hematopoiesis and
angiogenesis.11-15 However, the mechanism of action of PF4
remains poorly understood, because no cell surface receptor has
been yet identified. The basic nature of the C-terminus of PF4,
From the Institut des Vaisseaux et du Sang, Hôpital Lariboisière,
Paris, France; and Serbio Research Laboratories, Gennevilliers,
France; and the Institute of Hematology, Chinese Academy of Sciences,
Tianjin, China.
Submitted July 7, 1997; accepted November 26, 1997.
Supported by a grant from the Association pour la Recherche sur le
Cancer to Z.C.H.
Address reprint requests to Jacques P. Caen, MD, IVS-Hôpital
Lariboisière, 8 Rue Guy-Patin, 75475 Paris Cedex 10, France.
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.
r 1998 by The American Society of Hematology.
0006-4971/98/9108-0043$3.00/0
2772
MATERIALS AND METHODS
Reagents and Growth Factors
Unfragmented heparin was purchased from Serbio Laboratories
(France). Recombinant murine granulocyte-macrophage colonystimulating factor (rmGM-CSF) was provided by Beite Kaito (Paris,
France) and recombinant murine stem cell factor (rmSCF) was provided
by R & D (Oxford, UK). Both growth factors were diluted in
phosphate-buffered saline (PBS) 1 0.01% bovine serum albumin
(BSA) and stored at 220°C.
Blood, Vol 91, No 8 (April 15), 1998: pp 2772-2780
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THE PF4 CENTRAL DOMAIN INPAIRS HEMATOPOIESIS
2773
PF4 Purification
Heparin Binding Properties of the Peptides
Highly purified human PF4 extracted from platelet concentrates and
synthetic peptides related to PF4 were provided by Serbio (Gennevilliers, France). Briefly, human PF4 was prepared from fresh human
platelet concentrates, washed, and disrupted by three repetitive deepfreezing (280°C) and thawing (30 minutes at 37°C) cycles. Platelet
debris were removed after centrifugation at 5,000g and all the platelet
released proteins were recovered in the supernatant. This supernatant
was used for the preparation of PF4 as described by Handin and
Cohen,22 with the exception that a final gel filtration step was performed
on Superdex 75, in 0.05 mol/L Tris, 0.5 mol/L NaCl buffer at pH 7.5.
This preparation was highly purified (single band on sodium dodecyl
sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) and contained no fibronectin, fibrinogen, thrombospondin, or von Willebrand
factor. Both Western blot and direct enzyme-linked immunosorbent
assay (ELISA) analysis showed no contamination by human transforming growth factor b (TGFb).12,13 PF4 was originally stored in a glycine
buffer saline at a concentration of 100 µg/mL and then lyophilized and
reconstituted with 1 mL distilled water.
Various 125I-PF4 and 125I-p34-58 aliquots were incubated overnight
with heparin cross-linked to agarose (Bio-Rad, Ivry sur Seine, France).
One milliliter of NaCl at increasing concentrations (from 0 to 2.0
mol/L) was added to each sample. After 20 minutes of incubation at
room temperature, the heparin-associated PF4 and p34-58 were spun
down and radioactivity in the supernatants was quantified by g-counting
to determine the nonbound PF4 and p34-58 fractions.
Peptide Synthesis
Peptides were synthesized using standard solid-phase methodology
and purified by high-performance liquid chromatography (HPLC) using
a C18 column and a 0% to 80% linear acetonitrile gradient in 0.1%
trifluoroacetic acid. Amino acid sequences of the peptides are shown in
Fig 1.
Antibodies
Rabbit anti-PF4 polyclonal antibodies were immunopurified using a
PF4 column and then digested with pepsin to obtain the anti-PF4 F(ab8)2
fragments that were further purified by gel filtration using a Sephazyl
S100 column. This fraction was subsequently applied onto a p34-58
affinity column. Although still exhibiting high anti-PF4 reactivity, the
F(ab8)2 contained in the filtrate did not recognize the PF4 sequence
34-58, and the fraction was thus named anti-PF4(p34-58)2. The F(ab8)2
fragments bound onto p34-58 column were separately eluted from the
affinity column and designated as the anti-p34-58 fraction.
