Effects of S-Adenosyl-l -Methionine on Blood Platelet Activation

ISSN 0306-3623/97 $17.00 1 .00 PII S0306-3623(96)00571-X All rights reserved

Gen. Pharmac. Vol. 29, No. 4, pp. 651–655, 1997 Copyright  1997 Elsevier Science Inc. Printed in the USA.

Effects of S-Adenosyl-l-Methionine on Blood Platelet Activation J. P. De La Cruz,* M. Me´rida, J. A. Gonza´lez-Correa, P. Ortiz and F. Sa´nchez de la Cuesta Department of Pharmacology and Therapeutics, School of Medicine, University of Malaga, Campus de Teatinos s/n, 29071 Malaga, Spain [Tel: 134-52-131567; Fax: 134-52-131568] ABSTRACT. 1. In rats treated with S-adenosyl-L-methionine (SAMe), platelet aggregation in whole blood was inhibited by 50–57% with respect to control values. 2. In whole blood, SAMe also inhibited platelet aggregation, particularly when aggregation was induced with collagen (maximum inhibition 78.665.8% with 102 5 M SAMe). 3. The antiaggregant response was seen with SAMe in the range of concentrations from 1027 to 2 10 5 M, whereas concentrations in the range from 1024 to 1023 M had a progressively weaker effect. 4. Both red blood cells and leukocytes enhanced the antiaggregant effect of SAMe, as did simultaneous incubation with L-arginine. 5. The intraplatelet concentration of glutathione also was increased by SAMe. gen pharmac 29;4:651–655, 1997.  1997 Elsevier Science Inc. KEY WORDS. S-adenosyl-l-methionine, platelets, aggregation INTRODUCTION S-adenosyl-l-methionine (SAMe) is naturally present in all living organisms. In humans, SAMe is distributed throughout all tissues and body fluids and plays important roles in many biochemical enzymatic reactions. It thus contributes to the synthesis, activation and metabolism of hormones, neurotransmitters, nucleic acids, proteins, phospholipids and certain drugs (Giulidori et al., 1984). The molecule is produced endogenously from methionine and adenosine triphosphate (ATP) by SAMe-synthetase (Friedel et al., 1989). SAMe metabolism has been found to produce glutathione only in the liver, because it is the only organ expressing an active transulfuration pathway. A stable analog of this salt, obtained with ionic reactions involving p-toluene sulfonic acid and sulfuric acid, is currently used to treat certain types of liver disease (Friedel et al., 1989). Liver disease leads to alterations in hemostasis, particularly in platelet function (Laffi et al., 1986). The metabolism of SAMe by different organs produces adenosine and glutathione (Giulidori et al., 1984), substances that also modify platelet activation. The present study was therefore designed to investigate the effect of SAMe on platelet behavior. MATERIALS AND METHODS

Study design We used samples of human blood (in vitro studies) and experiments in Wistar rats (ex vivo and in vitro studies). Human blood samples were from healthy men (mean age 37.562.1 years) who had taken no medications during the previous 15 days or longer. All blood samples were obtained between 9:00 a.m. and 10:00 a.m. after an overnight fast. Animal studies were done in 50 male Wistar rats (250–275 g body weight). Forty animals (four groups of ten rats each) were *To whom correspondence should be addressed. Received 3 June 1996; accepted 14 November 1996.

treated with SAMe (5 or 10 mg/(kg day) SC for 15 days) or its solvent. At the end of this period, all rats were anesthetized with sodium diazepam IP, and 2 ml of venous blood was collected. The anticoagulant used consisted of 3.8% sodium citrate (1:10) with 10 U/ ml sodium heparin.

