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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 50-57
CHEMOKINES
Stromal cell-derived factor-1 and macrophage-derived
chemokine: 2 chemokines that activate platelets
M. Anna Kowalska,
Mariusz Z. Ratajczak,
Marcin Majka,
Jianguo Jin,
Satya Kunapuli,
Lawrence Brass, and
Mortimer Poncz
From the Department of Pediatrics, Children's Hospital of
Philadelphia; the Departments of Medicine and Pediatrics, University of
Pennsylvania School of Medicine; and the Department of Physiology,
Temple University School of Medicine, Philadelphia, PA.
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Abstract |
Platelets play roles in both thrombosis and inflammation,
and chemokines that are released at sites of inflammation could potentially activate platelets. Among the chemokine receptors expressed
on platelets, the CXCR4 is the receptor for chemokine stromal
cell-derived factor-1 (SDF-1), and the CCR4 is the receptor for
macrophage-derived chemokine (MDC). Of the chemokines tested, SDF-1 and
MDC were the only 2 that activated platelets. Both are weak agonists,
but they enhanced response to low-dose adenosine 5'-diphosphate
(ADP), epinephrine, or serotonin. When SDF-1 and MDC were added
together, full and brisk platelet aggregation occurred. Platelet
activation by these 2 chemokines appears to involve distinct pathways:
SDF-1 inhibited an increase in cyclic adenosine monophosphate (cAMP)
following prostaglandin (PG) I2, while MDC had no effect. In contrast, MDC, but not SDF-1, lead to Ca++
mobilization by platelets. Further, second-wave aggregation induced by
MDC in platelet-rich plasma was inhibited by aspirin, ADP scavenger creatine phosphate/creative phosphokinase (CP/CPK), and
ARL-66096, an antagonist of the ADP P2TAC receptor involved
in adenylyl cyclase inhibition. But the aggregation was not affected by
A3P5PS, an inhibitor of the ADP P2Y receptor. SDF-1-induced
aggregation was inhibited by aspirin, but it was only slightly affected
by CP/CPK, ARL-66096, or A3P5PS. Finally, the presence of chemokines in
platelets was determined. Reverse transcriptase-polymerase chain
reaction studies with platelet RNA did not detect the presence of SDF-1 or MDC. In summary, SDF-1 and MDC are platelet agonists that activate distinct intracellular pathways. Their importance in the development of
thrombosis at sites of inflammation needs to be further evaluated.
(Blood. 2000;96:50-57)
© 2000 by The American Society of Hematology.
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Introduction |
Chemokines are small cytokines that are important as
intermediates in the inflammatory response.1,2
The chemokines fall into 2 major families: the CXC (or  -chemokines)
and the CC (or  -chemokines). The designation of CC indicates that
the first 2 of 4 conserved cysteine residues fall immediately adjacent
to each other; in CXC, there is an intervening additional amino acid residue between the first 2 cysteine residues. Membrane receptors for
these chemokines are members of the G protein-coupled family of
receptors and have been designated CCR and CXCR, depending on the
family of chemokines to which they bind. Some receptors bind to
multiple related chemokines, while others, like CXCR4, bind to a single
ligand, in this case SDF-1.3-5
In addition to their importance in inflammation, chemokines have other
biological roles. In 1989 it was shown that platelet factor 4 (PF4)
inhibits megakaryocyte formation.6 Since then it has been
shown that in addition to PF4, other chemokines, specifically interleukin-8 (IL-8), macrophage inflammatory protein-1 (MIP-1), and MIP-1, also inhibit megakaryopoiesis.7 It appears
that maturing megakaryocytes express CXCR1 and CXCR2, the known IL-8 receptors.7 Further studies have shown the presence of
other chemokine receptors on the surface of hematopoietic
cells.8 Of great interest was the
demonstration by our group10 and others9,11 that CXCR4, a coreceptor for T-tropic human immunodeficiency virus-1 (HIV-1) infection, was present on CD34+ cells, early
megakaryocyte progenitors, megakaryocytes, and platelets. The presence
of CXCR4 on marrow progenitor cells was not unexpected, as
CXCR4 / knockout mice do not properly populate
their adult marrow space.12-14 This suggests that the SDF-1
ligand/receptor axis is central to hematopoietic progenitor cell
homing. Indeed, studies with megakaryocyte progenitor cells
demonstrated that SDF-1 can induce chemotaxis of these cells via
CXCR4.9-11
The role of SDF-1 on mature megakaryocytes and platelets is unclear. In
our hands, mature megakaryocytes did not show a similar chemotactic
response as megakaryocyte progenitors.10 SDF-1 did not
inhibit or stimulate megakaryopoiesis, and we could not demonstrate a
Ca++ flux in cells stimulated with SDF-1. Others have found
similar results.11 However, Wang et al9 have
reported a modest but consistent stimulation of megakaryocyte colony
formation by SDF-1 and suggested that SDF-1, along with
thrombopoietin, is involved in the normal regulation of
megakaryopoiesis. Also data showing that SDF-1 increases the growth of
murine megakaryocytic colonies in the presence of thrombopoietin
have been published recently.15 In addition, the
ability of SDF-1 to attract maturing megakaryocytes may be
important for platelet formation.16
The presence of CXCR4 on platelets and the ability of SDF-1 to activate
platelets were also studied.10 There were approximately 2000 copies of CXCR4 per platelet, with an SDF-1 KD of 24 nmol/L. Similar values have been reported on other cells
lines. Using washed platelets, platelet activation was not detected,
possibly because the CXCR4 receptor was residually present on the
surface of the platelet, and the lack of platelet activation was due to an intracellular signaling block in circulating platelets. However, with platelets in platelet-rich plasma (PRP), SDF-1 acted as a weak
platelet agonist.
