THROMBOSIS RESEARCH 60; 105-l 08,199O 0049-3848190 $3.00 + .OO Printed in the USA. Copyright (c) 1990 Pergamon Press pk. All rights reserved.
BRIEF
COMMUNICATION
ENDOTHELIN-1 DOES NOT AFFECT HUMAN PLATELET AGGREG.4TION IN WHOLE BLOOD AND PLATELET RICH PLASMA. B. Battistinil, J. G. Filep 1$2*,F. Herman2 AND P. Siroisl IDepartment
of Pharmacology, Faculty of Medecine, University of Sherbrooke, Sherbrooke, Quebec, Canada JlH 5N4 2Department of Pathophysiology, Semmelweis University, Medical School, Budapest, Hungary. (Received
1.8.1990;
accepted
in original form 6.8.1990
by Editor H. Vinazzer)
INTRODUCTION Endothelin-1 (ET-l, human-porcine endothelin) is a recently characterized peptide isolated from cultured vascular endothelial cells(l). ET1 is a potent vasoconstrictor (l), but it is also capable of stimulating the release of vasodilators, such as endothelium-derived relaxing factor and prostacyclin (2-5). ET-l was reported to inhibit ex vivo platelet aggregation in rabbits (6) and in vivo platelet aggregation in dogs (5) and rabbits (7). In the present experiments, we studied the effects of ET-l on aggregation of human platelets in whole blood and platelet rich plasma. MATERIALS AND METHODS Venous blood was obtained from male healthy volunteers (23-26 years old) who fasted overnight and had not taken any drugs for the previous two weeks. Blood was collected into trisodium citrate (final concentration 1 mM). Platelet aggregation in whole blood was monitored by measuring electric impedance using a Chronolog aggregometer (Chrono Log Corp., Havertown, PA) according to the method of Wilsoncroft et a1.(8). Platelet rich plama (PRP) and platelet poor plasma (PPP) were prepared and aggregation of platelets was monitored by measuring light transmission in the same aggregometer as described previously (9). Platelet count was determined in a coulter counter (Coulter Electronics Inc, Hialah, FL). PRP or whole blood were pre-incubated with ET-l(l-100 nM, Peninsula Labs, Belmont, CA) at 37 OC for 2 to 10 min prior to addition of ADP(Sigma,St-Louis, MO; 10 and 20 MM in whole blood; Key
words:
endothelin- 1, human
platelets, 105
whole
blood,
platelet-rich
plasma
106
ENDOTHELIN-1 AND HUMAN PLATELETS
2-6 PM in 4 pgg/ml in Switzerland; experiments 60-80% of was added addition of
Vol. 60, No. 1
PRP), collagen (Sigma, St-Louis, MO; 5-20 pg/ml in whole blood; lPRP) and platelet activating factor (C16-PAF, Bachem, Bubendorf, 1 and 3 FM in whole blood; 0.1 and 0.3 p M in PRP). Preliminary showed that all agents at the indicated concentrations evoked maximal platelet aggregation. In another set of experiments, ET-l at the time when nearly complete aggregation developed following the same concentrations of aggregatory agents. RESULTS
In the first set of experiments, ET-1 (0.1 - 100 nM) was added to human PRP or whole blood and incubated. In our experimental conditions, ET-l did not induce platelet aggregation. In the second set of experiment, human PRP or whole blood were preincubated for 2-10 min in the presence of the same concentration of ET-l as those previously described and then challenged with ADP, collagen or PAF. As shown in table 1, ET-1 failed to inhibit or potentiate Furthermore, when ET- 1 the aggregation induced by these three stimuli. (100 nM) was added to platelets aggregated by ADP, collagen or PAF, no disaggregation was observed in both PRP or whole blood (data. not shown). TABLE 1 Effect of Endothelin(ET-1) on ADP, Collagen and PAF -Induced Platelet Aggregation in Human Platelet-Rich Plasma and Whole Blood Platelet Rich Plasma (% Light Transmission) ----
ADP
__-----
-------------
Dose
+[ET-l]
1727 43?8 69+9
24+ 4 44 f 6 67k 10
Collagen 1 pg/ml 2 pg/ml
2725 52+7
28 ?: 2 53 t 5
4 pg/ml
73+7
74 f 6
3327 56+10
32 f 5 53 ?: 8
PAF
2UM 4pM 6UM
Control
0.1 MM 0.3 PM
Whole Blood @m-is) Dose 5 flM 10 PM 20 MM 5 I.rg/rnl 10 ug/ml 20 Mg/ml
1 PM 3 JIM
---
Control 4.20 f 0.22 7.25 f 0.49 7.50 f 0.37
+[ET-l] 4.45 f 0.30 7.14 + 0.38 7.66 + 0.61
11.11?1.71
11.71?0.03
15.23kO.83 16.82r1.90
1 1.33+0.86 13.96k2.57
3.91 6.36
f 0.77 f 0.64
3.90 f 0.45 5.94
+ 0.73
Values are means + SEM for 3-6 donors. Platelet count in PRP was adjusted Platelet count to 200 000 cells / ml with autologous platelet poor plasma. Platelet-rich plasma in whole blood was 206 000 f 4500 cells/u1 (n=6). and whole blood were preincubated with ET-1 (100 nM) for 10 min at 37°C.
