High concentrations of arachidonic acid induce platelet aggregation and serotonin release independent of prostagladin endoperoxides and thromboxane A2

Biochimica et Biophysica Acta 841 (1985) 283-291 Elsevier

283

BBA 22119

High concentrations of arachidonic acid induce platelet aggregation and s e r o t o n i n r e l e a s e i n d e p e n d e n t of p r o s t a g l a n d i n e n d o p e r o x i d e s and thromboxane A 2 Y o s h i a k i H a s h i m o t o a, C h i k a y u k i N a i t o b 9 Shoji K u m e a,,, H i r o k a z u K a t o a T s u y o s h i W a t a n a b c a, M i t s u n o b u K a w a m u r a b, T a m i o T c r a m o t o a, a n d Hiroshi Oka ~ aFirst Department of Internal Medicine and the Central Clinical Laboratory, Faculty of Medicine, University of Tokyo, 3-1, Hongo 7-chome, Bunkyo-ku, Tokyo ]13 and b Department of lnternal Medicine, The Tokyo Teishin Hospital, 14-23, Fujimi 2-chome, Chiyoda-ku, Tokyo 102 (Japan) (Received September 25th, 1984) (Revised manuscript received June 4th, 1985)

Key words: Arachidonic acid; Platelet aggregation; Serotonin release; Prostaglandin endoperoxide; Thromboxane A 2

We examined platelet aggregation and serotonin release, induced by less than 60 # M arachidonic acid, using washed platelet suspensions in the absence of albumin. The concentration of arachidonic acid used did not cause platelet lysis. Piatelet responses induced by less than 20 p M arachidonic acid were inhibited by aspirin, whereas those induced by above 30 /~M arachidonic acid were not inhibited, even by both aspirin and 5,8,11,14-eicosatetraynoic acid. Although pbosphatidic acid and 1,2-diacyiglycerol increased after the addition of arachidonic acid in aspirin-treated platelets, the amounts were not parallel to platelet aggregation. Oleic, linoleic and linolenic acids also induced platelet responses, while paimitic, stearic and arachidic acids did not. EDTA, dibutyryl cyclic AMP, apyrase and creatine phosphate/creatine phosphokinase brought about almost the same effects in platelet responses induced by the unsaturated fatty acids, other than arachodinic acid, as those induced by 40 p M arachodonic acid. These results suggest that the mechanism of the actions of more than 30 /~M arachodinic acid on platelets is the same as that of the other unsaturated fatty acids and is independent of prostaglandin endoperoxides, thromboxane A z and, perhaps, phosphatidic acid and 1,2-diacylglycerol. Introduction Platelets contain virtually no free arachodonic acid [1]. Free arachidonic acid is released from platelet membrane phospholipids as a result of activation of phospholipases [2] and it then metabolized via the cyclooxygenase pathway to prostaglandin endoperoxides and thromboxane * Present address: The Central Clinical Laboratory, Yamanashi Medical College, Shimokato, Tamaho, Nakakoma, Yamanashi 409-38, Japan.

A 2 , which induce platelet responses [3,4]. Several

investigators have observed that an addition of arachodonic acid to platelets results in platelet responses [5-9,13]. When arachodonic acid is added to platelet suspensions containing almumin, a part of the acid binds to albumin in the suspension medium [17] and the remainder enters the platelets. However, if a suspension medium contains no albumin, most of the arachidonic acid added to the suspension disappears promptly from the suspension medium (Hashimoto, Y., unpublished data), suggesting the rapid entrance of arachidonic

0304-4165/85/$03~30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

284 acid into platelets. We investigated platelet aggregation and serotonin release induced by adding arachodonic acid to washed platelet suspensions in the absence of albumin, aiming at exploring effects of "free" arachidonic acid on platelets. In this paper we report results suggesting that free arachidonic acid itself could induce platelet aggregation and serotonin release independently of prostaglandin endoperoxides and thromboxane A 2. Materials and Methods

