European Journal of Pharmacology, 60 (1979) 245--248 © Elsevier/North-Holland Biomedical Press
245
Short communication 6-KETO-PROSTAGLANDIN E~ INHIBITS THE AGGREGATION OF HUMAN PLATELETS PATRICK Y.-K. WONG, JOHN C. McGIFF, F R A N K F. SUN * and WARREN H. LEE
Departments of Pharmacology, University of Tennessee, Center for Health Science, Memphis, Tennessee 38163, New York Medical College, Valhalla, New York 10595, and * The Upjohn Company, Kalamazoo, Michigan 48001, U.S.A. Received 5 September 1979, accepted 16 October 1979
P.Y.-K. WONG, J.C. McGIFF, F.F. SUN and W.H. LEE, 6-Keto-prostaglandin E1 inhibits the aggregation of human platelets, European J. Pharmacol. 60 (1979) 245--248. 6-Keto-prostaglandin-E1 (6-keto-PGE1) was found to have a similar potency to prostacyclin (PGI2) as an inhibitor of platelet aggregation. It caused a time and concentration dependent inhibition of ADP, collagen and epinephrine induced platelet aggregation, the dose ratios for 70% inhibition by 6-keto-PGE1, PGI2 and PGE1 being approximately 1 : 1 : 13. In doses similar to those of PGI2, 6-keto-PGEl partially inhibited the release of [ 3H ]-serotonin from platelet-rich plasma induced by collagen. Prostacyclin
Prostaglandin E1
6-Keto-prostaglandin E1
1. Introduction Prostacyclin (PGI2), the bicyclic enol-ether derivative of prostaglandin endoperoxide (PGH2), is the most potent inhibitor of platelet aggregation so far described (Gryglewski et al., 1976). The antiaggregatory effect of PGI2 may be related to stimulation of platelet adenylate cyclase (Tateson et al., 1977). PGI2 is relatively short-lived; its biological activity in aqueous solution disappears within several minutes during the course of its transformation to a more stable product, 6-keto-PGF~a, which has little biological activity (Gryglewski et al., 1976}. The latter may be converted to a biologically active metabolite, 6-keto-PGEl, via the 9-hydroxyprostaglandin dehydrogenase pathway in the liver (Wong et al., 1979). Axen et al. (1979) first reported that 6-ketoPGE1 inhibits platelet aggregation. In this study, we have compared the relative potencies of authentic 6-keto-PGEl, PGI2 and PGE~ as inhibitors of ADP, epinephrine and collagen induced aggregation and studied the effects
Platelet aggregation
of 6-keto-PGEl and PGI2 on [3H]-serotonin release.
2. Materials and methods
2.1. Platelet preparation Blood was drawn from volunteers who had not taken acetylsalicylic acid or other drugs for the preceding 10 days. 9 parts of whole blood were mixed with 1 part of 3.8% sodium citrate to a total volume of 5 ml in a plastic tube. The platelet-rich plasma (PRP) was removed with a siliconized pipette after centrifuging blood at 150 g for 10 min. Plateletpoor plasma (PPP) was prepared by centrifuging the remaining blood at 12 000 g f o r 10 min. The final platelet count in PRP was adjusted to 2 X 10S/ml with PPP.
2.2. Platelet aggregation experiments These were performed with 0.5 ml aliquots of PRP stirred at 1,200 rpm at 37°C in a dual
246
P.Y.-K. WONG ET AL.
channel Payton aggregation module and transcribed on a linear recorder (Payton Associates, Buffalo, N.Y.). We studied the concentration of 6-keto-PGE1 and PGI2 required to inhibit platelet aggregation induced by ADP (6 pM), epinephrine (5.4 pM) and collagen (49 #g). PGE, and 6-keto-PGE1 were stored in dry acetone (1 mg/ml) a t - 2 5 ° C and diluted with Tris buffer (50 mM, pH 8.8) before use. Prostacyclin sodium salt was dissolved (1 mg/ml) in Tris buffer (50 mM, pH 8.8) and diluted to the required concentration prior to use with the same buffer. LEpinephrine and ADP were obtained from Sigma (St. Louis, Mo.). Calf skin collagen was obtained from Calbiochem. Corp. (LaJolla, Calif.). Serotonin Release: The PRP was labeled with [3H]-serotonin (5-hydroxytryptamine binoxalate, 28.5 mCi/pmole, New England Nuclear, Boston, Mass.). Platelets and [3H]A
serotonin (0.8 pCi/ml) were mixed and incubated at 37°C for 30 min. Between 85 and 90% of the [3H]-serotonin was taken up by the platelets. The platelets were divided into 0.5 ml aliquots for release studies. The aggregating agents were added to these aliquots which were then stirred at 1200 rpm for 15 min at 37°C. These were centrifuged for 2 min at room temperature and 0.1 ml of the supernate from each tube was transferred to 10 ml of scintillation fluid consisting of 0.4% omnifluor and 20% Triton-X 100 in toluene and counted in a Searle Mark III scintillation counter. Release of [3H]-serotonin was expressed as percentage of radioactivity in platelets as described by Ardie and Han (1974). % release = (A--B)/C--B) × 100 where A = radioactivity of the supernate of
B
6 KetoPGE1 L~EPINEPHRINE
C
6 Keto- COLLAGEN PGE1 [49ugl
6 Keto. A6DP PGF.1
lng
~'~
~
'~I~.'q'O'5n,
k ~
CONTROL
E
~ C O N T R O L '
PG~2 ADP t ~ .