Other chemokines were tested to see if they could activate washed
platelets or platelets in PRP. These include: the CXC chemokines IL-8,
PF4, neutrophil-activating peptide-2 (NAP-2), and epithelial cell-derived neutrophil attractant-78 (ENA-78); the CC chemokines human MDC, RANTES (regulated on activation normal T expressed and
secreted), MIP-1, monocyte chemoattractant protein-3 (MCP-3), and
thymus- and activation-related chemokine (TARC); and the
CX3C chemokine fractalkine. Among these chemokines, only
MDC stimulated Ca++ flux in washed platelets and induced
aggregation in both washed platelets and platelets in PRP. The platelet
effects of these chemokines were further characterized by their
interactions with other weak agonists and by the intracellular pathways
involved in their activation. The study examined the possibility that
these 2 chemokines may be present in platelets. The potential
biological consequences of the chemokines' ability to activate
platelets is discussed.
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Materials and methods |
Materials
Chemokines SDF-1 and MDC were purchased from 2 sources
(PeproTech Inc, Rocky Hill, NJ, and R&D Systems, Minneapolis, MN), and
there was no difference in chemokine potency between these 2 sources.
Other chemokines were also used (R&D Systems), and PF4 and NAP-2 were
synthesized in our laboratory as previously described.17
All other chemokines were purchased from R&D Systems. Rabbit anti-human
MDC IgG was purchased from PeproTech Inc. Adensosine diphosphate (ADP)
was purchased from Sigma Diagnostics (St. Louis, MO) and thrombin
receptor PAR1 activating peptide SFLLRN (TRAP) was from Bachem
(Torrance, CA). Carbacycline, a stable analog of prostaglandin
PGI2, was purchased from BIOMOL Research Laboratories, Inc.
(Plymouth Meeting, PA). All common reagents including acetylsalicylic acid (aspirin), apyrase (Grade VII), theophylline and inhibitor of
P2Y1PLC ADP receptor A3P5PS were from Sigma Co. The
inhibitor of P2YAC receptor, ARL 66096, was a gift from
Fisons (now Astra Zeneca, Loughborough, UK). All molecular biology
enzymes and tissue culture medium for growing megakaryocytes were from
GibcoBRL (Gaithersburg, MD). Delipidated, deionized, and
charcoal-treated BSA, iron-saturated transferrin, insulin, and 2 L-glutamine for megakaryocyte growth were from Sigma Co. Recombinant
human (rH) thrombopoietin (TPO) and rH interleukin-3 (IL-3) were
obtained from R&D Systems, Minneapolis, MN.
Isolation of human blood platelets
The platelets were isolated by differential centrifugation of whole
blood. Blood, obtained by venipuncture from healthy volunteers for
studies approved by the institutional human subject's review board,
was anticoagulated with 3.8% sodium citrate. PRP used for the study of
the platelets was isolated from plasma by blood centrifugation at 150g
for 20 minutes at room temperature. To obtain washed platelets, blood
was collected on acid-citrate-dextrose (ACD) and
centrifuged as above, and PGE1 was added to PRP to a final
concentration of 1 µmol/L. The platelets were then pelleted by PRP
centrifugation at 800g for 20 minutes at room temperature. This pellet
was resuspended and washed twice in a Tyrode's buffer: 134 mmol/L
sodium chloride (NaCl), 3 mmol/L potassium chloride (KCl), 0.3 mmol/L
sodium phosphate (NaH2 PO4) 2 mmol/L magnesium
dichloride (MgCl2), 5 mmol/L N-[2-Hydroxyethyl] piperazine-N'-[2-ethane-sulfonic acid (HEPES), 5 mmol/L D-(+) glucose
containing 1 mg/mL albumin, 5 U/mL apyrase, and 1 mmol/L EGTA
(ethyleneglycotetraacetic acid). After each wash, the platelets were
sedimented by centrifugation at 900g for 15 minutes at room temperature. The final pellet was resuspended in Tyrode's buffer (pH
7.5) to a concentration of 3 × 108 platelets per
mL. Following each preparation, the platelets were tested for their
ability to aggregate with ADP in the presence of fibrinogen and 1 mmol/L calcium dichloride (CaCl2) and used within 5 hours. When indicated, the platelets were incubated with 1 mmol/L aspirin for at least 10 minutes at room temperature.
Measurements of platelet activation
Platelet activation was measured using
3-4 × 108 platelets per mL of either freshly washed
platelets or platelets in PRP. Four different measurements were
completed: (1) extent of platelet aggregation, (2) activation of
IIb/3 platelet surface expression, (3) P-selectin expression on
the platelet surface, and (4) amount of PF4 released from the platelet
-granules.
For the first measurement, the extent of platelet aggregation was
quantified using an aggregometer (Chrono-Log, Havertown, PA),
as a light transmission change in platelet suspension,
stirred at 37°C in the presence of a specific agonist. The results
were expressed as a percentage of light transmission compared with unstimulated platelets. The second measurement, activation of IIb/3, was detected using the fluorescein isothiocyanate-labeled (FITC-labeled) monoclonal antibody (mAb) PAC-118 and a
fluorescence activated cell sorter (FACS) (FACS Star Plus II; Becton
Dickinson, San Jose, CA).