Materials. [14C]Serotonin (5-hydroxy[sidechain-2-14C]tryptamine creatinine sulphate) (58 m C i / m m o l ) and carrier-free [3ZP]orthophosphate were obtained from Amersham International, U.K. [1-14 C]Arachidonic acid (52.1 m C i / m m o l ) and [3H]arachidonic acid (87.4 C i / m m o l ) were from New England Nuclear, Boston, MA. Aspirin, apyrase, creatine phosphate/creatine phosphokinase, dibutyryl cyclic AMP, EDTA, fatty acids (99% pure), 1,2-dipalmitin (99% pure) and phosphatidic acid (99% pure) were from Sigma Chemical Co., St. Louis, MO. 5,8,11,14-Eicosatetraynoic acid was a gift from Nippon Roche Co., Tokyo. Fatty acids, aspirin and 5,8,11,14-eicosatetraynoic acid were dissolved in ethanol. Silica-gel(60)-precoated plates with a concentration zone were from E. Merk, Darmstadt. Lactate dehydrogenase assay kits were from Nippon Shoji Co., Tokyo. Platelet preparation. Venous blood was freshly drawn from healthy donors who had not taken any drugs for at least 2 weeks. The blood was mixed with 1 / 9 vol. of 3.8% (w/v) trisodium citrate and was centrifuged at 200 × g for 8 min at 22°C. The upper phase, platelet-rich plasma, was recovered and 1 / 6 vol. of an anticoagulant solution (2.2% trisodium citrate/2.2% glucose/0.8% citric acid (w/v)) was added. The mixture was centrifuged at 600 × g for 12 min. The platelet pellet was washed twice with 15 mM Tris-HC1 buffer (pH 6.5)/134 mM NaC1/5 mM glucose/1 mM EDTA (washing buffer), and then resuspended to 3 - 1 0 8 / m l in 15 mM Tris-HC1 buffer (pH 7.4)/134 mM N a C I / 5 mM glucose (suspension buffer).

Measurement of platelet aggregation and serotonin release. A washed platelet suspension

(3 - 108/ml) was labeled by incubating [14C]serotonin (0.1 # C i / m l platelet suspension) for 60 min at 30°C. The amount of [14C]serotonin in platelets reached a plateau (75 80% of the total count) at 60 min after the incubation. Aggregation of the [14C]serotonin-labeled platelets was monitored photometrically at 370(;` by use of Niko Biosscience four-channel aggregometer (model PAT-4A). The labeled platelet suspension (200 /al) and 1 ~tl of 200 mM CaC12 were stirred in the aggregometer at 37°C for 2 rain and were then stimulated with fatty acid. After 3 rain the reaction was terminated by adding 50/zl of 0.5% glutaraldehyde and cooling the sample in an ice bath. Serotonin release was calculated according to the method of Holmsen et al. [10] with a slight modification, i.e., % secreted = [(S t - Sc )/( T - S~ )] × 100, where T, ,% and S t stand for ~4C radioactivity in uncentrifuged cuvette contents, in supernatants of centrifuged cuvette contents not incubated with the fatty acid(s) and in supernatants of cuvette contents indubated for 3 min at 37°C with the fatty acid(s), respectively.

Preparation of [~H]arachidonic acid-labeled platelets. After centrifugation of the platelet-rich plasma, the pellet was suspended in 0.5 vol. of the suspension buffer containing 0.1% bovine serum albumin. The platelet suspension was incubated with [3H]arachidonic acid (0.5 ~ C i / m l ) at 37°C for 60 rain. The labeled platelets were washed twice with the washing buffer and resuspended to 3- 108/ml in the suspension buffer. Preparation of ~-'P-labeled platelets. After centrifugation of the platelet-rich plasma (25-40 ml), the pellet was suspended in 1.5 ml of the suspension buffer. The platelet suspension was incubated with [32P]orthophosphate (0.5 mCi) at 37°C for 60 min. The labeled platelets were washed once with the washing buffer and resuspended to 3- 108/ml in the suspension buffer.

