THROMBOSIS RESEARCH Printed in the United States
Vol. 9, PP- 387-405, 1976 Pergamon Press, Inc.
FACTORS AFFECTING PLATELET CYCLIC GMP LEVELS DURING AGGREGATION INDUCED BY COLLAGEN AND BY ARACHIDONIC ACID
T. Davies, M.M.L. Davidson, M.D. McClenaghan, A. Say and R.J. Haslam Department of Pathology, McMaster University, Hamilton, Ontario, Canada
(Received 12.7.1976; in revised form 20.8.1976. Accepted by Editor S. Niewiarowski)
ABSTRACT Platelet aggregation, [14C]5-HT re1ease, malondialdehyde (MDA) production and changes in platelet cyclic GMP were measured in stirred heparinized platelet-rich plasma after addition of aggregating agents in the presence and absence of specific inhibitors. Platelet cyclic GMP levels were monitored using a simple method for isolation of cyclic [~H]GMP from platelets containing [SH]GTP labelled by preincubation with r3H]guanine: the agents studied did not affect the specific radioactivity of platelet [3H]GTP. Addition of ADP or 5-HT increased platelet cyclic [BH]GMP, this effect being greater with ADP, which also caused more marked aggregation than 5-HT. These actions of ADP were blocked by creatine phosphate with creatine phosphokinase (CP+CPK) and those of 5-HT by methysergide. cp+ CPK,but not indomethacin,inhibited the increase in cyclic [3H]GMP caused by collagen at l()pg/ml, indicating that released ADP and not formation of prostaglandin endoperoxides was responsible for this increase. With collagen at 80 pg/ml, CP+CPK, methysergide and indomethacin, individually or in combination, did not block the increase in cyclic c3~&?@, indicating that factors in addition to ADP, 5-HT and endoperoxides or derivatives can mediate the increase. CP+CPK, alone or with methysergide, did not affect the increase in cyclic [~H]GMP caused by 1 mM arachidonic acid, while indomethacin abolished it, indicating that in this case endoperoxide formation, but not the action of released ADP, was essential. The results provide further evidence that collagen, unlike arachidonic acid, in' duces the release reaction by both indomethacin-sensitive and insensitive mechanisms. CP+CPK partially inhibited both the release of [14C]5-HT and production of MDA observed with collagen or arachidonic acid, suggesting that released ADP may potentiate the release reaction by enhancing endoperoxide synthesis. ADP also potentiated indomethacin-insensitive release. In all these studies the increases in cyclic [3~]~ levels correlated much more closely with platelet aggregation than with release of [14C]5-HT or MDA formation.
387
388
PLATELET CYCLIC GMP
Vo1.9,No.4
INTKODUCTION
A variety of agents that can induce platelet aggregation, either directly (e.g. ADP) or indirectly via the secretion of platelet granule constituents in the release reaction (e.g. collagen), have been shown to increase platelet cyclic GMP levels (l-7). St has been suggested that cyclic GMP may mediate or facilitate the release reaction (1,2,5) but, even if this is the case, other factors must also be required, as ADP can increase cyclic GMP levels without inducing release (4,5). An alternative hypothesis, that increases in cyclic GMP may be secondary to the release reaction and.could therefore potentially play a role in the aggregating actions of the various mediators released from platelets, including ADP, is equally plausible. The only evidence bearing on this point is the observation that acetylsalicylic acid and indomethacin, under conditions in which,they inhibited the release reaction induced by collagen by about 90X, did not block the associated increase in platelet cyclic GMP (2,5). Although this was consistent with a direct effect of collagen on cyclic GMP levels, there was still sufficient platelet aggregation in these experiments to suggest the release of mediators which might secondarily increase cyclic GMP levels. To investigate this problem more thoroughly, we have in this study examined the effects'of collagen on platelet function and cyclic GMP levels in the presence and absence of specific inhibitors of mediator action or formation added either individually or in combination. ADP, the release of which is responsible for most of the aggregation induced by collagen (8) and which also potentiates the release reaction (9), was removed by addition of creatine phosphate (CP) with creatine phosphokinase (CPK) (10). Moreover, ATP formed by this enzyme system specifically antagonizes the action of ADP on platelets ,The possible effects of 5-hydroxytryptamine (5-HT) released from (11). platelets we* blocked with methysergide (12). Finally, indomethacin was used to inhibit the synthesis of prostaglandin endoperoxides (13) and thromboxane A (14) which, when formed on addition of collagen to platelets, may have a d$rect aggregating action in addition to that of released ADP (15), as well as mediating the component of the release reaction which is sensitive to In view of previous evidence non-steroidal anti-inflammatory agents (16-18). (2,5) that prostaglandin endoperoxides and derivatives did not mediate the increases in platelet cyclic GMP caused by collagen, comparative experiments were carried out with their metabolic precursor, arachidonic acid, the aggregating and release-inducing effects of which can be completely blocked by inThe studies described in this paper permit a more dedomethacin (19,20). tailed analysis of the relationships between increased platelet cyclic GMP Some of levels and platelet function than has been possible previously. the results described in this paper have been reported briefly elsewhere (21). METHODS
Materials Human heparinized platelet-rich plasma (PKP) containing 3.5 - 4.7 x 1OS platelets/ml was prepared from the blood of donors who had not taken any drugs for at least one week (10). Suspensions of collagen fibres were also prepared as described previously (4). Arachidonic acid (Sigma Chemical Co., St. Louis, MO) was purified by application of a solution in Ehexane to columns of activated silicic acid (Unisil, Clarkson Chemical Co., Williamsport, PA) followed by elution with chloroform-benzene (l:l, v/v>.