Vol. 60, No. 1
ENDOTHELIN-1 AND HUMAN PLATELETS
107
DISCUSSION The present study demonstrates that ET-l, up to a concentration of 100 nM, (i) does not induce platelet aggregation by itself and (ii) neither inhibits nor potentiates platelet aggregation induced by ADP, collagen or PAF. The results obtained with human PRP are consonant with previous data showing that ET-1 did not affect rabbit and dog platelet aggregation in PRP Using whole blood, ET-l neither induced aggregation nor modified (2, 5-6). the aggregatory response to ADP, collagen or PAF. This latter results suggest that ET-l did not stimulate other blood borne cells (e.g. neutrophils) to release relaxing factors (like neutrophil-derived relaxing factor)(lO-11) which could exert an antiaggregatory action. In contrast, in vivo studies in rabbits and dogs showed that ET-l is capable to exert an antiaggregatory action probably through release of endothelium-derived relaxing factor (2) and PGI2 (5) from endothelial cells. The role of prostacyclin in mediating the in vivo antiaggregatory effect of ET-l is further supported by the findings that both indomethacin and acetylsalicylic acid attenuate the antiaggregatory action of ET-l in vivo (5-7). The present findings indicate that ET-1 does not act on human platelets in vitro, and suggest that human platelets do not possess ET-l receptors. Whether ET-l could inhibit aggregation of human platelets in vivo, remains to be studied. CONCLUSION Endothelin-1 (0.1 -100 nM) by itself does not induce aggregation of human platelets in platelet-rich plasma or whole blood. Furthermore, preincubation of platelets with ET-l neither modified the aggregatory response to ADP, collagen or platelet-activating factor nor caused disagregation in platelet-rich plasma and whole blood. These findings indicate that ET-l does not affect human platelet aggregation in vitro, and suggest that platelets do not possess ET-l receptors.
Acknowledgments We thank Ms. Nicole Levesque and Carole St-Onge for their expert technical assistance. This research was supported by the Medical Research Council of Canada. J.G.F. and P.S. are in receipt of MRC Fellowship and Scientist Award, respectively.
1.
YANAGISAWA M., MITSUI Y., vasoconstrictor m, 411-415,
M., KURIHARA H., KIMURA S., TOMOBE Y., KOBAYASHI YASAKI Y., GOT0 K., MASAKI T. A novel potent peptide produced by vascular endothelial cells Nature, 1988.
108
ENDOTHELIN-1 AND HUMAN PLATELETS
Vol. 60, No. 1
2.
DeNUCCI, G., THOMAS R., D’ORLEANS JUSTE P., ANTUNES E., WALDER C., WARNER T.D., VANE J.R. Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc. Natl. Acad. Sci.USA , a , 9797-9800, 1988.
3.
WARNER T.D., MITCHELL A.M., DeNUCCI G., VANE J.R. Endothelin-1 and endothelin-3 release EDRF from isolated perfused arterial vessels of the rat and rabbit. J. Cardiovasc. Pharmacol., 13Csu~~l. 5) , S85S88, 1989.