Measurement of metabolites from added [NC]arachidonic acid, and of [3:P]phosphatidic acid, [~H]phosphatidic acid, [3H]diacylglycerol and protein phosphorvlation. The unlabeled or labeled platelet suspension, stirred in I mM CaCI: solution in the aggregometer at 37°C for 2 min, was stimulated with ~4C-labeled or unlabeled arachidonic acid. At various times as indicated, the reaction was terminated by adding 8 vol. of chloro-

285

form/methanol (1:1). The lipids were extracted and separated by thin-layer chromatography [11]. Metabolites from [14C]arachidonic acid, and [32p]phosphatidic acid were localized by autoradiography. [3H]Phosphatidic acid and [3H]diacylglycerol were localized by co-chromatography with unlabeled standards which were visualized by iodine vapor. The spots were scraped off and counted in a Packard Prias liquid scintillation counter (model 400 CL/D). For measurement of protein phosphorylation, aliquots (200/tl) were quenched by adding 100/~1 3-fold concentrated Laemmli sample buffer [12] and incubated at 100°C for 3 min. The sample was subjected to sodium dodecyl sulfate polyacrylamide slab gel electrophoresis under the conditions descrtibed by Laemmli [12]. The separating and stacking gels contained 13 and 4.4% acrylamide, respectively. Gels were stained with Coomassie brilliant blue, dried and subjected to autoradiography. Detection of platelet lysis. The washed platelet suspension stirred in 1 mM CaCI= solution in the aggregometer at 37°C for 2 min was stimulated with various concentrations of fatty acids. After 3 min the suspension was transferred on the top of silicone oil in a tube and the platelets sedimented by immediate centrifugation for 30 s at 6000 x g in a Fisher centrifuge (Model 59). Lactate dehydrogenase activity of the upper phase was calculated as a percentage of the lactate dehydrogenase activity released from the platelets after freezing and thawing three times. Data anslysis. Tests of the statistical significance of differences were performed by using analysis of variance. Results

Platelet aggregation and serotonin release induced by arachidonic acid Less than 60 #M arachidonic acid did not induce lactate dehydrogenase loss, although, above 70 /~M, the loss was greater and dose-dependent. Therefore, we investigated platelet aggregation and serotonin release at concentrations of below 60 /~M arachidonic acid where platelet lysis was not observed and a rise in light transmission merely indicated platelet aggregation.

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Fig. 1. Platelet aggregation and serotinin release induced by various concentrrations of arachidonic acid. After preincubation for more than 5 min at room temperature with ethanol ( x ) or aspirin (500 # M ) (O), [14C]serotonin-labeled platelet suspensions, stirred in 1 m M CaC12 solution in the aggregometer at 37°C for 2 min, were stimulated with various concentrations of arachidonic acid for 3 min. The final concentration of ethanol in platelet suspensions was 0.75% in all experiments. Three separate experiments were performed. Although quantitative responses were different in each experiment, the general tendency of responses was almost the same. Therefore we present one typical experiment, Each dot in (a) represents the m a x i m u m platelet aggregation within 3 min and in (b) represents serotonin release at 3 rain after the addition of arachidonic acid.

At concentrations of less than 2 /~M, arachidonic acid induced platelet aggregation and serotonin release dose-dependently, while at concentrations between 2 and 20 /xM, they decreased dose-dependently. These platelet responses were suppressed by aspirin. However, more than 30/~M arachidonic acid again caused dose-dependent platelet aggregation and serotonin release, which

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© 60 Fig. 2. Effects of aspirin and 5,8,11,14-eicosatetraynoic acid on platelet aggregation induced by 40 # M arachidonic acid. After preincubation with ethanol (a), aspirin (500 p,M) (b), or aspirin (500 `aM)+5,8,11,14-eicosatetraynoic acid (2 ~ M ) (c) at room temperature for more than 5 rain, washed platelet suspensions, stirred in 1 m M CaCI 2 solution in the aggregometer at 37°C for 2 min, were stimulated with 40 `aM [14C]arachidonic acid. The final concentration of ethanol in platelet suspensions was 0.8% in all experiments. Although three separate experiments were performed, one typical experiment is presented.

were not suppressed by aspirin (Fig. 1) or even by aspirin and 5,8,11,14-eicosatetraynoic acid (Fig. 2). Although 5,8,11j4-eicosatetraynoic acid inhibited both cyclooxygenase and lipoxygenase [20,21], we used aspirin and 5,8,11,14-eicosatetraynoic acid to ensure the block of both enzymes by use of a very small dose of 5,8,11,14-eicosatetraynoic acid. In aspirin + 5,8,11,14-eicosatetraynoic acid-treated platelets, an irreversible aggregation was induced by 40 ~M arachidonic acid, while in untreated and in aspirin-treated platelets, a reversible reversible aggregation was induced (Fig. 2). In addition, the maximum values of the platelet aggregation and serotonin release, 3 min after the addition of 40 ktM arachidonic acid, were in increasing order: untreated < aspirin-treated < aspirin + 5,8,11,14eicosatetraynoic acid-treated platelets. Phosphorylation of M r 40 000 protein