The third measurement quantified surface expression on the platelets of
activation-dependent -granule membrane protein (CD62 or
P-selectin).19 The binding of phycoerythrin-conjugated
(PE-conjugated) mouse antihuman CD62P mAb (Becton Dickinson) to the
platelets was also measured using the FACS sorter. We also measured the amount of PF4 released from platelet -granules (Assera-Chrom PF4
kit; American Bioproduct Co, Parsippany, NJ). Aliquots of platelets in
PRP were stimulated for 5 minutes in an aggregometer cuvette with
various agonists followed by centrifugation at 4000 rpm for 3 minutes,
and the supernatants were measured for the amount of PF4 released from
the platelet -granules.
Measurement of thromboxane B2 formation in intact
platelets
Aliquots of washed platelets or platelets in PRP were
stimulated for 5 minutes in an aggregometer cuvette with various
agonists. This was then followed by freezing and thawing. The amount of thromboxane B2 (TxB2) formed in platelets was
measured using the BIOTRAK Thromboxane B2 Enzymeimmunoassay
System (Amersham International, Little Chalfont, England).
Measurement of cyclic AMP formation in intact platelets
Washed platelets were stimulated with 10 nmol/L carbacycline, the
stable analogue of PGI2, in the presence of 7 mmol/L
theophylline. We added 1 µmol/L ADP, 100 nmol/L SDF-1, or 100 nmol/L MDC for 5 minutes. The reaction was stopped by the addition of
an equal amount of ice-cold 10% trichloroacetic acid. The samples were centrifuged at 15 000 rpm for 5 minutes. The supernatants were extracted with 5 volumes of water-saturated ether and then lyophilized. Platelet cAMP concentrations were measured in the supernatants using
the 125I-cAMP radioimmunoassay kit (NEN; Life Science
Products, Inc, Boston, MA). These assays were completed in the presence
of 1 mmol/L aspirin to inhibit the cyclooxygenase pathway and to
abolish generation of TxA2.
Measurement of cytoplasmic Ca++ in platelets
Cytosolic-free Ca++ was determined after the platelets
were loaded with Fura-2/AM (Molecular Probes, Eugene, OR) for 30 minutes at room temperature.17 Excess Fura-2/AM was removed
by washing cells in wash buffer, and the platelets were resuspended in
resuspension buffer as described above. Fluorescence was recorded with
an Aminco-Bowman Series-2 Luminescence Spectrometer (SLM Instruments,
Inc, Urbana, IL). We stirred 1 mL aliquots of cells continuously in a
warmed holder during the period of changes in fluorescence recording. Fluorescence was monitored at 340 and 380 nm for excitation and 510 nm
for emission. The data were recorded as the relative ratio of
fluorescence excited at 340 and 380 nm, and concentration of mobilized
Ca++ was calculated using 224 nmol/L, the
known dissociation constant of the Fura-2:Ca++ complex.
Binding of 125I-MDC to platelets
We resuspended 8 × 107 platelets in 75 µL of
binding buffer (50 mmol/L HEPES (pH 7.4), 150 mmol/L sodium chloride
(NaCl), 5 mmol/L magnesium dichloride (MgCl2),
1 mmol/L calcium dichloride (CaCl2), and 5% BSA. MDC was
radiolabeled (IODO-Bead method; Pierce, Rockford, IL)
according to the manufacturer's instructions. Next, 5 nmol/L
125I-MDC (specific activity,
429.2 × 1010 Bq/mmol [116 Ci/mmol]) was added to
the cells with more cold MDC in an additional 25 µL binding buffer.