Vol.9,No.b
PLATELET CYCLIC GMP
After solvent evaporation in a stream of N2, the purified arachidonic acid was redissolved in hexane and stored at -20°C, after determination of its concentration (22). Immediately before experiments, hexane solutions of arachidonic acid were evaporated in a stream of N2. The arachidonic acid was then dissolved in ethanol, neutralized with Na2C0 and diluted with 0.154 M NaCl, to give a clear solution of 20 mM arach4donic acid containing 10% (v/v) ethanol. Cyclic [8-14C]GMP (56 mCi/mmole) and [side chain 2-14~]5-~~ (56 mCi/mmole) were obtained from Amersham/Searle Corp., Don Mills, Ontario. Creatine phosphate (CP), rabbit muscle creatine phosphokinase (approx. 150 units/ mg) (CPK), indomethacin, all nucleotides and neutral alumina (Type WN-3) were obtained from Sigma Chemical Co., 1,1,3,3-tetramethoxypropane from Aldrich Chemical Co., Milwaukee, WI, 2-thiobarbituric acid from Fisher Scientific Co., DowDon Mills, Ontario, and methysergide bimaleate from Sandoz Ltd., Basle. ex 50 cation exchange resin (AG 50 W-X8, ZOO-400 mesh, H+ form) was obtained from Bio-Rad Laboratories, Richmond, CA. Other materials, including [8-3H]guanine (3-6 Ci/mmole), were obtained from sources given elsewhere (2). Platelet aggregation and release of [14C15-HT. These were measured in incubation mixtures comprising 0.85 ml of PRP and a Einal total of 0.15 ml of additions in 0.154 M NaCl. Platelet aggregation was measured turbidometritally as the change in per cent transmission (AT) in the same incubation mixtures as were used for determination of cyclic [3H]GMP levels (2). These contained PRP that had been pre-incubated for 60 - 120 min with 2 w [3~]guanine and 0.2 pM 5-HT (unlabelled). Release of [14C]5-HT was measured, as described previously (lo), in incubation mixtures containing sam les of the same PRP that had, in contrast, been pre-incubated with 0.2 l&i [P 4C]5-HT and Inhibitors of aggregation or the release reaction 2 pM guanine (unlabelled). were added 5 min before the aggregating agent and were replaced by 0.154 M NaCl in controls. The incubation mixtures were stirred in an aggregometer (Payton Associates Ltd., Scarborough, Ontario) from 0.5 min before addition of All these procedures were performed at 37OC. the aggregating agent. Uptake of r3H]guanine by platelets. In each experiment incorporation of 3H into the platelets was measured (Z), both after 60 min pre-incubation No significant with [3H]guanine and at the end of the experimental period. change in the uptake of [3H]guanine occurred during this time (3 to 8% in different experiments). Measurement of cyclic [~H]GMP. Platelet cyclic [3H]GMP was isolated by a modification of a method of Jakobs, B&me and Schultz (23). Each incubation (1 ml) was terminfked by addition of 0.2 ml of ice-cold 3 N HC104 with Precipitated protein was re0.2 nmoles of cyclic [ C]GMP (1 pCi/mmole). The supernatant was applied to a column (7 mm inmoved by centrifugation. ternal diameter) containing 1.5 g neutral alumina that had been washed with The column was then washed with 12 ml of 0.5 N HC104 12 ml of 0.5 N HC104. followed by 12 ml of water, and cyclic GMF was eluted with 0.2 M ammonium The first 2 ml was discarded formate adjusted to pH 6.0 with formic acid. and the following 3 ml was collected, acidified with 0.05 ml of 3 N HCl and applied to a column (10 mm internal diameter) containing 5 ml (packed volume) of cation exchange resin (Dowex 50). Cyclic GMP was eluted with 0.05 N HCl; the first 6 ml was discarded and the following 8,ml was collected, lyophilised and counted for 3H and 14C by liquid scintillation spectrometry in a cocktail consisting of 1 ml of water and 7.5 ml of Quantafluor (Mallinckrodt The recovery of cyclic [14c]~ through the complete Inc., St. Louis, MO). The purity of the cyclic isolation procedure was in the range 45-55%.
