European Journal of Pharmacology, 194 ( 1991) 63 - 70 © 1991 Elsevier Science Pubfishers B.V. 0014-2999/91/$03.50 ADONIS 0014299991001806
63
EJP 51731
Interactions between prostaglandin E 2 and inhibitors of platelet aggregation which act through cyclic AMP S t e p h e n J. G r a y a n d S t a n H e p t i n s t a l l Department of Medicine, University Hospital, Nottingham NG7 2 UH, U.K. Received 8 March 1990, revised MS received 24 July 1990, accepted 27 November 1990
Prostaglandin (PG) E 2 potentiates platelet aggregation at low concentrations (10-8-10 -6 M). It also inhibits aggregation at a higher concentration (10 -s M), probably by acting through cyclic adenosine 3',5'-monophosphate (cyclic AMP). The mechanism of this biphasic effect of PGE 2 and its implications for thrombosis are not clearly understood. Using a sensitive cyclic AMP assay, in conjunction with platelet aggregation studies, we have examined the interactions between PGE 2 and other inhibitors of platelet aggregation which act through cyclic AMP. Low concentrations of PGE 2 reversed the inhibition of platelet aggregation and increase in cyclic AMP levels induced by PGI 2, PGD 2 and adenosine (which stimulate adenylate cyclase (AC) through separate and specific platelet receptors). In contrast, low concentrations of PGE 2 added to the inhibition of platelet aggregation and increase in cyclic AMP levels induced by forskolin (which stimulates AC directly) and AH-P 719 and DN-9693 (which inhibit cyclic AMP phosphodiesterase (PDE)). These results suggest that the biphasic effect of PGE 2 may be mediated by interaction with two separate platelet receptors. Low concentrations appear to potentiate aggregation by acting at a receptor which is directly coupled to an inhibitory guanine nucleotide-binding protein (Gi) , possibly the putative PG endoperoxide receptor. High concentrations of PGE 2 appear to inhibit aggregation by acting at an additional receptor, probably the PGI 2 receptor. The ease with which PGE 2 reverses the effects of PGI 2, PGD 2 and adenosine, but adds to the effects of AH-P 719 and DN-9693, suggests that PDE inhibitors might offer greater potential then these AC stimulators as an anti-thrombotic strategy. This could be particularly true in the microcirculation (including the coronary and cerebral microcirculation) and in the intra-renal circulation, where PGE 2 is the major prostaglandin synthesised. Prostaglandin E2; Platelet aggregation inhibitors; Adenosine cyclic monophosphate
1. Introduction
Cyclic adenosine 3',5'-monophosphate (cyclic AMP) has an important physiological role in blood platelets, acting as a second messenger to a wide range of pharmacological agents. Its formation in platelets is controlled by adenylate cyclase (AC) and its breakdown by cyclic A M P phosphodiesterase (PDE). Pharmacological agents which increase cyclic A M P levels in platelets (either by stimulating AC or inhibiting PDE) can inhibit platelet aggregation (Salzman, 1972; Haslam, 1975), and this forms the basis for their potential use in anti-thrombotic therapy. Examples of pharmacological agents which stimulate AC and inhibit platelet aggregation are prostaglandin (PG) 12, P G D 2, P G E 1, P G E 2 and adenosine (Huang and Detwiler, 1986; Hawiger et al., 1987). All of these
Correspondence to: S.J. Gray, Department of Clinical Pharmacology, Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, U.K.