The platelets were incubated at room temperature for 1 hour, and then
unbound radioactivity was removed by filtering cells through a Whatman
GF/C filter (Whatman International, Maidstone, England) presoaked in
polyethylenimine. The filters were washed 3 times with 4 mL wash buffer
comprising 50 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 5 mmol/L
MgCl2, and 1 mmol/L CaCl2. The filters were
counted in a Beckman Gamma 5500 Counter (Beckman Instruments, Palo
Alto, CA); the KD and the number of binding sites per
platelet were calculated as described earlier.10
Reverse transcriptase-polymerase chain reaction
Reverse transcriptase-polymerase chain reaction (RT-PCR)
was carried out as previously reported using messenger RNA (mRNA) that
was isolated from cells and platelets (Quick-Pre mRNA purification Kit;
Pharmacia, Piscataway, NJ).20 The total RNA was prepared using platelets obtained from 100 mL human blood, and the entire preparation was used for a single set of experiments. For these studies, the platelet-to-white-cell ratio was greater than
104:1. In addition, RNA was extracted from whole buffy coat
cells so that total RNA was rich in white cells. CD34+
cells were expanded in a serum-free liquid system as
described.20 Briefly, CD34+
AT mononuclear cells were
resuspended in 104 cells per mL Iscove's Modified
Dulbecco's medium (IMDM) supplemented with 25% artificial serum
containing 1% delipidated, deionized, and charcoal-treated BSA; 270 mg/mL iron-saturated transferrin; 20 mg/mL insulin; and 2 mmol/L
L-glutamine. The megakaryocyte colony-forming unit (CFU-Meg) growth was
stimulated with 50 ng/mL rHTPO and 10 ng/mL rHIL-3. Cultures were
incubated at 37°C in humidified atmosphere supplemented with 5%
carbon dioxide (CO2). Under these conditions, after 14 days, approximately 85% of the expanded cells were positive for
IIb/3.20 For RT-PCR analysis, cells were further
enriched for a population of positive IIb/3 cells (purity,
greater than 97%) by additional selection with immunomagnetic
beads (Miltenyl Biotec, Auburn, CA). Stromal cells were obtained as
described earlier.20
We reverse transcribed 0.5 µg mRNA with 500 U Moloney murine
leukemia virus RT by using 50 pmol 3'-antisense
oligonucleotide of each of the respective primer pairs. The resulting
first-strand complementary DNA (cDNA) was PCR-amplified using 5 units
Thermus aquaticus (Taq) polymerase and the primer pairs
indicated: CCR4 (sense [S]), 5'-ATG AAC CCC ACG GAT
ATSA GCA GAT-3'; CCR4 (antisense [AS]), 5'-CGA ACA CAG
CCA CTG ACC ATG TAG-3'; LFA-1 2 chain (S), 5'-ATC GAC
CTG TAC TAT CTG ATG GAC-3'; LFA-1 2 chain (AS), 5'-GCG ACC TGC ATC ATG GCG TCC AGC-3'; SDF-1 (S), 5'-AAC GCC AAG
GTC GTG GTC GTG CTG-3'; SDF-1 (AS), 5'-CAC ATG TTG AAC CTC
TTG TTT AAA AGC-3'; MDC (S), 5'-GCT CGC CTA CAG ACT GCA CTC
CTG-3'; MDC (AS), 5'-GCT TAT TGA GAA TCA TCT TCA CCC
AGG-3'; MIP-1 (S), 5'-CCT TGC TGT CCT CCT CTG
CAC-3'; MIP-1 (AS), 5'-CAC TCA GCT CCA GGT CGC
TGA-3'; RANTES (S), 5'-TCA TTG CTA CTG CCC TCT
GCG-3'; and RANTES (AS), 5'-CTC ATC TCC AAA GAG TTG
ATG-3'.
At the same time, -actin mRNAs were amplified using specific primers
as reported previously.21 DNA products were
size-fractionated on an agarose gel as previously
described.21
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Results |
Activation of human washed platelets by chemokines
We had previously found that washed platelets could not be activated
by SDF-1,10 and repeat studies confirmed this finding (Figure 1A). Using human washed platelets,
platelet aggregation studies were also done with other chemokines. The
CC chemokine MDC stimulated washed platelets with a small but
reproducible response (Figure 1A). A number of other CXC chemokines
(IL-8, NAP-2, PF4, and ENA-78) and CC chemokines (MIP-1 and MCP-3) did not activate platelets, even at concentrations greater than that needed
to activate other cell lines (data not shown).
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| Fig 1.
Platelet aggregation using chemokine agonists.
(A) Platelet aggregation data using human washed platelets. Platelets
were stimulated with 100 nmol/L chemokine (as shown) or 10 µmol/L ADP
in the presence of 1 mmol/L CaCl2 and 200 µg/mL
fibrinogen. (B) Ca++-flux data using Fura-2-loaded washed
platelets involving 30 nmol/L MDC activation. This activation was
inhibited by prior incubation of the platelets with 20 U/mL apyrase, as
shown in the middle of the figure. The activation specificity is shown
by the ability of 20 µg/mL anti-MDC antibody to block MDC-induced
platelet activation, as shown on the right side of the figure.
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Using human washed platelets loaded with Fura-2, the activation of
platelets by MDC was also seen with mobilization of intracellular Ca++ (Figure 1B, left side). SDF-1 did not
induce intracellular Ca++ mobilization in washed platelets
at a concentration at which we were able to induce such changes in
neutrophils.10 In parallel studies, MDC did not induce
Ca++ mobilization in neutrophils, as also demonstrated by
others22 (data not shown). MDC-induced Ca++
mobilization was inhibited partially by apyrase, an inhibitor of
ADP23 (Figure 1B, middle), thereby demonstrating the
importance of ADP release in MDC activation of platelets. The response
to MDC was specific: 20 µg/mL antihuman MDC antibody blocked the MDC
induction of Ca++ mobilization (Figure 1B, right side).
Platelet-binding studies with MDC
We had previously shown that SDF-1 binds to the CXCR4 receptor on
the surface of platelets, with approximately 2000 sites per platelet
and a KD of 24 nmol/L.10 We now examined
whether CCR4, the chemokine receptor which was shown to bind
MDC,24 is present on platelets. The CCR4-specific antibody
(a gift from R&D Systems) does not bind to CCR4 on primary cells.
Therefore, 125I-radiolabeled MDC was used to detect MDC
binding to platelet membrane,25 and the binding
characteristics of 125I-MDC were determined using
displacement studies with increasing concentrations of unlabeled MDC
(Figure 2A). It appears that there are
approximately 4000 MDC binding sites per platelet, with a KD of 30 nmol/L. This number of sites and affinity is
similar to that reported for SDF-1 and its receptor on
platelets.10 While the 125I-MDC binding was
completely inhibited by 1.25 µmol/L unlabeled MDC, the addition of
cold SDF-1 at the same concentration did not affect MDC binding (data
not shown).
