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Stimulation of human platelet adenylate cyclase by prostaglandin D2
vol.
THROMBOSIS RESEARCH Printed in the United
States
J, pp. 401-412, Pergamon Press,
1974
Inc.
STIMULATION OF HUMAN PLATELET ADENYLATE CYCLASE BY PROSTAGLANDIN D2
David C. B. Mills & Donald E. Macfarlane Thrombosis Research Center Temple University Health Sciences Center 3400 North Broad Street Philadelphia, Pennsylvania 19140 (Received
6.8.1974;
Accepted
ABSTRACT.
in revised form 9.8.1974. by Editor A.L. Copley)
Prostaglandin D2 (PGD2) stimulates the formation of cyclic AMP in human platelets measured as the incorporation of radioactivity from previously labelled intracellular nucleotides. In this action it is similar to, and more powerful than PGEl. Both inhibition of platelet aggregation and stimulation of cyclic AMP accumulation by PGD2 and by PGEl are potentiated by an inhibitor of platelet phosphodiesterase. A number of minor differences in the response of platelets to PGD2 and PGEl suggest the existence of at least two prostaglandin receptors influencing a single adenylate cyclase.
Introduction Smith et al. (1) have recently shown that prostaglandin D2 (PGD2) inhibits the aggregation of human platelets, and is more potent than prostaglandin El (PGEl).
Investigations in several laboratories indicate
that PGEl inhibits platelet aggregation by its effect on the platelet adenylate cyclasefcyclic AMP system (see refs. 2 and 3).
Although there
is a general similarity in the structures of PGD2 and PGEl, there are sufficient differences to make it unlikely that they can both stimulate an identical receptor, especially since several closely related prostaglandins have little or no inhibitory activity.
PGD2 is a possible
metabolite of arachidonic acid in human platelets and consequently may 401
02
PLATELET
ADENYLXTE
CTCLA3E:PGD2
be involved in a regulatory system controlling platelet aggregation.
We
have therefore examined the effects of PGD2 on the formation and accumulation of cyclic AMP in human platelets, and compared these effects with those of PGE1.
Materials and Methods Platelet rich plasma was prepared by differential centrifugation of titrated human venous blood (l/10 vol. 3.8% Na3 citrate).
Aggregation
was recorded photometrically at 37" (4) using 0.8ml samples of plasma in unsiliconized, flat bottomed glass tubes of 6mm internal diameter, and plated metal stirring bars (Payton Associates Ltd., Buffalo). added in 20 1 or less with Hamilton microsyringes. P
Drugs were
The rate of aggregation
was found by measuring the slope of the steepest part of the aggregation tracing.
Percentage inhibition of this rate was calculated using inter-
polated control values. Radioactive cyclic AMP levels in platelet rich plasma was estimated after incubation with 1 M [14C] adenine for 1 - 1 l/2 hrs at 37" to label P intracellular metabolic nucleotides.
Aliquots of 0.5ml were incubated with
drugs as indicated and the reaction stopped by addition of 0.8ml of 1 N HC104 containing 0.25 Foles
cyclic AMP and 1500 cpm of [3H] cyclic AMP to
act as internal standard for the subsequent recovery of [14C] cyclic AMP. After centrifugation, the supematants
were applied to columns of Dowex 50
exchange resin and washed with 1.5ml of water.
The eluate from a further
2.0 ml of water was collected and precipitated twice with barium hydroxide and zinc sulphate (2,5).
The samples were counted in a dioxane-napthalene-
butylPBD cocktail in an Intertechnique SL30 scintillation counter using optimised channel settings.
After correction for background, crossover
and recovery, the [14C] cyclic AMP counts were calculated as percentage of the total [14C] in the platelets.
v ‘0 1
PLATELET
. j,So.3
ADESYLATE
CYCLASE
: PGD,
PGEl and PGD2 were generous gifts from Dr. .I.E. Pike, Upjohn, Co., Kalamazoo, Michigan.
They were judged to be better than 95% pure by thin
layer chromatography*.
RA233 (2,6 bis (diethanolamino) - 4 - piperidino-
pyrimido [4,5d] pyrimidine) was a gift from Dr. J. W. Bell, Boehringer Ingelheim Ltd., Middlesex, England.
[U-14C] adenine (S.A. 281 ci/mole)
was obtained from Amersham/Searle Ltd., Arlington Heights, Ill.
[8-3~]
adenosine 3'..5' cyclic monophosphate (S.A. 14.2 ci/m mole) was obtained from Schwartz/Mann, Orangeburg, N.Y. Dowex 5OW-X4 exchange resin, lOO200 mesh was obtained from BioRad Laboratories, Richmond, Ca., and used in the Hi form.