4.
RAE G.A., TRYBULEC M., DeNUCCI G., VANE J.R. Endothelin-1 releases eicosanoids from rabbit isolated perfused kidney and spleen. L Cardiovasc. Pharmacol., 13(su~~1.51 , S89-S92, 1989.
5.
HERMAN F., MAGYAR K., CHABRIER P.E., BRAQUET P., FILEP J. Prostacyclin mediates antiaggregatory and hypotensive actions of endothelin in anaesthetized beagle dogs. Br. J. Pharmacol., 98 , 3840, 1989.
6.
THIEMERMANN C., LIDBURY P., THOMAS R., VANE J.R. Endothelin inhibits ex vivo platelet aggregation in the rabbit. Eur. J. Pharmacol., , 181-182, 1988. m
7.
THIEMERMANN C., MAY G.R., PAGE C.P., VANE J.R. Endothelin-1 inhibits platelet aggregation in vivo: a study with 11rindium-labelled platelets. Br. J. Pharmacol., 99 , 303-308, 1990.
8.
WILSONCROFT P.S., LOFTS F.J., GRIFFITS R.J., MOORE P.K. The effect of 6 oxo-PGE1 on human platelet aggregation in whole blood in vitro. L Pharm. Pharmacol,, 37 , 139, 1985.
9.
FILEP J., ROSENKRANZ B. Mechanism of vasopressin-induced aggregation. Thromb, Res., ti , 7-16, 1987.
10.
RIMELE T.J., STURM R.J., ADAMS L.M., HENRY D.E., HEASLIP R.J., WEICHMAN B.M., GRIMES D. Interaction of neutrophils with vascular smooth muscle identification of a neutrophil-derived relaxing factor. ,I. Pharmacol. Exper. Ther., 245(l) , 102-111, 1988.
11.
SCHMIDT H.H.H.W., SEIFERT R., BOHME E. Formation and release of nitric oxide from human neutrophils and HL-60 cells induced by a chemotactic peptide, platelet activating factor and leukotriene B4. FEBS Lett., 244(21 , 357-360, 1989.
platelet
BRIEF
COMMUNICATION
ENDOTHELIN-1 DOES NOT AFFECT HUMAN PLATELET AGGREG.4TION IN WHOLE BLOOD AND PLATELET RICH PLASMA. B. Battistinil, J. G. Filep 1$2*,F. Herman2 AND P. Siroisl IDepartment
of Pharmacology, Faculty of Medecine, University of Sherbrooke, Sherbrooke, Quebec, Canada JlH 5N4 2Department of Pathophysiology, Semmelweis University, Medical School, Budapest, Hungary. (Received
1.8.1990;
accepted
in original form 6.8.1990
by Editor H. Vinazzer)
INTRODUCTION Endothelin-1 (ET-l, human-porcine endothelin) is a recently characterized peptide isolated from cultured vascular endothelial cells(l). ET1 is a potent vasoconstrictor (l), but it is also capable of stimulating the release of vasodilators, such as endothelium-derived relaxing factor and prostacyclin (2-5). ET-l was reported to inhibit ex vivo platelet aggregation in rabbits (6) and in vivo platelet aggregation in dogs (5) and rabbits (7). In the present experiments, we studied the effects of ET-l on aggregation of human platelets in whole blood and platelet rich plasma. MATERIALS AND METHODS Venous blood was obtained from male healthy volunteers (23-26 years old) who fasted overnight and had not taken any drugs for the previous two weeks. Blood was collected into trisodium citrate (final concentration 1 mM). Platelet aggregation in whole blood was monitored by measuring electric impedance using a Chronolog aggregometer (Chrono Log Corp., Havertown, PA) according to the method of Wilsoncroft et a1.(8). Platelet rich plama (PRP) and platelet poor plasma (PPP) were prepared and aggregation of platelets was monitored by measuring light transmission in the same aggregometer as described previously (9). Platelet count was determined in a coulter counter (Coulter Electronics Inc, Hialah, FL). PRP or whole blood were pre-incubated with ET-l(l-100 nM, Peninsula Labs, Belmont, CA) at 37 OC for 2 to 10 min prior to addition of ADP(Sigma,St-Louis, MO; 10 and 20 MM in whole blood; Key
words:
endothelin- 1, human
platelets, 105
whole
blood,
platelet-rich
plasma
106
ENDOTHELIN-1 AND HUMAN PLATELETS
2-6 PM in 4 pgg/ml in Switzerland; experiments 60-80% of was added addition of
Vol. 60, No. 1