Fig 1. Amino acid sequences
of human PF4 and peptides
tested for inhibitory activity on
the formation of CFU-MK, CFUGM, and BFU-E from total cells
of bone marrow and purified
CD341 bone marrow cells.
Colony Assays of Hematopoietic Progenitors
Cell preparation. Total and CD341 bone marrow cells obtained
from Balb/c mice were used in the present study. The mice (6- to
8-week-old male mice) were purchased from IFFA CREDO Laboratories (L’Arbresle, France) and maintained under standard housing
conditions with water and commercial rodent chow. After mice were
killed by cervical dislocation, the femurs were removed and the total
bone marrow was expelled with 5 mL a medium (Eurobio, Paris,
France).
For the purification of CD341 cells, bone marrow cells were
incubated with biotinylated rat anti-CD34 MoAb (RAM34; Pharmingen, San Diego, CA) for 30 minutes on ice, washed twice in PBS-BSA,
and stained with fluorescein isothiocyanate (FITC)-conjugated streptavidin (Caltag, San Francisco, CA) for 30 minutes. Control cells were
stained with biotinylated rat-IgG2a (clone R35-95; Pharmingen) and
streptavidin-FITC. Viable cells were defined by forward (FSC) and side
(SSC) light-scattering properties and exclusion of propidium iodide on
dual-laser FACS Vantage cell sorter (Becton Dickinson Immunocytometry Systems, San Jose, CA). The visible laser output was set at 150 mW
with emission at 488 nm and the emitted light was collected with 530/30
bandpass filter. Cells were collected in a tube with a medium containing
10% aplastic anemia serum (AAS) obtained by blood collection from
pigs 5 days after 8 Gy total body irradiation and were counted and
reanalyzed for purity.
Colony-forming unit-megakaryocyte (CFU-MK) assay. Megakaryocytes and their progenitor cells were studied using a plasma clot
system.23,24 Briefly, 2 3 105 nucleated marrow cells or 24,000 CD341
marrow cells were cultured in at least triplicate in Petri dishes (35 mm)
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2774
LECOMTE-RACLET ET AL
in a total volume of 1 mL with 1% BSA (Sigma Chemical Co, St Louis
MO), 10% bovine citrated plasma (GIBCO, Cergy-Pontoise, France),
1 3 1024 mol/L 2-mercaptoethanol (Sigma Chemical), 0.34 mg CaCl2
(Prolabo, Paris, France), 15 U penicillin plus 15 µg streptomycin, and
10% AAS. PF4, various synthetic peptides, and antibodies were added
exogenously just before the plasma-clot assay. Heparin (5 IU/dish) was
added after the clot formation. The cultures were incubated at 37°C in a
humidified atmosphere of 5% CO2. After 7 days of culture, the dishes
were fixed with 1% paraformaldehyde and stained for acetylcholinesterase to determine the number of colonies derived from CFU-MK.
Identification of colonies was performed as previously described.23 A
CFU-MK–derived colony was defined as a cluster of three or more
cells. Megakaryocytes and CFU-MK were counted using a computerized automatic image analysis. Briefly, this analysis system was based
on acetylcholinesterase staining, a specific stain for murine bone
marrow megakaryocytes, and an image capturing instrument with a
computer program (see addendum).
Burst-forming unit-erythroid (BFU-E) and colony-forming unit–
granulocyte-macrophage (CFU-GM) assays. BFU-E and CFU-GM
were assayed using a methylcellulose system as previously described.24
A total of 1 3 105 marrow nucleated cells/mL or 16,000 CD341 marrow
cells/mL were plated in semisolid medium containing 0.8% methylcellulose, 10% AAS, 1 3 1024 mol/L 2-mercaptoethanol (Sigma Chemical), 10 ng/mL rmSCF, and 10 ng/mL rmGM-CSF for BFU-E and
CFU-GM assays. Quadruplicate cultures for each assay were incubated
at 37°C in a humidified atmosphere of 5% CO2. A BFU-E colony ($3
clusters of 20 cells) and CFU-GM colonies ($50 cells) were scored
under an inverted microscope at 5 days of culture.
