(Circulation. 1997;96:3888-3896.)
© 1997 American Heart Association, Inc.
Articles |
From the Institut für Kardiovaskuläre Physiologie, Klinikum der JWG-Universität (V.B.S.-K., B.F., R.B.) and Blutspendedienst Hessen (S.B., C.M.K.), Frankfurt am Main (Germany).
| Abstract |
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Methods and Results Thrombin receptor expression was assessed by
Northern and Western blot analyses and functionally by
measuring the release of 6-keto prostaglandin
F1
. Platelet-derived products (PDPs) released by
aggregating human platelets enhanced thrombin receptor mRNA levels
in a time- and concentration-dependent manner, an effect that was
potentiated by transient acidification of PDPs, which release bioactive
transforming growth factor (TGF)-ß1, and that was
slightly inhibited by ketanserin. Among several factors known to be
released by aggregating platelets, only TGF-ß1,
platelet-derived growth factorAB (PDGFAB),
and serotonin mimicked the PDP effect. The level of
membrane thrombin receptor protein was increased in
TGF-ß1treated VSMCs. Pretreatment of VSMCs with either
acidified PDP, or TGF-ß1 increased the
-thrombinstimulated release of 6-keto prostaglandin
F1
. This effect was blunted by incubating acidified PDP
with either a TGF-ßor a PDGF-neutralizing antibody.
Conclusions Aggregating human platelets stimulate the expression of thrombin receptors in VSMCs through the release of TGF-ß1, PDGFAB, and, to a lesser extent, serotonin. The upregulation of the thrombin receptor by products released by aggregating platelets may sustain the mitogenic activity of thrombin in the vascular wall at sites of injury.
Key Words: platelet-derived factors receptors thrombin muscle, smooth arteriosclerosis
| Introduction |
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Several lines of evidence also suggest that thrombin plays an important role in the development of restenosis after balloon angioplasty. First, in contrast to the platelet response, which rapidly subsides,16 thrombin activity remains elevated in balloon-injured vessels for weeks after injury.17 Second, thrombin is a potent SMC mitogen in vitro.9,18,19 This effect is mediated largely via the seven transmembranespanning thrombin receptor that requires proteolysis to produce a tethered ligand capable of activating the thrombin receptor.20,21 After activation, the thrombin receptor is rapidly subjected to homologous desensitization due to a combination of phosphorylation and proteolysis.22,23 Recovery of thrombin responsiveness is correlated with the replenishment of plasma membrane thrombin receptors by newly synthesized receptors and/or by the recruitment of a limited intracellular pool of receptors.2224 In addition to its mitogenic effect, thrombin induces VSMC migration as well as the synthesis of inflammatory cytokines, which are hallmarks in atherogenesis.9,25 Third, the pattern of PDGF ligand and PDGF receptor gene expression after balloon injury in vivo closely follows the pattern seen when cultured VSMCs are treated with thrombin but not with a variety of other growth factors and vasoactives molecules that have been implicated in restenosis.26 Fourth, thrombin inhibitors (D-Phe-Pro-ArgCH2Cl, hirudin, desulfatohirudin, and hirulog) reduced restenosis after balloon angioplasty of arteries in rabbits and also after coronary stent angioplasty in minipigs.2731 Moreover, an increased thrombin receptor expression is found in atherosclerotic and balloon-injured arteries, predominantly in areas of active SMC proliferation and areas rich in macrophages.32,33 The continuous supply of thrombin-activatable receptors to the plasma membrane of VSMCs creates the potential for thrombin to exert its proarteriosclerotic effect throughout the development of vascular lesions. Although the stimulus responsible for the enhanced smooth muscle thrombin receptor expression at sites of vascular injury remains to be determined, this upregulation shortly after the injury suggests a role for events occurring during the initial thrombotic and hemostatic response. Consistent with such an idea, 5-HT, a product released by aggregating platelets, has been shown to increase thrombin receptor expression in cultured VSMCs.34 To understand the role of platelets as potential modulators of the VSMC thrombin receptor expression, the effect of aggregating human platelets on thrombin receptor expression in cultured rat and human VSMCs was examined. The present findings demonstrate that products released by aggregating platelets stimulate vascular smooth muscle thrombin receptor expression, an effect that is mediated mainly by TGF-ß1 and PDGFAB and possibly also by 5-HT.
| Methods |
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,9
-epoxymethano-prostaglandin
F2
) was provided by Upjohn. All plastic ware
was obtained from Greiner GmbH. Deoxycytidine
5'-[
-32P]triphosphate (3000 Ci/mmol)
was obtained from Hartmann Analytic.