Results PGD2 inhibited ADP-induced aggregation in a dose dependent fashion. In the experiment shown in Fig. 1, PGD2 was compared with PGEl as an inhibitor of aggregation induced by a low (2.5F) of ADP using titrated PRP from one donor.
and a high (5OF)
concentration
The tracings show that PGD2 was
more effective than PGEl especially at high concentrations both of prostaglandins and of ADP and that the pattern of inhibition was slightly different.
PGEl not only reduces the rate and extent of aggregation, but also
gives rise to a marked delay between the addition of ADP and the onset of aggregation.
This delay was not seen with PGD2 at the low concentration
of ADP, and was less marked than with PGEl at the high concentration. The percentage inhibition of the rate of aggregation in this experiment is plotted in Fig. 2 against the concentration of prostaglandins. Two other experiments of this type were performed on different donors, and the effect of the phosphodiesterase inhibitor RA233 (25 ) on the inhibYi ition of aggregation was assessed.
This concentration of RA233 produced
* Chromatography kindly performed by Dr. J. B. Smith.
Olc
PLATELET
ADENYLATE
Vol.j,No.3
CYCLASE:PGD2
I
,---AOP2$M,
POP 5oyy
125
”
PGEl
Fig. 1.
1 Min PGO2
PGEl
PGO2
Superimposed aggregation tracings showing the inhibition of ADP-induced aggregation by PGEl and PGD2 added 30 set before ADP at the concentration (nM) shown at the end of each tracing. c- Control.
D2
ADP=2.5 M r
I
PROSTAGLANDIN CONCENTRATION (FM)
Fig. 2.
Percent inhibition of rate of aggregation plotted against the concentration of prostaglandin added (log scale). PGD2 (&,a) or PGEl (h,O) were added 30 set before ADP, final concentration 2.5uM (0,O) or 50uM (A,a).
PMTELET
ADENYLATE
CYCLASE:pGD,
4Oj
Table I Relative Potencies of PGD2 and PGEl as Inhibitors of Platelet Aggregation Experiment No.
RA233** )IM
ED50" (ti) PGEl PGD2
1
0
182
105
2
0
95
29
3
0
225
427
1
25
32
26
2
25
20
3
25
48
Potency Ratio*** PGD2:PGE1
1.74
Degree of potentiation by RX233 (fold) PGEl
PGD2
1.2
5.6
4.0
9
2.2
4.8
3.1
76
0.63
4.8
5.6
3.3 0.54
* ED50 = concentration giving 50% inhibition of rate of aggregation induced by ~OJ.IM ADP added 30 sec. later. **
RA233 was added 15 set before prostaglandins.
*** Potency ratio = ED50 for PGE1/ED50 for PGD2.
no measurable inhibition in its own right, but produced a parallel shift in the log dose response curves.
The results (Table I) show that there was
a considerable independent variation in the potency of both PGEl and PGD2 between donors, whereas RA233 caused an equal and consistent potentiation of the effects of both prostaglandins. The addition of PGD2 to platelets prelabelled with [14C] adenine resulted in an increase in the [14C] cyclic AMP content of the platelets.
In
the experiment shown in Fig. 3, prelabelled platelets were incubated with either PGEl or PGD2 for 30 set with or without the phosphodiesterase inhibitor RA233.
The level of cyclic AMP'is plotted against the concen-
tration of the prostaglandin, and shows that PGD2 was'slightly more powerful than PGEl and that the effects of both prostaglandins were equally potentiated by RA233.
PLATELET
406
ADENYLATE
CYCLASE:PGD2
33
0
I
0.1
I
1-o
I
10
PROSTAGLANDIN CONCENTRATION (F)
Fig. 3.
Radioactivity of cyclic AMP as percent of total [14C] radioactivity measured 30 set after addition of different concentrations of PGE (a; A> or PGD2 (0; 0) plotted on a logarithmic scale. & en symbols: prostaglandins only. Filled symbols: prostaglandins added together with RA233, 0.2 mM.
The interaction of the two prostaglandins was studied in the experiments shown in Fig. 4.
The increase in cyclic AMP level observed in
platelets exposed to either prostaglandin gave linear and parallel log dose response curves.
When the two prostaglandins were mixed and added simul-
taneously, the observed increase in cyclic AMP was close to that expected from a simple additive effect of two agonists acting through the same mechanism. The increase in the level of cyclic AMP in platelets exposed to PGEl has a very characteristic time course, reaching a peak in 20 - 30 set and thereafter declining to a plateau (6). different response:
Fig. 5 shows that PGD2 produced a
the peak level was reached later and the subsequent
fall was less pronounced.
Vol.
PLATELET
j,X0.3
ADEXYLXTE
ho7
CYCLASE:PGD,
0.8 -
%
0.6
-
," 6 G zj 0.4 u q * g
0.2 -
2 E 0'
I
I
I
0.05
0.1
0.2
I
0.4
CONCENTRATION OF TOTAL PROSTAGLANDIN (~lkl>
Fig. 4.