390
PLATELET
CYCLIC
GMP
Vol.9,No.4
[311]GMI' lsolutod was dctcrmlucd 111occuslonirlsumplcs Iron1Lhe 3H:14C ratio in product GMP separated chromatographically after treatment of the presumed cyclic [3H]GMP with cyclic nucleotide phos hodiesterase (2). By this method, the radiochemical purity of the cyclic [3f H GMP isolated from both control and aggregated platelets was at least 95%. Amount of r3H]GTP in platelets. Samples (0.9 ml) of PRP labelled with [3H]guanine as above, were stirred for 2 min with various additions (0.1 ml), then mixed with 0.2 ml of ice-cold 3 N HC104 and 0.25 ml of 10 mM GTP and cooled to O°C. Protein was removed by centrifugation, the supernatants were neutralized with 6 N KOH, KC104 was removed by further centrifugation and 20 ul samples of neutralized supernatant were subjected to thin layer chromatography in two dimensions (2). GTP was eluted with water (2), samples were counted for 3H and the radioactivity found was corrected for recovery of the nucleotide, as determined from the extinctions of the eluates at 252 run. Specific radioactivity of L3~]c~p. Samples (4.5 ml) of PRP labelled with PHJguanine were shaken for 2 min with various additions (0.5 ml), then mixed with 1 ml of ice-cold 60% (w/v) trichloracetic acid and cooled to O'C. Precipitated protein was removed by centrifugation and nucleotides were isolated from the supernatants by adsorption to and elution from charcoal (24). The nucleotides were then separated by thin layer chromatography in two dimensions (2). GTP was eluted with water; the amounts present in the eluates were determined from their extinctions at 252 nm and the 3H present was counted by liquid scintillation. Malondialdehyde (MDA) production. This was used as a measure of platelet cycle-oxygenase activity (25,26). Mixtures consisting of 0.85 ml of PRP and 0.15 ml of additions in 0.154 M NaCl were incubated with stirring in an aggregometer. Incubations were terminated by addition of 0.2 ml of 30% (w/v) trichloracetic acid. Precipitated protein was removed by centrifugation and 0.7 ml of each supernatant was mixed with 0.35 ml of 1% (w/v) 2thiobarbituric acid in 0.01 N NaOH, heated for 15 min at lOO'C, cooled and clarified by further centrifugation. Extinctions at 532 nm were measured and MDA formation was calculated from the increments seen in the presence of aggBecause the recovery of MDA from plasma was incomplete regating agents. (83 - 93%), a standard curve was constructed for each experiment by addition to the particular plasma of known amounts of MDA, prepared by hydrolysis of 1,1,3,3-tetramethoxypropane as described elsewhere (27). Statistics. The significance of experimentally-induced changes in platelet cyclic [~H]GMP or in MUA production in replicate samples of the same PRP were evaluated by unpaired Student's t tests.
RESULTS
Effects of AUP and of 5-HT. Incubation of PRP that had been preincubated with L'HJguanine with either ADP (0.1 - 4.0 PM) or 5-HT (0.3 - 10.0 pi) was accompanied by platelet aggregation and increases of cyclic [3~]~ In 0.5 min incubations with ADP, maximal above the basal level (Table 1). increases in cyclic [3H]GMP of 105 - 148% were seen in 3 separate experiments (one of which is shown in Table 1) on addition of ADP at a final concentration of 0.4 uM or greater, while half-maximal increases were seen with 0.2 PM. In 2 min incubations with ADP concentrations over 0.4 uM, larger increases in cyclic [3H]GMP (200 - 250%) have previously been.observed (4,5). The
;f . z" 0; r; g
TABLE 1
Extent of aggregation
5yclic c HlrmP
Levels
NS
Vol. 9, PP- 387-405, 1976 Pergamon Press, Inc.
