Br. J. Pharmacol. (1990), 99, 303-308
IVMacmillan Press Ltd, 1990
Endothelin- inhibits platelet aggregation in vivo: a study with H lindium-labelled platelets 1Christoph Thiemermann, *Gillian R. May, *Clive P. Page & John R. Vane The William Harvey Research Institute, St Bartholomew's Hospital Medical College, Charterhouse Square, London ECIM 6BQ and *Department of Pharmacology, King's College, University of London, Manresa Road, London SW3 6LX 1 A non-invasive technique for the scintigraphic determination of "'indium-labelled platelet aggregation stimulated with submaximal doses of adenosine diphosphate (ADP, 56ugkg-' i.v.), collagen (lOOI gkg'- i.v.), platelet-activating factor (PAF, 0.1 ygkg'- i.v.) or thrombin (18 iukg'- i.v.) was used to investigate the platelet-inhibitory effects of endothelin 1 (ET-1) in anaesthetized rabbits in vivo. 2 ET-1 (1 nmol kg-' i.v.) inhibited ADP-stimulated platelet aggregation in vivo; a maximum inhibition of 78% of the control value was reached at 3 min, with 45% inhibition at 15 min, and a return to control values at 30min after injection of the peptide. 3 ET-1 (1 nmol kg- i.v.) inhibited in vivo platelet aggregation in response to collagen or PAF by 86% and 52%, respectively, but had no effect on thrombin-induced platelet aggregation. 4 Indomethacin (5 mgkg' i.v.) abolished the ET-1-induced inhibition of ADP-stimulated platelet aggregation and significantly potentiated and prolonged the pressor response brought about by ET-1. 5 In conclusion, the data demonstrate that ET-1 potently inhibits platelet aggregation in the anaesthetized rabbit in vivo by releasing a hypotensive and anti-aggregatory cyclo-oxygenase product, presumably prostacycin, into the circulation.
Introduction The endothelins are a family of three structurally related, but pharmacologically distinct vasoactive peptides, containing 21 amino acid residues with 2 sets of intrachain disulphide bridges (Inoue et al., 1989). Endothelin-1 (ET-1), the most widely studied member of this family, was isolated from conditioned medium of cultured vascular endothelial cells (Yanagisawa et al., 1988a) and is now well characterized as a potent constrictor of arterial (Yanagisawa & Masaki, 1989) and venous vessels (D'Orleans-Juste et al., 1989) both in vitro and in vivo. In addition, ET-1 releases vasodilator autacoids including prostacyclin (PGI2) and endothelium-derived relaxing factor (EDRF) from perfused organs and vessels of a variety of species (De Nucci et al., 1988; Warner et al., 1989) including the rabbit (Rae et al., 1989). We have previously demonstrated that ET-1 inhibits platelet aggregation in the anaesthetized rabbit when measured ex vivo in response to ADP (Thiemermann et al., 1988). However, in vitro and ex vivo tests of platelet function correlate poorly with the in vivo activation of platelets and their participation in vascular thrombosis (Packham & Mustard, 1980; Smith, 1981; Dewanjee, 1984). More suitable experimental approaches for investigating platelet activation in vivo include: (1) determination of shortened platelet survival and/or increased turnover by means of radioactive-labelled platelets (Harker, 1978) or continuous platelet counting (Smith & Freuler, 1974); (2) measurement of circulating platelet aggregate ratios (Wu & Hoak, 1974); (3) the use of filter-loop techniques (Hornstra, 1980; Bee et al., 1980) and (4) the determination of increased plasma levels of platelet factor 4, 0-thromboglobulin (Kaplan & Osen, 1981), or thromboxane B2 (Granstrom et al., 1976). In the present study a non-invasive technique for the scintigraphic determination of in vivo platelet aggregation with "'indium-labelled platelets (Page et al., 1982) was used to investigate the potential inhibitory effects of ET-1 on platelet aggregation induced by i.v. injection of adenosine 5'diphosphate (ADP), collagen, platelet activating factor (PAF) or thrombin. A further aim was to determine whether the platelet-inhibitory or the haemodynamic effects of the peptide 1
Author for correspondence.
are mediated by an increased formation of endogenous prostaglandins in vivo.
Methods Animal selection and exclusion criteria The study was carried out on male rabbits (New Zealand White SPF Rabbits, Regal, Great Bookham, U.K.) weighing 1.9 to 3.5kg; they received a standard diet and water ad libitum. All data obtained from animals which died due to the injection of an agonist of platelet aggregation were excluded from the study.
l'Indium-labelling of platelets Rabbit platelets were labelled in vitro with "'In-oxine (Thakur et al., 1976). Details of this procedure have been previously described (Page et al., 1982; Oyekan & Botting, 1986) and validated for the rabbit (May et al., 1989). Briefly, blood (15 ml) was obtained from the marginal ear vein, collected into trisodium citrate (3.8% w/v) and subsequently centrifuged for 12min at 200g to produce platelet-rich plasma (PRP). After the addition of prostaglandin El (PGEl, 300ngmlP1), the PRP was centrifuged for 15 min at 640g and the platelet pellet resuspended in 1.5 ml of buffer solution (calcium-free Tyrode solution containing 300ngml-l PGEJ). The platelets were incubated for 90s at 370C with "'In-oxine (25-50 pCi). After a further centrifugation step (640g for 15min), the supernatent which contained free "'In-oxine was removed and the platelet pellet resuspended in 1 ml of buffer.
Measurement of in vivo platelet aggregation with an automated isotope monitoring system Animals were anaesthetized with a combination of diazepam (5 mgkg-' i.p.) and Hypnorm (0.4 ml kg-' i.m. containing 0.315mgml-1 fentanyl citrate and 10mgml-1 fluanisone). "'In-labelled platelets (suspended in 1 ml buffer) were administered via a cannula in the marginal ear vein and allowed to equilibrate for I h. Circulating I"'In-labelled platelets were continuously monitored in the thoracic and hind limb regions by one inch
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crystal scintillation probes located over the thorax (to measure platelet accumulation in the pulmonary circulation) and over the hind limb (to measure peripheral thrombocytopenia). Counts were estimated with a dual channel spectrometer adjusted for the 174keV and 247keV peaks used (Nuclear Enterprises NE 461) and logged with the aid of a special application interface with a microcomputer. In vivo platelet aggregation in response to i.v. administration of various platelet agonists was measured as an increase in "'In platelet radioactivity in the pulmonary vasculature and as a concurrent fall in "'In radioactivity in the femoral region (peripheral thrombocytopenia). Maximal changes in radioactive counts over thoracic and hind limb region were calculated, corrected for blood pool "'In radioactivity (baseline value) and expressed as % increase and % decrease, respectively.
The following compounds were used: ADP, indomethacin, bovine serum albumin (BSA), Tyrode salt solution and thrombin (Sigma, Poole, Dorset); diazepam (Valium, Roche, Welwyn Welwyn Garden City); Hypnorm (Janssen Pharmaceutical Ltd., Oxford); collagen (Hormon-Chemie, Munich, F.R.G.); endothelin-1 (Peptide Research Institute, Osaka, Japan); PAF (Novabiochem, Nottingham); pentobarbitone sodium (Sagatal, May & Baker, Dagenham); "'indium-oxine (Amersham International plc). ET-1 was dissolved in phosphate-buffered saline (pH 7.4). PAF was dissolved in 0.25% BSA and collagen was suspended in isotonic glucose solution (pH 2.7). Indomethacin was dissolved in 0.5% (w/v) sodium bicarbonate. All other compounds were prepared in physiological saline.