Radioactivity of cyclic AMP as a percent of total [14C] radioactivity plotted against concentration of added prostaglandin (log scale). Samples of labelled PRP were incubated for 20 sec. with either PGEl, (A--O); PGD2 (0-O) or a 1:l mixture of PGEl and PGD2 (C----.).
The aggregating agents ADP (6) and adrenaline (3,7) inhibit the stimulation of cyclic AMP accumulation by PGEl and also rapidly reduce elevated cyclic AMP levels of platelets incubated with PGEl.
Fig. 6
shows the effects of the simultaneous addition of different concentrations of one of these aggregating agents and either PGEl or PGD2 on the formation of cyclic AMP by platelets.
The results show that adrenaline was a
more powerful inhibitor of the effects of the prostaglandins than was ADP, and inhibited the effects of PGEl and PGD2 equally. active inhibitor of PGD2 than of PGEl.
ADP was a less
408
PLATELET
ADENYLATE
CYCLASE:PGD;?
Vo1.5,No.3
-oPG020+6JJM -*PGEl 2pM
-l210 TME Fig. 5.
(SEC)
Radioactivity of cyclic AMP as percentage of total [14C] radioactivity plotted against the time of incubation with PGD2 (O.Sj& 0) or PGEi (~JIM,A).
a
PGEl
0 PGD2 A-A
PGEl
O-0
PGD2
b--A
PGEl
0-a
PGD2
+ ADP
+ ADRENALINE
I
LJ
0.1
0.3
I
1.0
CONCENTIuTiON OF ADP OR ADRENALINE
Fig. 6.
I
30 $M)
Radioactivity of cyclic AMP as percent total [I%] radioactivity plotted against concentration of added ADP or adrenaline (log scale). Samples of labelled PRP were incubated for 30 set with PGEl (2yM,A, A) or with PGD2 (O.S#, o,.) containing either ADP (O-@,A-A) or adrenaline (~--0,&-d) at the concentration shown.
PLATELET
ADENYLATE
CYCLASE
rrog
: PGD2
Exposure of platelets to the sulphydryl reagent N-ethylmaleimide (NEM) enhances the formation of cyclic AMP in response to PGEl (by a mean of 73%, range 57 - 91% in five experiments) and blocks the inhibitory effect of ADP NEM at 0.4mM also blocked the inhibition by ADP of the formation of
(6).
cyclic AMP by platelets in response to PGD2, but did not have as much effect on the cyclic AMP level in platelets exposed to PGD2 alone (in three experiments the increase was 29X, 16% and -5%).
NEM does not completely
block the effect of adrenaline on PGEl stimulated platelets (6) and it did not relieve the inhibition by adrenaline of the effect of PGD2.
Discussion Many studies have indicated that the inhibition of platelet aggregation by PGEl is mediated by the stimulation of adenylate cyclase and the consequent accumulation of cyclic AMP (8,9).
We have shown that PGD2 also
stimulates the formation of cyclic AMP and that it is at least as powerful as PGEl both as an inhibitor of aggregation and as a stimulator of cyclic AMP production.
Both the inhibition of aggregation and stimulation of
cyclic AMP formation were potentiated by the phosphodiesterase inhibitor BA233.
This suggests that PGD2 exerts its inhibition of aggregation
through the cyclic AMP mechanism. Several close analogues of PGEl i.e. PGAl, PGFld,
PGF lTj, (10)
have less then 1% of its activity on human platelets, indicating a high degree of specificity of the PGEl receptor for the substituents on the ring.
PGE2 can antagonize the action of PGEl on the adenylate cyclase
of disrupted platelets (11) and can both enhance and inhibit aggregation (12), so that the PGEl receptor is also sensitive to the presence of a 5,6 double bond in the carboxyl side chain.
In the light of this spec-
ificity, the structure of PGD2 makes it unlikely that it acts on the PGEl receptor, and our studies reveal several minor differences in the
PLATELET
$10
ADENYLATE
CYCLASE:PGD2
Vo1.5,No.3
actions of PGEl and PGD2 that support the view that they operate on different receptors.
Firstly, the aggregation tracings show that PGD2 delayed
the onset of aggregation less than did PGEl. cyclic AMP production was different.
Secondly, the time course of
Thirdly, NEM enhanced the cyclic AMP
response to PGEl more than the response to PGD2, and fourthly, ADP was a less powerful antagonist of cyclase stimulation by PGD2 than by PGEl. Lastly we found that the ability of PGEl and PGD2 to inhibit aggregation of different donors' platelets varied independently.
A further indication
that PGEl and PGD2 stimulate different receptors is provided by the species differences reported by Smith et al. (1):
PO2
does not inhibit aggrega-
tion of rat platelets, and is ten times less potent than PGEl in rabbits. The differences in the time course of cyclic AMP production and the aggregation tracings could be explained if PGD2 were able to cause inhibition of phosphodiesterase as well as to stimulate the cyclase. were so, PGD2 should potentiate the action of PGEl.