Fig 2. Effect of PF4 and various peptides (p47-55, p47-58, p47-70,
and p58-70) of PF4 on the formation CFU-MK. Values are expressed as
mean 6 SEM of triplicate determination obtained from three separate
experiments. *P F .01 as compared with control value determined by
the Student’s t-test. One hundred percent (100%) corresponds to the
number of megakaryocyte colonies (75 colonies/mL) in control cultures.
Statistical Analysis
Results were expressed as the mean 6 SEM for data from 3 or more
separate experiments. The significance of the difference between groups
was determined by the Student’s t-test. * and # indicate P , .05 and P ,
.01, respectively.
RESULTS
Effects of PF4-Related Peptides on Megakaryocyte Colony
Formation From Bone Marrow Cells
In an attempt to detect new functional domains of PF4,
various peptides were synthesized (Fig 1). The longer peptide
p17-58 contains two cysteines and one of the sequences DLQ, a
functional motif located in position 54-56 of PF4. The peptide
p34-58 corresponds to the C-terminal part of p17-58, and thus it
still includes the two cysteines and the DLQ motif. Other
peptides synthesized included four peptides of C-terminal
region of PF4, the p47-70, p47-58, p47-55, and p58-70, which
have been previously characterized by us.19
The capacity of these peptides to interfere with megakaryocytopoiesis was examined in vitro in comparison with PF4. It was
found that p47-58 and p47-70, like PF4, significantly inhibited
megakaryocyte colony formation from bone marrow cells to
30%, whereas p58-70 did not, suggesting that p47-58 and
p47-70 retain the inhibitory activity of PF4 but p58-70 does not
possess such an activity (Fig 2). Figure 3 shows the results
obtained from the experiments using a range of concentrations
of p17-58 and p34-58 compared with PF4 and p47-58. As seen
in Fig 3, a dose of at least 130 nmol/L (1 µg/mL) was required
for PF4 to exert a significant inhibitory effect on the growth of
CFU-MK. In contrast, 60-fold lower concentrations (2.2 nmol/L)
of peptides p17-58 and p34-58 were sufficient to induce a
similar effect on megakaryocytopoiesis. Further shortening of
the peptide resulted in a loss of activity, because p47-58
inhibited the growth of CFU-MK at the same molar concentrations as PF4, suggesting that the central region of PF4 contains a
functional domain responsible for a strong growth inhibitory
activity. Because no statistical significant difference in the
inhibitory activity was detected between peptides p17-58 and
p34-58, further characterization of the central domain of PF4
was performed only using the shorter active peptide p34-58.
Structural Determinants for the Activity of p34-58
The motif DLQ is implicated in the inhibitory action of PF4.
Thus, we synthesized a new peptide in which the DLQ in
position 54-56 was mutated to ALA (Fig 1). In comparison to
the native peptide, this peptide has lost most of the inhibitory
activity on MK colony formation, suggesting that this motif was
essential for the effect of the peptide (Fig 4). This is consistent
with our previous observation on the peptide 47-58, in which
the partial deletion of the DLQ sequence also resulted in the
inactivation of peptide 47-55 (Fig 2).
Because p34-58 contains two Cys (36 and 52), we could not
discard the formation of dimeric or cyclic peptides with
different inhibitory activities. To test this possibility, other
peptides were synthesized in which the Cys36 or both Cys were
replaced by Ser. As shown in Table 1, there was no difference in
the effective inhibitory concentrations of p34-58S36 and p3458S36S52 when compared with the native peptide. To examine
the formation or intramolecular and intermolecular disulfide
bridges, the native p34-58 peptide and its two mutated forms
(p34-58S36 and p34-58S36S52) were analyzed by fast protein
liquid chromatography (FPLC) using a TSK 3000 column. All
peptides eluted at a molecular weight of about 2,500 Daltons,
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THE PF4 CENTRAL DOMAIN INPAIRS HEMATOPOIESIS
2775
Fig 3. Effect of PF4 and various peptides (p17-58,
p34-58, and p47-58) of PF4 (0, 0.2, 2.2, 22, 76.5, 130,
and 630 nmol/L) on the formation CFU-MK. Values
are expressed as the mean 6 SEM of triplicate
determination obtained from three separate experiments. *P F .01 as compared with control value
determined by the Student’s t-test. One hundred
percent (100%) corresponds to the number of megakaryocyte colonies (70 colonies/mL) in control cultures.