agents act through surface-located receptors that are coupled to the catalytic sub-unit of AC by a guanine nucleotide-binding regulatory protein (G protein) (Levitski, 1987). Forskolin is another agent that stimulates AC, but unlike the above agents it acts directly on the catalytic sub-unit of the enzyme (Seamon and Daly, 1981). Examples of pharmacological agents that inhibit P D E and inhibit platelet aggregation are methylxanthines (such as theophylline and 3-isobutyl-1methylxanthine), A H - P 719 and DN-9693 (Sakuma and Ashida, 1985; Huang and Detwiler, 1986; Sills and Heptinstall, 1986). The latter is a synthetic quinazolinone derivative that is currently undergoing early clinical trials as an inhibitor of platelet and leukocyte function in patients following acute myocardial infarction (Daiichi Seiyaku Co., personal communication). P G E 2 is one of the most abundant naturally occurring prostaglandins which is known to influence cyclic A M P formation and platelet aggregation. It is also the exception amongst the above group of agents which act through cyclic AMP, in that it has been reported to both stimulate and inhibit platelet aggregation depend-
64 ing on its concentration (Shio and Ramwell, 1971; Salzman et al., 1972; G r a y and Heptinstall, 1985). The mechanism of this biphasic effect of P G E 2 is not clearly understood, but in view of its stimulatory effect on platelet aggregation, P G E 2 may be involved in the pathogenesis of thrombosis. In this investigation we have used a sensitive cyclic A M P assay, in conjunction with platelet aggregation studies, to examine the effects of P G E 2 and other agents which inhibit platelet aggregation by acting through cyclic AMP. We have also examined the interaction between P G E 2 and these pharmacological agents, which raise cyclic A M P levels either by stimulating AC or inhibiting PDE. These studies were carried out to gain information on the mechanism by which P G E 2 stimulates platelet aggregation, and also to examine the possible implications of the interactions as far as antithrombotic therapy with these agents is concerned.
2. Materials and methods
2.1. Collection of blood samples Blood samples were taken by clean venepuncture from an antecubital vein of healthy volunteers. All subjects denied ingestion of drugs known to inhibit P G synthesis within the previous 10 days. Aliquots of blood (9 ml) were placed in polystyrene tubes containing 1 ml of 3.8% ( w / v ) trisodium citrate dihydrate as anticoagulant.
2.2. Preparation of platelet suspensions Platelet-rich plasma (PRP) was prepared by centrifuging blood at 160 x g for 10 min, and platelet-poor plasma (PPP) by re-centrifuging the blood at 1200 × g for 10 min after the PRP had been removed. The platelet count in the PRP was determined using a Coulter counter (Model F N) and subsequently adjusted using autologous PPP to give 3 x 108 platelets ml -a. Throughout the preparation and use of PRP and PPP, the samples were kept under an atmosphere of 5% CO 2 in air to prevent p H changes due to CO 2 diffusion. All experiments were completed within 3 h of venepuncture.
2.3. Platelet aggregation in PRP When the effect of a single pharmacological agent was under investigation, aliquots (460 /~1) of P R P were placed in polystyrene tubes containing 20 ~tl of the drug. The tubes were then pre-incubated in a water bath for 2 rain at 3 7 ° C before being transferred with metal stirrer bars to a platelet aggregometer (Adams et al., 1975), also maintained at 37°C. When the effect of a
combination of two pharmacological agents was under investigation, aliquots (440 F1) of PRP were pre-incubated for 2 min with both drugs (2 X 20 #1) before being transferred to the aggregometer. The samples were stirred in the aggregometer at 1000 rpm and incubated for a further 1 min before an aliquot of a solution of adenosine diphosphate (ADP) (1-5 # M ) was added, and the change in light absorbance was recorded for 6 min. Following platelet shape change, aggregation to A D P in PRP occurs in distinct phases depending on the concentration of the aggregating agent used. At low concentrations it is reversible (1st phase aggregation), and at higher concentrations it becomes irreversible and is associated with the release of contents from platelet granules (2nd phase aggregation). In this study we have used reversible (1st phase) aggregation to examine the effects of P G E 2 alone, thus allowing both potentiation and inhibition of aggregation to be observed. We have used maximal irreversible (lst and 2nd phase) aggregation to examine the effects of other inhibitors of platelet aggregation alone, and also to examine the effects of P G E 2 in the presence of inhibitors of platelet aggregation.