If this
When mixtures of PGEl
and PGD2 were added simultaneously, the cyclic AMP increase was no greater than predicted if they both act on the same mechanism.
In other experi-
ments, supramaximal concentrations of PCXl (~OJ.IM) and PGD2 (5@
were added
separately and together to platelets, and the cyclic AMP increase produced by the mixture was no higher than that produced by either prostaglandin alone.
PO2
(5~1M)was also added one minute after PGEl (10~M)
and this caused no further increase in cyclic AMP.
These experiments show
that PGD2 does not directly or indirectly inhibit platelet phosphodiesterase. Although there is this evidence that there are two prostaglandin receptors capable of stimulating cyclic AMP production in human platelets, the experiments in which the two prostaglandins were added simultaneously and sequentially to platelets indicate that they probably stimulate the same adenylate cyclase, for otherwise the mixture of pros-
vcll.J,xo.3
PLATELET
XDENYLATE
CYCLASE:PGD,
taglandins would produce an increase of cyclic AMP levels above those obtained with either separately. It has been difficult to explain the function of the cyclic AMP mediated inhibitory system in platelets, because the most active naturally occurring activator of this system, PGBl, is not found in sufficient quality -in vivo to have a significant effect.
PGD2 could be formed from arach-
idonic acid via the cyclic endoperoxide precursors of prostaglandins, and there is evidence of an enzyme which catalyzes the formation of D-type prostaglandins in other tissues (13).
Thus platelets themselves may be
able to generate sufficient PGD2 to regulate their own function via the cyclic AMP system.
PGEl has too many actions to be of any clinical value
as an antithrombotic agent.
PGD2 is known to be inactive in several
biological systems (13) and is therefore a potentially useful agent; but pharmacological studies on human tissues are clearly required to determine the likely side effects of the use of PGD2 or its derivatives in man.
References 1.
SMITH, .J.B., SILVER, M.J., INGERMAN, C.M. & KOCSIS, J.J. (1974) Prostaglandin D2 inhibits the aggregation of human platelets. Thromb. Res. s: 291.
2.
MILLS, D.C.B. & SMITH, J.B. (1971) The influence on platelet aggregation of drugs that effect the accumulation of adenosine 3':5' - cyclic monophosphate in platelets. Biochem. J. 121: 185.
3.
HASLAM, R.J. (1973) Interactions of the pharmacological receptors of blood platelets with adenylate cyclase. Ser. Haematol. a: 333.
4.
MILLS, D.C.B. & ROBERTS, G.C.K. (1967) Effects of adrenaline on human blood platelets. J. Physiol. 193: 443.
5.
KRISHNA, G., WEISS, B. & BRODIE, B.B. (1968) A simple, sensitive method for the assay of adenyl cyclase. J. Pharmac. Exp. Therap. 163: 379.
6.
MILLS, D.C.B. & SMITH, J.B. (1972) The control of platelet responsiveness by agents that influence cyclic AMP metabolism. Ann. N.Y. Acad. Sci. 201: 391.
412
PLATELET
ADENYLATE
CYCWSE:PGDP
Vo1.5,No.3
7.
ROBISON, G.A., ARNOLD, A. & HARTMANN, R-C. (1969) Divergent effects of epinephrine and prostaglandin El on the level of cyclic AMP in human blood platelets. Pharmacol. Res. Comm. -1: 325.
8.
MILLS, D.C.B. (1974) Factors influencing platelet adenylate cyclase. In Sherry, S. C Scriabine, A. Eds. Platelets and Thrombosis. Balzmore:University Park Press.
9.
SMITH, J.B. & MACFARLANE, D.E. (1974) Platelets. In Prostaglandins, Volume 2. Ed. by Ramwell, P.W. Plenum Press, N.Y. -
10.
IRION, E. & BLOMB;a;CK, M. (1969) Prostaglandins in Platelet Aggregation. Stand. J. Clin. Lab. Invest. -24: 141.
11.
SALZMAN, E.W. (1973) Prostaglandins in the function of blood platelets. -In Les Prostaglandines. Ed. by E. Baulieu. INSERM, Paris.
12.
SHIO, H., RAMWELL, P.W. & JESSUP, S.J. (1972) Prostaglandin E2: Effects on aggregation, shape change and cyclic AMP of rat platelets. Prostaglandins 1: 29.
13.
NUGTEREN, D.H. & HAZELHOF, E. (1973) Isolation and properties of intermediates in prostaglandin biosynthesis. Biochim. Biophys. Acta. 326: 448.
Acknowledgments We thank Ms. Shelia Gardner for skilled technical assistance, and we thank Dr. J.B. Smith for valuable discussions. NIH Grant HL 14217.