corresponding to the monomeric form. In addition, mass
spectrometry analysis demonstrated for all peptides a molecular
weight of 2,600 to 2,700 Daltons, confirming the monomeric
presentation. This analysis also demonstrated that Cys residues,
when present, are in the reduced form. Lastly, reduction studies,
followed by a hydrophobic analysis, failed to show any
modification of the molecular presentations of these peptides.
All these data suggest that these peptides are in the monomeric
form and that the Cys residues, when present, are not oxidized.
Inhibitory Action of PF4 and Its Central Domain on Other
Hematopoietic Lineages
The effects of p34-58 on the growth of other hematopoietic
lineages were next analyzed. Murine bone marrow cells were
seeded into methylcellulose in the presence of appropriate
growth factors and the number of colonies was determined in
the presence or the absence of PF4 or p34-58. As reported in Fig
5, the peptide p34-58 impaired to a similar extent the growth of
CFU-MK as well as BFU-E and CFU-GM at a concentration of
at least 2.2 3 1029 mol/L, optimally 8.64 3 1028 mol/L.
Effect of PF4 and p34-58 on Colony Formation From Purified
CD341 Marrow Cells
We next studied the effects of PF4 and p34-58 on the growth
of purified CD341 progenitor population. The murine bone
marrow CD341 population was enriched 17- to 20-fold by flow
cytometry sorting. The purity of the isolated CD341 population
was 70% to 80%. The enriched CD341 population was cultured
as described above, and its capacity to form colonies in the
presence of increasing doses of PF4 or its derived peptides was
examined.
As seen in Fig 6, PF4 and p34-58 retained their ability to
Table 1. Effect of Native and Mutated p34-58 on CFU-MK, CFU-GM,
and BFU-E Growth
CFU-MK
CFU-GM
BFU-E
Control
100 6 2.81
100 6 3.55 100 6 4.42
p34-58 (22 nmol/L)
63.4 6 3.1*
64.1 6 2.9*
65 6 5.9*
p34-58S36 (22 nmol/L)
70.3 6 2.2*
59.3 6 4.1*
62 6 3.7*
p34-58S36S52 (22 nmol/L) 68.0 6 3.67* 64.5 6 5*
68.3 6 3.6*
Fig 4. Effect of PF4 and various peptides (p34-58 and p34-58ALA)
on the formation CFU-MK. Values are expressed as the mean 6 SEM
of triplicate determination obtained from three separate experiments. *P F .01 as compared with control value determined by the
Student’s t-test. One hundred percent (100%) corresponds to the
number of megakaryocyte colonies (52 colonies/mL) in control cultures.
Values are expressed as the mean 6 SEM of triplicate determination
obtained from three separate experiments. One hundred percent
(100%) corresponds to the control value (PBS) (52 CFU-MK/mL, 57
CFU-GM/mL, and 56 BFU-E/mL).
*P , .01 as compared with control value determined by the
Student’s t-test.
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2776
LECOMTE-RACLET ET AL
Effect of Heparin on the Inhibitory Properties of PF4 and Its
Derived Peptides
Although the PF4 C-terminal heparin binding domain is not
present in the p34-58 peptide, we tested if heparin could still
modulate the inhibitory action of the peptide. As seen in Table
2, the addition of heparin (5 IU/dish) almost completely
neutralized the in vitro inhibitory effect of PF4 on the growth of
CFU-MK. On the contrary, heparin had no effect on the
inhibitory activity of p34-58 (at 2.2 nmol/L).