FACTORS AFFECTING PLATELET CYCLIC GMP LEVELS DURING AGGREGATION INDUCED BY COLLAGEN AND BY ARACHIDONIC ACID
T. Davies, M.M.L. Davidson, M.D. McClenaghan, A. Say and R.J. Haslam Department of Pathology, McMaster University, Hamilton, Ontario, Canada
(Received 12.7.1976; in revised form 20.8.1976. Accepted by Editor S. Niewiarowski)
ABSTRACT Platelet aggregation, [14C]5-HT re1ease, malondialdehyde (MDA) production and changes in platelet cyclic GMP were measured in stirred heparinized platelet-rich plasma after addition of aggregating agents in the presence and absence of specific inhibitors. Platelet cyclic GMP levels were monitored using a simple method for isolation of cyclic [~H]GMP from platelets containing [SH]GTP labelled by preincubation with r3H]guanine: the agents studied did not affect the specific radioactivity of platelet [3H]GTP. Addition of ADP or 5-HT increased platelet cyclic [BH]GMP, this effect being greater with ADP, which also caused more marked aggregation than 5-HT. These actions of ADP were blocked by creatine phosphate with creatine phosphokinase (CP+CPK) and those of 5-HT by methysergide. cp+ CPK,but not indomethacin,inhibited the increase in cyclic [3H]GMP caused by collagen at l()pg/ml, indicating that released ADP and not formation of prostaglandin endoperoxides was responsible for this increase. With collagen at 80 pg/ml, CP+CPK, methysergide and indomethacin, individually or in combination, did not block the increase in cyclic c3~&?@, indicating that factors in addition to ADP, 5-HT and endoperoxides or derivatives can mediate the increase. CP+CPK, alone or with methysergide, did not affect the increase in cyclic [~H]GMP caused by 1 mM arachidonic acid, while indomethacin abolished it, indicating that in this case endoperoxide formation, but not the action of released ADP, was essential. The results provide further evidence that collagen, unlike arachidonic acid, in' duces the release reaction by both indomethacin-sensitive and insensitive mechanisms. CP+CPK partially inhibited both the release of [14C]5-HT and production of MDA observed with collagen or arachidonic acid, suggesting that released ADP may potentiate the release reaction by enhancing endoperoxide synthesis. ADP also potentiated indomethacin-insensitive release. In all these studies the increases in cyclic [3~]~ levels correlated much more closely with platelet aggregation than with release of [14C]5-HT or MDA formation.
387
388
PLATELET CYCLIC GMP
Vo1.9,No.4
INTKODUCTION
A variety of agents that can induce platelet aggregation, either directly (e.g. ADP) or indirectly via the secretion of platelet granule constituents in the release reaction (e.g. collagen), have been shown to increase platelet cyclic GMP levels (l-7). St has been suggested that cyclic GMP may mediate or facilitate the release reaction (1,2,5) but, even if this is the case, other factors must also be required, as ADP can increase cyclic GMP levels without inducing release (4,5). An alternative hypothesis, that increases in cyclic GMP may be secondary to the release reaction and.could therefore potentially play a role in the aggregating actions of the various mediators released from platelets, including ADP, is equally plausible. The only evidence bearing on this point is the observation that acetylsalicylic acid and indomethacin, under conditions in which,they inhibited the release reaction induced by collagen by about 90X, did not block the associated increase in platelet cyclic GMP (2,5). Although this was consistent with a direct effect of collagen on cyclic GMP levels, there was still sufficient platelet aggregation in these experiments to suggest the release of mediators which might secondarily increase cyclic GMP levels. To investigate this problem more thoroughly, we have in this study examined the effects'of collagen on platelet function and cyclic GMP levels in the presence and absence of specific inhibitors of mediator action or formation added either individually or in combination. ADP, the release of which is responsible for most of the aggregation induced by collagen (8) and which also potentiates the release reaction (9), was removed by addition of creatine phosphate (CP) with creatine phosphokinase (CPK) (10). Moreover, ATP formed by this enzyme system specifically antagonizes the action of ADP on platelets ,The possible effects of 5-hydroxytryptamine (5-HT) released from (11). platelets we* blocked with methysergide (12). Finally, indomethacin was used to inhibit the synthesis of prostaglandin endoperoxides (13) and thromboxane A (14) which, when formed on addition of collagen to platelets, may have a d$rect aggregating action in addition to that of released ADP (15), as well as mediating the component of the release reaction which is sensitive to In view of previous evidence non-steroidal anti-inflammatory agents (16-18). (2,5) that prostaglandin endoperoxides and derivatives did not mediate the increases in platelet cyclic GMP caused by collagen, comparative experiments were carried out with their metabolic precursor, arachidonic acid, the aggregating and release-inducing effects of which can be completely blocked by inThe studies described in this paper permit a more dedomethacin (19,20). tailed analysis of the relationships between increased platelet cyclic GMP Some of levels and platelet function than has been possible previously. the results described in this paper have been reported briefly elsewhere (21). METHODS
Materials Human heparinized platelet-rich plasma (PKP) containing 3.5 - 4.7 x 1OS platelets/ml was prepared from the blood of donors who had not taken any drugs for at least one week (10). Suspensions of collagen fibres were also prepared as described previously (4). Arachidonic acid (Sigma Chemical Co., St. Louis, MO) was purified by application of a solution in Ehexane to columns of activated silicic acid (Unisil, Clarkson Chemical Co., Williamsport, PA) followed by elution with chloroform-benzene (l:l, v/v>.