The work was supported by
THROMBOSIS RESEARCH Printed in the United
States
J, pp. 401-412, Pergamon Press,
1974
Inc.
STIMULATION OF HUMAN PLATELET ADENYLATE CYCLASE BY PROSTAGLANDIN D2
David C. B. Mills & Donald E. Macfarlane Thrombosis Research Center Temple University Health Sciences Center 3400 North Broad Street Philadelphia, Pennsylvania 19140 (Received
6.8.1974;
Accepted
ABSTRACT.
in revised form 9.8.1974. by Editor A.L. Copley)
Prostaglandin D2 (PGD2) stimulates the formation of cyclic AMP in human platelets measured as the incorporation of radioactivity from previously labelled intracellular nucleotides. In this action it is similar to, and more powerful than PGEl. Both inhibition of platelet aggregation and stimulation of cyclic AMP accumulation by PGD2 and by PGEl are potentiated by an inhibitor of platelet phosphodiesterase. A number of minor differences in the response of platelets to PGD2 and PGEl suggest the existence of at least two prostaglandin receptors influencing a single adenylate cyclase.
Introduction Smith et al. (1) have recently shown that prostaglandin D2 (PGD2) inhibits the aggregation of human platelets, and is more potent than prostaglandin El (PGEl).
Investigations in several laboratories indicate
that PGEl inhibits platelet aggregation by its effect on the platelet adenylate cyclasefcyclic AMP system (see refs. 2 and 3).
Although there
is a general similarity in the structures of PGD2 and PGEl, there are sufficient differences to make it unlikely that they can both stimulate an identical receptor, especially since several closely related prostaglandins have little or no inhibitory activity.
PGD2 is a possible
metabolite of arachidonic acid in human platelets and consequently may 401
02
PLATELET
ADENYLXTE
CTCLA3E:PGD2
be involved in a regulatory system controlling platelet aggregation.
We
have therefore examined the effects of PGD2 on the formation and accumulation of cyclic AMP in human platelets, and compared these effects with those of PGE1.
Materials and Methods Platelet rich plasma was prepared by differential centrifugation of titrated human venous blood (l/10 vol. 3.8% Na3 citrate).
Aggregation
was recorded photometrically at 37" (4) using 0.8ml samples of plasma in unsiliconized, flat bottomed glass tubes of 6mm internal diameter, and plated metal stirring bars (Payton Associates Ltd., Buffalo). added in 20 1 or less with Hamilton microsyringes. P
Drugs were
The rate of aggregation
was found by measuring the slope of the steepest part of the aggregation tracing.
Percentage inhibition of this rate was calculated using inter-
polated control values. Radioactive cyclic AMP levels in platelet rich plasma was estimated after incubation with 1 M [14C] adenine for 1 - 1 l/2 hrs at 37" to label P intracellular metabolic nucleotides.
Aliquots of 0.5ml were incubated with
drugs as indicated and the reaction stopped by addition of 0.8ml of 1 N HC104 containing 0.25 Foles
cyclic AMP and 1500 cpm of [3H] cyclic AMP to
act as internal standard for the subsequent recovery of [14C] cyclic AMP. After centrifugation, the supematants
were applied to columns of Dowex 50
exchange resin and washed with 1.5ml of water.
The eluate from a further
2.0 ml of water was collected and precipitated twice with barium hydroxide and zinc sulphate (2,5).
The samples were counted in a dioxane-napthalene-
butylPBD cocktail in an Intertechnique SL30 scintillation counter using optimised channel settings.
After correction for background, crossover
and recovery, the [14C] cyclic AMP counts were calculated as percentage of the total [14C] in the platelets.
v ‘0 1
PLATELET
. j,So.3
ADESYLATE
CYCLASE
: PGD,
PGEl and PGD2 were generous gifts from Dr. .I.E. Pike, Upjohn, Co., Kalamazoo, Michigan.
They were judged to be better than 95% pure by thin
layer chromatography*.
RA233 (2,6 bis (diethanolamino) - 4 - piperidino-
pyrimido [4,5d] pyrimidine) was a gift from Dr. J. W. Bell, Boehringer Ingelheim Ltd., Middlesex, England.
[U-14C] adenine (S.A. 281 ci/mole)
was obtained from Amersham/Searle Ltd., Arlington Heights, Ill.
[8-3~]
adenosine 3'..5' cyclic monophosphate (S.A. 14.2 ci/m mole) was obtained from Schwartz/Mann, Orangeburg, N.Y. Dowex 5OW-X4 exchange resin, lOO200 mesh was obtained from BioRad Laboratories, Richmond, Ca., and used in the Hi form.
