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Fibrinolytic activity of endothelin-3
THROMBOSIS RESEARCH 55; 797-799, 1989 0049-3848/89 $3.00 t .OO Printed in the USA. Copyright (c) 1989 Pergamon Press plc. All rights reserved.
MINI-REPORT FIBRINOLYTIC
ACTIVITY
OF ENDOTHELIN-3
R. Korbut, P. Lidbury, G.R. Thomas, and J.R. Vane The William Harvey Research Institute, St. Bartholomew's Hospital Medical College, Charterhouse Square, London EClM 6BQ, U.K. (Received 15.6.1989; accepted in original form 30.6.1989 by Editor-in Chief B. Blomb'a'ck)
The vasoconstrictor endothelin-1 peptide (ET-l) releases prostacyclin (PGI2) and endothelium-derived relaxing factor (EDRF) from isolated tissues as well as eicosanoids (most likely PGI ) in vivo (1). PGI2 stimulates fibrinolysis in vivo (2), whi3 e the role of EDRF In the fibrinolytic system remains unknown. Here we report on activation of the fibrinolytic system in rabbits by an intra-arterial injection of endothelin-3 (ET-3). This peptide, at doses which do not increase systemic blood pressure, is eguipotent with ET-l in releasing EDRF (3,4). In vitro, and ex vivo fibrinolytic activity of ET-3 was assayed by the euglobulin clot lysis time (ECLT) using the ET-3 (Peptide method of von Kaulla et al. (5). In vitro, Ins_$ itute In -3. Corp., Osaka, Japan) at concentrati;;;ob:;Oimn to 10 M was instilled onto whole blood, 10 fraction or applied directly to euglobulin clots prepared in duplicates. The time required for complete lysis of the clots ET-3 did not activate fibrinolysis in vitro was measured. (control 11523 min vs 10e7M ET-3 115+3 min; mean+S.E.M. n=6). were carr?ed out on 22 New Zealand Ex vivo experiments white _jabbits anaesthetized with sodium pentobarbitone (20-30 Blood was sampled and ECLT estimated before i.v.). mg kg and 1, 5, -151 30 and 60 min. after ila. injection of ET-3 Research (10 nmol kg Wellcome (3nmol kg ) or PG12 The same procedure was u:ed for rabbits which Laboratories). were pretreated with indomethacin (15 pm01 kg", i.v., Sigma The results are U.K.) 20 min. prior to ET-3 injection. expressed as % control.
Key words: endothelin-3,
fibrinolysis, 797
prostacvclin
ENDOTHELIN-3, FIBRINOLYSIS
798
Vol. 55, No. 6
During the 60 min. experimental period, no changes in ECLT were observed in animals treated with vehicle (saline or indomethacin (15 pm01 kg" i.v., n=2). ET-3 (3 Eli' k?T-;i: caused significant, bi-phasic shortening of ECLT (fig. 1 ). ECLT(%Control) lOO-
80-
60-
40-
20T;u.*tl Tl_qrelated fibrinolytic effect of ill. injected ET-3 + ). f n=3, A )or PG12 (10 nmol kg group received indomethacin '(li-4Lmol kgseparate an&al l.V., 20 min prior to injection of ET-3 1 (3 nmoln;;~l)P All data are expressed as mean % of control + S.E.M. of n observations.
The first increase in fibrinolysis was observed at 5 min and then the fibrinolytic activity remained elevated until 30 min. At 60 min. after injection of the peptide a second phase of fibrinolysis occurred. Pretreatment of the rabbits with indomethacin significantly inhibited (two-way analysis of variance followed by a least significance difference procedure - LSD, pcO.05) the shortening of ECLT observed at 15, 30 and 60 min. after ET-3 injection. However, it did not modify the decrease in ECLT occurring at 5 min. ET-3 in a dose of 3 nmol caused two transient falls in systemic blood pressure. kg The first fall, from 109 + 3mmHg to 88 + 2mmHg (pCO.05, n = 6) r was observed at 20 set after injection, and a second fall from 112 + 4mmHg to 93 + 4mmHg (~~0.05, n = 6) at 5 min. Indomethacin did not alter the first, but completely abolished the second depressor effect of ET-3. The fibrinolytic effect of PGI (10 nmol kg" i.a.) was time-dependent. No shortening of EE LT was observed at 5 and 15 min, however within 30 and 60 min. after injection of PG12, fibrinolysis was significantly activated (p
Vol. 55, No. 6
ENDOTHELIN-3, FIBRINOLYSIS
799
ECLT is believed to reflect the level of blood plasminogen activator (PA) released from the endothelium, was which originally proposed as the mechanism for the fibrinolytic action of PGI Indomethacin does not (2). inhibit the first phase of 2f. ibrinolysis induced by ET-3, so the mechanism of fibrinolytic activity of ET-3 is only partly explained by the release of PG12. ET-3 may directly stimulate the release of PA from the endothelium, or ET-3 induced release of EDRF may be involved in the fibrinolytic activity of this peptide. Thus, ET-3 is a potent fibrinolytic agent, producing a long-lasting effect by, at least in part, an eicosanoid-mediated mechanism.