PRP), collagen (Sigma, St-Louis, MO; 5-20 pg/ml in whole blood; lPRP) and platelet activating factor (C16-PAF, Bachem, Bubendorf, 1 and 3 FM in whole blood; 0.1 and 0.3 p M in PRP). Preliminary showed that all agents at the indicated concentrations evoked maximal platelet aggregation. In another set of experiments, ET-l at the time when nearly complete aggregation developed following the same concentrations of aggregatory agents. RESULTS
In the first set of experiments, ET-1 (0.1 - 100 nM) was added to human PRP or whole blood and incubated. In our experimental conditions, ET-l did not induce platelet aggregation. In the second set of experiment, human PRP or whole blood were preincubated for 2-10 min in the presence of the same concentration of ET-l as those previously described and then challenged with ADP, collagen or PAF. As shown in table 1, ET-1 failed to inhibit or potentiate Furthermore, when ET- 1 the aggregation induced by these three stimuli. (100 nM) was added to platelets aggregated by ADP, collagen or PAF, no disaggregation was observed in both PRP or whole blood (data. not shown). TABLE 1 Effect of Endothelin(ET-1) on ADP, Collagen and PAF -Induced Platelet Aggregation in Human Platelet-Rich Plasma and Whole Blood Platelet Rich Plasma (% Light Transmission) ----
ADP
__-----
-------------
Dose
+[ET-l]
1727 43?8 69+9
24+ 4 44 f 6 67k 10
Collagen 1 pg/ml 2 pg/ml
2725 52+7
28 ?: 2 53 t 5
4 pg/ml
73+7
74 f 6
3327 56+10
32 f 5 53 ?: 8
PAF
2UM 4pM 6UM
Control
0.1 MM 0.3 PM
Whole Blood @m-is) Dose 5 flM 10 PM 20 MM 5 I.rg/rnl 10 ug/ml 20 Mg/ml
1 PM 3 JIM
---
Control 4.20 f 0.22 7.25 f 0.49 7.50 f 0.37
+[ET-l] 4.45 f 0.30 7.14 + 0.38 7.66 + 0.61
11.11?1.71
11.71?0.03
15.23kO.83 16.82r1.90
1 1.33+0.86 13.96k2.57
3.91 6.36
f 0.77 f 0.64
3.90 f 0.45 5.94
+ 0.73
Values are means + SEM for 3-6 donors. Platelet count in PRP was adjusted Platelet count to 200 000 cells / ml with autologous platelet poor plasma. Platelet-rich plasma in whole blood was 206 000 f 4500 cells/u1 (n=6). and whole blood were preincubated with ET-1 (100 nM) for 10 min at 37°C.
Vol. 60, No. 1
ENDOTHELIN-1 AND HUMAN PLATELETS
107
DISCUSSION The present study demonstrates that ET-l, up to a concentration of 100 nM, (i) does not induce platelet aggregation by itself and (ii) neither inhibits nor potentiates platelet aggregation induced by ADP, collagen or PAF. The results obtained with human PRP are consonant with previous data showing that ET-1 did not affect rabbit and dog platelet aggregation in PRP Using whole blood, ET-l neither induced aggregation nor modified (2, 5-6). the aggregatory response to ADP, collagen or PAF. This latter results suggest that ET-l did not stimulate other blood borne cells (e.g. neutrophils) to release relaxing factors (like neutrophil-derived relaxing factor)(lO-11) which could exert an antiaggregatory action. In contrast, in vivo studies in rabbits and dogs showed that ET-l is capable to exert an antiaggregatory action probably through release of endothelium-derived relaxing factor (2) and PGI2 (5) from endothelial cells. The role of prostacyclin in mediating the in vivo antiaggregatory effect of ET-l is further supported by the findings that both indomethacin and acetylsalicylic acid attenuate the antiaggregatory action of ET-l in vivo (5-7). The present findings indicate that ET-1 does not act on human platelets in vitro, and suggest that human platelets do not possess ET-l receptors. Whether ET-l could inhibit aggregation of human platelets in vivo, remains to be studied. CONCLUSION Endothelin-1 (0.1 -100 nM) by itself does not induce aggregation of human platelets in platelet-rich plasma or whole blood. Furthermore, preincubation of platelets with ET-l neither modified the aggregatory response to ADP, collagen or platelet-activating factor nor caused disagregation in platelet-rich plasma and whole blood. These findings indicate that ET-l does not affect human platelet aggregation in vitro, and suggest that platelets do not possess ET-l receptors.