1rain' ~
lng O.Sng
2.Sng
D
lng
~
Sng
~-O.25ng CONTROL
6KPGE1 AOP 4 t
/
2.5ng
F
PGE1 lng ~" 0.Sng
ADP
\~---~
5ng
CONTROL
Fig. 1. Upper panel: inhibition by 6 keto-PGEl of (A) epinephrine (5.4 pM) and (B) collagen (49 pg)-induced platelet aggregation and (C) reversal of ADP-induced platelet aggregation by 6 keto-PGE1 added 1 rain after the addition of ADP (6 pM). Lower panel: superimposed aggregation tracings showing the inhibition of ADP-induced aggregation by increasing doses of (D) 6 keto-PGE1, (E) PGI2 and (F) PGE1, added 1 rain before the addition of the inducing agent.
6-KETO-PGEI INHIBITS PLATELET A G G R E G A T I O N
the test sample, B = radioactivity of the supernate of the control, C = total radioactivity of the control sample.
247
release of labeled [3H]-serotonin from PRP. The same dose of PGI2 produced similar inhibition of [3H]-serotonin release from platelets (table 1).
3. Results 4. Discussion Epinephrine (5.4 pM) and collagen (49/~g) induced platelet aggregation were inhibited by 6-keto-PGEl in a dose
245
Short communication 6-KETO-PROSTAGLANDIN E~ INHIBITS THE AGGREGATION OF HUMAN PLATELETS PATRICK Y.-K. WONG, JOHN C. McGIFF, F R A N K F. SUN * and WARREN H. LEE
Departments of Pharmacology, University of Tennessee, Center for Health Science, Memphis, Tennessee 38163, New York Medical College, Valhalla, New York 10595, and * The Upjohn Company, Kalamazoo, Michigan 48001, U.S.A. Received 5 September 1979, accepted 16 October 1979
P.Y.-K. WONG, J.C. McGIFF, F.F. SUN and W.H. LEE, 6-Keto-prostaglandin E1 inhibits the aggregation of human platelets, European J. Pharmacol. 60 (1979) 245--248. 6-Keto-prostaglandin-E1 (6-keto-PGE1) was found to have a similar potency to prostacyclin (PGI2) as an inhibitor of platelet aggregation. It caused a time and concentration dependent inhibition of ADP, collagen and epinephrine induced platelet aggregation, the dose ratios for 70% inhibition by 6-keto-PGE1, PGI2 and PGE1 being approximately 1 : 1 : 13. In doses similar to those of PGI2, 6-keto-PGEl partially inhibited the release of [ 3H ]-serotonin from platelet-rich plasma induced by collagen. Prostacyclin
Prostaglandin E1
6-Keto-prostaglandin E1
1. Introduction Prostacyclin (PGI2), the bicyclic enol-ether derivative of prostaglandin endoperoxide (PGH2), is the most potent inhibitor of platelet aggregation so far described (Gryglewski et al., 1976). The antiaggregatory effect of PGI2 may be related to stimulation of platelet adenylate cyclase (Tateson et al., 1977). PGI2 is relatively short-lived; its biological activity in aqueous solution disappears within several minutes during the course of its transformation to a more stable product, 6-keto-PGF~a, which has little biological activity (Gryglewski et al., 1976}. The latter may be converted to a biologically active metabolite, 6-keto-PGEl, via the 9-hydroxyprostaglandin dehydrogenase pathway in the liver (Wong et al., 1979). Axen et al. (1979) first reported that 6-ketoPGE1 inhibits platelet aggregation. In this study, we have compared the relative potencies of authentic 6-keto-PGEl, PGI2 and PGE~ as inhibitors of ADP, epinephrine and collagen induced aggregation and studied the effects
Platelet aggregation
of 6-keto-PGEl and PGI2 on [3H]-serotonin release.