ACKNOWLEDGEMENTS The authors wish to thank Dr R. Botting for editorial help. The William Harvey Research Institute is supported by a grant from Glaxo Group Research Limited.
1. DE NUCCI, G., THOMAS, R., D'ORLEANS-JUSTE, P., ANTUNES, E., WALDER, C., WARNER, T.D. AND VANE, J.R. Pressor effects of are limited by its removal in the circulating endothelin pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc. Natl. Acad. Sci. USA. 85, 9797-9800, 1988. 2. UTSUNOMIYA, T., KRAUSZ, M.M., VALERI, C.R., SHEPRO, D. AND embolism Treatment of pulmonary with HBCHTMAN, H.B. prostacyclin. Surserv. 88, 25-30, 1980. 3. WARNER, T.D., DE NUCCI, G. AND VANE, J.R. Rat endothelin is vasodilator in the isolated perfused mesentery of the rat. Eur.J. Pharmacol. 159, 325-326, 1989. 4. SPOKES, R.A., GHATEI, M.A. AND BLOOM, S.R. Studies with pressure and on rat blood endothelin-3 and endothelin-1 isolated tissues: Evidence for multiple endothelin receptor subtypes. J.Cardiovasc.Pharmacol. 13 (Suppl.5), 191-192, 1989. 5. KAULLA VON, K.N. AND SCHULTZ, R.L. Methods for evaluation of human fibrinolysis: Studies with two combined techniques. Am. J. Clin. Pathol. 29, 104-112, 1958.
MINI-REPORT FIBRINOLYTIC
ACTIVITY
OF ENDOTHELIN-3
R. Korbut, P. Lidbury, G.R. Thomas, and J.R. Vane The William Harvey Research Institute, St. Bartholomew's Hospital Medical College, Charterhouse Square, London EClM 6BQ, U.K. (Received 15.6.1989; accepted in original form 30.6.1989 by Editor-in Chief B. Blomb'a'ck)
The vasoconstrictor endothelin-1 peptide (ET-l) releases prostacyclin (PGI2) and endothelium-derived relaxing factor (EDRF) from isolated tissues as well as eicosanoids (most likely PGI ) in vivo (1). PGI2 stimulates fibrinolysis in vivo (2), whi3 e the role of EDRF In the fibrinolytic system remains unknown. Here we report on activation of the fibrinolytic system in rabbits by an intra-arterial injection of endothelin-3 (ET-3). This peptide, at doses which do not increase systemic blood pressure, is eguipotent with ET-l in releasing EDRF (3,4). In vitro, and ex vivo fibrinolytic activity of ET-3 was assayed by the euglobulin clot lysis time (ECLT) using the ET-3 (Peptide method of von Kaulla et al. (5). In vitro, Ins_$ itute In -3. Corp., Osaka, Japan) at concentrati;;;ob:;Oimn to 10 M was instilled onto whole blood, 10 fraction or applied directly to euglobulin clots prepared in duplicates. The time required for complete lysis of the clots ET-3 did not activate fibrinolysis in vitro was measured. (control 11523 min vs 10e7M ET-3 115+3 min; mean+S.E.M. n=6). were carr?ed out on 22 New Zealand Ex vivo experiments white _jabbits anaesthetized with sodium pentobarbitone (20-30 Blood was sampled and ECLT estimated before i.v.). mg kg and 1, 5, -151 30 and 60 min. after ila. injection of ET-3 Research (10 nmol kg Wellcome (3nmol kg ) or PG12 The same procedure was u:ed for rabbits which Laboratories). were pretreated with indomethacin (15 pm01 kg", i.v., Sigma The results are U.K.) 20 min. prior to ET-3 injection. expressed as % control.