-Thrombin (specific clotting
activity of 3488.6 U/mg) was kindly provided by Dr Fenton II (Albany,
NY). Male Wistar rats were obtained from Charles River Wiga
Deutschland GmbH, Sulzfeld, Germany.
Platelet Preparation
One volume of platelet-rich plasma
(2.6x1010 platelets) was collected into 25%
(vol/vol) acid-citrate-dextrose buffer (citric acid 71
mmol/L, trisodium citrate 85 mmol/L, glucose
111 mmol/L, pH 4.5) and centrifuged at
180g for 10 minutes. Thereafter, the platelet-rich
plasma was collected and centrifuged at 700g for 10
minutes. Platelets were resuspended in buffer A (25 mL; NaCl
130 mmol/L,
KH2PO4 3.9
mmol/L, Na2HPO4
3.9 mmol/L,
NaH2PO4 22
mmol/L, glucose 5.5. mmol/L,
CaCl2 1 mmol/L;
pH 6.5) and centrifuged at 700g for 10 minutes.
After two washing steps with buffer A, platelets were resuspended
in 7.5 mL of buffer B [
tris(hydroxymethyl)aminomethane-HCl 0.02 mol/L (pH
7.4), NaCl 0.14 mol/L, glucose 5 mmol/L,
CaCl2 1 mmol/L; platelet
buffer].
Preparation and Treatment of Platelet-Derived Products
Preparations of platelet-derived products were generated
by incubating suspensions of washed platelets
(3.5x109 platelets/mL) at 37°C for 2
minutes. Platelet suspensions were then stimulated with
-thrombin (1 U/mL) for 2 minutes. Thereafter, the proteolytic
activity of
-thrombin was terminated by the addition of 10 U/mL
thrombin-inactivating hirudin. Platelet buffer was processed in a
manner similar to platelet suspensions. Platelet aggregates
were removed by centrifugation at 700g for 5
minutes followed by a second centrifugation at
48 000g for 30 minutes, and the platelet-derived
product and platelet buffer preparations were collected and
stored in aliquots at -70°C until being used. The protein content of
platelet-derived product preparations amounted to 260 to 370
µg protein/mL (22 different preparations). In some instances, the
platelet-derived product preparation and platelet buffer
were transiently acidified (a condition known to release bioactive
TGF-ß135) by addition of HCl
(10N) to pH 2.0 to 2.5 for 30 minutes at 22°C, followed by
neutralization to pH 7.4 with NaOH (2N). In some experiments, active
PDGFAB and TGF-ß1
present in the platelet-derived product preparation were
neutralized by incubating the aggregating platelet-conditioned
buffer with the IgG fraction (0.2 mg/mL) of either PDGF- or
TGF-ßneutralizing polyclonal antibodies for 60 minutes at 22°C,
respectively.
Cell Culture
SMCs were isolated by elastase and collagenase
digestion of thoracic aortas from male Wistar rats and from one piece
of a human aorta.36,37 Human cells were kindly
provided by Dr T. Scott-Burden, Texas Heart Institute, Houston. Rat and
human VSMCs were cultured serially in minimum essential medium
containing L-glutamine 2 mmol/, TES 5
mmol/L, HEPES 5 mmol/L (the latter two both at
pH 7.3), penicillin 100 U/mL, streptomycin 50 µg/mL, and 10%
fetal bovine serum. All experiments were performed on VSMCs between 5
and 20 passages. When VSMCs reached confluence, the culture medium was
replaced by serum-free medium containing 0.1% fatty acidfree bovine
serum albumin for 2 days before treatment.