The heparin-binding affinity experiments were performed as
another approach to determine the relationship between heparin
and PF4 as well as related peptides. The results show that PF4,
as previously reported, was effectively retained onto heparincoated beads and dissociated from heparin by NaCl at a starting
concentration of 0.5 mol/L. At 0.75 mol/L, NaCl eluted 50% of
bound PF4. In contrast, the peptides p34-58 and p17-58 did not
bind to heparin.
Fig 5. Effect of peptide p34-58 (0, 0.2, 2.2, 22, 76.5, 130, and 630
nmol/L) on the formation CFU-MK, CFU-GM, and BFU-E. Values are
expressed as the mean 6 SEM of triplicate determination obtained
from three separate experiments. *P F .01 as compared with control
value determined by the Student’s t-test. One hundred percent
(100%) corresponds to the number of colonies derived from CFU-MK
(70 colonies/mL), CFU-GM (84/mL), and BFU-E (64/mL).
inhibit the growth of CFU-MK as well as CFU-GM and BFU-E
colonies derived from purified CD341 cells, similar to their
inhibition of colonies derived from total bone marrow cells.
Statistically significant inhibition induced by p34-58 was shown
at concentrations of 2.2 nmol/L or greater for progenitors of
three lineages, whereas that induced by PF4 was seen at 130
nmol/L (1 µg/mL) for CFU-MK and 650 nmol/L (5 µg/mL) for
CFU-GM and BFU-E, respectively. These data indicate that
p34-58, like PF4, acts directly on hematopoietic progenitors.
However, its inhibitory activity is approximately 60-fold for
CFU-MK and 300-fold for CFU-GM and BFU-E higher than
PF4.
Effect of Various Anti-PF4 Antibodies on CFU-MK Growth
It was found from three experiments that the anti-p34-58, the
F(ab8)2 fragments recognizing specifically the peptide p34-58,
represents only approximately 1.5% of initial immunopurified
anti-PF4 polyclonal antibody, suggesting that in the native PF4
molecule this sequence may be masked. Specificity of the
reactivities of the anti-p34-58 and the anti-PF4(p34-58)2
[the anti-PF4 F(ab8)2 antibody depleted of the anti-p34-58
fraction] were documented by ELISA (results not shown).
The two F(ab8)2 fragment preparations still recognized native PF4 coated in the microassay plate. The anti-p34-58
fraction strongly reacted with insolubilized p34-58 peptide,
whereas the anti-PF4(p34-58)2 recognized whole PF4 but not
p34-58. When added to the cultures, the anti-PF4 (p34-58)2
neutralized the inhibitory effect of PF4 on the growth of
megakaryocyte progenitor cells but not that of p34-58. In
contrast, the antip34-58 F(ab8)2 fragments did not block the
effect of PF4 but effectively reversed the inhibition induced by
p34-58 (Fig 7).
DISCUSSION
The present work provides evidence for a new functional
domain in the central region of PF4, which is implicated in the
hematopoietic inhibitory properties of this chemokine and is
active in a heparin-independent manner. This is based on the
following observations. (1) The peptide p34-58 possesses a 60to 300-fold increased inhibitory activity than PF4 on hematopoietic colony formation from either bone marrow or purified
CD341 cells. (2) This peptide similarly inhibits the growth of
CFU-MK as well as CFU-GM and BFU-E, whereas PF4 is
more active on CFU-MK. (3) Unlike PF4, p34-58 lacks affinity
for heparin and its inhibitory activity cannot be abrogated by
heparin. (4) The antibody against p34-58 neutralizes the inhibitory activity of p34-58 but not that of native PF4, suggesting
that the region 34-58 is poorly or not accessible on the native
molecule. (5) Preservation of the DLQ motif in position 54-56
is essential for the activity of p34-58.