Key words: endothelin-3,
fibrinolysis, 797
prostacvclin
ENDOTHELIN-3, FIBRINOLYSIS
798
Vol. 55, No. 6
During the 60 min. experimental period, no changes in ECLT were observed in animals treated with vehicle (saline or indomethacin (15 pm01 kg" i.v., n=2). ET-3 (3 Eli' k?T-;i: caused significant, bi-phasic shortening of ECLT (fig. 1 ). ECLT(%Control) lOO-
80-
60-
40-
20T;u.*tl Tl_qrelated fibrinolytic effect of ill. injected ET-3 + ). f n=3, A )or PG12 (10 nmol kg group received indomethacin '(li-4Lmol kgseparate an&al l.V., 20 min prior to injection of ET-3 1 (3 nmoln;;~l)P All data are expressed as mean % of control + S.E.M. of n observations.
The first increase in fibrinolysis was observed at 5 min and then the fibrinolytic activity remained elevated until 30 min. At 60 min. after injection of the peptide a second phase of fibrinolysis occurred. Pretreatment of the rabbits with indomethacin significantly inhibited (two-way analysis of variance followed by a least significance difference procedure - LSD, pcO.05) the shortening of ECLT observed at 15, 30 and 60 min. after ET-3 injection. However, it did not modify the decrease in ECLT occurring at 5 min. ET-3 in a dose of 3 nmol caused two transient falls in systemic blood pressure. kg The first fall, from 109 + 3mmHg to 88 + 2mmHg (pCO.05, n = 6) r was observed at 20 set after injection, and a second fall from 112 + 4mmHg to 93 + 4mmHg (~~0.05, n = 6) at 5 min. Indomethacin did not alter the first, but completely abolished the second depressor effect of ET-3. The fibrinolytic effect of PGI (10 nmol kg" i.a.) was time-dependent. No shortening of EE LT was observed at 5 and 15 min, however within 30 and 60 min. after injection of PG12, fibrinolysis was significantly activated (p
Vol. 55, No. 6
ENDOTHELIN-3, FIBRINOLYSIS
799
ECLT is believed to reflect the level of blood plasminogen activator (PA) released from the endothelium, was which originally proposed as the mechanism for the fibrinolytic action of PGI Indomethacin does not (2). inhibit the first phase of 2f. ibrinolysis induced by ET-3, so the mechanism of fibrinolytic activity of ET-3 is only partly explained by the release of PG12. ET-3 may directly stimulate the release of PA from the endothelium, or ET-3 induced release of EDRF may be involved in the fibrinolytic activity of this peptide. Thus, ET-3 is a potent fibrinolytic agent, producing a long-lasting effect by, at least in part, an eicosanoid-mediated mechanism.
ACKNOWLEDGEMENTS The authors wish to thank Dr R. Botting for editorial help. The William Harvey Research Institute is supported by a grant from Glaxo Group Research Limited.
1. DE NUCCI, G., THOMAS, R., D'ORLEANS-JUSTE, P., ANTUNES, E., WALDER, C., WARNER, T.D. AND VANE, J.R. Pressor effects of are limited by its removal in the circulating endothelin pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc. Natl. Acad. Sci. USA. 85, 9797-9800, 1988. 2. UTSUNOMIYA, T., KRAUSZ, M.M., VALERI, C.R., SHEPRO, D. AND embolism Treatment of pulmonary with HBCHTMAN, H.B. prostacyclin. Surserv. 88, 25-30, 1980. 3. WARNER, T.D., DE NUCCI, G. AND VANE, J.R. Rat endothelin is vasodilator in the isolated perfused mesentery of the rat. Eur.J. Pharmacol. 159, 325-326, 1989. 4. SPOKES, R.A., GHATEI, M.A. AND BLOOM, S.R. Studies with pressure and on rat blood endothelin-3 and endothelin-1 isolated tissues: Evidence for multiple endothelin receptor subtypes. J.Cardiovasc.Pharmacol. 13 (Suppl.5), 191-192, 1989. 5. KAULLA VON, K.N. AND SCHULTZ, R.L. Methods for evaluation of human fibrinolysis: Studies with two combined techniques. Am. J. Clin. Pathol. 29, 104-112, 1958.