Acknowledgments We thank Ms. Nicole Levesque and Carole St-Onge for their expert technical assistance. This research was supported by the Medical Research Council of Canada. J.G.F. and P.S. are in receipt of MRC Fellowship and Scientist Award, respectively.
1.
YANAGISAWA M., MITSUI Y., vasoconstrictor m, 411-415,
M., KURIHARA H., KIMURA S., TOMOBE Y., KOBAYASHI YASAKI Y., GOT0 K., MASAKI T. A novel potent peptide produced by vascular endothelial cells Nature, 1988.
108
ENDOTHELIN-1 AND HUMAN PLATELETS
Vol. 60, No. 1
2.
DeNUCCI, G., THOMAS R., D’ORLEANS JUSTE P., ANTUNES E., WALDER C., WARNER T.D., VANE J.R. Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc. Natl. Acad. Sci.USA , a , 9797-9800, 1988.
3.
WARNER T.D., MITCHELL A.M., DeNUCCI G., VANE J.R. Endothelin-1 and endothelin-3 release EDRF from isolated perfused arterial vessels of the rat and rabbit. J. Cardiovasc. Pharmacol., 13Csu~~l. 5) , S85S88, 1989.
4.
RAE G.A., TRYBULEC M., DeNUCCI G., VANE J.R. Endothelin-1 releases eicosanoids from rabbit isolated perfused kidney and spleen. L Cardiovasc. Pharmacol., 13(su~~1.51 , S89-S92, 1989.
5.
HERMAN F., MAGYAR K., CHABRIER P.E., BRAQUET P., FILEP J. Prostacyclin mediates antiaggregatory and hypotensive actions of endothelin in anaesthetized beagle dogs. Br. J. Pharmacol., 98 , 3840, 1989.
6.
THIEMERMANN C., LIDBURY P., THOMAS R., VANE J.R. Endothelin inhibits ex vivo platelet aggregation in the rabbit. Eur. J. Pharmacol., , 181-182, 1988. m
7.
THIEMERMANN C., MAY G.R., PAGE C.P., VANE J.R. Endothelin-1 inhibits platelet aggregation in vivo: a study with 11rindium-labelled platelets. Br. J. Pharmacol., 99 , 303-308, 1990.
8.
WILSONCROFT P.S., LOFTS F.J., GRIFFITS R.J., MOORE P.K. The effect of 6 oxo-PGE1 on human platelet aggregation in whole blood in vitro. L Pharm. Pharmacol,, 37 , 139, 1985.
9.
FILEP J., ROSENKRANZ B. Mechanism of vasopressin-induced aggregation. Thromb, Res., ti , 7-16, 1987.
10.
RIMELE T.J., STURM R.J., ADAMS L.M., HENRY D.E., HEASLIP R.J., WEICHMAN B.M., GRIMES D. Interaction of neutrophils with vascular smooth muscle identification of a neutrophil-derived relaxing factor. ,I. Pharmacol. Exper. Ther., 245(l) , 102-111, 1988.
11.
SCHMIDT H.H.H.W., SEIFERT R., BOHME E. Formation and release of nitric oxide from human neutrophils and HL-60 cells induced by a chemotactic peptide, platelet activating factor and leukotriene B4. FEBS Lett., 244(21 , 357-360, 1989.
platelet