Vol.9,No.b
PLATELET CYCLIC GMP
After solvent evaporation in a stream of N2, the purified arachidonic acid was redissolved in hexane and stored at -20°C, after determination of its concentration (22). Immediately before experiments, hexane solutions of arachidonic acid were evaporated in a stream of N2. The arachidonic acid was then dissolved in ethanol, neutralized with Na2C0 and diluted with 0.154 M NaCl, to give a clear solution of 20 mM arach4donic acid containing 10% (v/v) ethanol. Cyclic [8-14C]GMP (56 mCi/mmole) and [side chain 2-14~]5-~~ (56 mCi/mmole) were obtained from Amersham/Searle Corp., Don Mills, Ontario. Creatine phosphate (CP), rabbit muscle creatine phosphokinase (approx. 150 units/ mg) (CPK), indomethacin, all nucleotides and neutral alumina (Type WN-3) were obtained from Sigma Chemical Co., 1,1,3,3-tetramethoxypropane from Aldrich Chemical Co., Milwaukee, WI, 2-thiobarbituric acid from Fisher Scientific Co., DowDon Mills, Ontario, and methysergide bimaleate from Sandoz Ltd., Basle. ex 50 cation exchange resin (AG 50 W-X8, ZOO-400 mesh, H+ form) was obtained from Bio-Rad Laboratories, Richmond, CA. Other materials, including [8-3H]guanine (3-6 Ci/mmole), were obtained from sources given elsewhere (2). Platelet aggregation and release of [14C15-HT. These were measured in incubation mixtures comprising 0.85 ml of PRP and a Einal total of 0.15 ml of additions in 0.154 M NaCl. Platelet aggregation was measured turbidometritally as the change in per cent transmission (AT) in the same incubation mixtures as were used for determination of cyclic [3H]GMP levels (2). These contained PRP that had been pre-incubated for 60 - 120 min with 2 w [3~]guanine and 0.2 pM 5-HT (unlabelled). Release of [14C]5-HT was measured, as described previously (lo), in incubation mixtures containing sam les of the same PRP that had, in contrast, been pre-incubated with 0.2 l&i [P 4C]5-HT and Inhibitors of aggregation or the release reaction 2 pM guanine (unlabelled). were added 5 min before the aggregating agent and were replaced by 0.154 M NaCl in controls. The incubation mixtures were stirred in an aggregometer (Payton Associates Ltd., Scarborough, Ontario) from 0.5 min before addition of All these procedures were performed at 37OC. the aggregating agent. Uptake of r3H]guanine by platelets. In each experiment incorporation of 3H into the platelets was measured (Z), both after 60 min pre-incubation No significant with [3H]guanine and at the end of the experimental period. change in the uptake of [3H]guanine occurred during this time (3 to 8% in different experiments). Measurement of cyclic [~H]GMP. Platelet cyclic [3H]GMP was isolated by a modification of a method of Jakobs, B&me and Schultz (23). Each incubation (1 ml) was terminfked by addition of 0.2 ml of ice-cold 3 N HC104 with Precipitated protein was re0.2 nmoles of cyclic [ C]GMP (1 pCi/mmole). The supernatant was applied to a column (7 mm inmoved by centrifugation. ternal diameter) containing 1.5 g neutral alumina that had been washed with The column was then washed with 12 ml of 0.5 N HC104 12 ml of 0.5 N HC104. followed by 12 ml of water, and cyclic GMF was eluted with 0.2 M ammonium The first 2 ml was discarded formate adjusted to pH 6.0 with formic acid. and the following 3 ml was collected, acidified with 0.05 ml of 3 N HCl and applied to a column (10 mm internal diameter) containing 5 ml (packed volume) of cation exchange resin (Dowex 50). Cyclic GMP was eluted with 0.05 N HCl; the first 6 ml was discarded and the following 8,ml was collected, lyophilised and counted for 3H and 14C by liquid scintillation spectrometry in a cocktail consisting of 1 ml of water and 7.5 ml of Quantafluor (Mallinckrodt The recovery of cyclic [14c]~ through the complete Inc., St. Louis, MO). The purity of the cyclic isolation procedure was in the range 45-55%.