2.4. Measurement of platelet cyclic A M P levels The method used to measure cyclic A M P in platelets is a modification of that originally described by Haslam and McClenaghan (1981). The assay is based on measurement of the changes in platelet [3H]cyclic A M P that occur after incubation of pharmacological agents with platelets whose metabolic pool of adenine nucleotides has been 3H-labelled. This is achieved by pre-incubating the cells with [3H]adenine. Aliquots (440-480 /~1) of PRP were placed in polystyrene tubes containing 10/~1 of [2,8-3H]adenine (final concentration 1.2 /~M; 2 /~Ci ml -a of PRP) and the samples were covered with a mixture of 5% CO 2 in air. The tubes were then capped and incubated in a water bath at 37 ° C. After 90 min (when adenine uptake had reached a maximum) the tubes were removed from the water bath and up to 50 /~1 of solutions of the pharmacological agent(s) under investigation were added. The sample tubes were then returned to the water bath for a further 8 min incubation at 37 ° C. In some experiments, a solution of A D P (20 /~1) was added 2 min into this incubation period. The final volume in the tubes was always 500 #1. The reaction was stopped by adding 2 ml of ice-cold 15% ( w / v ) trichloroacetic acid containing 1000 cpm of [a4C] cyclic A M P to each sample tube. The [a4C]cyclic A M P was added to monitor and correct for variations in recovery of [3H]cyclic A M P during the subsequent isolation procedure. The contents of the tubes were mixed thoroughly using a vortex mixer and stored at - 2 0 ° C until the cyclic A M P was isolated and assayed using
65
2.6. Statistics
sequential alumina and Dowex 50 column chromatography as described by Haslam and McClenaghan (1981). After correction for background radiation, spillover of 14C into the 3H channel of the scintillation counter and [14C]cyclic AMP recovery, the [3H]cyclic A M P was expressed as a percentage of the total 3H that had been incorporated into the platelets during the labelling procedure.
Differences between sets of data were analysed statistically using the paired t-test. Approximate normality of distribution of the data was checked using a normal probability plot (Statgraphics; Statistical Graphics Corp.). P-values of less than 0.05 were considered to be significant.
2.5. Drugs and solutions 3. Results PGE 2, P G D 2, PGI 2, adenosine and A D P were purchased from the Sigma Chemical Co. Stock solutions of PGE 2 and P G D 2 (in ethanol), PGI 2 (in 0.05 M Tris buffer, pH 9.5) and A D P and adenosine (in saline) were stored at - 2 0 ° C. A stock solution of forskolin (Calbiochem-Behring Corp.) in ethanol was stored at - 2 0 ° C . AH-P 719 and DN-9693 were gifts from Boehringer Ingelheim and the Daiichi Seiyaku Co., respectively. Stock solutions were prepared in saline and stored at - 2 0 o C. Saline (150 mM NaC1) was infusion saline prepared by Trevanol Laboratories. [2,8- 3H]adenine (specific activity 12.8-19.6 Ci m M - 1) was purchased from New England Nuclear and was diluted in a solution of unlabelled adenine (57.7/~M; Sigma Chemical Co.) to achieve the concentration and specific activity required to label the platelets (1.2 /zM; 2 ~Ci m1-1 of PRP). [Adenine-UJ4C]adenosine 3',5'cyclic phosphate ammonium salt ([14C]cyclic AMP; specific activity 276-286 mCi mM -1) was purchased from Amersham International.
300
PGE 2 had a biphasic effect on ADP-induced platelet aggregation, potentiating aggregation at low concentrations (10-8-10 -6 M) and inhibiting aggregation at a higher concentration (10 -5 M). In contrast, PGI 2 (an AC stimulator) and A H - P 719 and DN-9693 (PDE inhibitors) only inhibited platelet aggregation (fig. 1). On a molar basis, PGI 2 was the most potent inhibitor, followed by AH-P 719 and then DN-9693. Despite the fact that low concentrations of P G E 2 (10-8-10 -6 M) potentiated platelet aggregation, we did not observe a reduction in basal platelet cyclic A M P levels at these concentrations. The effect of P G E 2 was solely to increase platelet cyclic A M P levels at concentrations of >/10 -8 M. PGI 2, A H - P 719 and DN9693 also increased platelet cyclic A M P levels and, once again, on a molar basis PGI 2 was the most potent, followed by A H - P 719 and then DN-9693 (fig. 2). When we examined the interaction between P G E 2 and other pharmacological agents which act through
100 [
/
o *-' C
80
.
8 '~
200 .
60
C
.o ¢e
=
40 100 20
W
0
I
I
I
I
I
9
8
7
6
5
0
I
I
I
I
I
I
10
9
8
7
6
5
- I o g l o concentration (M)
Fig. 1. Effect of a range of concentrations of PGE2 (Q), PGI2 (O), AH-P 719 (o) and DN-9693(Ill) on platelet aggregation induced by 1-5 lgM ADP in PRP. The concentration of ADP used was that required to induce a reversible aggregationwhen examining the effects of PGE2, and maximum aggregation when examining the effects of the other agents. The results are expressed as a percentage of the aggregatoryresponse to ADP alone, and are the means+ S.D. of at least four observations. An asterisk indicates a significant difference from the control (P < 0.05 or lower).