Results PGD2 inhibited ADP-induced aggregation in a dose dependent fashion. In the experiment shown in Fig. 1, PGD2 was compared with PGEl as an inhibitor of aggregation induced by a low (2.5F) of ADP using titrated PRP from one donor.
and a high (5OF)
concentration
The tracings show that PGD2 was
more effective than PGEl especially at high concentrations both of prostaglandins and of ADP and that the pattern of inhibition was slightly different.
PGEl not only reduces the rate and extent of aggregation, but also
gives rise to a marked delay between the addition of ADP and the onset of aggregation.
This delay was not seen with PGD2 at the low concentration
of ADP, and was less marked than with PGEl at the high concentration. The percentage inhibition of the rate of aggregation in this experiment is plotted in Fig. 2 against the concentration of prostaglandins. Two other experiments of this type were performed on different donors, and the effect of the phosphodiesterase inhibitor RA233 (25 ) on the inhibYi ition of aggregation was assessed.
This concentration of RA233 produced
* Chromatography kindly performed by Dr. J. B. Smith.
Olc
PLATELET
ADENYLATE
Vol.j,No.3
CYCLASE:PGD2
I
,---AOP2$M,
POP 5oyy
125
”
PGEl
Fig. 1.
1 Min PGO2
PGEl
PGO2
Superimposed aggregation tracings showing the inhibition of ADP-induced aggregation by PGEl and PGD2 added 30 set before ADP at the concentration (nM) shown at the end of each tracing. c- Control.
D2
ADP=2.5 M r
I
PROSTAGLANDIN CONCENTRATION (FM)
Fig. 2.
Percent inhibition of rate of aggregation plotted against the concentration of prostaglandin added (log scale). PGD2 (&,a) or PGEl (h,O) were added 30 set before ADP, final concentration 2.5uM (0,O) or 50uM (A,a).
PMTELET
ADENYLATE
CYCLASE:pGD,
4Oj
Table I Relative Potencies of PGD2 and PGEl as Inhibitors of Platelet Aggregation Experiment No.
RA233** )IM
ED50" (ti) PGEl PGD2
1
0
182
105
2
0
95
29
3
0
225
427
1
25
32
26
2
25
20
3
25
48
Potency Ratio*** PGD2:PGE1
1.74
Degree of potentiation by RX233 (fold) PGEl
PGD2
1.2
5.6
4.0
9
2.2
4.8
3.1
76
0.63
4.8
5.6
3.3 0.54
* ED50 = concentration giving 50% inhibition of rate of aggregation induced by ~OJ.IM ADP added 30 sec. later. **
RA233 was added 15 set before prostaglandins.
*** Potency ratio = ED50 for PGE1/ED50 for PGD2.
no measurable inhibition in its own right, but produced a parallel shift in the log dose response curves.
The results (Table I) show that there was
a considerable independent variation in the potency of both PGEl and PGD2 between donors, whereas RA233 caused an equal and consistent potentiation of the effects of both prostaglandins. The addition of PGD2 to platelets prelabelled with [14C] adenine resulted in an increase in the [14C] cyclic AMP content of the platelets.
In
the experiment shown in Fig. 3, prelabelled platelets were incubated with either PGEl or PGD2 for 30 set with or without the phosphodiesterase inhibitor RA233.
The level of cyclic AMP'is plotted against the concen-
tration of the prostaglandin, and shows that PGD2 was'slightly more powerful than PGEl and that the effects of both prostaglandins were equally potentiated by RA233.
PLATELET
406
ADENYLATE
CYCLASE:PGD2
33
0
I
0.1
I
1-o
I
10
PROSTAGLANDIN CONCENTRATION (F)
Fig. 3.
Radioactivity of cyclic AMP as percent of total [14C] radioactivity measured 30 set after addition of different concentrations of PGE (a; A> or PGD2 (0; 0) plotted on a logarithmic scale. & en symbols: prostaglandins only. Filled symbols: prostaglandins added together with RA233, 0.2 mM.
The interaction of the two prostaglandins was studied in the experiments shown in Fig. 4.
The increase in cyclic AMP level observed in
platelets exposed to either prostaglandin gave linear and parallel log dose response curves.
When the two prostaglandins were mixed and added simul-
taneously, the observed increase in cyclic AMP was close to that expected from a simple additive effect of two agonists acting through the same mechanism. The increase in the level of cyclic AMP in platelets exposed to PGEl has a very characteristic time course, reaching a peak in 20 - 30 set and thereafter declining to a plateau (6). different response:
Fig. 5 shows that PGD2 produced a
the peak level was reached later and the subsequent
fall was less pronounced.
Vol.
PLATELET
j,X0.3
ADEXYLXTE
ho7
CYCLASE:PGD,
0.8 -
%
0.6
-
," 6 G zj 0.4 u q * g
0.2 -
2 E 0'
I
I
I
0.05
0.1
0.2
I
0.4
CONCENTRATION OF TOTAL PROSTAGLANDIN (~lkl>
Fig. 4.