Stimulation of platelet aggregation in vivo
Statistical comparison
Pulmonary accumulation of "IIn-labelled platelets was monitored in response to an i.v. bolus injection (total volume 0.3 ml) of ADP, collagen, PAF or thrombin into the marginal ear vein of the anaesthetized animal. All agonists used in this study induce a dose-related increase in "'In-platelet radioactivity in the pulmonary vasculature of the rabbit (May et al., 1989). Thus, submaximal doses of ADP (56 pg kg-' i.v.), collagen (100pgkg-' i.v.), PAF (0.1yg kg-' i.v.) or thrombin (18iukg-' i.v.) were used to investigate the potential antiaggregatory effects of ET-1. In animals that received several injections of ADP, a minimum time interval of 10min was left between each injection. In animals that had more than one injection of collagen or PAF a time interval of at least 1 h was left between each injection. Only one dose of thrombin was given per animal, since the responses did not necessarily return to preinjection values within 60 min (May et al., 1989).
All values in the text, figures and tables are expressed as the mean + s.e.mean of n observations. Where repeated measurements were taken over the course of the experimental period the results were analysed by a two-way analysis of variance, while end-point experiments were analysed by a one-way analysis of variance. Analysis of variance were followed by a least significance difference procedure (LSD) to determine the nature of the response (SPSS Inc., Chicago, Il., U.S.A.). A P value of < 0.05 was considered statistically significant.
Haemodynamic measurements The haemodynamic effects of ET-1 were investigated in a separate group of rabbits to minimize the number of interventions (i.e. vascular trauma associated with surgery; vascular instrumentation by catheters) resulting in endothelial cell damage, enhanced thromboxane B2 plasma levels, as well as thrombocytopenia which may therefore artificially influence platelet function in vivo (Schror & Thiemermann, 1986). Ten minutes before surgery, the animals were premedicated with Hypnorm (0.1mlkg-' i.m.). General anaesthesia was induced with sodium pentobarbitone (30mgkg-' i.v.) and maintained with supplementary doses of the anaesthetic as required. The rabbits were intubated and ventilated with air from a Harvard ventilator at a rate of 36-40 strokes per min and a tidal volume of 18-20ml. These ventilation parameters maintain physiological blood gas values (Thiemermann et al., 1989a). Body temperature was maintained at 38 + 1°C by means of a rectal probe thermometer attached to a homeothermic blanket control unit (CFP 8185). The left femoral vein was cannulated for drug administration. A catheter was placed in the left ventricle via the right common carotid artery for left ventricular pressure recordings made from a Bell & Howell 40-422-0001 pressure transducer. Haemodynamic parameters, including heart rate, left ventricular systolic (LVP) and left ventricular contractile force dp/dt,,, xwere continuously recorded on a 4-channel Grass 7D polygraph recorder (Quincy, Mass., U.S.A.). However, detailed analysis was only performed at time 0 (baseline measurement), 1, 5, 15, 30 and 60min. Heart rate and contractile force (dp/dt,,,) were automatically calculated from left ventricular systolic pulse curves by means of a Grass 7P4H tachograph and a Grass 7P20C differentiator, respectively. Four experimental groups were studied: vehicle controls (n = 3); indomethacin controls (n = 3), ET-1 (n = 6) and ET-1 after indomethacin (n = 6).
Drugs
Results
Endothelin-) inhibits platelet aggregation in vivo The accumulation of "I In-labelled platelets in the pulmonary vasculature in response to ADP (56upgkg-' i.v.; n = 6) was rapid and transient, reached a maximum within 30s and returned to preinjection levels within 5 min. Subsequent injection of ADP (56 yg kg'- i.v.; n = 4) at 15 min intervals (for 60min) produced reproducible responses indicating that a desensitization of the platelets to repetitive injections of ADP did not occur (Table 1). The time-course of the effects of ET-1 against ADP-induced platelet aggregation in vivo is deplicted in Figure 1. ET-1 (1 nmol kg-' i.v.; n = 6) inhibited the maximal increase in "In-platelet radioactivity in the thoracic region in response to ADP by approximately 80% and 45% of the control value at 3 min and 15 min after injection of the peptide, respectively (P < 0.01; Figure la). However, at 30 min after injection of the peptide, the platelet-inhibitory effect of ET-1 was no longer evident (P > 0.05).
Table 1 Reproducibility of changes in "'In-platelet radioactivity over thoracic and hind limb region in response to adenosine diphosphate (ADP) in anaesthetized rabbits 111 n-platelet radioactivity (% change) Hind limb region Thoracic region Time (% decrease in counts) (% increase in counts) (min) 0 15 30 45 60
38 + 4 40 + 5 37 + 4 36 ± 6 37 ± 5
26 ± 3 27 + 5 25 + 5 24 + 6 26 ± 5
Values are expressed as mean ± s.e.mean of 4 observations. Successive injections of ADP (56pgkg-'; i.v. at 15min intervals) produced consistent changes in "' In-platelet radioactivity in both thoracic (accumulation in the lung) and hind limb regions (peripheral thrombocytopenia).
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Time (min) Figure 1 Time course of the effects of endothelin-l (ET-1) on in vivo platelet aggregation induced by ADP (56ugkg-', i.v.) in the anaesthetized rabbit. In vivo platelet aggregation was measured as % increase in "'In-platelet radioactivity in the thoracic region (a) and % decrease in "'In-platelet radioactivity in the femoral region (b) in response to successive injections of ADP (56pgkg-', i.v. at 0, 3, 15, 30, 45 and 60min). Different groups of animals received either ET-1 (@, l nmolkg-', n = 6) or indomethacin plus ET-1 (U, 5mgkg-' i.v., n = 4). Indomethacin attenuated the anti-aggregatory effect of ET-1 at 3 and 10min after injection of the peptide. (c) Depicts the changes in left ventricular systolic pressure (LVP) in animals that received either vehicle (0, control group; n = 6), ET-1 (0, nmol kg-' i.v., n = 6) or indomethacin plus ET-l (U, Smgkg-' i.v., n = 6). ET-l was given at time 0. Data are expressed as mean of n observations; vertical lines show s.e.mean. *P < 0.05 when compared to vehicle-control.