Analytical techniques We used an electrical impedance method described by Cardinal and Flower (1980) in a Chrono-Log model 540S aggregometer (Chrono-Log Corp., Haverton, PA, USA). Whole blood and platelet-enriched plasma (PRP) were tested. The latter was obtained by centrifuging whole blood at 180g for 10 min at 208C. The platelet count was adjusted to 270,000–300,000 platelets/ml with autologous platelet-poor plasma, which was obtained by centrifuging whole blood at 1800g for 15 min at 208C. Aggregation was tested (initial curve) in each sample after incubation for 5 min at 378C in the vehicle used for SAMe (isotonic saline solution, pH 7.4). Aggregation curves were then obtained after incubation for 5 min at 378C in the presence of different concentrations of SAMe (range 1027–1023 M, pH 7.4). Aggregation was induced with adenosine diphosphate (ADP) (2.5 mmol/l), collagen (1.0 mmol/l) (Menarini Diagno´stica, Barcelona, Spain) or arachidonic acid (400 mmol/l) (Bio Data Corp, USA). In each experiment, the change in electrical impedance was recorded 10 min after the aggregant was added. Concentration-effect curves were drawn from the percentage of aggregation obtained at each concentration of SAMe with respect to samples incubated without the drug. Aggregometry was done with samples of whole blood, PRP and PRP enriched with red blood cells (RBCs) or leukocytes. Red blood cells were obtained from the pellet remaining after centrifugation for PRP; the pellet was washed two or three times with isotonic saline solution enriched with 9 g/l of glucose (pH 7.4). Leukocytes were obtained by centrifuging whole blood on a Ficoll-Hypaque PLATELET AGGREGOMETRY.

652

J. P. De La Cruz et al. TABLE 1. Maximum intensity of platelet aggregation (Imax) in whole blood induced by 10 mg/ ml collagen, and platelet thromboxane B2 (TxB2), and aortic 6-keto-PGF1a production, from SAMe-treated rats

Saline (n 5 10) Vehicle (n 5 10) SAMe 5 mg/(kg day) (n 5 10) 10 mg/(kg day) (n 5 10) a

Imax (ohms)

TxB2 (ng/ml)

6-keto-PGF1a (ng/mg aorta)

5.16 6 0.76 4.90 6 0.91

224 6 19 245 6 6.3

58.6 6 7.8 45.9 6 9.0

2.21 6 0.60b

260 6 34

52.3 6 7.5

2.55 6 0.41b

272 6 23

71.5 6 6.1a

P , 0.05, b P , 0.01 versus saline. 6-keto-PGF1a: 6-keto-prostaglandin F1a.

density gradient (densities of 1,077 and 1,119) according to the method described by Boyum (1968). In some experiments, we tested the effects of adenosine or nitric oxide (NO) on the antiaggregant effect of SAMe. Samples of PRP were incubated with adenosine, adenosine-deaminase (an enzyme that destroys adenosine), l-arginine (a precursor of NO synthesis) or N-methyl-l-arginine (NMLA, an inhibitor of NO synthase). [14C]ADENOSINE UPTAKE BY ERYTHROCYTES. Adenosine uptake capacity was measured as described by Roos and Pleger (1972). Washed RBCs were diluted to a concentration of 200,000/ml and distributed in aliquots of 500 ml. The cells were incubated with SAMe vehicle or SAMe solution (at concentrations ranging from 1027 to 1023 M) for 5 min at 378C. Then [14C]adenosine (specific activity 55 mCi/ml) was added (Amersham International, USA) to a final concentration of 1 mmol/l. After incubation for 0–30 min at 378C, the reaction was stopped by immersing the sample in ice and adding glutaraldehyde to a final concentration of 4%. The samples were centrifuged at 10,000g for 3 min, and radioactivity was measured in the supernatant and the pellet. The results were expressed as the percentage of [14C]adenosine absorbed by RBCs.

TOTAL GLUTATHIONE DETERMINATIONS. Total glutathione in RBCs, platelets and plasma was quantified with the method described by Anderson (1989). Briefly, part of the washed RBC preparation for the adenosine uptake experiments was diluted in metaphosphoric acid (final concentration 5%) and then centrifuged at 2500g for 10 min at 48C. Part of the platelet-poor plasma was processed in the same way. Platelets in PRP were washed in buffer containing (in mmol/l): 113 NaCl, 4 Na2HPO4, 24 NaH2PO4, 4 KHPO4, and 5 glucose. Then 50 nmol/l PGE1 and 50 mg/ml apyrase were added, and the mixture was centrifuged at 650g for 15 min at 48C. The pellet was resuspended in the same buffer without PGE1, and 0.5 mg/ml bovine serum albumin was added until a concentration of 250,000–300,000 platelets/ml was obtained. Metaphosphoric acid (5%) was then added. In all samples, total glutathione was quantified by spectrophotometry after the addition of 143 mmol/l phosphate-buffered saline, 6.3 mmol/l EDTA, 0.248 mg/ml NADPH, 6 mmol/l 5,5’-dithiobis(2-nitrobenzoic) acid, 2.5 ml glutathione reductase (266 U/ml) and 10 ml of supernatant of the sample under analysis. The change in absorbance at 412 nm was determined by comparison with a reduced glutathione (GSH) standard curve.