Northern Blot Analysis
The level of thrombin receptor mRNA was assessed by Northern
blot analysis as described previously.34
Briefly, total RNA (20 to 25 µg) was size fractionated by
electrophoresis on 1% agarose gels and then transferred to nylon
membranes (Hybond-N). After prehybridization, the Northern blots were
hybridized with a 32P-labeled 2000bplong
Pst I restriction fragment from a Chinese hamster thrombin
receptor cDNA clone (kindly provided by Dr E. Van Obberghen-Schilling,
Nice, France). The RNA loading in each lane was determined by either
methylene blue staining or by use of a
32P-labeled probe for 18S ribosomal RNA.
Autoradiography was performed with Fuji RX film with
intensifying screens (DuPont de Nemours) at -70°C. The
autoradiographs were analyzed by scanning densitometry.
Thrombin receptor mRNA levels were normalized to their respective 18S
ribosomal RNA levels and expressed in arbitrary units as a fold
increase of the signal obtained with untreated cells.
Western Blot Analysis
VSMCs were lysed in bidistilled water by five cycles of
freeze/thaw. An equal volume of homogenization
buffer (Tris-HCl 100 mmol/L [pH 7.4]; KCl 2.3%
[wt/vol]; ethylenediaminetetraacetic acid 2 mmol/L;
DL-dithiothreitol 0.2 mmol/L;
phenylmethylsulfonyl fluoride 8.8 mg/L; and 2
mg/L each of leupeptin, pepstatin A, trypsin
inhibitor, antipain, chymostatin, and aprotinin) was added
to cell homogenates, and cell membranes were prepared by
centrifugation at 15 000g for 10 minutes at
4°C. The pellets were resuspended in Laemmli buffer and subjected to
8% SDS-PAGE. The separated proteins were electrophoretically
transferred to nitrocellulose membranes. Nitrocellulose blots were
incubated overnight at 4°C with a monoclonal antibody against human
thrombin receptor (dilution 1:500; Biodesign International) and then
with a secondary polyclonal goat anti-mouse antibody conjugated to
horseradish peroxidase. Thrombin receptor immunoreactivity was
visualized by exposing an x-ray film to blots incubated with the ECL
reagent.
Release of 6-Keto Prostaglandin F1
VSMCs were incubated at 37°C in fresh serum-free culture
medium in the absence and presence of either platelet-derived
products, buffer, or TGF-ß1 for 6 hours.
The conditioned medium was then removed, and the cells were washed
twice with a HEPES-Tyrode solution. After an additional 30-minute
equilibration period, VSMCs were incubated either with solvent or
-thrombin. Thereafter, aliquots were removed from the incubation
medium, and the amount of 6-keto prostaglandin
F1
present in each sample was determined
by use of a commercially available radioimmunoassay. At the end of the
experiment, the number of VSMCs per well was determined after enzymatic
dispersion with trypsin by a cell counter (Schärfe System).
Determination of TGF-ß1 and
PDGFAB
The contents of TGF-ß1 and
PDGFAB in platelet-derived product
preparations and in transiently acidified platelet-derived
product preparations were determined by use of commercially
available immunoassays (Quantikine human TGF-ß1
and human PDGFAB immunoassays, respectively). The
TGF-ß1 immunoassay detects
TGF-ß1 with no significant cross-reactivity or
interference with other growth factors such as EGF, basic FGF, PDGF,
and IGF-I, and the PDGF immunoassay detects the
PDGFAB isoform with 10% cross-reactivity with
PDGFAA and 2% cross-reactivity with
PDGFBB.
Statistical Analysis
Results are shown as mean±SEM. Statistical analyses
were performed with the use of the Student's paired t test
(two-tailed) or an ANOVA followed by Fisher's protected least
significant difference test to identify differences between two
treatments. A value of P<.05 was considered to be
statistically significant.
| Results |
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5-HT, a major nonpeptidergic substance that is stored at high
concentration in the dense granules of platelets and released
during their degranulation,6 has been shown to
stimulate thrombin receptor expression in VSMCs through the activation
of 5-HT2 receptors.34
Therefore, ketanserin (a 5-HT2 receptor
antagonist) was used in an attempt to identify the role of
5-HT in the stimulatory effect of platelet-derived products.