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| Fig 2.
Presence of CCR4 on megakaryocytes and platelets.
(A) Unlabeled MDC competition study of 125I-MDC binding to
nonactivated platelets. The number of binding sites (approximately 4000 sites per platelet), with a kd of 30 nmol/L, were determined as
described in "Material and methods." (B) RT-PCR data from
megakaryocytes and platelets shows the presence of a detectable CCR4
message in both. The anticipated 500-base pair (500-bp) band is seen
in both platelet and megakaryocyte RT-PCR. Platelet preparation was the
same for both Figures 2 and 3.
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Expression of CCR4 and chemokines in platelets
Using oligonucleotides specific for CCR4 receptor, we were able to
show the presence of the CCR4 message in platelets and megakaryocytes. RT-PCR of platelet and megakaryocyte total RNA demonstrated the presence of a 500-base pair (500-bp) band
corresponding to the predicted product of CCR4 cDNA amplification
(Figure 2B). There were no observed bands in the same condition when
the reverse transcription step was omitted. Contamination with white
cell RNA was ruled out by the absence of an LFA-1 2 chain message, which was negative for the same platelet RNA preparation (Figure 3).
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| Fig 3.
Studies examining whether the messages of either SDF-1 or
MDC are detectable in platelet total RNA.
Results are given for (A) RT-PCR of human platelets and (B) RT-PCR of
stromal cells. The expected size for each band is indicated.
Lane 1: An SDF-1 message (281 bp) is present in stromal
cells but not in platelets. Lane 2: An MDC message (270 bp) is also
absent in platelets. Lanes 3-5 represent the following messages: Lane
3: A RANTES message (238 bp); Lane 4: An IL-8 message (536 bp); and
Lane 5: An MIP-1 message (254 bp). Lane 6: The LFA-1 message (353 bp) is a white cell-specific message showing that the preparation is
clean of contaminating white cell messages at the level of RT-PCR
examined. Line 7: The ubiquitous message from -actin (309 bp) was
used as a general positive control for the RT-PCR studies.
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A number of chemokines are stored in platelet -granules. In
particular, PF4 and platelet basic protein (PBP), which is cleaved into
-thromboglobulin (TG) and further cleaved into NAP-2, are platelet-specific. They are made and stored at high concentrations in
the -granules of maturing megakaryocytes.26 In addition, platelet -granules contain demonstrable amounts of other chemokines such as IL-8,27 ENA-78, MCP-3, and RANTES.28 It
is possible that the release of these chemokines is one mechanism by
which platelets contribute to inflammation. We wondered whether SDF-1 and MDC could also be synthesized by megakaryocytes. RT-PCR studies with gel-filtered platelet total RNA did not detect the presence of
SDF-1 or MDC (Figure 3A), while it did detect the presence of RANTES
and MIP1- messages. The SDF-1 messages were detected in a positive
control of total RNA extracted from hematopoietic stromal cells (Figure
3B).
Characterization of platelets in PRP activated by chemokines
To examine the effect of these chemokines on platelet activation in
a more physiological environment, platelet activation in PRP was
studied. Full aggregation was achieved with 100 nmol/L MDC (Figure
4A). At concentrations of at least 10 nmol/L, MDC induced a dose-dependent biphasic activation of
platelets with full aggregation, although the minimal concentration of
MDC required for full activation was donor-dependent. The related
chemokine TARC, which binds to the same receptor as MDC,24
induced only a primary wave of platelet aggregation, even at
concentrations of 100 nmol/L (data not shown).
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| Fig 4.
Characterization of the intracellular mechanism(s) by
which SDF-1 and MDC activate platelets in PRP.
The figure demonstrates the ability of 500 µmol/L aspirin to inhibit
the secondary wave of plasma platelet aggregation stimulated by 100 nmol/L (A) MDC (B) or SDF-1. The inclusion of the ADP scavenger, 10 mmol/L creatine phosphate, plus 10 U/mL creatine phosphokinase (CP/CPK)
inhibits platelet activation stimulated by (C) 100 nmol/L MDC but not by (D) 100 nmol/L SDF-1.
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With the exception of SDF-1, none of the other
chemokines tested activated platelets in PRP (data not shown). Although
SDF-1 did not cause aggregation of washed platelets, it appears that in
platelets in PRP, 100 nmol/L SDF-1 induced a biphasic curve of
aggregation (Figure 4B). This discrepancy may be due to the fact that
full aggregation of platelets in PRP by MDC or SDF-1 may result from a
synergistic effect of TxA2, released ADP, and/or the
presence of these or other agonists (eg, epinephrine or serotonin) in
the plasma. To test this possibility, platelet stimulation by MDC was
carried out in the presence of aspirin, which inhibits TxA2
formation. Preincubation of platelets in PRP with aspirin led to
reversible platelet aggregation after MDC stimulation, similar to the
effect in washed platelets (Figure 4A). Preexposure of the platelets to
aspirin did not effect initial platelet activation by SDF-1 in PRP, and
a full second wave was not present. However, in contrast to MDC,
primary aggregation was not completely reversible, and small
aggregates with less variability in the tracing were observed
(Figure 4B). Thus, it appears that chemokine secondary aggregation of
platelets in PRP is dependent upon TxA2
formation.29
We tested whether ADP release is important for platelet
activation by either MDC or SDF-1. The presence of the ADP scavenger, creatinine phosphate 10 mmol/L plus 10 U/mL creatinine phosphokinase (CP/CPK), markedly inhibited MDC activation (Figure 4C), but only modestly decreased SDF-1 stimulation (Figure 4D) in PRP. This suggests
that the presence of released ADP is an important part of the response
to MDC stimulation only. When both TxA2 and ADP were
eliminated by the simultaneous addition of aspirin and CP/CPK, the initial platelet response to the stimulation by SDF-1
and MDC was the same as the platelet response in the presence of
aspirin only (data not shown). Thus, it appears that the presence of
TxA2 and ADP in PRP may at least partially explain the
difference seen between these 2 chemokines regarding platelet
activation using washed platelets and platelets in PRP. For SDF-1, the
blockage of the ADP effect and TxA2 generation did not result in a loss of first-wave aggregation, as seen with the washed platelets in Figure
1A. It may be that either TxA2 was already present or that some other
agonist in the plasma, such as serotonin, was responsible for the
enhanced response of platelets in PRP following SDF-1 stimulation in
the presence of both CP/CPK and aspirin.
