15-hydroperoxyeicosapentaenoic acid inhibits arachidonic acid metabolism in rabbit platelets more potently than eicosapentaenoic acid

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ELSEVIER

Biochimica et Biophysica Acta 1300 (1996) 171-176

Biochi~ic~a et BiophysicaA~ta

15-Hydroperoxyeicosapentaenoic acid inhibits arachidonic acid metabolism in rabbit platelets more potently than eicosapentaenoic acid Masahide Tsunomori, Yohko Fujimoto, Emiko Muta, Hiroko Nishida, Satoru Sakuma, Tadashi Fujita * Department of Hygienic Chemistry, Osaka Universi~" of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-11, Japan

Received 14 July 1995; revised 3 November 1995; accepted 7 December 1995

Abstract The effect of 15-hydroperoxy-5,8,11,13,15-eicosapentaenoic acid (15-HPEPE), a hydroperoxy adduct of eicosapentaenoic acid (EPA), on the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), thromboxane (TX) B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) from exogenous arachidonic acid in washed rabbit platelets was examined. 15-HPEPE inhibited 12-HETE, TXB2 and HHT formation at concentrations ranging from 2 to 8 ~M. The inhibitory effect of 15-HPEPE was dose-dependent (12-HETE, 16.0-82.9% inhibition; TXB 2, 16.7-57.2% inhibition; HHT, 4.6-52.0% inhibition). EPA inhibited the production of these three metabolites, but the inhibitory effect was kept low (20-100 /zM: 12-HETE, 8.3-31.1% inhibition; TXB 2, 18.9-49.5% inhibition; HHT, 12.5-41.7% inhibition) as compared with 15-HPEPE. Experiments utilizing 15-hydroxy-5,8,11,13,15-eicosapentaenoic acid and hydroxyl radical scavengers (dimethyl sulfoxide and mannitol) revealed that 15-HPEPE exerted its effect in the form of the hydroperoxy adduct. These results suggest that 15-HPEPE has the potential to modulate the activities of the cyclo-oxygenase and 12-1ipoxygenase in platelets. This may also be one convincing mechanism for the anti-thrombotic and anti-atherosclerotic actions of EPA. Kevwords: 15-Hydroperoxy-5,8,11,13,15-icosapentaenoic acid; Icosapentaenoic acid; Arachidonic acid metabolism; Lipid hydroperoxide; Platelet

1. I n t r o d u c t i o n

In platelets, arachidonic acid (AA) is converted into thromboxane (TX) A 2 and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) by the cyclo-oxygenase pathway and into 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12HETE) by the 12-1ipoxygenase pathway [1]. TXA 2 is a potent vasoconstrictor and inducer of platelet aggregation and rapidly breaks down to form the stable end-product, TXB 2. 12-HETE has been reported to give rise to platelet aggregation [2,3], and neutrophil [4] and aortic smooth muscle cell migration [5]. So, it seems likely that 12-HETE

Abbreviations: 15-HPEPE, 15-hydroperoxy-5,8,11,13,15-eicosapentaenoic acid; 15-HEPE, 15-hydroxy-5,8,11,13,15-eicosapentaenoic acid; 12-HPETE, 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid; 12-HETE, 12-hydroxy-5,8,10,14-eicosatetraenoic acid; 15-HPETE, 15-hydroperoxy5,8,11,13-eicosatetraenoic acid; 13-HPODE, 13-hydroperoxy-9,11-octadecadienoic acid; TX, thromboxane; HHT, 12-hydroxy-5,8,10-heptadecatrienoic acid; AA, arachidonic acid; ADAM, 9-anthryldiazomethane; PG, prostaglandin; DMSO, dimethyl sulfoxide. * Corresponding author. Fax: +81 726 901005. 0005-2760/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 0 0 5 - 2 7 6 0 ( 9 5 ) 0 0 2 4 3 - X