The ADP-induced accumulation of "'In-labelled platelets in the pulmonary vasculature was associated with a significant fall in "'In-platelet radioactivity in the hind limb region (Figure lb). This peripheral thrombocytopenia in response to ADP was also inhibited by ET-1; the time-course of the ET-1mediated reduction in both ADP-induced thrombocytopenia and accumulation of "'In-platelet radioactivity in the lung showed a very similar pattern (Figure la and b). To elucidate whether the inhibitory effect of ET-1 on platelet accumulation is mediated by the release of antiaggregatory prostaglandins, a separate group of animals was treated with the cyclo-oxygenase inhibitor indomethacin (5mg kg-, i.v.; n = 6) 20min before injection of peptide. Indomethacin did not affect the responses to ADP alone (hind limb: 26+4% and 25+5%; lung: 39+5% and 39+6% before and after indomethacin, respectively), but abolished the inhibition of ADP-induced platelet aggregation brought about by ET-1 (Figure la and b). Figure 2 shows the dose-dependence of the plateletinhibitory effect of ET-1 in vivo. Lower doses of the peptide
(0.01-O.1nmolkg-') had no effect on the accumulation of "'In-labelled platelets in the lung in response to ADP (n = 4;
therefore not investigated in the present study. The collagen (100pgkg-'; n = 5)-induced platelet accumulation in vivo was transient, reached a maximum (of 43 + 6%) within 3min and returned to preinjection levels within 1025min. ET-1 (3min before injection of collagen) significantly attenuated the maximal increase in "'In-labelled platelet radioactivity in the thoracic region and the fall in "'Inplatelet radioactivity in the hind limb region (n = 6; P < 0.01; Figure 3). Furthermore, ET-1 inhibited the PAF-stimulated (0.1 pgkg-'; n = 5) accumulation of "'In-platelet radioactivity in the pulmonary vasculature and the concurrent thrombocytopenia by about 50% and 40%, respectively (n = 5; P < 0.01; Figure 3). The degree of "'In-platelet accumulation in the lung after stimulation with thrombin (18 iu kg'-; n = 5) reached a maximum of 35 + 5% within 2 min. The corresponding fall in "'In-platelet radioactivity in the hind limb region amounted to 28 + 6%, but these responses returned to preinjection levels within 30-60min. ET-1 reduced the thrombin-stimulated
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Figure 3 Endothelin-l (ET-1)-induced inhibition of platelet accumulation in the lung induced by collagen and platelet-activating factor (PAF) in the anaesthetized rabbit. In vivo platelet aggregation was measured as % increase of In-platelet radioactivity in the thoracic region. Different groups of animals received collagen (solid column; 100lugkg-' i.v.), PAF (open column; 0.1 pgkg-' i.v.); ET-1 plus collagen (hatched column) or ET-1 plus PAF (stippled column). ET-1 (1 nmol kg-' i.v.) was given 3 min before PAF or collagen. Data are expressed as mean of 5-6 observations; vertical bars show s.e.mean. *P < 0.01 when ET-1-treated animals were compared to the respective controls. "
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Table 2 Haemodynamic data in rabbits for heart rate (HR) and left ventricular contractile force (dp/dtx) in control group endothelin-1 (ET-1) group and ET-1 plus indomethacin (Indo) group Time (min)
1 1 5 15 30 60
Con
272 + 5 273+4 272+3
271±3 263 + 2 266 + 4
HR (beats min -1) ET
263 ± 6 251 + 16 234 ± 18* 237 ± 16* 238 ± 3* 255 ± 9
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dp/dt,,, (lOmmHgs-1) Indo
264 ± 5 244 + 13* 213 ± 7* 220 + 9* 235 + 9* 244 ± 7
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408 + 400 + 408 + 409 + 400 + 396 +
17 16 17 16 16 18
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Indo
396 ± 35 488 + 66* 450 ± 56 429 + 51 367 + 37 363 + 35
383 + 17 504 + 35* 500 ± 47* 458 ± 31 413 + 27 375 + 22
Values are given as mean + s.e.mean of 6 observations for each group; *P < 0.05 when compared to control at the same time points.
platelet accumulation in the pulmonary vasculature and the respective thrombocytopenia to 22 + 6% and 17 + 3%, respectively, but this effect was not significant (n = 7; P > 0.05).
Haemodynamic effects of endothelin-J Values for LVP, HR and left ventricular contractile force dp/dtmx measured during the course of the experiment are given in Figure Ic and Table 2. Baseline (time 0) haemodynamic data were similar in all animal groups studied (P > 0.05). In control animals, neither vehicle (phosphate-buffered saline; n = 3) nor indomethacin (n = 3) had any effect on any of the haemodynamic parameters measured. Thus, the results of both groups were combined and will be referred to as the control group. ET-1 caused a significant increase in LVP from 98 + 6mmHg to 124 + 7mmHg (P < 0.01; n = 6; Figure ic) which returned to normal values within 30min. Pretreatment of the animals with indomethacin both potentiated and prolonged the rise in LVP brought about by ET-1 (P < 0.05 at 5 to 60min when compared to ET-1), so that the LVP was still significantly elevated in indomethacin-ET-1 treated animals at 30min (P < 0.01 vs control) and 60 min (P < 0.05 vs control) after injection of the peptide (Figure ic). In addition, ET-1 caused a reduction in heart rate and an increase in left ventricular contractile force, which were not significantly influenced by pretreatment of the animals with indomethacin (Table 2).
Discussion In the present study, we investigated the platelet-inhibitory effects of ET-1 by measuring the accumulation of "'indiumlabelled platelets in the lung in response to various agonists of platelet aggregation. This technique is a measure of in vivo platelet aggregation (Page et al., 1982; Oyekan & Botting, 1986; May et al., 1989). Our data clearly demonstrate that ET-1 inhibits in vivo platelet aggregation in response to ADP, collagen or PAF in the anaesthetized rabbit. The weak inhibition of thrombinstimulated platelet aggregation by ET-1 was not unexpected, for thrombin-generated fibrin stabilizes the growing platelet mass resulting in deposition of irreversible platelet aggregates in the microcirculation (Niewiarowski et al., 1972). This is reflected by our finding that the thrombin-induced accumulation of "'In-labelled platelets in the pulmonary vasculature is not completely reversible and associated with a long-lasting thrombocytopenia. The platelet-inhibitory effect of ET-1 in vivo is transient, lasting for about 15-30min, and is most likely due to the release of anti-aggregatory autacoids, principally prostacyclin, into the circulation, for ET-1 does not inhibit platelet aggregation in vitro (De Nucci et al., 1988). The view that the inhibition of platelet aggregation caused by ET-1 is mediated by prostacyclin is supported by our finding here that indomethacin abolished the anti-aggregatory effect of the peptide and by our previous finding that, like exogenous prostacyclin