Additional measures of platelet activation
We also measured other parameters of platelet activation in both
washed platelets and platelets in PRP. Neither SDF-1 nor MDC could
stimulate TxB2 formation in washed platelets, further supporting the fact that these chemokines are weak agonists (data not
shown). When PRP was stimulated under aggregating conditions by various
agonists, including 100 nmol/L SDF-1 and MDC, there was formation of
TxB2, and PF4 was released from -granules (Figure 5A). However, platelet
stimulation by the 2 chemokines in nonaggregating conditions (unstirred
platelets in PRP) did not lead to activation, as indicated by
measurements of either surface expression of P-selectin or activation
of the IIb/3 receptor by the active-complex dependent mAb
PAC-117 (Figure 5B). This contrasts with the results from the stronger agonist, the TRAP peptide, which activates the PAR-1 thrombin receptor on human platelets.30 These findings
further demonstrate that these chemokines are weak platelet agonists. In addition, the secondary aggregation and release reaction observed in
PRP require close platelet-to-platelet contact, which takes place as
aggregates form in a rapidly stirred system and may be due in part to
the synergistic effects of TxA2 and released
ADP.31
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| Fig 5.
Characterization of platelet activation by SDF-1, MDC,
ADP, and TRAP.
(A) TxB2 formation (gray bars) and PF4 release from
platelets (hatched bars) after plasma platelet stimulation with
agonists under aggregating conditions. (B) Surface P-selectin
expression (white bars) and II/3 receptor activation (black bars)
after plasma platelet stimulation with various agonists under
nonaggregating conditions. Each chemokine shown was
tested at 100 nmol/L; ADP was tested at 10 µmol/L, and TRAP was
tested at 25 µmol/L. Data represents the mean ± SEM for 3 experiments completed in duplicate.
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Effect of chemokines on the inhibition of adenylyl
cyclase in platelets
Elevated concentrations of cAMP can inhibit platelet response to
stimulation with various agonists. Agents, such as PGI2, stimulate adenylyl cyclase and increase platelet cAMP
levels.32 A major intracellular effect of ADP on platelets
is the inhibition of the cAMP levels, which leads to platelet
aggregation.33 This is achieved by activation of
the G i protein, which leads to the
inhibition of adenylyl cyclase.34 We investigated the ability of SDF-1 and MDC to inhibit the PG-stimulated adenylyl cyclase
activity in washed platelets (Figure 6).
Platelet cAMP concentrations measured in the presence of theophylline
rose after stimulation with 10 nmol/L carbacycline, the stable analog
of PGI2, and this increase was inhibited by 1 µmol/L ADP.
Like ADP, 100 nmol/L SDF-1 inhibited cAMP levels, but 100 nmol/L MDC
had little effect on this assay. Other chemokines tested, including RANTES, MCP-3, ENA-78, PF4, and Fractalkine2 (all at a
concentration of 100 nmol/L), had no significant effect on the cAMP
inhibition in platelets (data not shown). Thus, comparing chemokines
tested, SDF-1 appears to be unique in strongly inhibiting adenylyl
cyclase activity in platelets. Most of the chemokine receptors are
coupled through pertussis toxin-sensitive Gi proteins,
although coupling can also occur through the Gq
proteins.1 Our results suggest that, in
platelets, the SDF-1 receptor CXCR4 is coupled through a
Gi protein. On the other hand, the MDC receptor CCR4 may
be coupled to a different G protein, which does not signal by
inhibiting adenylyl cyclase.
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| Fig 6.
Effect of SDF-1 and MDC on PGI2-stimulated
adenylyl cyclase activity.