66
0.06[
0.3
63
EJP 51731
Interactions between prostaglandin E 2 and inhibitors of platelet aggregation which act through cyclic AMP S t e p h e n J. G r a y a n d S t a n H e p t i n s t a l l Department of Medicine, University Hospital, Nottingham NG7 2 UH, U.K. Received 8 March 1990, revised MS received 24 July 1990, accepted 27 November 1990
Prostaglandin (PG) E 2 potentiates platelet aggregation at low concentrations (10-8-10 -6 M). It also inhibits aggregation at a higher concentration (10 -s M), probably by acting through cyclic adenosine 3',5'-monophosphate (cyclic AMP). The mechanism of this biphasic effect of PGE 2 and its implications for thrombosis are not clearly understood. Using a sensitive cyclic AMP assay, in conjunction with platelet aggregation studies, we have examined the interactions between PGE 2 and other inhibitors of platelet aggregation which act through cyclic AMP. Low concentrations of PGE 2 reversed the inhibition of platelet aggregation and increase in cyclic AMP levels induced by PGI 2, PGD 2 and adenosine (which stimulate adenylate cyclase (AC) through separate and specific platelet receptors). In contrast, low concentrations of PGE 2 added to the inhibition of platelet aggregation and increase in cyclic AMP levels induced by forskolin (which stimulates AC directly) and AH-P 719 and DN-9693 (which inhibit cyclic AMP phosphodiesterase (PDE)). These results suggest that the biphasic effect of PGE 2 may be mediated by interaction with two separate platelet receptors. Low concentrations appear to potentiate aggregation by acting at a receptor which is directly coupled to an inhibitory guanine nucleotide-binding protein (Gi) , possibly the putative PG endoperoxide receptor. High concentrations of PGE 2 appear to inhibit aggregation by acting at an additional receptor, probably the PGI 2 receptor. The ease with which PGE 2 reverses the effects of PGI 2, PGD 2 and adenosine, but adds to the effects of AH-P 719 and DN-9693, suggests that PDE inhibitors might offer greater potential then these AC stimulators as an anti-thrombotic strategy. This could be particularly true in the microcirculation (including the coronary and cerebral microcirculation) and in the intra-renal circulation, where PGE 2 is the major prostaglandin synthesised. Prostaglandin E2; Platelet aggregation inhibitors; Adenosine cyclic monophosphate
1. Introduction
Cyclic adenosine 3',5'-monophosphate (cyclic AMP) has an important physiological role in blood platelets, acting as a second messenger to a wide range of pharmacological agents. Its formation in platelets is controlled by adenylate cyclase (AC) and its breakdown by cyclic A M P phosphodiesterase (PDE). Pharmacological agents which increase cyclic A M P levels in platelets (either by stimulating AC or inhibiting PDE) can inhibit platelet aggregation (Salzman, 1972; Haslam, 1975), and this forms the basis for their potential use in anti-thrombotic therapy. Examples of pharmacological agents which stimulate AC and inhibit platelet aggregation are prostaglandin (PG) 12, P G D 2, P G E 1, P G E 2 and adenosine (Huang and Detwiler, 1986; Hawiger et al., 1987). All of these
Correspondence to: S.J. Gray, Department of Clinical Pharmacology, Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, U.K.
agents act through surface-located receptors that are coupled to the catalytic sub-unit of AC by a guanine nucleotide-binding regulatory protein (G protein) (Levitski, 1987). Forskolin is another agent that stimulates AC, but unlike the above agents it acts directly on the catalytic sub-unit of the enzyme (Seamon and Daly, 1981). Examples of pharmacological agents that inhibit P D E and inhibit platelet aggregation are methylxanthines (such as theophylline and 3-isobutyl-1methylxanthine), A H - P 719 and DN-9693 (Sakuma and Ashida, 1985; Huang and Detwiler, 1986; Sills and Heptinstall, 1986). The latter is a synthetic quinazolinone derivative that is currently undergoing early clinical trials as an inhibitor of platelet and leukocyte function in patients following acute myocardial infarction (Daiichi Seiyaku Co., personal communication). P G E 2 is one of the most abundant naturally occurring prostaglandins which is known to influence cyclic A M P formation and platelet aggregation. It is also the exception amongst the above group of agents which act through cyclic AMP, in that it has been reported to both stimulate and inhibit platelet aggregation depend-
64 ing on its concentration (Shio and Ramwell, 1971; Salzman et al., 1972; G r a y and Heptinstall, 1985). The mechanism of this biphasic effect of P G E 2 is not clearly understood, but in view of its stimulatory effect on platelet aggregation, P G E 2 may be involved in the pathogenesis of thrombosis. In this investigation we have used a sensitive cyclic A M P assay, in conjunction with platelet aggregation studies, to examine the effects of P G E 2 and other agents which inhibit platelet aggregation by acting through cyclic AMP. We have also examined the interaction between P G E 2 and these pharmacological agents, which raise cyclic A M P levels either by stimulating AC or inhibiting PDE. These studies were carried out to gain information on the mechanism by which P G E 2 stimulates platelet aggregation, and also to examine the possible implications of the interactions as far as antithrombotic therapy with these agents is concerned.