(Tateson et al., 1977; Gorman et al., 1977), inhibition of ex vivo platelet aggregation by ET-1 is associated with increased platelet cyclic AMP levels, and that this increase is ablated by indomethacin (Thiemermann et al., 1989b). Bult et al. (1980) have shown that lower doses of indomethacin (2.5mgkg-' i.v.) are sufficient to inhibit prostacyclin formation (measured as 6-keto-PGFg by radioimmunoassay) in the rabbit. Even when the rabbits were subjected to endotoxin (a potent stimulus of endogenous prostacyclin formation), this low dose of indomethacin abolished the rise in plasma 6-keto-PGF1. for at least 3 h. In addition to inhibiting platelet aggregation, ET-1 caused a marked increase in left ventricular systolic pressure resulting in a reflex bradycardia. The pressor effect was potentiated and prolonged by indomethacin, substantiating that a concurrent release of vasodilator prostanoids, i.e. prostacyclin, into the circulation ameliorates the direct vasopressor effects of ET-1 (De Nucci et al., 1988; Thiemermann et al., 1988; Walder et al., 1989). Another possibility is that the platelet-inhibitory effects of ET-1 are mediated by endothelium-derived relaxing factor (EDRF; Furchgott & Zawadzki, 1980), for ET-1 releases EDRF in vitro (Warner et al., 1989) and EDRF inhibits platelet aggregation (Azuma et al., 1986; Furlong et al., 1987) and adhesion (Radomski et al., 1987; Sneddon & Vane, 1988) in vitro and platelet aggregation in vivo (Bhardwaj et al., 1988). However, it is unlikely that a stimulation of EDRF release by ET-1 in vivo accounts for the potent anti-platelet effects of the peptide for it is ablated by indomethacin, while the plateletinhibitory effect of EDRF is not modified by indomethacin. Furthermore, inhibition of ex vivo platelet aggregation by ET-1 is not associated with an increase in platelet cyclic GMP levels (Thiemermann et al., 1989b). The deposition of "'In-labelled platelets in the pulmonary vasculature is positively correlated with the number of circulating platelets and pulmonary blood flow (Hanson et al., 1983). Thus, an ET-1 induced thrombocytopenia and/or a reduction in pulmonary blood flow may theoretically contribute to the observed platelet-inhibitory effect of the peptide. Interestingly, the sequestration of platelets within the lung is not due to pulmonary vasoconstriction brought about by ET-1 for indomethacin potentiated the systemic pressor effects of the peptide, whilst preventing the effects of ET-1 on accumulation of "'In-labelled platelets in the lung or the concurrent peripheral thrombocytopenia in response to ADP. What mechanisms, then, can account for the ET-1-induced stimulation of endogenous prostacyclin formation? A variety of other pressor agents including angiotensin II (Cooper & Malik, 1986), vasopressin (Cooper & Malik, 1986), noradrenaline (Stewart et al., 1984; Jeremy et al., 1985b; Cooper & Malik, 1986) and the thromboxane A2 analogue U46619 (Jeremy et al., 1985a) stimulate vascular prostacyclin synthesis (Stewart et al., 1984; Jeremy et al., 1985ab; Cooper & Malik, 1986), suggesting that prostacyclin release may occur as a defence mechanism in response to elevation of arterial blood pressure. However, in contrast to ET-1, endothelin-3 releases prostacyclin and inhibits ex vivo platelet aggregation in the anaesthetized rabbit without causing a significant pressor
ENDOTHELIN-1 INHIBITS PLATELET AGGREGATION IN VIVO
effect (Lidbury et al., 1989). Thus, it seems unlikely that the endothelin-induced release of prostacyclin in vivo occurs as a response to elevated blood pressure, but rather may be a direct receptor-mediated event. As there is very little difference between the platelet-inhibitory effects of ET-1 and ET-3 (Lidbury et al., 1989), but a marked difference between them as pressor agents (Yanagisawa et al., 1988b; Lidbury et al., 1989; Walder et al., 1989) it may well be that there is a functional difference between the putative endothelin receptors responsible for the release of prostacyclin and those mediating the constriction of vascular smooth muscle. A similar suggestion has been made about the receptors mediating vasocon-
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striction and EDRF release in response to ET-1 in perfused vascular beds (Warner et al., 1989). Thus, our work reinforces the possibility that there are subclasses of endothelin receptors, some of which release anti-aggregatory and antihypertensive autocoids whereas others cause the pressor effects. The William Harvey Research Institute is supported by a research grant from Glaxo Group Research Limited. C.T. is a research fellow of the Thyssen Stiftung, Koln, F.R.G. Part of this work was carried out while C.T. was in receipt of a research grant from the Deutsche Forschungsgemeinschaft, Bonn, F.R.G.
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WALDER, C., WARNER, T.D. & VANE, J.R. (1988). Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endotheliumderived relaxing factor. Proc. Nati. Acad. Sci. U.S.A., 85, 97979800. DEWANJEE, M.K. (1984). Cardiac and vascular imaging with labeled platelets and leukocytes. Sem. Nucl. Med., 14, 154-187. D'ORLEANS, P., FINET, M., DE NUCCI, G. & VANE, J.R. (1989). Pharmacology of endothelin-1 in isolated vessels: Effect of nicardipine, methylene blue, haemoglobin, and gossypol. J. Cardiovasc. Pharmacol., 13 (Suppl. 5), S19-S22. FURCHGOTT, R.F. & ZAWADZKI, J.V. (1980). The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature, 286, 373-376. FLURLONG, B., HENDERSON, A.H., LEWIS, M.J. & SMITH, J.A. (1987). Endothelium-derived relaxing factor inhibits in vitro platelet aggregation. Br. J. Pharmacol., 90, 687-692. GORMAN, R.R., BUNTING, S. & MILLER, O.V. (1977). Modulation of human platelet adenylate cyclase by prostacyclin (PGX). Prostaglandins, 13, 377-388. GRANSTRAM, E., KINDHAL, H. & SAMUELSSON, B. (1976). A method for measuring the unstable thromboxane A2: Radioimmunoassay of the derived mono-O-methyl-thromboxane B2. Prostaglandins, 12,929-941. HARKER, L.A. (1978). Platelet survival time: Its measurement and use. Prog. Hemost. Thromb., 4, 321-347. HANSON, S.R., KOTZE, H.F. & HARKER, L.A. (1983). "'In-platelet deposition onto collagen surface: relative importance of platelet count and blood flow rate. Circulation, 68 (Suppl. 3), 105. HORNSTRA, G. (1980). A method to determine the degree of ADPinduced platelet aggregation in circulating rat blood (Filter Loop Technique). Br. J. Pharmacol., 69, 321-329. INOUE, A., YANAGISAWA, M., KIMURA, S., KASUYA, Y., MIYAUCHI, T., GOTO, K. & MASAKI, T. (1989). The human endothelin family: Three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc. Natl. Acad. Sci. U.S.A., 86, 2863-2867. JEREMY, J.Y, MIKHAILIDIS, D.P. & DANDONA, P. (1985a). Thromboxane A2 analogue (U46619) stimulates vascular prostacyclin synthesis. Eur. J. Pharmacol., 107, 259-262. JEREMY, J.Y., MIKHAILIDIS, D.P. & DANDONA, P. (1985b). Adrenergic modulation of vascular prostacyclin synthesis. Eur. J. Pharmacol., 114, 33-40. KAPLAN, K.L. & OWEN, J. (1981). Plasma levels of P-thromboglobulin and platelet factor 4 as indices of platelet activation in vivo. Blood, 57, 199-202.
LIDBURY, P.S., THIEMERMANN, C., THOMAS, G.R. & VANE, J.R.