Radioactivity of cyclic AMP as a percent of total [14C] radioactivity plotted against concentration of added prostaglandin (log scale). Samples of labelled PRP were incubated for 20 sec. with either PGEl, (A--O); PGD2 (0-O) or a 1:l mixture of PGEl and PGD2 (C----.).
The aggregating agents ADP (6) and adrenaline (3,7) inhibit the stimulation of cyclic AMP accumulation by PGEl and also rapidly reduce elevated cyclic AMP levels of platelets incubated with PGEl.
Fig. 6
shows the effects of the simultaneous addition of different concentrations of one of these aggregating agents and either PGEl or PGD2 on the formation of cyclic AMP by platelets.
The results show that adrenaline was a
more powerful inhibitor of the effects of the prostaglandins than was ADP, and inhibited the effects of PGEl and PGD2 equally. active inhibitor of PGD2 than of PGEl.
ADP was a less
408
PLATELET
ADENYLATE
CYCLASE:PGD;?
Vo1.5,No.3
-oPG020+6JJM -*PGEl 2pM
-l210 TME Fig. 5.
(SEC)
Radioactivity of cyclic AMP as percentage of total [14C] radioactivity plotted against the time of incubation with PGD2 (O.Sj& 0) or PGEi (~JIM,A).
a
PGEl
0 PGD2 A-A
PGEl
O-0
PGD2
b--A
PGEl
0-a
PGD2
+ ADP
+ ADRENALINE
I
LJ
0.1
0.3
I
1.0
CONCENTIuTiON OF ADP OR ADRENALINE
Fig. 6.
I
30 $M)
Radioactivity of cyclic AMP as percent total [I%] radioactivity plotted against concentration of added ADP or adrenaline (log scale). Samples of labelled PRP were incubated for 30 set with PGEl (2yM,A, A) or with PGD2 (O.S#, o,.) containing either ADP (O-@,A-A) or adrenaline (~--0,&-d) at the concentration shown.
PLATELET
ADENYLATE
CYCLASE
rrog
: PGD2
Exposure of platelets to the sulphydryl reagent N-ethylmaleimide (NEM) enhances the formation of cyclic AMP in response to PGEl (by a mean of 73%, range 57 - 91% in five experiments) and blocks the inhibitory effect of ADP NEM at 0.4mM also blocked the inhibition by ADP of the formation of
(6).
cyclic AMP by platelets in response to PGD2, but did not have as much effect on the cyclic AMP level in platelets exposed to PGD2 alone (in three experiments the increase was 29X, 16% and -5%).
NEM does not completely
block the effect of adrenaline on PGEl stimulated platelets (6) and it did not relieve the inhibition by adrenaline of the effect of PGD2.
Discussion Many studies have indicated that the inhibition of platelet aggregation by PGEl is mediated by the stimulation of adenylate cyclase and the consequent accumulation of cyclic AMP (8,9).
We have shown that PGD2 also
stimulates the formation of cyclic AMP and that it is at least as powerful as PGEl both as an inhibitor of aggregation and as a stimulator of cyclic AMP production.
Both the inhibition of aggregation and stimulation of
cyclic AMP formation were potentiated by the phosphodiesterase inhibitor BA233.
This suggests that PGD2 exerts its inhibition of aggregation
through the cyclic AMP mechanism. Several close analogues of PGEl i.e. PGAl, PGFld,
PGF lTj, (10)
have less then 1% of its activity on human platelets, indicating a high degree of specificity of the PGEl receptor for the substituents on the ring.
PGE2 can antagonize the action of PGEl on the adenylate cyclase
of disrupted platelets (11) and can both enhance and inhibit aggregation (12), so that the PGEl receptor is also sensitive to the presence of a 5,6 double bond in the carboxyl side chain.
In the light of this spec-
ificity, the structure of PGD2 makes it unlikely that it acts on the PGEl receptor, and our studies reveal several minor differences in the
PLATELET
$10
ADENYLATE
CYCLASE:PGD2
Vo1.5,No.3
actions of PGEl and PGD2 that support the view that they operate on different receptors.
Firstly, the aggregation tracings show that PGD2 delayed
the onset of aggregation less than did PGEl. cyclic AMP production was different.
Secondly, the time course of
Thirdly, NEM enhanced the cyclic AMP
response to PGEl more than the response to PGD2, and fourthly, ADP was a less powerful antagonist of cyclase stimulation by PGD2 than by PGEl. Lastly we found that the ability of PGEl and PGD2 to inhibit aggregation of different donors' platelets varied independently.
A further indication
that PGEl and PGD2 stimulate different receptors is provided by the species differences reported by Smith et al. (1):
PO2
does not inhibit aggrega-
tion of rat platelets, and is ten times less potent than PGEl in rabbits. The differences in the time course of cyclic AMP production and the aggregation tracings could be explained if PGD2 were able to cause inhibition of phosphodiesterase as well as to stimulate the cyclase. were so, PGD2 should potentiate the action of PGEl.