RESULTS

Ex vivo experiments Table 1 shows the results of aggregometric determinations in animals treated for 15 days with vehicle or SAMe [5 or 10 mg/(kg day) SC]. Platelet aggregation in whole blood was significantly reduced at both doses of SAMe. The vehicle had no significant effect in any of the experiments.

In vitro experiments

FIGURE 1. Maximum intensity of platelet aggregation (Imax) in whole blood induced by 10 mg/ml collagen in rats treated with SAMe for 15 days. *P,0.01 versus saline-treated and nontreated rats.

When PRP was incubated with SAMe, we found no significant inhibition of platelet aggregation induced by collagen or by arachidonic acid. When ADP was the inducing agent, maximum inhibition was 18.161.9% after incubation with 10 mmol/l SAMe (Fig. 1). When the concentration of SAMe was 1023 M, platelet aggregation increased significantly with collagen (by 25.663.9%) or with arachidonic acid (by 36.866.2%). In whole blood, SAMe significantly inhibited platelet aggregation induced by ADP (maximum inhibition 40.362.8% with SAMe 1025 M), collagen (maximum inhibition 78.665.8% with SAMe 102 6 M) or arachidonic acid (maximum inhibition 34.664.1% with SAMe 102 6 M). The effect was dose dependent in the range between 1027 and 1025 M. How-

SAMe and Platelet Activity

653

TABLE 2. Maximum intensity of platelet aggregation (Imax) in whole blood induced by 2.5 mmol/l ADP, PRP and PRP plus different proportions of RBCs or leukocytes (LK), before and after incubation with SAMe (10 mmol/l) SAMe (mmol/l) 0 Whole blood PRP PRP 1 10% RBC PRP 1 20% RBC PRP 1 30% RBC PRP 1 40% RBC PRP 1 1,750 LK/ml PRP 1 3,500 LK/ml PRP 1 7,000 LK/ml

7.2 6.4 7.6 7.3 7.2 7.1 7.6 6.8 5.4

6 6 6 6 6 6 6 6 6

10 0.4 0.6 0.4 0.5 1.1 0.6 0.7 0.6 0.4

4.6 6.1 7.1 7.7 6.0 3.5 7.5 5.9 2.5

6 6 6 6 6 6 6 6 6

% inhibition 0.2a 0.3 0.5 0.5 0.5 0.1a 0.7 0.5 0.4a

36.5 4.7 1.7 2.0 15.8 43.7 1.3 14.1 36.1

6 6 6 6 6 6 6 6 6

2.9 0.6b 0.4 0.4 1.9c 3.3d 0.3 1.5c 3.7d

Values of Imax arre expressed in ohms. Each value is the mean 6 SEM of six to ten independent experiments. a P , 0.01 versus samples without SAMe. b P , 0.001 versus inhibition in whole blood. c P , 0.01, d P , 0.001 versus inhibition in PRP.

ever, the antiaggregant effect diminished gradually with increasing concentrations ranging from 1025 to 1023 M. The antiaggregant effect of SAMe was significantly enhanced by incubating PRP in the presence of RBCs or leukocytes (Table 2). When PRP was incubated with 40% RBCs or with 7,000 leukocytes/ ml, inhibition was similar to that found in the presence of whole blood. Adenosine and adenosine deaminase had no effect on the antiaggregant capacity of SAMe in experiments with PRP. However, in the presence of l-arginine, the antiaggregant effect increased, whereas the NO synthetase inhibitor N-methyl-l-arginine (NMLA) decreased this effect (Table 3). [14C]Adenosine uptake by erythrocytes was inhibited by SAMe in the range of 1027 to 1024 M but was increased by concentrations ranging from 1024 to 1023 M (Fig. 2). Total glutathione increased in platelets and RBCs (Fig. 3) when samples were treated with SAMe in the range of 1027 to 102 5 M but was decreased by concentrations ranging from 102 4 to 102 3 M.