(1989). Endothelin-3: Selectivity as an anti-aggregatory peptide in vivo. Eur. J. Pharmacol., 166, 335-338. MAY, G.R., HERD, C., BUTLER, K.D. & PAGE, C.P. (1989). A radioisotopic method for evaluating platelet aggregation in vivo in the rabbit. Br. J. Pharmacol., 96, 277P. NIEWIAROWSKI, S., REGOECZI, E. & STEWART, G.T. (1972). Platelet interaction with polymerizing fibrin. J. Clin. Invest., 51, 685-700. OYEKAN, A.O. & BOTTING, J.H. (1986). A minimally invasive technique for the study of intravascular platelet aggregation in anaesthetized rats. J. Pharmacol. Meth., 15, 271-277. PACKHAM, M.A. & MUSTARD, J.F. (1980). Pharmacology of plateletaffecting drugs. Circulation, 62, V26-V41. PAGE, C.P., PAUL, W. & MORLEY, J. (1982). An in vivo model for studying platelet aggregation and disaggregation. Thromb. Haemostas., 47, 210-213. RADOMSKI, M.W., PALMER, R.M.J. & MONCADA, S. (1987). Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet, ii, 1057-1058. RAE, G.A., TRYBULEC, M. DE NUCCI, G. & VANE, J.R. (1989). Endothelin-1 releases eicosanoids from rabbit isolated perfused kidney and spleen. J. Cardiovasc. Pharmacol., 13 (Suppl. 5), S89S92. SCHROR, K. & THIEMERMANN, C. (1986). Treatment of acute myocardial ischaemia with a selective antagonist of thromboxane receptors (BM 13.177). Br. J. Pharmacol., 87, 631-637. SMITH, G.M. (1981). A study of intravascular platelet aggregation by continuous platelet counting. Trends Pharmacol. Sci., 2, 105-107. SMITH, G.M. & FREULER, F. (1974). The measurement of intravascular aggregation by continuous platelet counting. Bibl. Anat., 12, 257265. SNEDDON, J.M. & VANE, J.R. (1988). Endothelium-derived relaxing factor reduces platelet adhesion to bovine endothelial cells. Proc. Nati. Acad. Sci. U.S.A., 85, 2800-2804. STEWART, D., PUTNEY, E. & FITCHETT, D. (1984). Norepinephrinestimulated vascular prostacyclin synthesis. Receptor-dependent calcium channel control prostaglandin output. Can. J. Physiol. Pharmacol., 62, 1341-1347. TATESON, J.E., MONCADA, S. & VANE, J.R. (1977). Effects of prostacyclin (PGX) on cyclic AMP concentrations in human platelets. Prostaglandins, 13, 389-399. THAKUR, M.L., WELCH, MJ., JOIST, J.H. & COLEMAN, R.E. (1976). Indium-1 1-labeled platelets: Studies on preparations and evaluation of in vitro and in vivo functions. Thromb. Res, 9, 345-357. THIEMERMANN, C., LIDBURY, P.S., THOMAS, G.R. & VANE, J.R. (1988). Endothelin inhibits ex vivo platelet aggregation in the rabbit. Eur. J. Pharmacol., 158, 181-182. THIEMERMANN, C., LIDBURY, P.S., THOMAS, G.R. & VANE, J.R.
(1989a). Endothelin-1 releases prostacyclin and inhibits ex vivo platelet aggregation in the anaesthetized rabbit. J. Cardiovasc. Pharmacol., 13 (Suppl. 5), S138-S141. THIEMERMANN, C., THOMAS, G.R. & VANE, J.R. (1989b). Defibrotide reduces infarct size in a rabbit model of acute myocardial ischaemia and reperfusion. Br. J. Pharmacol., 97, 401-408. YANAGISAWA, M., KURIHARA, H., KIMURA, S., TOMOBE, Y., KOBAYASHI, M., MITSUI, Y., YAZAKI, Y., GOTO, K. & MASAKI, T. (1988a). A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature, 332, 411-415. YANAGISAWA, M., INOUE, A., ISHIKAWA, T., KASUYA, Y., KIMURA, S., KUMAGAYE, S., NAKAJIMA, K., WATANABE, T.X., SAKAKIBARA, S., GOTO, K. & MASAKI, T. (1988b). Primary structure, synthesis, and biological activity of rat endothelin, an endothelium-derived vasoconstrictor peptide. Proc. Nati. Acad. Sci. U.S.A., 85, 6964-6967.
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YANAGISAWA, M. & MASAKI, T. (1989). Endothelin, a novel endothelium-derived peptide. Biochem. Pharmacol., 38, 1877-1883. WALDER, C.E., THOMAS, G.R., THIEMERMANN, C. & VANE, J.R. (1989). The haemodynamic effects of endothelin in the pithed rat. J. Cardiovasc. Pharmacol., 13 (Suppl. 5), S93-S97. WARNER, T.D., MITCHELL, J.A., DE NUCCI, G. & VANE, J.R. (1989).
Endothelin-1 and endothelin-3 release EDRF from isolated perfused arterial vessels of the rat and the rabbit. J. Cardiovasc. Pharmacol., 13 (Suppl. 5), S85-S88. WU, K.K. & HOAK, J.C. (1974). A new method for the quantitative detection of platelet aggregates in patients with arterial insufficiency. Lancet, ii, 924-926. (Received August 7, 1989 Revised September 22, 1989 Accepted October 11, 1989)
IVMacmillan Press Ltd, 1990
Endothelin- inhibits platelet aggregation in vivo: a study with H lindium-labelled platelets 1Christoph Thiemermann, *Gillian R. May, *Clive P. Page & John R. Vane The William Harvey Research Institute, St Bartholomew's Hospital Medical College, Charterhouse Square, London ECIM 6BQ and *Department of Pharmacology, King's College, University of London, Manresa Road, London SW3 6LX 1 A non-invasive technique for the scintigraphic determination of "'indium-labelled platelet aggregation stimulated with submaximal doses of adenosine diphosphate (ADP, 56ugkg-' i.v.), collagen (lOOI gkg'- i.v.), platelet-activating factor (PAF, 0.1 ygkg'- i.v.) or thrombin (18 iukg'- i.v.) was used to investigate the platelet-inhibitory effects of endothelin 1 (ET-1) in anaesthetized rabbits in vivo. 2 ET-1 (1 nmol kg-' i.v.) inhibited ADP-stimulated platelet aggregation in vivo; a maximum inhibition of 78% of the control value was reached at 3 min, with 45% inhibition at 15 min, and a return to control values at 30min after injection of the peptide. 3 ET-1 (1 nmol kg- i.v.) inhibited in vivo platelet aggregation in response to collagen or PAF by 86% and 52%, respectively, but had no effect on thrombin-induced platelet aggregation. 4 Indomethacin (5 mgkg' i.v.) abolished the ET-1-induced inhibition of ADP-stimulated platelet aggregation and significantly potentiated and prolonged the pressor response brought about by ET-1. 5 In conclusion, the data demonstrate that ET-1 potently inhibits platelet aggregation in the anaesthetized rabbit in vivo by releasing a hypotensive and anti-aggregatory cyclo-oxygenase product, presumably prostacycin, into the circulation.
Introduction The endothelins are a family of three structurally related, but pharmacologically distinct vasoactive peptides, containing 21 amino acid residues with 2 sets of intrachain disulphide bridges (Inoue et al., 1989). Endothelin-1 (ET-1), the most widely studied member of this family, was isolated from conditioned medium of cultured vascular endothelial cells (Yanagisawa et al., 1988a) and is now well characterized as a potent constrictor of arterial (Yanagisawa & Masaki, 1989) and venous vessels (D'Orleans-Juste et al., 1989) both in vitro and in vivo. In addition, ET-1 releases vasodilator autacoids including prostacyclin (PGI2) and endothelium-derived relaxing factor (EDRF) from perfused organs and vessels of a variety of species (De Nucci et al., 1988; Warner et al., 1989) including the rabbit (Rae et al., 1989). We have previously demonstrated that ET-1 inhibits platelet aggregation in the anaesthetized rabbit when measured ex vivo in response to ADP (Thiemermann et al., 1988). However, in vitro and ex vivo tests of platelet function correlate poorly with the in vivo activation of platelets and their participation in vascular thrombosis (Packham & Mustard, 1980; Smith, 1981; Dewanjee, 1984). More suitable experimental approaches for investigating platelet activation in vivo include: (1) determination of shortened platelet survival and/or increased turnover by means of radioactive-labelled platelets (Harker, 1978) or continuous platelet counting (Smith & Freuler, 1974); (2) measurement of circulating platelet aggregate ratios (Wu & Hoak, 1974); (3) the use of filter-loop techniques (Hornstra, 1980; Bee et al., 1980) and (4) the determination of increased plasma levels of platelet factor 4, 0-thromboglobulin (Kaplan & Osen, 1981), or thromboxane B2 (Granstrom et al., 1976). In the present study a non-invasive technique for the scintigraphic determination of in vivo platelet aggregation with "'indium-labelled platelets (Page et al., 1982) was used to investigate the potential inhibitory effects of ET-1 on platelet aggregation induced by i.v. injection of adenosine 5'diphosphate (ADP), collagen, platelet activating factor (PAF) or thrombin. A further aim was to determine whether the platelet-inhibitory or the haemodynamic effects of the peptide 1
Author for correspondence.
are mediated by an increased formation of endogenous prostaglandins in vivo.