Preincubation of VSMCs with ketanserin (1 µmol/L) for 15
minutes affected the response to platelet-derived products (10
µg protein/mL for 4 hours) only slightly (the stimulatory effect was
reduced from a 2.9±1.1-fold to a 2.5±0.9-fold increase over basal
level; Fig 3
), indicating that 5-HT is
not a major stimulus for the expression of thrombin receptor mRNA by
platelet-derived products.
|
Thrombin-activated human platelets secrete
TGF-ß1 predominantly in a biologically latent
form in which the precursor peptide (the latency-associated peptide)
remains covalently associated with the mature 25-kD
TGF-ß1 dimer.38
Biologically active TGF-ß1 is released from the
latency-associated peptide in vitro by transient acidification or
alkalinization.35 To determine whether
platelet-derived TGF-ß1 regulates thrombin
receptor expression, platelet-derived products were transiently
acidified (pH 2 to 2.5 for 30 minutes) before their addition to the
VSMCs. Transient acidification enhanced the stimulatory effect of
platelet-derived products on the expression of thrombin
receptor mRNA by
180% (Fig 4
). This
increase, however, just failed to attain statistical significance
(P<.07). Exposure of cells to the same volume of
transiently acidified buffer only minimally affected the level of
thrombin receptor mRNA (Fig 4
).
|
Effect of Peptidergic and Nonpeptidergic Platelet-Derived
Substances on the Expression of Thrombin Receptor mRNA
To further identify putative mediators of the stimulatory effect
of platelet-derived products, the effect of several exogenous
platelet-derived molecules was investigated at concentrations that
have been shown previously to evoke maximal activation of
VSMCs.34,36,39,40 Exposure of rat VSMCs for 4
hours to either TGF-ß1 (the only isoform
present in human platelets41; 10
ng/mL), PDGFAB (the predominant isoform in
human platelets42; 30 ng/mL), or 5-HT
(1 µmol/L) increased the steady-state level of thrombin
receptor mRNA, whereas IGF-I (30 ng/mL), EGF (30 ng/mL),
platelet factor-4 (250 ng/mL), and the
thromboxane A2 mimetic U46619 (3
µmol/L) were without effect (Fig 5
). The stimulatory effect of
TGF-ß1 was the most pronounced (13.8±1.6-fold
increase), followed by PDGFAB (5.9±2.3-fold
increase) and 5-HT (4.0±1.5-fold increase). Similar to the findings
obtained with rat VSMCs, exposure of human VSMCs for 4 hours to either
TGF-ß1 (10 ng/mL),
PDGFAB (30 ng/mL), or 5-HT (1
µmol/L) also increased (but to a lesser extent) the
level of expression of thrombin receptor mRNA, whereas IGF-I (30
ng/mL) and EGF (30 ng/mL) were without significant effect
(Fig 6
).
|
Effect of TGF-ß1 on the Expression of Thrombin
Receptor Protein
Western blot analysis using a monoclonal antibody raised
against the human thrombin receptor was performed to determine whether
the TGF-ß1induced expression of thrombin
receptor in human VSMCs also occurs at the protein level. A strong
immunoreactive band of 60 kD was detected in membrane preparations from
TGF-ß1 (10 ng/mL for either 6 or 24
hours)treated VSMCs, whereas only a faint band was found with control
VSMCs (Fig 7
). An immunoreactive band of
60 kD was also detected in membrane preparations from primary cultures
of human umbilical vein endothelial cells (data not
shown); the observed size of the thrombin receptor is
consistent with previous findings.43
|
Release of 6-Keto Prostaglandin F1
The effect of platelet-derived products and of the most
potent inducer of thrombin receptor expression,
TGF-ß1, on the functional expression of the
receptor was next examined. The level of thrombin receptors was
assessed functionally by the release of 6-keto
prostaglandin F1
evoked by a
submaximally effective concentration of
-thrombin.34,44 Exposure of rat VSMCs to
-thrombin (30 nmol/L) for 20 minutes caused a
consistent and substantial release of 6-keto
prostaglandin F1
into the
incubation medium (Fig 8
). In good
agreement with the 4.3-fold and 13.8-fold increases in thrombin
receptor mRNA levels evoked by acidified platelet-derived
products and TGF-ß1,
-thrombin elicited
a 3.0- and 6.3-fold greater release of 6-keto prostaglandin
F1
in VSMCs that had been pretreated with
acidified platelet-derived products (10 µg protein/mL) and
TGF-ß1 (10 ng/mL) for 6 hours,
respectively (Fig 8
). Similarly to acidified platelet-derived
products, treatment of VSMCs with platelet-derived products
also increased the
-thrombinstimulated release of 6-keto
prostaglandin F1
(data not shown).