40/~M arachidonic acid induced the phosphorylation of M r 40000 protein and this phosphorylation was not inhibited by aspirin. On the contrary, the phosphorylation of the protein induced by 2 /~M arachidonic acid was inhibited by aspirin (Fig. 3).

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Fig. 3. Autoradiograph of sodium dodecyl sulfate polyacrylamide slab gel of )~P-labeled platelets. After preincubation for more than 5 rain at room temperature with ethanol or aspirin (500 `aM), ~2P-laheled platelet suspensions incubated in 1 mM CaCI~ solution in the aggregometer at 37°C for 2 rain were stimulated with ethanol (1), I U / m l of thrombin (2), 2 tLM arachidonic acid (3). 20 `aM arachidonic acid (4) or 40 `aM arachidonic acid (5) for l rain. The final concentration of ethanol in platelet suspensions was 0.75% in all experiments.

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Arachidonic acid (t,M) Fig. 4. Effect of aspirin (500 `aM) on thromboxane B2 synthesis from exogenous arachidonic acid. After preincubation for more than 5 rain at room temperature with ethanol ( × ) or aspirin (500 a M ) (O), platelet suspensions stirred in 1 mM CaCI 2 solution in the aggregometer at 37°C for 2 rain were stimulated with various concentrations of [14C]arachidonic acid for 3 min. The final concentration of ethanol in platelet suspensions was 0.75% in all experiments. Results are expressed as the mean +_ S.E. of three separate experiments.

287

Formation of phosphatidic acid or 1,2-diacylglycerol by addition of arachidonic acid Arachidonic acid increased phosphatidic acid and 1,2-diacylglycerol production dose-dependently at concentrations of less than 20-30 /zM and the production reached a plateau at concentrations of more than 30 t~M arachidonic acid in aspirin-treated platelets. On the other hand, platelet aggregation increased dose-dependently at concentrations of more than 30 ~tM arachidonic acid in aspirin-treated platelets (Fig. 6). In addition, as shown in Table I, the maximum platelet aggregation increased in the order of: untreated < aspirin-treatred < aspirin + 5,8,11,14-eicosatetraynoic acid-treated platelets, whereas, the formation of [3H]phosphatidic acid and [3H]diacylglycerol was not affected by either aspirin, or aspirin + 5,8,11,14-eicosatetraynoic acid.

Metablism of added arachidonic acid The level of tromboxane ]~2 formed from exogenous arachidonic acid increased dose-dependently and reached a plateau at concentrations of more than 30 /~M. The thromboxane Bz synthesis was markedly suppressed by aspirin (Fig. 4). Moreover, the formation of 12-hydroxy-5,8,10,14eicosatetraenoic acid, a lipoxygenase product, was inhibited by 5,8,11,14-eicosatetraynoic acid (Fig. 5b). The added arachidonic acid was scarcely metabolized in aspirin + 5,8,11,14-eicosatetraynoic acid-treated platelets and most of the arachidonic acid remained unchanged. On the other hand, most of the added arachidonic acid was converted to metabolites in untreated or aspirin-treated platelets (Fig. 5). As the amount of arachidonic acid in the medium, 30 s after the addition of 40 /~M arachidonic acid to aspirin + 5,8,11,14-eicosatetraynoic acid-treated platelet suspensions, was already only 9.3 + 3.1% (the mean + S.D., n = 3) of the added arachidonic acid, most of the added arachidonic acid which remained unchanged 3 min after the addition of 40 /~M arachidonic acid to aspirin + 5,8,11,14-eicosatetraynoic acid-treated platelet suspensions would exist in platelets.