2. Materials and methods
2.1. Collection of blood samples Blood samples were taken by clean venepuncture from an antecubital vein of healthy volunteers. All subjects denied ingestion of drugs known to inhibit P G synthesis within the previous 10 days. Aliquots of blood (9 ml) were placed in polystyrene tubes containing 1 ml of 3.8% ( w / v ) trisodium citrate dihydrate as anticoagulant.
2.2. Preparation of platelet suspensions Platelet-rich plasma (PRP) was prepared by centrifuging blood at 160 x g for 10 min, and platelet-poor plasma (PPP) by re-centrifuging the blood at 1200 × g for 10 min after the PRP had been removed. The platelet count in the PRP was determined using a Coulter counter (Model F N) and subsequently adjusted using autologous PPP to give 3 x 108 platelets ml -a. Throughout the preparation and use of PRP and PPP, the samples were kept under an atmosphere of 5% CO 2 in air to prevent p H changes due to CO 2 diffusion. All experiments were completed within 3 h of venepuncture.
2.3. Platelet aggregation in PRP When the effect of a single pharmacological agent was under investigation, aliquots (460 /~1) of P R P were placed in polystyrene tubes containing 20 ~tl of the drug. The tubes were then pre-incubated in a water bath for 2 rain at 3 7 ° C before being transferred with metal stirrer bars to a platelet aggregometer (Adams et al., 1975), also maintained at 37°C. When the effect of a
combination of two pharmacological agents was under investigation, aliquots (440 F1) of PRP were pre-incubated for 2 min with both drugs (2 X 20 #1) before being transferred to the aggregometer. The samples were stirred in the aggregometer at 1000 rpm and incubated for a further 1 min before an aliquot of a solution of adenosine diphosphate (ADP) (1-5 # M ) was added, and the change in light absorbance was recorded for 6 min. Following platelet shape change, aggregation to A D P in PRP occurs in distinct phases depending on the concentration of the aggregating agent used. At low concentrations it is reversible (1st phase aggregation), and at higher concentrations it becomes irreversible and is associated with the release of contents from platelet granules (2nd phase aggregation). In this study we have used reversible (1st phase) aggregation to examine the effects of P G E 2 alone, thus allowing both potentiation and inhibition of aggregation to be observed. We have used maximal irreversible (lst and 2nd phase) aggregation to examine the effects of other inhibitors of platelet aggregation alone, and also to examine the effects of P G E 2 in the presence of inhibitors of platelet aggregation.
2.4. Measurement of platelet cyclic A M P levels The method used to measure cyclic A M P in platelets is a modification of that originally described by Haslam and McClenaghan (1981). The assay is based on measurement of the changes in platelet [3H]cyclic A M P that occur after incubation of pharmacological agents with platelets whose metabolic pool of adenine nucleotides has been 3H-labelled. This is achieved by pre-incubating the cells with [3H]adenine. Aliquots (440-480 /~1) of PRP were placed in polystyrene tubes containing 10/~1 of [2,8-3H]adenine (final concentration 1.2 /~M; 2 /~Ci ml -a of PRP) and the samples were covered with a mixture of 5% CO 2 in air. The tubes were then capped and incubated in a water bath at 37 ° C. After 90 min (when adenine uptake had reached a maximum) the tubes were removed from the water bath and up to 50 /~1 of solutions of the pharmacological agent(s) under investigation were added. The sample tubes were then returned to the water bath for a further 8 min incubation at 37 ° C. In some experiments, a solution of A D P (20 /~1) was added 2 min into this incubation period. The final volume in the tubes was always 500 #1. The reaction was stopped by adding 2 ml of ice-cold 15% ( w / v ) trichloroacetic acid containing 1000 cpm of [a4C] cyclic A M P to each sample tube. The [a4C]cyclic A M P was added to monitor and correct for variations in recovery of [3H]cyclic A M P during the subsequent isolation procedure. The contents of the tubes were mixed thoroughly using a vortex mixer and stored at - 2 0 ° C until the cyclic A M P was isolated and assayed using
65
2.6. Statistics
sequential alumina and Dowex 50 column chromatography as described by Haslam and McClenaghan (1981). After correction for background radiation, spillover of 14C into the 3H channel of the scintillation counter and [14C]cyclic AMP recovery, the [3H]cyclic A M P was expressed as a percentage of the total 3H that had been incorporated into the platelets during the labelling procedure.