PF4 has previously been demonstrated to be an important
negative regulator of hematopoiesis, particularly megakaryocytopoiesis.11-13 The exact mechanism of action of PF4 remains to
be elucidated. The basic nature of the C-terminus of PF4 confers
to the molecule a high affinity for heparin and other sulfated
glycans, which was considered to be involved in the mechanism
of action of PF4. Accordingly, heparin abrogates the inhibitory
effect of PF4 on myeloid colony formation. But other domains
of PF4 may be implicated in the functionality of this hematopoietic regulator. Recent studies on structure-function relationships
of chemokines have shown that the DLQ motifs of PF4 located
in positions 7-9 and 54-56 are necessary for this protein to
inhibit myeloid progenitor proliferation.21
Several approaches were therefore made to determine functional domains of PF4 implicated in regulating negatively
hematopoiesis. First, the effects of the peptides corresponding
to the central and C-terminal domains of PF4 were studied
under the same experimental conditions for culturing hematopoietic progenitors. We found in our system that p47-70 and
p47-58 retained the inhibitory activity of PF4 but p58-70 was
not active. To detect other functional determinants on PF4, we
synthesized a longer peptide overlapping the sequence 17-58.
This peptide significantly inhibited megakaryocyte colony
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THE PF4 CENTRAL DOMAIN INPAIRS HEMATOPOIESIS
2777
Fig 6. Effect of PF4 (0, 76.5, 130, 630, and 1,300
nmol/L) and p34-58 (0, 0.2, 2.2, 22, 76.5, and 130
nmol/L) on the formation of CFU-MK (A), CFU-GM
(B), and BFU-E (C) from purified CD341 of bone
marrow. Values are expressed as the mean 6 SEM of
triplicate determination obtained from three separate experiments. *P F .01 as compared with control
value determined by the Student’s t-test. One hundred percent (100%) corresponds to 60 CFU-MK
colonies/mL, 70 CFU-GM colonies/mL, and 60 BFU-E/
mL, which were grown from control cultures.
formation from murine bone marrow at lower concentrations
than the native molecule. In an attempt to define the sequence
responsible for this strong activity, a shorter peptide including
region 34-58 (p34-58) was designed and activity of this peptide
was compared with p17-58, p47-58, and PF4. The p34-58
induced a similar inhibition of CFU-MK at comparable molar
concentrations as p17-58. Maximum inhibition induced by
p17-58 or p34-58 was similar to that caused by PF4, but it
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2778
LECOMTE-RACLET ET AL
Fig 7. Effect of anti-p34-58 and anti-PF4 (p34-58)2 antibodies in
presence of PF4 or p34-58 on the formation of and CFU-MK from total
cells of bone marrow. Values are expressed as the mean 6 SEM of
triplicate determination obtained from three independent experiments. *P F .01 as compared with control value determined by the
Student’s t-test. One hundred percent (100%) corresponds to 91
CFU-MK colonies/mL of control cultures.
occurred at an approximately 60-fold lower molar concentration. The peptide p34-58 contains the motif DLQ at position
54-56. This sequence may be involved in the inhibitory
properties of the peptide, because the mutation DLQ = ALA
suppresses the inhibitory properties of the peptide. Furthermore,
the partial deletion of the DLQ motif in the peptide p47-55
resulted in a complete loss of activity when compared with the
peptide p47-58.19 Indeed, PF4 contains two DLQ sequences
located at positions 7-9 and 54-56. Mutation of the first DLQ
motif has been demonstrated to completely suppress the inhibitory action of PF4 on hematopoietic colony formation.21 In the
same work, participation of the DLQ(54-56) in the activity of
PF4 was not well established. Thus, our results support for the
first time the functional importance of the DLQ motif located in
position 54-56 within the region 34-58.
Although all the active peptides tested in our experiments
Table 2. Effect of PF4 and p34-58 onCFU-MK Growth in the Presence
or Absence of Heparin
Control
PF4
p34-58
0 U/mL Heparin
5 U/mL Heparin
100 6 6.6
66.7 6 2.1*
65.8 6 3.7*
112 6 6.6
99.2 6 2.9
67.9 6 7.1*
Values are expressed as the mean 6 SEM of triplicate determination
obtained from three separate experiments. One hundred percent
(100%) corresponds to the control value (PBS) (45 CFU-MK/mL).
Formation of CFU-MK from PF4 (630 nmol/L) or p34-58 (22 nmol/L)
treated bone marrow cells. Heparin was added after plasma-clot
formation.