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Fig. 5. Effects of aspirin and 5,8,11,14-eicosatetraynoic acid on the metabolism of arachidonic acid. After preincubated with ethanol (C)), aspirin (500 #M) ( :,<) or aspirin (500/~M)+5,8,11,14-eicosatetraynoic acid (2 ~tM) (A) at room temperature for more than 4 min, washed platelet suspensions were stimulated with 40 # M []4C]arachidonic acid as described in the legend to Fig. 2. At indicated times, reactions were terminated, and [14C]arachidonic acid metabolites and unmetabolized [14C]arachidonic acid were measured. Platelets used in these experiments were the same as the ones used in the experiments of Fig. 2. The final concentrations of ethanol in platelet Suspensions were 0.8% in all experiments. HETE, 12-hydroxy-5,8,10,14-eicosatetraenoic acid.

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EFFECTS OF ASPIRIN AND 5,8,11,14-EICOSAT E T R A Y N O I C A C I D ON PLATELET A G G R E G A T I O N A N D F O R M A T I O N OF [3H]PHOSPHATIDIC A C I D A N D [ 3 H ] D I A C Y L G L Y C E R O L BY T H E A D D I T I O N OF 40 #M ARACHIDONIC ACID

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(500 #M) and 5+8,11,14-eicosatetraynoic acid (2 #M) at room temperature for more than 5 rain, [3H]arachidonic acid-labeled platelet suspensions were incubated in 1 m M CaCI 2 solution in thc aggregometer at 37°C for 2 min and then stimulated with 40 ftM arachidonic acid for 2 rain. The final concentration of cthanol was 0.8~ in all experiments. Platelet aggregation represents the m a x i m u m within 2 min. Results are expressed as the m e a n + S . D , of one experiment performed in quaduplicates. Although three separate cxperiments were performed, one typical experiment is presented. Results of three experiments showed the same tendency

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Fig. 6. Effects of various concentrations of arachidonic acid on the formation of phosphatidic acid or 1,2-diacylglycerol and on aggregation in aspirin-treated washed platelets. After preincubation with aspirin (500/~M) at room temperature for more than 5 rain, 32p-labeled or [3H]arachidonic acid-labeled platelet suspensions stirred in 1 m M CaCI 2 solution in the aggregometer at 37°C for 2 rain were stimulated with various concentrations of arachidonic acid. After 2 min, the reactions were terminated and [32p]phosphatidic acid or [3H]diacylglycerol measured. [32p]Phosphatidic acid or [3H]diacylglycerol (o) is expressed as a percentage of the control value. Platelet aggregation ((D) represents the m a x i m u m within 2 rain. The final concentration of ethanol in platelet suspensions was 0.75% in all experiments.

" P < 0.0l compared with the control. h p < 0.001 compared with the control. " P < 0.001 compared with the aspirin-treated platelets.

platelet aggregation and serotonin release dose-dependently at concentrations of more than 30 /zM. On the other hand, the saturated fatty acids tested did not induce platelet aggregation and serotonin release at concentrations of below 100/zM.

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Fig. 7. Platelet aggregation and serotonin release induced by oleic, linoleic or linolenic acid. [14C]Serotonin-labeled platelet suspensions stirred in 1 m M CaCI 2 solution in the aggregometer at 37°C for 2 min were stimulated with various concentrations of oleic, linoleic or linolenic acid for 3 min. The final concentration of ethanol in platelet suspensions was 0.5% in all experiments. Although three separate experiments were performed, one typical experiment is presented. Platelet aggregation ( × ) represents the m a x i m u m within 3 min and serotonin release ( O ) represents that at 3 min. None of the fatty acid induced lactate dehydrogenase loss from platelets at the concentrations tested.

289

T A B L E 11 EFFECTS O F V A R I O U S INHIBITORS ON P L A T E L E T A G G R E G A T I O N A N D SEROTON1N RELEASE I N D U C E D BY A R A C H I D O N 1 C A C I D (2 /~M, 40 ~M), OLEIC A C I D (60 /~M), LINOLEIC A C I D ( 6 0 / t M ) A N D L I N O L E N I C A C I D (60 /~M) After preincubation with each inhibitor at a room temperature for 5 min, [14 C]serotonin-labeled platelet suspensions were incubated in 1 m M CaC12 solution in the aggregometer at 37°C for 2 min and then stimulated with fatty acid. The m a x i m u m platelet aggregation within 3 min, and serotonin release at 3 min, were compared with those of the control ( + + + + ). ( - ) indicates that platelet aggregation or serotonin was not induced. The final concentration of ethanol was 0.5% in all experiments. AA, arachidonic acid; OA, oleic acid; LA, linileic acid; LN, linolenic acid; A, aggregation; R, serotonin release; CP, creatine phosphate; CPK, creatine phosphokinase Addition None (control) EDTA (1 mM) Dibutyryl cyclic A M P (2 mM) Apyrase (2 m g / m l ) C P / C P K (3 m M / 1 5 units per ml)