Methods Animal selection and exclusion criteria The study was carried out on male rabbits (New Zealand White SPF Rabbits, Regal, Great Bookham, U.K.) weighing 1.9 to 3.5kg; they received a standard diet and water ad libitum. All data obtained from animals which died due to the injection of an agonist of platelet aggregation were excluded from the study.
l'Indium-labelling of platelets Rabbit platelets were labelled in vitro with "'In-oxine (Thakur et al., 1976). Details of this procedure have been previously described (Page et al., 1982; Oyekan & Botting, 1986) and validated for the rabbit (May et al., 1989). Briefly, blood (15 ml) was obtained from the marginal ear vein, collected into trisodium citrate (3.8% w/v) and subsequently centrifuged for 12min at 200g to produce platelet-rich plasma (PRP). After the addition of prostaglandin El (PGEl, 300ngmlP1), the PRP was centrifuged for 15 min at 640g and the platelet pellet resuspended in 1.5 ml of buffer solution (calcium-free Tyrode solution containing 300ngml-l PGEJ). The platelets were incubated for 90s at 370C with "'In-oxine (25-50 pCi). After a further centrifugation step (640g for 15min), the supernatent which contained free "'In-oxine was removed and the platelet pellet resuspended in 1 ml of buffer.
Measurement of in vivo platelet aggregation with an automated isotope monitoring system Animals were anaesthetized with a combination of diazepam (5 mgkg-' i.p.) and Hypnorm (0.4 ml kg-' i.m. containing 0.315mgml-1 fentanyl citrate and 10mgml-1 fluanisone). "'In-labelled platelets (suspended in 1 ml buffer) were administered via a cannula in the marginal ear vein and allowed to equilibrate for I h. Circulating I"'In-labelled platelets were continuously monitored in the thoracic and hind limb regions by one inch
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crystal scintillation probes located over the thorax (to measure platelet accumulation in the pulmonary circulation) and over the hind limb (to measure peripheral thrombocytopenia). Counts were estimated with a dual channel spectrometer adjusted for the 174keV and 247keV peaks used (Nuclear Enterprises NE 461) and logged with the aid of a special application interface with a microcomputer. In vivo platelet aggregation in response to i.v. administration of various platelet agonists was measured as an increase in "'In platelet radioactivity in the pulmonary vasculature and as a concurrent fall in "'In radioactivity in the femoral region (peripheral thrombocytopenia). Maximal changes in radioactive counts over thoracic and hind limb region were calculated, corrected for blood pool "'In radioactivity (baseline value) and expressed as % increase and % decrease, respectively.
The following compounds were used: ADP, indomethacin, bovine serum albumin (BSA), Tyrode salt solution and thrombin (Sigma, Poole, Dorset); diazepam (Valium, Roche, Welwyn Welwyn Garden City); Hypnorm (Janssen Pharmaceutical Ltd., Oxford); collagen (Hormon-Chemie, Munich, F.R.G.); endothelin-1 (Peptide Research Institute, Osaka, Japan); PAF (Novabiochem, Nottingham); pentobarbitone sodium (Sagatal, May & Baker, Dagenham); "'indium-oxine (Amersham International plc). ET-1 was dissolved in phosphate-buffered saline (pH 7.4). PAF was dissolved in 0.25% BSA and collagen was suspended in isotonic glucose solution (pH 2.7). Indomethacin was dissolved in 0.5% (w/v) sodium bicarbonate. All other compounds were prepared in physiological saline.
Stimulation of platelet aggregation in vivo
Statistical comparison
Pulmonary accumulation of "IIn-labelled platelets was monitored in response to an i.v. bolus injection (total volume 0.3 ml) of ADP, collagen, PAF or thrombin into the marginal ear vein of the anaesthetized animal. All agonists used in this study induce a dose-related increase in "'In-platelet radioactivity in the pulmonary vasculature of the rabbit (May et al., 1989). Thus, submaximal doses of ADP (56 pg kg-' i.v.), collagen (100pgkg-' i.v.), PAF (0.1yg kg-' i.v.) or thrombin (18iukg-' i.v.) were used to investigate the potential antiaggregatory effects of ET-1. In animals that received several injections of ADP, a minimum time interval of 10min was left between each injection. In animals that had more than one injection of collagen or PAF a time interval of at least 1 h was left between each injection. Only one dose of thrombin was given per animal, since the responses did not necessarily return to preinjection values within 60 min (May et al., 1989).
All values in the text, figures and tables are expressed as the mean + s.e.mean of n observations. Where repeated measurements were taken over the course of the experimental period the results were analysed by a two-way analysis of variance, while end-point experiments were analysed by a one-way analysis of variance. Analysis of variance were followed by a least significance difference procedure (LSD) to determine the nature of the response (SPSS Inc., Chicago, Il., U.S.A.). A P value of < 0.05 was considered statistically significant.
Haemodynamic measurements The haemodynamic effects of ET-1 were investigated in a separate group of rabbits to minimize the number of interventions (i.e. vascular trauma associated with surgery; vascular instrumentation by catheters) resulting in endothelial cell damage, enhanced thromboxane B2 plasma levels, as well as thrombocytopenia which may therefore artificially influence platelet function in vivo (Schror & Thiemermann, 1986). Ten minutes before surgery, the animals were premedicated with Hypnorm (0.1mlkg-' i.m.). General anaesthesia was induced with sodium pentobarbitone (30mgkg-' i.v.) and maintained with supplementary doses of the anaesthetic as required. The rabbits were intubated and ventilated with air from a Harvard ventilator at a rate of 36-40 strokes per min and a tidal volume of 18-20ml. These ventilation parameters maintain physiological blood gas values (Thiemermann et al., 1989a). Body temperature was maintained at 38 + 1°C by means of a rectal probe thermometer attached to a homeothermic blanket control unit (CFP 8185). The left femoral vein was cannulated for drug administration. A catheter was placed in the left ventricle via the right common carotid artery for left ventricular pressure recordings made from a Bell & Howell 40-422-0001 pressure transducer. Haemodynamic parameters, including heart rate, left ventricular systolic (LVP) and left ventricular contractile force dp/dt,,, xwere continuously recorded on a 4-channel Grass 7D polygraph recorder (Quincy, Mass., U.S.A.). However, detailed analysis was only performed at time 0 (baseline measurement), 1, 5, 15, 30 and 60min. Heart rate and contractile force (dp/dt,,,) were automatically calculated from left ventricular systolic pulse curves by means of a Grass 7P4H tachograph and a Grass 7P20C differentiator, respectively. Four experimental groups were studied: vehicle controls (n = 3); indomethacin controls (n = 3), ET-1 (n = 6) and ET-1 after indomethacin (n = 6).