*P , .01 as compared with control value determined by the
Student’s t-test.
contain the DLQ motif at position 54-56, effective doses of
p47-58 were more similar to the native PF4 and lower than the
longer peptides. Consequently, differences between p34-58 and
p47-58 may be related to other structural features. PF4 contains
three large loops that participate in joining the three b-sheet
strands and the C-terminal a-helix.25 The p34-58 sequence
forms two complete b-sheet strands in the PF4 molecule,
whereas p47-58 includes only the sequence of the last b-sheet.
The secondary structure of a PF4 peptide overlapping the
domain 38-57 has been solved and shows that the native
conformation is conserved.26,27 Thus, it is possible that the
structures of peptides analyzed in the present study are quite
similar to the native. Moreover, we have observed that the Cys
36 and 52, which do not associate together to form a disulfide
bridge in the native PF4, remain in a reduced state in the peptide
p34-58. These findings can also explain why the native as well
as the two mutated peptides p34-58S36 and p34-58S36S52
keep the same enhanced activity.
Members of CXC chemokine family share a significant
overall homology up to 25% to 40% and similar threedimensional conformations. When the sequence 34-58 of PF4 is
compared with the equivalent domain of other CXC chemokines, homologies increase to 65% to 70%. IL-8 also manifests
hematopoietic inhibitory activity and acts synergistically with
PF4 in inhibiting cell proliferation.21,28 Thus, we studied the
biological importance of the central region of other chemokines
and particularly IL-8. But, contrary to PF4, a peptide corresponding to the region 34-58 of IL-8 did not retain the inhibitory
activity of the chemokine (results not shown). This finding
suggests that the functionality of this region may be specific for
PF4. This is consistent with the lack of activity on myeloid
proliferation of a recombinant chimeric PF4 molecule in which
the domain 51-60 was replaced by the corresponding IL-8
sequence.21
To examine the role of the domain 34-58 in the PF4 molecule,
a second series of experiments was performed using two
purified antibodies against PF4 and p34-58, respectively. Purified F(ab8)2 anti-p34-58 completely abrogated the inhibitory
action of peptide p34-58, but it could not reverse the negative
effect of PF4. This difference can be related to the accessibility
of the antibody to its epitope within the whole PF4 molecule.
Indeed, the anti-p34-58 was purified from a total F(ab8)2
anti-PF4 polyclonal antibody and was found to be present only
in limiting amounts (,1.5%). Previous observations on threedimensional structures of PF4 have pointed out that the majority
of 34-58 sequence is inside of PF4 tetramer.25 Taken together,
these data indicate that the p34-58 sequence is usually masked
within whole PF4 molecule, probably explaining why the whole
PF4 is much less active than its central peptides such as p17-58
and p34-58 observed in the present study. Indeed, it has been
reported that the cleavage of PF4 at position between 16 and 17
results in a dramatic increase in endothelial cell proliferation
inhibition.15 This observation could be related to an exposition
of the masked activity of the PF4 34-58 domain.
We have previously shown that PF4 directly impaired MK
development from human cord blood CD341 progenitor cells.25
In this study, we tested the p34-58 on an enriched CD341
population purified from murine bone marrow cells. Basically,
no difference in the inhibitory profiles of either PF4 or p34-58
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
THE PF4 CENTRAL DOMAIN INPAIRS HEMATOPOIESIS
was observed on both enriched CD341 cells and total bone
marrow cells. It seems possible that p34-58 as well as PF4 may
act directly on the growth of hematopoietic progenitors. It was
interesting to note that the inhibitory effect of p34-58 was not
restricted to the megakaryocytic lineage, because it also impaired the growth of BFU-E and CFU-GM from bone marrow
cells in vitro. Concerning PF-4 specificity, it has been previously demonstrated by Gewirtz et al11 and our group12,13 and
confirmed by the present work that a higher concentration is
required to inhibit the development of erythropoietic or macrophage-granulopoietic colonies, compared with its effect on the
megakaryocyte lineage. These results suggest that p34-58, in
addition to its increased inhibitory activity, may act on hematopoiesis through a mechanism that is not exactly the same as
PF4.