the higher dose of SAMe (10 mg/(kg day). In humans, the recommended dose for parenteral administration is 800–1,000 mg/day, which is similar to the higher dose used in our experimental animals (equivalent to 700 mg/day in a person weighing 70 kg). We can thus conclude that SAMe, given at a dose considered within the therapeutic range, inhibits platelet aggregation. This effect does not appear to be caused by the inhibition of platelet thromboxane synthesis. Rather, the increase in the vascular production of prostacyclin, an endogenous prostanoid with antiaggregant and vasodilating effects (Moncada et al. 1976) may be involved in this effect. In vitro experiments to further study the inhibition of platelet aggregation showed that the effect of SAMe on platelets was dependent on the incubation medium (PRP or whole blood) and the range of concentrations used. In PRP, the effect was unclear, whereas, in whole blood, there was an evident antiaggregant effect. When collagen was used as the inducing agent, the antiaggregant effect was approximately twice as intense as when ADP or arachidonic acid was used. Concentrations of SAMe in the range of 1027 to 102 6 M led to an antiaggregant effect, whereas concentrations of 1025 to 102 3 M were less antiaggregant, and, at 1023 M, SAMe had no antiaggregant effect. We have found no published studies of the effects of SAMe on platelet aggregation; because we cannot compare our data with

DISCUSSION Our results show that, after the parenteral administration of SAMe in rats, platelet aggregation is inhibited and the vascular synthesis of prostacyclin is stimulated; these effects were especially clear at

TABLE 3. Maximum intensity of platelet aggregation (Imax) in PRP induced by 2.5 mmol/l ADP, before and after incubation with SAMe (10 mmol/l). Influence of cAMP or cGMP stimulation/ inhibition SAMe (mmol/l)

PRP 1 Adenosine (0.5 mmol/l) 1 Adenosine-deaminase (10 UI/ml) 1 l-arginine (100 mmol/l) 1 NMLA (300 mmol/l)

0

10

% inhibition

6.8 6 0.2 5.3 6 0.2

6.3 6 0.1 4.9 6 0.3

5.1 6 1.1 7.1 6 0.6

7.0 6 0.4

6.4 6 0.4

8.3 6 0.6

5.8 6 0.6

4.3 6 0.5a

20.1 6 1.3b

6.9 6 0.5

6.8 6 0.9

1.4 6 0.5c

Values of Imax are expressed in ohms. Each value is the mean 6 SEM of eight to ten independent experiments. a P , 0.01 versus samples without SAMe. b P , 0.001, c P , 0.05 versus inhibition in PRP.

654

J. P. De La Cruz et al. those of other experiments, it is difficult to explain why the effect of SAMe changes with increasing concentration. We postulate that, at high concentrations (1024 to 1023 M), the mechanism of methylation is unspecific, and the antiaggregant capacity of SAMe is therefore reduced. In this connection, some studies have shown that under certain conditions, SAMe can have the opposite effect to that expected. For example, Muriel et al. (1994) found that SAMe can increase transaminase levels in liver disease. Gonza´lez-Correa et al. (1995) reported that, at 10–20 mg/(kg day), SAMe reduced liver glutathione in rats with biliary stasis, whereas a dose of 5 mg/(kg day) increased glutathione levels. However, in humans, the range of therapeutic concentrations in plasma is 1027 to 1025 M; on the basis of our results in rats, the predominant effect of SAMe is apparently to inhibit platelet aggregation. Moreover, the commercial preparation of SAMe might contain small amounts of other molecules such as S-adenosylhomocysteine, homocysteine, methyl-thiadenosine, and so forth, and, at the high concentrations of SAMe used in the present experiments (1023–1024), these contaminants might be responsible for the biphasic curves observed on platelet behavior. Different effects in PRP and whole blood have been found with drugs such as dipyridamole (Gresele et al., 1983), mopidamole, aspirin, triflusal and pentoxifylline (De La Cruz et al., 1986, 1988, 1993, 1994). Red blood cells and leukocytes affect platelet function (Santos et al., 1986; Schattner et al., 1990) through two basic mechanisms: RBCs absorb adenosine, thus removing an antiaggregant molecule from the medium, whereas leukocytes release NO, which has an antiaggregant effect (Moncada and Higgs, 1991). Our findings show that both RBCs and leukocytes enhance the antiaggregant effect of SAMe (Table 2) and that the effect is greatest when the concentration of cells is similar to that found in whole blood. The inhibition of erythrocyte uptake of [14C]adenosine followed a pattern similar to that found with aggregometry, with a maximum inhibition of 26.564.8%. However, the antiaggregant effect of SAMe was apparently not affected by the presence or absence of adenosine in the incubation medium (Table 3). This argues against the adenosine-platelet cAMP pathway as the main site of action of SAMe. When platelets were incubated simultaneously with NO and l-arginine, the antiaggregant effect of SAMe was enhanced, whereas inhibition of NO synthase significantly reduced the antiag-