Negligible amounts of 6-keto prostaglandin
F1
were released in the absence of
-thrombin from VSMCs treated with acidified buffer and acidified
platelet-derived products as well as from control cells,
whereas a slight but significant release was found in
TGF-ß1pretreated VSMCs (from 1.2±0.3 to
5.4±5.9 ng per million cells; Fig 8
).
|
Selectively neutralizing antibodies were next used to identify the
active component(s) in the acidified platelet-derived product
preparations. Treatment of acidified platelet-derived product
preparations with either a TGF-ßneutralizing antibody or a
PDGF-neutralizing antibody significantly reduced their stimulatory
effect by 60% and 52%, respectively, whereas the control IgGs were
inactive (Fig 9
).
|
Determination of TGF-ß1 and PDGFAB Levels
in Platelet-Derived Product Preparations
The levels of TGF-ß1 and
PDGFAB in the platelet-derived product
preparations were determined by use of specific ELISAs.
TGF-ß1 in platelet-derived product
preparations amounted to 25.4±4.8 pg/µg protein (Table
). As
expected, transient acidification of platelet-derived product
preparations increased the level of TGF-ß1 by
23-fold (582.6±54.9 pg/µg protein; Table
). The level of
PDGFAB in platelet-derived product
preparations amounted to 342.9±19.8 pg/µg protein, whereas in
transiently acidified platelet-derived product preparations, it
was slightly reduced to 255.4±19.0 pg/µg protein (Table
).
|
| Discussion |
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|
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Although the platelet-derived product serotonin
significantly increased thrombin receptor expression, it is not a major
mediator of the stimulatory effect of platelet-derived
products, because receptor expression was reduced only slightly by
the 5-HT2 receptor antagonist
ketanserin used at a concentration that fully abolished the response to
serotonin (Reference 3434 and present findings).
Moreover, a role for thromboxane A2
can be ruled out because its analogue U46619 did not induce thrombin
receptor expression. The present study identified
TGF-ß1 as the most potent inducer of thrombin
receptor expression (more than a 10-fold increase in thrombin receptor
mRNA and a substantial increase in the level of membrane thrombin
receptor protein), followed by PDGFAB, which
elicited an
5-fold increase in thrombin receptor mRNA, whereas IGF-I
and EGF (all growth factors were tested at a concentration that has
been shown previously to evoke maximal activation of cultured
VSMCs34,36,39,40) were inactive. The hypothesis
that TGF-ß1 and PDGFAB
are the physiologically relevant mediators of
thrombin receptor expression by aggregating platelets is supported
by the following findings: (1) Transient acidification, which releases
biologically active TGF-ß1 from its
latency-associated peptide, increased by
180% the stimulatory
effect of platelet-derived products. Probably because of the
heterogeneous amount of TGF-ß1
present in the different preparations of platelet-derived
products studied (the TGF-ß1 content
amounted to between 3.6 and 7.7. ng/10 µg protein; Table
),
this increase did not reach statistical significance. (2) Selective
TGF-ßand PDGF-neutralizing antibodies were able to prevent the
stimulatory effect of acidified platelet-derived products on
the
-thrombinstimulated release of 6-keto
prostaglandin F1
. (3) The maximal
concentrations of TGF-ß1 and
PDGFAB determined in the different
platelet-derived product preparations were within the range of
those eliciting biological responses in cultured
VSMCs.39,49,50 In addition to strong
acidification and alkalinization, activation of latent TGF-ß can also
be effected in vitro by proteases such as plasmin, cathepsin D, or
trypsin, which degrade the latency-associated
peptide.51,52 The formation of significant
amounts of plasmin from its circulating precursor
plasminogen occurs in vivo during activation of the
fibrinolytic pathway. This conversion depends on the presence of
plasminogen activators that are derived from
both the blood and the injured arterial
wall.53,54 It is therefore conceivable that
plasmin might be generated within the vicinity of aggregating
platelets at sites of vascular injury and hence may provide a
mechanism for the local activation of TGF-ß1.