Washed platelets were stimulated with 10 nmol/L carbacycline, a stable
analog of PGI2, resulting in an elevated level of
cAMP in the treated platelets (represented as 100%). ADP (1 µmol/L) inhibited 70% of this increase. Similarly, 100 nmol/L SDF-1
inhibited 60% of the increased cAMP levels, while the same
concentration of MDC had no significant affect on this assay. Data
presented is the mean ± SEM for 5 experiments.
|
|
Chemokine enhancement of agonist-stimulated platelet
activation
Because SDF-1 and MDC appear to activate different signal
transduction pathways, we examined whether MDC can synergize with other
weak platelet agonists. Aggregation of washed platelets induced by MDC
was potentiated by epinephrine, a weak platelet agonist coupled to the
2A receptor, which mediates inhibition of adenylyl
cyclase35 (Figure 7A). MDC
could also synergize with low concentrations of ADP, although using
washed platelets, neither 100 nmol/L MDC nor 1 µmol/L ADP alone
resulted in full aggregation. However, the addition of a chemokine with
the same amount of ADP resulted in vigorous platelet aggregation (data not shown). The synergism between MDC and ADP contrasts with our previously published observation that low concentrations of ADP do not
potentiate SDF-1 aggregation.10 However, as shown in Figure
7B, SDF-1 can synergize with serotonin (5HT), a weak platelet agonist.
Serotonin activates a platelet receptor that is coupled to activation
of phospholipase C (PLC).36 Our results using SDF-1 and 5HT
are similar to the findings of Jin and Kunapuli37 (Figure
7B). They showed that weak agonists, such as low
concentrations of ADP and 5HT, can synergize in
platelet activation. This suggests that SDF-1 and 5HT activate
platelets, in part, by nonoverlapping pathways.38 In fact,
our studies suggest that SDF-1 and MDC may be such a pair of
nonoverlapping weak agonists. While 100 nmol/L of each chemokine alone
cannot activate washed platelets, the 2 together resulted in platelet
activation (Figure 7C).
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| Fig 7.
Synergistic effect of SDF-1 and MDC on washed platelet
aggregation.
(A) The synergistic effect of MDC and epinephrine (Epi)
on platelet aggregation. At the first arrow, 1 µmol/L
epinephrine was added, followed by 100 nmol/L MDC or SDF-1. (B) The
synergistic effect of 100 nmol/L SDF-1 and 5 µmol/L 5HT. (C)
Similarly, neither 100 nmol/L SDF-1 nor MDC induced platelet
aggregation, but the 2 agonists added together induced full aggregation
of washed platelets.
|
|
Effect of ADP receptor antagonists on chemokine-induced aggregation
in platelets in PRP
CP/CPK, a scavenger of ADP, inhibited the secondary wave of plasma
platelet aggregation stimulated by MDC. We therefore tested MDC-stimulated platelet aggregation in the presence of inhibitors of
the other pathways of ADP-induced signal transduction. The second wave
of MDC-induced plasma platelet aggregation was inhibited by 1 µmol/L
ARL-66096, an inhibitor of the proposed ADP P2TAC receptor
involved in adenylyl cyclase inhibition (Figure
8A).37 However, aggregation was
not affected by 100 µmol/L A3P5PS, an inhibitor of the P2Y1 ADP
receptor involved in PLC activation (Figure 8B). In contrast,
SDF-1-induced aggregation was only slightly affected by either ADP
antagonists ARL-66096 or A3P5PS (Figure 8C and D). These data are
consistent with the ability of SDF-1 to primarily activate platelets
through decreasing cAMP levels, which requires granular release to lead
to PLC activation and full platelet aggregation. MDC primarily
activates aggregation through PLC and Ca++ mobilization and
requires other agonists to inhibit adenylyl cyclase and fully aggregate
platelets. Thus, it appears that initial platelet activation by SDF-1
and MDC involves distinct pathways. We hypothesize that part of this
complementary activation of platelets (Figure 7C) is due to the fact
that SDF-1 predominantly activates platelets by inhibiting
adenylyl cyclase activity, and MDC predominantly activates platelets by
stimulating PLC activity.
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| Fig 8.
Effect of ADP antagonists ARL 66096 and A3P5PS on plasma
platelet aggregation induced by SDF-1 and MDC.
Platelets in PRP were preincubated with a
specific ADP antagonist and stimulated with 100 nmol/L MDC. (A) The
second wave of MDC-induced platelet aggregation was inhibited by 1 µmol/L ARL 66096, (B) while 100 µmol/L A3P5PS had only a slight
inhibitory effect. In contrast, aggregation induced by SDF-1 is only
slightly affected by (C) ARL 66096 or (D) A3P5PS.
|
|
| |
Discussion |
It has become clear that chemokine receptors are present on
multiple hematopoietic lineages including the megakaryocytic lineage. We and others have demonstrated that a number of chemokine receptors, including the HIV coreceptor CXCR4, are present on developing megakaryocytes.9-11 The roles these receptors play during
megakaryopoiesis and platelet formation and function are presently
being explored by a number of laboratories. Such roles can include
stimulation or inhibition of megakaryocyte formation, platelet
formation, or platelet biology. In our previous studies, we were
surprised to find that although the SDF-1 receptor CXCR4 is present on
platelets, SDF-1 did not activate washed platelets in the presence of
apyrase.10 We concluded that the CXCR4 receptor was
important at a much earlier stage of megakaryopoiesis and that CXCR4
was only residually present on platelets. The loss or change of a
cellular response to the binding of a chemokine by a particular
chemokine receptor is well documented. For example, D'Apuzzo et
al5 have shown that during B-cell differentiation,
intracellular signaling through CXCR4 by SDF-1 changes.
Ca++ mobilization by SDF-1 was observed in pro-B and pre-B
cell lines, but cell lines representing higher stages of maturation
were unresponsive.