PF4 has high affinity for heparin and its activity to modulate
cell proliferation as well as its binding to cell surface can be
abrogated by heparin.17,18,29 C-terminal peptides including the
major heparin domain still retained the inhibitory activity of the
native molecule in hematopoietic11 and endothelial cell systems.14,15 These data suggest that PF4 and its related C-terminal
peptides act on cell proliferation by heparin-dependent manner.
To explore further mechanism of action of PF4 and its related
peptides, the present work has studied the relationship between
progenitor proliferation inhibitory activity and heparin binding
properties of PF4 as well as related peptides. Although the
inhibitory activity of PF4 on hematopoiesis is completely
heparin-dependent, p34-58, unlike PF4, lacked affinity for
heparin and its inhibitory activity on megakaryocytopoiesis
could not be abrogated by heparin. Our results indicate that the
p34-58 functions as an inhibitor of hematopoiesis in a heparinindependent manner.
The phenomenon that the progenitors of megakaryocytic
lineage are more sensitive to the action of PF4 than those of
other hematopoietic lineages is an interesting issue worthy of
further elucidation. It has been shown that heparin and several
other GAGs can significantly stimulate in vitro and in vivo
megakaryocytopoiesis.30-32 Endogenous GAGs are also implicated in the regulation of megakaryocyte growth, because
treatment of cells with heparinase and chondroitinase or by the
inhibition of proteoglycan synthesis inhibits the growth of
megakaryocytic cells.18,30 However, at the same concentration,
the GAGs failed to stimulate granulopoiesis in the presence of
GM-CSF or IL-3,30,31 suggesting that hematopoietic progenitors
of different lineage could have different sensitivity to GAGs.
Concerning its high affinity for GAGs, PF4, unlike p34-58,
which lacks affinity for heparin, may inhibit megakaryocytopoiesis more efficiently than granulopoiesis and erythropoiesis by
interaction with endogenous GAGs in culture.
All these observations have led to establishment of a
hypothetical model of action of PF4. Chemokine PF4 is able to
inhibit directly the growth of hematopoietic progenitors through
two action pathways. The first pathway is related to its
heparin-binding properties. PF4 binds to GAGs present on
cellular surface via its C-terminal cationic tail. Such a binding
will block the interaction of cells with some growth factors,
resulting in an inhibition of cell proliferation. This pathway is
similar to the interaction of FGF-2 with cell surface heparin-like
molecules, a model pointing out the action of FGF-2 dependent
2779
of its heparin binding properties.33 PF4 contains another
important functional domain in its central region, which lacks
affinity for heparin and acts in a heparin-independent manner.
This domain is usually masked in whole PF4 molecule. This
first interaction of PF4 with heparin-like molecules on cell
surface may cause a change of conformation of this molecule,
which facilitates a subsequent contact of the central function
domain of PF4 with cells and induces ultimately inhibition of
proliferation. Artificial removal of C-terminal and N-terminal
regions from PF4 results in a full exposure of this functional
domain and thus gives rise to a potent inhibitor of hematopoiesis.
In conclusion, the present study has provided evidence for a
new functional domain in the central region of PF4, implicated
in the inhibitory properties of this chemokine. Our findings can
have important implications for the understanding of the
mechanism of action of PF4 in modulating proliferation of
hematopoietic progenitors. The future identification of the
receptor of PF4 would therefore allow determination of the
exact mechanism of action of PF4.
ADDENDUM
The full description of the computerized automatic image
analysis system used to quantify megakaryocyte colonies is
now in press.34
ACKNOWLEDGMENT
The authors thank Dr Jack Levin for his critical review of the
manuscript and Valérie Drouet for her helpful technical assistance.
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1998 91: 2772-2780
New Insights Into the Negative Regulation of Hematopoiesis by Chemokine
Platelet Factor 4 and Related Peptides
Laurence Lecomte-Raclet, Mònica Alemany, Anabelle Sequeira-Le Grand, Jean Amiral, Gérard Quentin,
Anne Marie Vissac, Jacques P. Caen and Zhong Chao Han
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