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Effects of various inhibitors on platelet aggregation and serotonin release induced by arachidonic acid (2 I~M, 40 IxM), oleic acid (60 I~M), linoleic acid (50 I~M) and linolenic acid (60 txM) We used EDTA (1 mM), dibutyryl cyclic AMP (2 mM), apyrase (2 mg/ml) and creatine phosphate (3 mM)/creatine phosphokinase (15 units/ml) as inhibitors. Platelet aggregation and serotonin release induced by 2 ~tM arachidonic acid were completely inhibited by dibutyryl cyclic AMP, apyrase and creatine phosphate/creatine phosphokinase. EDTA also completely inhibited platelet aggregation but partially inhibited serotonin release induced by 2 /tM arachidonic acid. On the other hand, platelet aggregation and serotonin release induced by 40 /~M arachidonic acid were not inhibited by apyrase and creatine phosphate/creatine phosphokinase. EDTA did not prevent serotonin release, but completely inhibited platelet aggregation induced by 40/tM arachidonic acid. Dibutyryl cyclic AMP partially prevented platelet aggregation but scarcely affected serotonin release induced by 40 /zM arachidonic acid. The effects of these inhibitors on platelet responses induced by oleic, linoleic and linolenic acid were almost the same as those induced by 40 ffM arachidonic acid (Table II).

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Fig. 8. Combined effects of arachidonic and oleic acid on platelets pretreated with aspirin (500 /~M) and 5,8,11,14eicosatetraynoic acid (2 /tM). After preincubation with aspirin (500 /tM)+5,8,11,14-eicosatetraynoic acid (2 /~M) at room temperature for more than 5 min, [14qserotonin-labeled platelet suspensions stirred in 1 m M CaCl 2 solution in the aggregometer at 37°C for 2 min were stimulated with 10 ~M arachidonic acid (A), 10 ~tM arachidonic a c i d + 1 0 p.M oleic acid (B), 10 ~ M arachidonic acid + 20/zM oleic acid (C), or 10 p.M arachidonic a c i d + 3 0 # M oleic acid (D) for 3 min. The final concentration of ethanol in platelet suspensions was 1.0% in all experiments. Lactate dehydrogenase loss was not induced. Platelet aggregation and serotonin release were not induced by less than 15 p,M arachidonic acid or less than 30 /~M oleic acid alone.

290

Combined effects of two kinds of unsaturated fat O' acid on platelets The combination of two kinds of unsaturated fatty acid increased the effects on platelets. Fig. 8 shows combined effects of arachidonic acid and oleic acid on platelets treated with aspirin (500 /~M) and 5,8,11,14-eicosatetraynoic acid (2 /~M). Platelet aggregation and serotonin release were not induced by below 15 t~M arachidonic acid or below 30 /~M oleic acid, whereas these were induced by the simultaneous addition of 10 /~M arachidonic acid and 10 ~M oleic acid. Moreover, platelet responses increased as a function of the concentration of the oleic acid added to a platelet suspension if the concentration of arachidonic acid was held constant at 10/~M. Discussion