FIGURE 2. Dose-inhibition curves of platelet aggregation in PRP (open circles) and whole blood (solid circles) induced by ADP (top), collagen (middle) or arachidonic acid (bottom), after incubation with SAMe. *P,0.05, **P,0.01, ***P,0.0001, versus PRP values. Each point is the mean6SEM of eight to ten independent experiments.

FIGURE 3. Concentration dependence of inhibition of erythrocyte uptake of 1 mmol/l [14C]-adenosine after incubation with SAMe. *P,0.05 versus saline. Each point is the mean6SEM of eight to ten independent experiments.

SAMe and Platelet Activity

655 with 1025 M SAMe). Under all experimental conditions, the antiaggregant response was seen when SAMe was used within the range of concentrations from 1027 to 1025 M, whereas concentrations in the range from 1024 to 102 3 had a progressively weaker effect. Both red blood cells and leukocytes enhanced the antiaggregant effect of SAMe, as did simultaneous incubation with l-arginine. The intraplatelet concentration of glutathione also was increased by SAMe. This study was supported in part by Laboratorios Boehringer Ingelheim Espan˜a. We thank the Hermandad de Donantes de Sangre, Hospital Cli´nico Universitario de Ma´laga, for help in obtaining blood samples. Our thanks also to Antonio Pino Blanes, for his expert technical assistance, and Karen Shashok, for translating the original manuscript into English.

References

FIGURE 4. Accumulation of glutathione in platelets or RBCs after incubation with saline (black) or SAMe 131027 M (upward diagonal), 131026 M (diamond hatch), 131025 M (downward diagonal), 131024 M (square hatch) or 131023 M (white). *P,0.01, **P,0.001, versus saline. Each point is mean6SEM of eight to ten independent experiments.

gregant effect. We therefore surmise that SAMe may impede platelet aggregation by affecting leukocytes or by interfering with the platelet NO-cAMP pathway. Nitric oxide production is influenced by glutathione (Moncada and Higgs, 1991), and SAMe modifies glutathione levels in other tissues (Gonzalez-Correa et al., 1995). Our results in platelets and RBCs show that SAMe increases intracellular glutathione (Fig. 3) and that the variations in this effect with different concentrations of SAMe are consistent with the results of other assays. Therefore, the increased levels of glutathione found in platelets after SAMe administration are probably due not to SAMe being converted into glutathione but to other actions of this molecule such as reduced glutathione utilization or increased synthesis from cysteine. These results suggest that the increase of glutathione could affect the antiaggregant action of NO, as well as the synthesis of aggregant and antiaggregant prostaglandins. Our findings show that SAMe, used in a range of concentrations comparable to those used in human clinical practice, inhibits platelet function. This effect may result from the sum of several mechanisms, including mainly an increase in glutathione mobilization together with stimulation of the NO–platelet cAMP pathway. Further studies should clarify the nature of the effects of SAMe on platelet activity. SUMMARY We studied the effect of the subcutaneous administration of 5 or 10 mg/(kg day) of SAMe on platelet function in rats ex vivo and in human blood and rat aortic rings in vitro. In treated animals, both doses inhibited platelet aggregation in whole blood by 50–57% with respect to control values. In vitro experiments showed that SAMe inhibited platelet aggregation in whole blood, particularly when aggregation was induced with collagen (maximum inhibition 78.665.8%

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