2. Materials and methods
2.1. Platelet preparation Blood was drawn from volunteers who had not taken acetylsalicylic acid or other drugs for the preceding 10 days. 9 parts of whole blood were mixed with 1 part of 3.8% sodium citrate to a total volume of 5 ml in a plastic tube. The platelet-rich plasma (PRP) was removed with a siliconized pipette after centrifuging blood at 150 g for 10 min. Plateletpoor plasma (PPP) was prepared by centrifuging the remaining blood at 12 000 g f o r 10 min. The final platelet count in PRP was adjusted to 2 X 10S/ml with PPP.
2.2. Platelet aggregation experiments These were performed with 0.5 ml aliquots of PRP stirred at 1,200 rpm at 37°C in a dual
246
P.Y.-K. WONG ET AL.
channel Payton aggregation module and transcribed on a linear recorder (Payton Associates, Buffalo, N.Y.). We studied the concentration of 6-keto-PGE1 and PGI2 required to inhibit platelet aggregation induced by ADP (6 pM), epinephrine (5.4 pM) and collagen (49 #g). PGE, and 6-keto-PGE1 were stored in dry acetone (1 mg/ml) a t - 2 5 ° C and diluted with Tris buffer (50 mM, pH 8.8) before use. Prostacyclin sodium salt was dissolved (1 mg/ml) in Tris buffer (50 mM, pH 8.8) and diluted to the required concentration prior to use with the same buffer. LEpinephrine and ADP were obtained from Sigma (St. Louis, Mo.). Calf skin collagen was obtained from Calbiochem. Corp. (LaJolla, Calif.). Serotonin Release: The PRP was labeled with [3H]-serotonin (5-hydroxytryptamine binoxalate, 28.5 mCi/pmole, New England Nuclear, Boston, Mass.). Platelets and [3H]A
serotonin (0.8 pCi/ml) were mixed and incubated at 37°C for 30 min. Between 85 and 90% of the [3H]-serotonin was taken up by the platelets. The platelets were divided into 0.5 ml aliquots for release studies. The aggregating agents were added to these aliquots which were then stirred at 1200 rpm for 15 min at 37°C. These were centrifuged for 2 min at room temperature and 0.1 ml of the supernate from each tube was transferred to 10 ml of scintillation fluid consisting of 0.4% omnifluor and 20% Triton-X 100 in toluene and counted in a Searle Mark III scintillation counter. Release of [3H]-serotonin was expressed as percentage of radioactivity in platelets as described by Ardie and Han (1974). % release = (A--B)/C--B) × 100 where A = radioactivity of the supernate of
B
6 KetoPGE1 L~EPINEPHRINE
C
6 Keto- COLLAGEN PGE1 [49ugl
6 Keto. A6DP PGF.1
lng
~'~
~
'~I~.'q'O'5n,
k ~
CONTROL
E
~ C O N T R O L '
PG~2 ADP t ~ .
1rain' ~
lng O.Sng
2.Sng
D
lng
~
Sng
~-O.25ng CONTROL
6KPGE1 AOP 4 t
/
2.5ng
F
PGE1 lng ~" 0.Sng
ADP
\~---~
5ng
CONTROL
Fig. 1. Upper panel: inhibition by 6 keto-PGEl of (A) epinephrine (5.4 pM) and (B) collagen (49 pg)-induced platelet aggregation and (C) reversal of ADP-induced platelet aggregation by 6 keto-PGE1 added 1 rain after the addition of ADP (6 pM). Lower panel: superimposed aggregation tracings showing the inhibition of ADP-induced aggregation by increasing doses of (D) 6 keto-PGE1, (E) PGI2 and (F) PGE1, added 1 rain before the addition of the inducing agent.
6-KETO-PGEI INHIBITS PLATELET A G G R E G A T I O N
the test sample, B = radioactivity of the supernate of the control, C = total radioactivity of the control sample.
247
release of labeled [3H]-serotonin from PRP. The same dose of PGI2 produced similar inhibition of [3H]-serotonin release from platelets (table 1).
3. Results 4. Discussion Epinephrine (5.4 pM) and collagen (49/~g) induced platelet aggregation were inhibited by 6-keto-PGEl in a dose