If this
When mixtures of PGEl
and PGD2 were added simultaneously, the cyclic AMP increase was no greater than predicted if they both act on the same mechanism.
In other experi-
ments, supramaximal concentrations of PCXl (~OJ.IM) and PGD2 (5@
were added
separately and together to platelets, and the cyclic AMP increase produced by the mixture was no higher than that produced by either prostaglandin alone.
PO2
(5~1M)was also added one minute after PGEl (10~M)
and this caused no further increase in cyclic AMP.
These experiments show
that PGD2 does not directly or indirectly inhibit platelet phosphodiesterase. Although there is this evidence that there are two prostaglandin receptors capable of stimulating cyclic AMP production in human platelets, the experiments in which the two prostaglandins were added simultaneously and sequentially to platelets indicate that they probably stimulate the same adenylate cyclase, for otherwise the mixture of pros-
vcll.J,xo.3
PLATELET
XDENYLATE
CYCLASE:PGD,
taglandins would produce an increase of cyclic AMP levels above those obtained with either separately. It has been difficult to explain the function of the cyclic AMP mediated inhibitory system in platelets, because the most active naturally occurring activator of this system, PGBl, is not found in sufficient quality -in vivo to have a significant effect.
PGD2 could be formed from arach-
idonic acid via the cyclic endoperoxide precursors of prostaglandins, and there is evidence of an enzyme which catalyzes the formation of D-type prostaglandins in other tissues (13).
Thus platelets themselves may be
able to generate sufficient PGD2 to regulate their own function via the cyclic AMP system.
PGEl has too many actions to be of any clinical value
as an antithrombotic agent.
PGD2 is known to be inactive in several
biological systems (13) and is therefore a potentially useful agent; but pharmacological studies on human tissues are clearly required to determine the likely side effects of the use of PGD2 or its derivatives in man.
References 1.
SMITH, .J.B., SILVER, M.J., INGERMAN, C.M. & KOCSIS, J.J. (1974) Prostaglandin D2 inhibits the aggregation of human platelets. Thromb. Res. s: 291.
2.
MILLS, D.C.B. & SMITH, J.B. (1971) The influence on platelet aggregation of drugs that effect the accumulation of adenosine 3':5' - cyclic monophosphate in platelets. Biochem. J. 121: 185.
3.
HASLAM, R.J. (1973) Interactions of the pharmacological receptors of blood platelets with adenylate cyclase. Ser. Haematol. a: 333.
4.
MILLS, D.C.B. & ROBERTS, G.C.K. (1967) Effects of adrenaline on human blood platelets. J. Physiol. 193: 443.
5.
KRISHNA, G., WEISS, B. & BRODIE, B.B. (1968) A simple, sensitive method for the assay of adenyl cyclase. J. Pharmac. Exp. Therap. 163: 379.
6.
MILLS, D.C.B. & SMITH, J.B. (1972) The control of platelet responsiveness by agents that influence cyclic AMP metabolism. Ann. N.Y. Acad. Sci. 201: 391.
412
PLATELET
ADENYLATE
CYCWSE:PGDP
Vo1.5,No.3
7.
ROBISON, G.A., ARNOLD, A. & HARTMANN, R-C. (1969) Divergent effects of epinephrine and prostaglandin El on the level of cyclic AMP in human blood platelets. Pharmacol. Res. Comm. -1: 325.
8.
MILLS, D.C.B. (1974) Factors influencing platelet adenylate cyclase. In Sherry, S. C Scriabine, A. Eds. Platelets and Thrombosis. Balzmore:University Park Press.
9.
SMITH, J.B. & MACFARLANE, D.E. (1974) Platelets. In Prostaglandins, Volume 2. Ed. by Ramwell, P.W. Plenum Press, N.Y. -
10.
IRION, E. & BLOMB;a;CK, M. (1969) Prostaglandins in Platelet Aggregation. Stand. J. Clin. Lab. Invest. -24: 141.
11.
SALZMAN, E.W. (1973) Prostaglandins in the function of blood platelets. -In Les Prostaglandines. Ed. by E. Baulieu. INSERM, Paris.
12.
SHIO, H., RAMWELL, P.W. & JESSUP, S.J. (1972) Prostaglandin E2: Effects on aggregation, shape change and cyclic AMP of rat platelets. Prostaglandins 1: 29.
13.
NUGTEREN, D.H. & HAZELHOF, E. (1973) Isolation and properties of intermediates in prostaglandin biosynthesis. Biochim. Biophys. Acta. 326: 448.
Acknowledgments We thank Ms. Shelia Gardner for skilled technical assistance, and we thank Dr. J.B. Smith for valuable discussions. NIH Grant HL 14217.
The work was supported by