Drugs
Results
Endothelin-) inhibits platelet aggregation in vivo The accumulation of "I In-labelled platelets in the pulmonary vasculature in response to ADP (56upgkg-' i.v.; n = 6) was rapid and transient, reached a maximum within 30s and returned to preinjection levels within 5 min. Subsequent injection of ADP (56 yg kg'- i.v.; n = 4) at 15 min intervals (for 60min) produced reproducible responses indicating that a desensitization of the platelets to repetitive injections of ADP did not occur (Table 1). The time-course of the effects of ET-1 against ADP-induced platelet aggregation in vivo is deplicted in Figure 1. ET-1 (1 nmol kg-' i.v.; n = 6) inhibited the maximal increase in "In-platelet radioactivity in the thoracic region in response to ADP by approximately 80% and 45% of the control value at 3 min and 15 min after injection of the peptide, respectively (P < 0.01; Figure la). However, at 30 min after injection of the peptide, the platelet-inhibitory effect of ET-1 was no longer evident (P > 0.05).
Table 1 Reproducibility of changes in "'In-platelet radioactivity over thoracic and hind limb region in response to adenosine diphosphate (ADP) in anaesthetized rabbits 111 n-platelet radioactivity (% change) Hind limb region Thoracic region Time (% decrease in counts) (% increase in counts) (min) 0 15 30 45 60
38 + 4 40 + 5 37 + 4 36 ± 6 37 ± 5
26 ± 3 27 + 5 25 + 5 24 + 6 26 ± 5
Values are expressed as mean ± s.e.mean of 4 observations. Successive injections of ADP (56pgkg-'; i.v. at 15min intervals) produced consistent changes in "' In-platelet radioactivity in both thoracic (accumulation in the lung) and hind limb regions (peripheral thrombocytopenia).
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Figure 2 Dose-dependent inhibition of platelet accumulation in the lung by endothelin-I (ET-1). In vivo platelet aggregation was measured as % increase in ..In-platelet radioactivity in the thoracic region in response to ADP (56pgkg-' i.v.). ET-1 was administered i.v. 3min before ADP; the time interval between successive injections of ET-1 was at least 60min. Concentrations of ET-1 below 0.1 nmol kg'- did not influence platelet aggregation, whereas concentrations of 0.3 nmol kg'-I and nmol kg'- of the peptide induced a significant inhibition. Data are expressed as mean of 4 observations; vertical lines show s.e.mean. UP < 0.05 when compared to control.
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Time (min) Figure 1 Time course of the effects of endothelin-l (ET-1) on in vivo platelet aggregation induced by ADP (56ugkg-', i.v.) in the anaesthetized rabbit. In vivo platelet aggregation was measured as % increase in "'In-platelet radioactivity in the thoracic region (a) and % decrease in "'In-platelet radioactivity in the femoral region (b) in response to successive injections of ADP (56pgkg-', i.v. at 0, 3, 15, 30, 45 and 60min). Different groups of animals received either ET-1 (@, l nmolkg-', n = 6) or indomethacin plus ET-1 (U, 5mgkg-' i.v., n = 4). Indomethacin attenuated the anti-aggregatory effect of ET-1 at 3 and 10min after injection of the peptide. (c) Depicts the changes in left ventricular systolic pressure (LVP) in animals that received either vehicle (0, control group; n = 6), ET-1 (0, nmol kg-' i.v., n = 6) or indomethacin plus ET-l (U, Smgkg-' i.v., n = 6). ET-l was given at time 0. Data are expressed as mean of n observations; vertical lines show s.e.mean. *P < 0.05 when compared to vehicle-control.
The ADP-induced accumulation of "'In-labelled platelets in the pulmonary vasculature was associated with a significant fall in "'In-platelet radioactivity in the hind limb region (Figure lb). This peripheral thrombocytopenia in response to ADP was also inhibited by ET-1; the time-course of the ET-1mediated reduction in both ADP-induced thrombocytopenia and accumulation of "'In-platelet radioactivity in the lung showed a very similar pattern (Figure la and b). To elucidate whether the inhibitory effect of ET-1 on platelet accumulation is mediated by the release of antiaggregatory prostaglandins, a separate group of animals was treated with the cyclo-oxygenase inhibitor indomethacin (5mg kg-, i.v.; n = 6) 20min before injection of peptide. Indomethacin did not affect the responses to ADP alone (hind limb: 26+4% and 25+5%; lung: 39+5% and 39+6% before and after indomethacin, respectively), but abolished the inhibition of ADP-induced platelet aggregation brought about by ET-1 (Figure la and b). Figure 2 shows the dose-dependence of the plateletinhibitory effect of ET-1 in vivo. Lower doses of the peptide
(0.01-O.1nmolkg-') had no effect on the accumulation of "'In-labelled platelets in the lung in response to ADP (n = 4;
therefore not investigated in the present study. The collagen (100pgkg-'; n = 5)-induced platelet accumulation in vivo was transient, reached a maximum (of 43 + 6%) within 3min and returned to preinjection levels within 1025min. ET-1 (3min before injection of collagen) significantly attenuated the maximal increase in "'In-labelled platelet radioactivity in the thoracic region and the fall in "'Inplatelet radioactivity in the hind limb region (n = 6; P < 0.01; Figure 3). Furthermore, ET-1 inhibited the PAF-stimulated (0.1 pgkg-'; n = 5) accumulation of "'In-platelet radioactivity in the pulmonary vasculature and the concurrent thrombocytopenia by about 50% and 40%, respectively (n = 5; P < 0.01; Figure 3). The degree of "'In-platelet accumulation in the lung after stimulation with thrombin (18 iu kg'-; n = 5) reached a maximum of 35 + 5% within 2 min. The corresponding fall in "'In-platelet radioactivity in the hind limb region amounted to 28 + 6%, but these responses returned to preinjection levels within 30-60min. ET-1 reduced the thrombin-stimulated
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Figure 3 Endothelin-l (ET-1)-induced inhibition of platelet accumulation in the lung induced by collagen and platelet-activating factor (PAF) in the anaesthetized rabbit. In vivo platelet aggregation was measured as % increase of In-platelet radioactivity in the thoracic region. Different groups of animals received collagen (solid column; 100lugkg-' i.v.), PAF (open column; 0.1 pgkg-' i.v.); ET-1 plus collagen (hatched column) or ET-1 plus PAF (stippled column). ET-1 (1 nmol kg-' i.v.) was given 3 min before PAF or collagen. Data are expressed as mean of 5-6 observations; vertical bars show s.e.mean. *P < 0.01 when ET-1-treated animals were compared to the respective controls. "
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Table 2 Haemodynamic data in rabbits for heart rate (HR) and left ventricular contractile force (dp/dtx) in control group endothelin-1 (ET-1) group and ET-1 plus indomethacin (Indo) group Time (min)
1 1 5 15 30 60
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272 + 5 273+4 272+3
271±3 263 + 2 266 + 4
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263 ± 6 251 + 16 234 ± 18* 237 ± 16* 238 ± 3* 255 ± 9
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264 ± 5 244 + 13* 213 ± 7* 220 + 9* 235 + 9* 244 ± 7
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396 ± 35 488 + 66* 450 ± 56 429 + 51 367 + 37 363 + 35
383 + 17 504 + 35* 500 ± 47* 458 ± 31 413 + 27 375 + 22
Values are given as mean + s.e.mean of 6 observations for each group; *P < 0.05 when compared to control at the same time points.
platelet accumulation in the pulmonary vasculature and the respective thrombocytopenia to 22 + 6% and 17 + 3%, respectively, but this effect was not significant (n = 7; P > 0.05).