Several chemokines were studied for their ability to activate
platelets, thereby leading to platelet aggregation. Among the chemokines tested, only SDF-1 and MDC were platelet agonists. Both were
effective in inducing aggregation of platelets in PRP. These chemokines
bind to platelets by way of their respective receptors using similar
dissociation constants and a similar number of binding sites. However,
these 2 weak agonists did not appear to activate platelets in an
identical fashion. MDC, but not SDF-1, induced intracellular
Ca++ mobilization in Fura-2-loaded washed platelets
(Figure 1B), thereby supporting an important PLC-dependent
pathway.41 SDF-1, but not MDC, inhibited
prostaglandin-induced cAMP levels in washed platelets (Figure 6).
In plasma platelets, both SDF-1 and MDC induced biphasic aggregation
(Figure 4A and 4B). However, the presence of aspirin inhibited MDC from
inducing full aggregation in platelets in PRP, and only reversible
first-wave aggregation was detected (Figure 4A). This finding suggests
a role for TxA2 formation in MDC-induced full platelet
aggregation. In contrast, while TxA2 formation seems to be
important for full secondary wave aggregation induced by SDF-1 in
platelets in PRP, stable small aggregates were still observed in the
presence of aspirin (Figure 4B). Further, ADP release appears to be
important for MDC activation of platelets in PRP, much more so than for
SDF-1 (Figure 4 C, D). Thus, our data are consistent with the fact
that MDC primarily activates platelets through PLC and Ca++
mobilization, thereby requiring other agonists to lead to adenylyl cyclase inhibition and full platelet aggregation. SDF-1 primarily activates platelets through decreasing cAMP levels, thereby requiring granular release to lead to PLC activation and full platelet aggregation.
Weak agonists that activate platelets by different pathways can
synergize and result in vigorous platelet activation when used in a
combination.37 For example, ADP and 5HT or epinephrine can
activate platelets synergistically. We found that SDF-1 could act
synergistically with 5HT, but not with epinephrine (Figure 5). On the
other hand, MDC could act synergistically with low concentrations of
ADP and epinephrine. Furthermore, SDF-1 and MDC could markedly
synergize each other's response using washed platelets
(Figure 7C). In washed platelets, SDF-1 alone had no effect, and MDC
was only able to achieve a weak primary wave. Together, these 2 chemokines achieved full and brisk platelet aggregation.
Significant differences in G-protein coupling have been noted among the
different chemokine receptors. Many of these receptors and other
7-transmembrane receptors are coupled only to the Gi protein, but some of them are coupled to other G proteins such as
Gq and/or G16.39,40 For
example, in platelets, the thrombin receptor PAR1, another
7-transmembrane receptor, has been shown to couple to both the
Gq protein, which activates PLC, and to the
Gi protein, which inhibits adenylyl
cyclase.34 A platelet-activation model has
been proposed to explain the complex activation of platelets by
ADP.37 In this model, intracellular Ca++
mobilization and inositol triphosphate (IP3) formation is modulated by
the 7-transmembrane purogenic P2Y1 receptor coupled through the
Gq protein to PLC, while inhibition of adenylyl cyclase is mediated through the P2YAC receptor coupled to the
Gi protein. In addition, the receptor-operated
Ca++ channel P2X1 facilitates rapid
Ca++ influx by ADP.41 Our results suggest that
the SDF-1 and MDC receptors are also coupled to distinct G proteins and
therefore trigger different signal transduction pathways. We propose
that the SDF-1 receptor CXCR4 links to the Gi protein
and inhibits adenylyl cyclase activity, while the MDC receptor CCR4 is
coupled to a different G protein, perhaps Gq, and leads
to mobilization of Ca++ from intracellular stores.
The biological importance of chemokine affects on platelets needs to be
determined. SDF-1 was originally cloned from a bone marrow stromal cell
line. It has been characterized as a pre-B-cell growth-stimulating
factor and chemotactic factor for monocytes, T lymphocytes, and
CD34+ human progenitor cells.3,42,43 Further
studies clearly showed that SDF-1 can be expressed by a large number of
tissues.44 On the other hand, MDC is a CC chemokine
synthesized by macrophages and dendritic cells.45
Therefore, both chemokines may exist at sites of vasculitis or at
atherogenic lesions. The presence of either chemokine would enhance
chemotactic attraction of inflammatory cells and contribute to the
activation of platelets at sites of injury, thereby leading to either
thrombosis and/or acceleration of injury to the vascular integrity.
Additionally, details of platelet activation at an inflammation site
may differ based on the chemokines being expressed. Thus it is possible
that different vascular injuries with different pools of
released chemokines may result in different degrees of
local activation of circulating platelets and risks of developing thrombosis.
| |
Acknowledgments |
We thank Dr Benjamin Doranz of the Department
of Pathology and Laboratory Medicine, University of Pennsylvania,
Philadelphia, PA, for the 125I-labeled MDC and R&D Systems,
Minneapolis, MN, for the CCR4-specific antibody.
| |
Footnotes |
Submitted October 21, 1999; accepted February 23, 2000.
Supported in part by grants HL61796 (M.A.K., M.R., and M.P.) and
HL40387 (L.B. and M.P.) from the National Institutes of
Health, Bethesda, MD.
Reprints: M. Anna Kowalska, Children's Hospital of
Philadelphia, 34th Street & Civic Center Blvd, ARC, Room 314I,
Philadelphia, PA 19104; e-mail: kowalska{at}emailchop.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction