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Circulation. 1997;96:3888-3896

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(Circulation. 1997;96:3888-3896.)
© 1997 American Heart Association, Inc.

Articles

Aggregating Human Platelets Stimulate the Expression of Thrombin Receptors in Cultured Vascular Smooth Muscle Cells via the Release of Transforming Growth Factor-ß1 and Platelet-Derived Growth FactorAB

Valérie B. Schini-Kerth, PhD; Steffen Bassus; Beate Fisslthaler, PhD; Carl M. Kirchmaier, MD; ; Rudi Busse, MD, PhD

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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 *Abstract
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Background Thrombin and the thrombin receptor have been implicated in the proliferation of vascular smooth muscle cells (VSMCs) observed after angioplasty and in atherosclerosis. Because thrombin receptor activation is an irreversible proteolytic event, the marked upregulation of the smooth muscle cell thrombin receptor after vascular injury may account for the maintained mitogenic activity of thrombin. The present study was designed to determine whether aggregating platelets stimulate thrombin receptor expression in cultured VSMCs and, if so, to identify the mediators.

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{alpha}. 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-ß1–treated VSMCs. Pretreatment of VSMCs with either acidified PDP, or TGF-ß1 increased the {alpha}-thrombin–stimulated release of 6-keto prostaglandin F1{alpha}. This effect was blunted by incubating acidified PDP with either a TGF-ß–or 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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 *Introduction
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The migration and proliferation of VSMCs and the deposit of extracellular matrix by these cells are considered to be major events in the development of restenosis after angioplasty as well as in the development of atherosclerotic lesions. Although the precise stimulus responsible for this activation of quiescent medial SMCs is not known, experimental evidence supports a role for several vasoactive factors (for review, see Reference 11 ). Balloon angioplasty is associated with an activation of the coagulation cascade, platelets, and the formation of a mural thrombus. Aggregating platelets release several growth factors such as PDGFAB, EGF, IGF, and TGF-ß1, as well as vasoactive molecules such as serotonin (5-HT), ADP, and thromboxane A2.2–8 In addition, the activation of the coagulation cascade generates SMC mitogens such as thrombin, factor XII, factor X, factor Xa, and protein S.9–11 Moreover, the arterial wall itself responds to balloon injury by generating growth factors such as PDGF, basic FGF, IGF-I, and TGF-ß.12–15 These blood- and vascular wall–derived growth factors are likely to act in concert to maintain SMCs in a proliferative and secretory state.

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 transmembrane–spanning 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.22–24 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.27–31 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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Materials
5-HT hydrochloride, human platelet factor-4, bovine fraction V albumin, TES, and HEPES were obtained from Sigma Chemical Co. Ketanserin tartrate was obtained from Research Biochemicals Inc. Recombinant human PDGFAB, TGF-ß–neutralizing antibody, and normal rabbit IgG and goat IgG were from R&D Systems; recombinant human TGF-ß1 from Boehringer Mannheim; hirudin (HBW-023) from Hoechst; PDGF-neutralizing antibody from Upstate Biotechnology Inc; monoclonal antibody against human thrombin receptor from Biodesign International; recombinant human IGF-I, basic FGF, and EGF from PreproTech EC Ltd; minimum essential medium containing Earle's salts, trypsin, and fetal bovine serum from PAN Systems; penicillin and streptomycin from Gibco BRL; and U46619 (9,11-dideoxy-11{alpha},9{alpha}-epoxymethano-prostaglandin F2{alpha}) was provided by Upjohn. All plastic ware was obtained from Greiner GmbH. Deoxycytidine 5'-[{alpha}-32P]triphosphate (3000 Ci/mmol) was obtained from Hartmann Analytic. {alpha}-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 {alpha}-thrombin (1 U/mL) for 2 minutes. Thereafter, the proteolytic activity of {alpha}-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-ß–neutralizing 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 acid–free 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 2000–bp–long 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{alpha}
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 {alpha}-thrombin. Thereafter, aliquots were removed from the incubation medium, and the amount of 6-keto prostaglandin F1{alpha} 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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 up arrowIntroduction
 up arrowMethods
 *Results
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Effect of Platelet-Derived Products on the Expression of Thrombin Receptor mRNA
In serum-deprived quiescent rat and human aortic VSMCs, a low level of thrombin receptor mRNA could be detected under basal conditions (Figs 1Down and 6Down). The steady-state level of the transcript was markedly increased after exposure of rat VSMCs to products released by aggregating washed human platelets, whereas exposure of the cells to the same volume of buffer had only minimal effects (Figs 1Down and 2Down). The stimulatory effect of platelet-derived products was transient. After a delay of 30 minutes, the steady-state level of thrombin receptor mRNA was increased by 5.1±2.8-fold at 1 hour, 4.9±1.9-fold at 2 hours, 4.3±1.4-fold at 4 hours, and 3.2±0.95-fold at 8 hours (three or four independent experiments). Thereafter, the signal returned to baseline within the next 7 hours (Fig 1Down). A 4-hour treatment period was chosen for the further characterization of the action of platelet-derived products. The stimulatory effect of platelet-derived products was concentration dependent, with a significant increase being obtained at concentrations equivalent to or greater than 2.5 µg protein/mL (Fig 2Down). Increasing the platelet-derived product concentration up to 20 µg protein/mL did not further increase the steady-state level of thrombin receptor mRNA (Fig 2Down).



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  		Figure 1. Northern blot showing the time course of thrombin receptor mRNA expression induced by platelet-derived products. Quiescent rat aortic SMCs were treated as indicated in "Methods." Hybridization was performed either with the thrombin receptor cDNA probe (top) or with the probe for 18S ribosomal RNA (center). Thrombin receptor mRNA levels and 18S ribosomal RNA levels were quantified by densitometry, and changes in thrombin receptor mRNA levels relative to 18S ribosomal levels are shown (bottom). Similar observations were obtained in three additional experiments.



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  		Figure 6. Northern blot analysis showing the effect of different exogenous platelet products on the expression of thrombin receptor mRNA in quiescent human SMCs (4-hour treatment period). Hybridization was performed either with the thrombin receptor cDNA probe (top) or with the probe for 18S ribosomal RNA (center). Thrombin receptor mRNA levels and 18S ribosomal RNA levels were quantified by densitometry, and changes in thrombin receptor mRNA levels relative to 18S ribosomal levels are shown (bottom). Similar observations were obtained in two additional experiments.



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  		Figure 2. Concentration-dependent effect of platelet-derived products on the expression of thrombin receptor mRNA. Quiescent rat aortic SMCs were exposed to platelet-derived products or buffer (same volume as for 20 µg protein/mL) for 4 hours. Results are shown as mean±SEM values from three to seven different experiments.

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 3Down), indicating that 5-HT is not a major stimulus for the expression of thrombin receptor mRNA by platelet-derived products.



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  		Figure 3. Effect of the 5-HT2 receptor antagonist ketanserin on the induction of thrombin receptor mRNA evoked by platelet-derived products (PDP). Quiescent rat aortic SMCs were incubated with ketanserin for 15 minutes before the addition of platelet-derived products (10 µg protein/mL) or the same volume of buffer (control) for 4 hours. Results are shown as mean±SEM values obtained from five different experiments.

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 {approx}180% (Fig 4Down). 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 4Down).



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  		Figure 4. Effect of transient acidification of platelet-derived products (PDP) on the induction of thrombin receptor mRNA. Quiescent rat aortic SMCs were exposed to either platelet-derived products (10 µg protein/mL) or the same volume of buffer and to acidified platelet-derived products (Ac. PDP; 10 µg protein/mL) or the same volume of acidified buffer (Ac. buffer) for 4 hours. Results are shown as mean±SEM values from six different experiments.

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 5Down). 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 6Up).



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  		Figure 5. Effect of several exogenous platelet products on the expression of thrombin receptor mRNA in quiescent rat aortic SMCs (4-hour treatment period). Results are shown as mean±SEM values. TGF-ß1, 10 ng/mL; PDGFAB, 30 ng/mL; IGF-I, 30 ng/mL; EGF, 30 ng/mL; 5-HT, 1 µmol/L; platelet factor-4 (PF4), 250 ng/mL; and U46619, 3 µmol/L.

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-ß1–induced 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 7Down). 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



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  		Figure 7. Western blot showing the effect of TGF-ß1 on the expression of thrombin receptor protein in human aortic SMCs. Quiescent SMCs were either untreated or exposed to TGF-ß1 (10 ng/mL) for 6 or 24 hours. SMC membrane proteins (50 µg) were separated on SDS-PAGE, transferred to nitrocellulose membrane, and incubated with a monoclonal antibody raised against human thrombin receptor. Similar observations were made in two additional experiments.

Release of 6-Keto Prostaglandin F1{alpha}
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{alpha} evoked by a submaximally effective concentration of {alpha}-thrombin.34,44 Exposure of rat VSMCs to {alpha}-thrombin (30 nmol/L) for 20 minutes caused a consistent and substantial release of 6-keto prostaglandin F1{alpha} into the incubation medium (Fig 8Down). 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, {alpha}-thrombin elicited a 3.0- and 6.3-fold greater release of 6-keto prostaglandin F1{alpha} 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 8Down). Similarly to acidified platelet-derived products, treatment of VSMCs with platelet-derived products also increased the {alpha}-thrombin–stimulated release of 6-keto prostaglandin F1{alpha} (data not shown). Negligible amounts of 6-keto prostaglandin F1{alpha} were released in the absence of {alpha}-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-ß1–pretreated VSMCs (from 1.2±0.3 to 5.4±5.9 ng per million cells; Fig 8Down).