Altogether, the present findings in conjunction with the previous
ones indicate that aggregating platelets are potential
endogenous stimulators of vascular smooth muscle thrombin
receptor expression after balloon catheter injury of arteries mainly
via the release of TGF-ß1 and
PDGFAB. Because PDGF induced a synergistic
increase in growth response of VSMCs to TGF-ß1
and serotonin,50,55 it is likely that
these platelet-derived factors may also act synergistically to
induce vascular smooth muscle thrombin receptor expression.
Previous studies using either electron microscopy or 51Cr-labeled platelet binding have shown that the vast majority of platelet adhesion and activation to the denuded subendothelium occurs within the first 24 hours after in vivo balloon catheter angioplasty and that thereafter, the injured blood vessel regains its patency.17,45,56 Hence, aggregating platelets may provide a stimulus for the early enhanced expression of the thrombin receptor at sites of balloon catheter injury but are unlikely to explain its upregulation found throughout the development of intimal thickening.33 One possibility is that the long-term upregulation of thrombin receptor expression is controlled by endogenous regulatory molecules derived from the damaged artery wall itself and possibly also from plasma. Indeed, an increased production of TGF-ß1 and PDGF-A has been found in the balloon catheterinjured rat carotid artery during the development of intimal thickening.12,14 Because the long-term production of TGF-ß1 occurs concomitantly with that of tissue-type and urokinase-type plasminogen activators,54 it is not unlikely that circulating plasminogen is converted to plasmin within the vicinity of the secreted latent TGF-ß1, resulting in the local generation of biologically active TGF-ß1 during the formation of intimal thickening. A role for PDGFAA is further supported by the fact that this PDGF isoform, like PDGFAB, increased, albeit modestly, thrombin receptor mRNA expression in cultured VSMCs.33 Basic FGF, which has been implicated in the proliferative response to balloon catheter injury,57 is another potential endogenous stimulus because this growth factor increased thrombin receptor mRNA expression in cultured VSMCs.33,58 Although SMCs are the major source of TGF-ß1, PDGF, and basic FGF at sites of vascular injury, these factors are also produced and secreted by monocytes and lymphocytes present in the neointima and by surrounding endothelial cells.59,60 Moreover, because exposure of cultured VSMCs to thrombin increased thrombin receptor mRNA expression,61 factors derived from blood may act in concert with those released by the injured arterial wall to stimulate thrombin receptor expression.
In conclusion, the present study demonstrates that aggregating human platelets stimulate the expression of thrombin receptors in cultured VSMCs mainly through the release of TGF-ß1 and PDGFAB. These findings identify activation of platelets at sites of endothelial denudation after balloon catheterization of arteries as a crucial event for the increased smooth muscle thrombin receptor expression during the early phase of the vascular response to injury. Thereafter, the long-term upregulation of smooth muscle thrombin receptor expression during restenosis may be due to factors generated within the injured artery wall itself and derived from blood. The continuous generation of new thrombin receptors at sites of vascular injury supports the notion that thrombin exerts its proarteriosclerotic effects throughout the development of vascular lesions.
| Selected Abbreviations and Acronyms |
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| Acknowledgments |
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| Footnotes |
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Received July 7, 1997; revision received August 14, 1997; accepted August 29, 1997.
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