The study indicates that high concentration (30-50/~M) of arachidonic acid can induce platelet aggregation and serotonin release independent of prostaglandin endoperoxides and thromboxane A 2. Some investigators have already reported that thromboxane A2-dependent platelet aggregation and serotonin release induced by a low concentration of arachidonic acid yielded a bell-shaped concentration response curve in platelet-rich plasma [7] and in a gel-filtered [7,9] or washed platelet suspension [8,13]. Although the cause of this turnoff of the platelet responses was not clear, it was suggested that it was due to an increase in cyclic AMP in platelets [9] or an increase in lipoxygenase products [13]. A high concentration of arachidonic acid induced the phosphorylation of M r 40000 protein in the same way as a low concentration of arachidonic acid [19] or other agents, such as thrombin [14,15], collagen [15], divalent cation ionophore A23187 [15] and platelet activating factor. [16,18], supporting evidence that the platelet responses were not due to membrane damage. Neither were the thromboxane A2-independent platelet responses, induced by a high concentration of arachidonic acid, due to lipoxygenase products because the responses were not inhibited by 5,8,11,14-eicosatetraynoic acid. The results indicate that a high concentration of arachidonic acid itself activates platelets. Also the unsaturated fatty acids tested, other than arachidonic acid, activated

platelets, whereas the saturated fatty acids tested did not. In addition, EDTA, dibutyryl cyclic AMP, apyrase and creatine phosphate/creatine phosphokinase brought about almost the same effects on platelet responses induced by the other unsaturated fatty acids as those on the responses induced by a high concentration of arachidonic acid. These results suggest that the mechanism of the action of a high concentration of arachidonic acid is the same as that of the other unsaturated fatty acids, and unsaturated bonds are needed to activate platelets. Haslam [22] and Hoak et al. [17] reported that even saturated fatty acids induced human platelet aggregation. In addition, Hoak et al. [17] reported that unsaturated fatty acids induced platelet aggregation even in platelet-rich plasma. On the other hand, we could not observe platelet aggregation after the addition of 8 mM or less arachidonic acid to aspirin-treated platelet rich plasma, although 8 mM arachidonic acid caused serotonin release without lactate dehydrogenase loss. The reasons for the discrepancies between our results and theirs are unknown. The mechanism by which arachidonic acid itself induces platelet responses is not clear. Although it had been recently reported that accumulation of phosphatidic acid or 1,2-diacylglycerol in platelets induced responses [16,18,19], platelet responses induced by a high concentration of arachidonic acid could not be attributed to accumulation of phosphatidic acid a n d / o r 1,2-diacylglycerol, because there was no significant correlation between platelet aggregation and the amount in platelets formed by the addition of a high concentration of arachidonic acid. More than 30 /~M arachidonic acid induced irreversible platelet aggregation in aspirin + 5,8,11,14-eicosatetraynoic acid-treated platelets which scarcely metabolized arachidonic acid via the cyclooxygenase and the lipoxygenase pathway, but induced reversible platelet aggregation in untreated and aspirin-treated platelets which metabolized the fatty ac.id. Moreover, oleic, linoleic or linolenic acids, which platelets scarcely metabolized via the pathways, induced irreversible platelet aggregation even in untreated platelets. These results suggest that a high concentration of unsaturated fatty acid itself might activate platelets directly. Because unsaturated fatty acids work additively

291 o n p l a t e l e t s to i n d u c e r e s p o n s e s , the a m o u n t s of u n s a t u r a t e d fatty acids in p h o s p h o l i p i d s of p l a t e lets [1,23] m a y b e e n o u g h , as a w h o l e , to i n d u c e platelet responses independent of prostaglandin e n d o p e r o x i d a s e s a n d t h r o m b o x a n e A 2. H o w e v e r , it r e m a i n s u n k n o w n w h e t h e r the f a t t y a c i d s a c t i v a t e platelets under some physiological conditions. F u r t h e r studies will b e n e e d e d o n the m e c h a n i s m of the effects of a h i g h c o n c e n t r a t i o n o f a r a c h i d o n i c a c i d a n d o t h e r u n s a t u r a t e d fatty acids o n platelets.

Acknowledgements W e t h a n k D r s . M a k o t o K i n o s h i t a of o u r d e p a r t m e n t a n d H i r o s h i H a y a s h i of the T o k y o T e i s h i n H o s p i t a l , for h e l p f u l d i s c u s s i o n s a n d M i s s A s a m i T a j i m a for h e r e x c e l l e n t t e c h n i c a l assistance. T h i s w o r k has b e e n s u p p o r t e d in p a r t b y a g r a n t for scientific r e s e a r c h f r o m the M i n i s t r y of E d u c a t i o n , S c i e n c e a n d C u l t u r e of J a p a n .

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