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  		Figure 8. Effect of acidified (Ac.) platelet-derived products (PDP) and TGF-ß1 pretreatment on the {alpha}-thrombin–stimulated release of 6-keto prostaglandin F1{alpha}. Quiescent rat aortic SMCs were incubated in serum-free medium in (A) the presence of acidified platelet-derived products (10 µg protein/mL) or acidified buffer and (B) in the presence of TGF-ß1 (10 ng/mL) or vehicle (solvent) for 6 hours. Thereafter, the medium was replaced three times to wash out the factors. The cells were then incubated with {alpha}-thrombin or vehicle (solvent). After 20 minutes, an aliquot was taken from the incubation medium, and the amount of 6-keto prostaglandin F1{alpha} present in each sample was determined by radioimmunoassay. Results are shown as mean±SEM of (A) three and (B) four different experiments performed in quadruplicate. *Significantly greater release of 6-keto prostaglandin F1{alpha} than the respective control. #Significant effect of pretreatment with acidified-platelet products and TGF-ß1.

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-ß–neutralizing antibody or a PDGF-neutralizing antibody significantly reduced their stimulatory effect by 60% and 52%, respectively, whereas the control IgGs were inactive (Fig 9Down).



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  		Figure 9. Effect of TGF-ß (TGF-ß Ab)–and PDGF-neutralizing (PDGF Ab) antibodies on the acidified platelet-derived product (Ac. PDP) potentiation of {alpha}-thrombin–stimulated release of 6-keto prostaglandin F1{alpha} in rat aortic SMCs. Quiescent SMCs were incubated for 6 hours in serum-free medium in the presence of acidified (Ac.) buffer or transiently acidified platelet-derived products that were either untreated or had been exposed to antibodies (0.2 mg/mL) or control IgGs (0.2 mg/mL) for 60 minutes before the experiment. Thereafter, the medium was replaced three times. The cells were then incubated with {alpha}-thrombin or vehicle (solvent). After 20 minutes, an aliquot was taken from the incubation medium, and the amount of 6-keto prostaglandin F1{alpha} present in each sample was determined by radioimmunoassay. Results are shown as mean±SEM of five different experiments performed in quadruplicate. *Significant inhibitory effect of TGF-ß Ab and PDGF Ab.

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 (TableDown). As expected, transient acidification of platelet-derived product preparations increased the level of TGF-ß1 by {approx}23-fold (582.6±54.9 pg/µg protein; TableDown). 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 (TableDown).


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  		Table 1. TGF-ß1 and PDGFAB Contents in Platelet-Derived Product Preparations Before and After Acidification


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
After balloon catheter injury to arteries, platelets are immediately recruited and activated at the newly exposed subendothelium with the release of a variety of growth factors and vasoactive molecules.16,45 These platelet-derived factors, together with growth factors in plasma, are major mediators of the migratory and proliferative response of VSMCs to balloon injury as well as of the local vasospasm.46–48 Recently, we showed that serotonin increased the expression of thrombin receptors in cultured VSMCs,34 implying a role for aggregating platelets in the early enhanced expression of the thrombin receptor at sites of vascular injury. The present findings extend these previous observations by showing that activation of platelets released sufficient amounts of the various platelet-derived factors to induce thrombin receptor expression, as indicated by the increased steady-state level of thrombin receptor mRNA in cultured VSMCs. The increased thrombin receptor mRNA level may result from increased transcription of the thrombin receptor gene and/or stabilization of its mRNA. Moreover, the clear association of thrombin responsiveness of VSMCs with their steady-state level of thrombin receptor mRNA suggests that aggregating platelets most likely also increase the number of plasma membrane activatable thrombin receptors in VSMCs.

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 {approx}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 {approx}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; TableUp), this increase did not reach statistical significance. (2) Selective TGF-ß–and PDGF-neutralizing antibodies were able to prevent the stimulatory effect of acidified platelet-derived products on the {alpha}-thrombin–stimulated release of 6-keto prostaglandin F1{alpha}. (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 catheter–injured 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
 
EGF = epidermal growth factor
FGF = fibroblast growth factor
5-HT = serotonin
IGF = insulin-like growth factor
PDGF = platelet-derived growth factor
SMC = smooth muscle cell
TGF = transforming growth factor
VSMC = vascular smooth muscle cell


*    Acknowledgments
 
This study was supported by grants from the Deutsche Forschungsgemeinschaft (Schi 389/2–3) and from the Institut de Recherches Internationales Servier (Paris, France). The authors thank Dr E. Van Obberghen-Schilling (Université de Nice, France) for kindly providing the thrombin receptor cDNA and for helpful discussions and Edeltraud Thielen, Mechtild Piepenbrock Gyamfi, and Michaela Stächele for technical assistance.


*    Footnotes
 
Reprint requests to V.B. Schini-Kerth, PhD, Institut für Kardiovaskuläre Physiologie, Klinikum der JWG-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany.

Received July 7, 1997; revision received August 14, 1997; accepted August 29, 1997.


*    References
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