Haemodynamic effects of endothelin-J Values for LVP, HR and left ventricular contractile force dp/dtmx measured during the course of the experiment are given in Figure Ic and Table 2. Baseline (time 0) haemodynamic data were similar in all animal groups studied (P > 0.05). In control animals, neither vehicle (phosphate-buffered saline; n = 3) nor indomethacin (n = 3) had any effect on any of the haemodynamic parameters measured. Thus, the results of both groups were combined and will be referred to as the control group. ET-1 caused a significant increase in LVP from 98 + 6mmHg to 124 + 7mmHg (P < 0.01; n = 6; Figure ic) which returned to normal values within 30min. Pretreatment of the animals with indomethacin both potentiated and prolonged the rise in LVP brought about by ET-1 (P < 0.05 at 5 to 60min when compared to ET-1), so that the LVP was still significantly elevated in indomethacin-ET-1 treated animals at 30min (P < 0.01 vs control) and 60 min (P < 0.05 vs control) after injection of the peptide (Figure ic). In addition, ET-1 caused a reduction in heart rate and an increase in left ventricular contractile force, which were not significantly influenced by pretreatment of the animals with indomethacin (Table 2).
Discussion In the present study, we investigated the platelet-inhibitory effects of ET-1 by measuring the accumulation of "'indiumlabelled platelets in the lung in response to various agonists of platelet aggregation. This technique is a measure of in vivo platelet aggregation (Page et al., 1982; Oyekan & Botting, 1986; May et al., 1989). Our data clearly demonstrate that ET-1 inhibits in vivo platelet aggregation in response to ADP, collagen or PAF in the anaesthetized rabbit. The weak inhibition of thrombinstimulated platelet aggregation by ET-1 was not unexpected, for thrombin-generated fibrin stabilizes the growing platelet mass resulting in deposition of irreversible platelet aggregates in the microcirculation (Niewiarowski et al., 1972). This is reflected by our finding that the thrombin-induced accumulation of "'In-labelled platelets in the pulmonary vasculature is not completely reversible and associated with a long-lasting thrombocytopenia. The platelet-inhibitory effect of ET-1 in vivo is transient, lasting for about 15-30min, and is most likely due to the release of anti-aggregatory autacoids, principally prostacyclin, into the circulation, for ET-1 does not inhibit platelet aggregation in vitro (De Nucci et al., 1988). The view that the inhibition of platelet aggregation caused by ET-1 is mediated by prostacyclin is supported by our finding here that indomethacin abolished the anti-aggregatory effect of the peptide and by our previous finding that, like exogenous prostacyclin
(Tateson et al., 1977; Gorman et al., 1977), inhibition of ex vivo platelet aggregation by ET-1 is associated with increased platelet cyclic AMP levels, and that this increase is ablated by indomethacin (Thiemermann et al., 1989b). Bult et al. (1980) have shown that lower doses of indomethacin (2.5mgkg-' i.v.) are sufficient to inhibit prostacyclin formation (measured as 6-keto-PGFg by radioimmunoassay) in the rabbit. Even when the rabbits were subjected to endotoxin (a potent stimulus of endogenous prostacyclin formation), this low dose of indomethacin abolished the rise in plasma 6-keto-PGF1. for at least 3 h. In addition to inhibiting platelet aggregation, ET-1 caused a marked increase in left ventricular systolic pressure resulting in a reflex bradycardia. The pressor effect was potentiated and prolonged by indomethacin, substantiating that a concurrent release of vasodilator prostanoids, i.e. prostacyclin, into the circulation ameliorates the direct vasopressor effects of ET-1 (De Nucci et al., 1988; Thiemermann et al., 1988; Walder et al., 1989). Another possibility is that the platelet-inhibitory effects of ET-1 are mediated by endothelium-derived relaxing factor (EDRF; Furchgott & Zawadzki, 1980), for ET-1 releases EDRF in vitro (Warner et al., 1989) and EDRF inhibits platelet aggregation (Azuma et al., 1986; Furlong et al., 1987) and adhesion (Radomski et al., 1987; Sneddon & Vane, 1988) in vitro and platelet aggregation in vivo (Bhardwaj et al., 1988). However, it is unlikely that a stimulation of EDRF release by ET-1 in vivo accounts for the potent anti-platelet effects of the peptide for it is ablated by indomethacin, while the plateletinhibitory effect of EDRF is not modified by indomethacin. Furthermore, inhibition of ex vivo platelet aggregation by ET-1 is not associated with an increase in platelet cyclic GMP levels (Thiemermann et al., 1989b). The deposition of "'In-labelled platelets in the pulmonary vasculature is positively correlated with the number of circulating platelets and pulmonary blood flow (Hanson et al., 1983). Thus, an ET-1 induced thrombocytopenia and/or a reduction in pulmonary blood flow may theoretically contribute to the observed platelet-inhibitory effect of the peptide. Interestingly, the sequestration of platelets within the lung is not due to pulmonary vasoconstriction brought about by ET-1 for indomethacin potentiated the systemic pressor effects of the peptide, whilst preventing the effects of ET-1 on accumulation of "'In-labelled platelets in the lung or the concurrent peripheral thrombocytopenia in response to ADP. What mechanisms, then, can account for the ET-1-induced stimulation of endogenous prostacyclin formation? A variety of other pressor agents including angiotensin II (Cooper & Malik, 1986), vasopressin (Cooper & Malik, 1986), noradrenaline (Stewart et al., 1984; Jeremy et al., 1985b; Cooper & Malik, 1986) and the thromboxane A2 analogue U46619 (Jeremy et al., 1985a) stimulate vascular prostacyclin synthesis (Stewart et al., 1984; Jeremy et al., 1985ab; Cooper & Malik, 1986), suggesting that prostacyclin release may occur as a defence mechanism in response to elevation of arterial blood pressure. However, in contrast to ET-1, endothelin-3 releases prostacyclin and inhibits ex vivo platelet aggregation in the anaesthetized rabbit without causing a significant pressor
ENDOTHELIN-1 INHIBITS PLATELET AGGREGATION IN VIVO
effect (Lidbury et al., 1989). Thus, it seems unlikely that the endothelin-induced release of prostacyclin in vivo occurs as a response to elevated blood pressure, but rather may be a direct receptor-mediated event. As there is very little difference between the platelet-inhibitory effects of ET-1 and ET-3 (Lidbury et al., 1989), but a marked difference between them as pressor agents (Yanagisawa et al., 1988b; Lidbury et al., 1989; Walder et al., 1989) it may well be that there is a functional difference between the putative endothelin receptors responsible for the release of prostacyclin and those mediating the constriction of vascular smooth muscle. A similar suggestion has been made about the receptors mediating vasocon-
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striction and EDRF release in response to ET-1 in perfused vascular beds (Warner et al., 1989). Thus, our work reinforces the possibility that there are subclasses of endothelin receptors, some of which release anti-aggregatory and antihypertensive autocoids whereas others cause the pressor effects. The William Harvey Research Institute is supported by a research grant from Glaxo Group Research Limited. C.T. is a research fellow of the Thyssen Stiftung, Koln, F.R.G. Part of this work was carried out while C.T. was in receipt of a research grant from the Deutsche Forschungsgemeinschaft, Bonn, F.R.G.
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