390
PLATELET
CYCLIC
GMP
Vol.9,No.4
[311]GMI' lsolutod was dctcrmlucd 111occuslonirlsumplcs Iron1Lhe 3H:14C ratio in product GMP separated chromatographically after treatment of the presumed cyclic [3H]GMP with cyclic nucleotide phos hodiesterase (2). By this method, the radiochemical purity of the cyclic [3f H GMP isolated from both control and aggregated platelets was at least 95%. Amount of r3H]GTP in platelets. Samples (0.9 ml) of PRP labelled with [3H]guanine as above, were stirred for 2 min with various additions (0.1 ml), then mixed with 0.2 ml of ice-cold 3 N HC104 and 0.25 ml of 10 mM GTP and cooled to O°C. Protein was removed by centrifugation, the supernatants were neutralized with 6 N KOH, KC104 was removed by further centrifugation and 20 ul samples of neutralized supernatant were subjected to thin layer chromatography in two dimensions (2). GTP was eluted with water (2), samples were counted for 3H and the radioactivity found was corrected for recovery of the nucleotide, as determined from the extinctions of the eluates at 252 run. Specific radioactivity of L3~]c~p. Samples (4.5 ml) of PRP labelled with PHJguanine were shaken for 2 min with various additions (0.5 ml), then mixed with 1 ml of ice-cold 60% (w/v) trichloracetic acid and cooled to O'C. Precipitated protein was removed by centrifugation and nucleotides were isolated from the supernatants by adsorption to and elution from charcoal (24). The nucleotides were then separated by thin layer chromatography in two dimensions (2). GTP was eluted with water; the amounts present in the eluates were determined from their extinctions at 252 nm and the 3H present was counted by liquid scintillation. Malondialdehyde (MDA) production. This was used as a measure of platelet cycle-oxygenase activity (25,26). Mixtures consisting of 0.85 ml of PRP and 0.15 ml of additions in 0.154 M NaCl were incubated with stirring in an aggregometer. Incubations were terminated by addition of 0.2 ml of 30% (w/v) trichloracetic acid. Precipitated protein was removed by centrifugation and 0.7 ml of each supernatant was mixed with 0.35 ml of 1% (w/v) 2thiobarbituric acid in 0.01 N NaOH, heated for 15 min at lOO'C, cooled and clarified by further centrifugation. Extinctions at 532 nm were measured and MDA formation was calculated from the increments seen in the presence of aggBecause the recovery of MDA from plasma was incomplete regating agents. (83 - 93%), a standard curve was constructed for each experiment by addition to the particular plasma of known amounts of MDA, prepared by hydrolysis of 1,1,3,3-tetramethoxypropane as described elsewhere (27). Statistics. The significance of experimentally-induced changes in platelet cyclic [~H]GMP or in MUA production in replicate samples of the same PRP were evaluated by unpaired Student's t tests.
RESULTS
Effects of AUP and of 5-HT. Incubation of PRP that had been preincubated with L'HJguanine with either ADP (0.1 - 4.0 PM) or 5-HT (0.3 - 10.0 pi) was accompanied by platelet aggregation and increases of cyclic [3~]~ In 0.5 min incubations with ADP, maximal above the basal level (Table 1). increases in cyclic [3H]GMP of 105 - 148% were seen in 3 separate experiments (one of which is shown in Table 1) on addition of ADP at a final concentration of 0.4 uM or greater, while half-maximal increases were seen with 0.2 PM. In 2 min incubations with ADP concentrations over 0.4 uM, larger increases in cyclic [3H]GMP (200 - 250%) have previously been.observed (4,5). The
;f . z" 0; r; g
TABLE 1
Extent of aggregation
5yclic c HlrmP
Levels
NS