Differences between sets of data were analysed statistically using the paired t-test. Approximate normality of distribution of the data was checked using a normal probability plot (Statgraphics; Statistical Graphics Corp.). P-values of less than 0.05 were considered to be significant.
2.5. Drugs and solutions 3. Results PGE 2, P G D 2, PGI 2, adenosine and A D P were purchased from the Sigma Chemical Co. Stock solutions of PGE 2 and P G D 2 (in ethanol), PGI 2 (in 0.05 M Tris buffer, pH 9.5) and A D P and adenosine (in saline) were stored at - 2 0 ° C. A stock solution of forskolin (Calbiochem-Behring Corp.) in ethanol was stored at - 2 0 ° C . AH-P 719 and DN-9693 were gifts from Boehringer Ingelheim and the Daiichi Seiyaku Co., respectively. Stock solutions were prepared in saline and stored at - 2 0 o C. Saline (150 mM NaC1) was infusion saline prepared by Trevanol Laboratories. [2,8- 3H]adenine (specific activity 12.8-19.6 Ci m M - 1) was purchased from New England Nuclear and was diluted in a solution of unlabelled adenine (57.7/~M; Sigma Chemical Co.) to achieve the concentration and specific activity required to label the platelets (1.2 /zM; 2 ~Ci m1-1 of PRP). [Adenine-UJ4C]adenosine 3',5'cyclic phosphate ammonium salt ([14C]cyclic AMP; specific activity 276-286 mCi mM -1) was purchased from Amersham International.
300
PGE 2 had a biphasic effect on ADP-induced platelet aggregation, potentiating aggregation at low concentrations (10-8-10 -6 M) and inhibiting aggregation at a higher concentration (10 -5 M). In contrast, PGI 2 (an AC stimulator) and A H - P 719 and DN-9693 (PDE inhibitors) only inhibited platelet aggregation (fig. 1). On a molar basis, PGI 2 was the most potent inhibitor, followed by AH-P 719 and then DN-9693. Despite the fact that low concentrations of P G E 2 (10-8-10 -6 M) potentiated platelet aggregation, we did not observe a reduction in basal platelet cyclic A M P levels at these concentrations. The effect of P G E 2 was solely to increase platelet cyclic A M P levels at concentrations of >/10 -8 M. PGI 2, A H - P 719 and DN9693 also increased platelet cyclic A M P levels and, once again, on a molar basis PGI 2 was the most potent, followed by A H - P 719 and then DN-9693 (fig. 2). When we examined the interaction between P G E 2 and other pharmacological agents which act through
100 [
/
o *-' C
80
.
8 '~
200 .
60
C
.o ¢e
=
40 100 20
W
0
I
I
I
I
I
9
8
7
6
5
0
I
I
I
I
I
I
10
9
8
7
6
5
- I o g l o concentration (M)
Fig. 1. Effect of a range of concentrations of PGE2 (Q), PGI2 (O), AH-P 719 (o) and DN-9693(Ill) on platelet aggregation induced by 1-5 lgM ADP in PRP. The concentration of ADP used was that required to induce a reversible aggregationwhen examining the effects of PGE2, and maximum aggregation when examining the effects of the other agents. The results are expressed as a percentage of the aggregatoryresponse to ADP alone, and are the means+ S.D. of at least four observations. An asterisk indicates a significant difference from the control (P < 0.05 or lower).
66
0.06[
0.3