as well as TXA 2 is involved in the initiation and propagation of thrombotic and atherosclerotic disorders. One of polyunsaturated fatty acids, eicosapentaenoic acid (EPA, 2 0 : 5 ( n - 3)) is present in large amounts in certain marine fish. A low incidence of thrombotic and atherosclerotic events by an intake of EPA has been reported by Dyerberg, Bang and their colleagues [6-10]. This could be explained partially by the interaction between this fatty acid and platelet AA metabolism [8-11]. It has been reported that EPA ingested inhibits AA metabolism in platelets [9-11]. However, the mechanism by which EPA inhibits the platelet AA metabolism has not yet been understood fully . TXA 2 and 12-HETE are formed from AA through hydroperoxide intermediates prostaglandin (PG) G 2 and 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12HPETE). Previous studies have shown that PGG 2 activates cyclo-oxygenase activity [12] and that 12-HPETE can regulate cyclo-oxygenase and lipoxygenase activities [13]. Recently, we have shown that a hydroperoxy intermediate of linoleic acid, 13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE), formed by cyclo-oxygenase or lipoxygenase,

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can be a selective inhibitor of platelet cyclo-oxygenase activity [14]. These observations imply that hydroperoxy intermediates of fatty acids can modify platelet cyclooxygenase a n d / o r lipoxygenase activity. On the other hand, several investigators [15-17] have reported that EPA is metabolized into various products including its hydroperoxy adducts. In aorta [16] and neutrophils [17], EPA is converted to 15-hydroperoxy5,8,11,13,15-eicosapentaenoic acid (15-HPEPE) by 15lipoxygenase. Wong et al. [18] have found that 15-HPEPE is further transformed to 5-series lipoxins by porcine leukocyte 5-1ipoxygenase. Lam and Wong [17] have shown that 5-series lipoxins have regulatory activities, which are of interest in vascular and inflammatory events. In platelets, it has also been shown that 15-1ipoxygenase or 15-1ipoxygenase-like activity exists [19-22]. Thus, it is possible that 15-HPEPE is produced from EPA in the cells. The present study was therefore undertaken to investigate whether 15-HPEPE can affect the two enzymatic pathways of AA in platelets.

2. Materials and methods

2.1. Materials TXB 2 and sodium salts of AA and EPA were purchased from Sigma (St. Louis, MO, USA) and HHT was from Cayman (Michigan, USA). 12-HETE, 15-HPEPE and 15hydroxy-5,8,11,13,15-eicosapentaenoic acid (15-HEPE) were obtained from Cascade Biochem (Berkshire, England) and 9-anthryldiazomethane (ADAM) was from Funakoshi Pharmaceutical (Tokyo, Japan). All other reagents were of analytical grade.

15-HEPE. 15-HPEPE and 15-HEPE were dissolved in ethanol. The final concentration of ethanol in platelet suspension was held constant at 2.5% ( v / v ) in all experiments. Ethanol at 2.5% had no effect on AA metabolism in platelets. AA (40 /zM) was subsequently added to the platelet suspension, and the mixture was incubated at 37°C lbr 5 rain. The reaction was terminated by quickly adding an appropriate amount of 0.25 N HCI to bring the pH of the reaction mixture to 3.0. In experiments utilizing mannitol or dimethyl sulfoxide (DMSO), the platelet suspension was preincubated for 5 rain at 37°C in buffer A with or without mannitol or DMSO prior to the incubation with AA for 5 rain, and 15-HPEPE was added at t = 4 rain to the preincubations.

2.4. Measurement of 12-HETE, TXB2, HHT. 15-HEPE and AA After incubation, the reaction mixture was extracted with 3 ml of ethyl acetate. 12-HETE, TXB 2, HHT, 15HEPE and AA in the extracted lipid were simultaneously determined by a HPLC method as described in our recent studies [23,24]. Briefly, 12-HETE, HHT and 15-HEPE were separated in normal-phase chromatography and simultaneously quantitated by employing a UV spectrophotometric detector. TXB~ and AA were measured after esterification using ADAM. TXB 2 and AA esterified with ADAM were separated in reverse-phase chromatography and simultaneously quantitated by employing a fluorescence spectrofluorometer. Our previous study utilizing indomethacin, an inhibitor of cyclo-oxygenase [25], and quercetin, an inhibitor of lipoxygenase [26,27], has demonstrated the capacity of the present in vitro system to simultaneously detect changes in the activities of platelet cyclo-oxygenase and lipoxygenase [28].

2.2. Preparation of platelets 2.5. Statistical analysis Blood was withdrawn into a 3.8% solution of trisodium citrate (9:1, v / v ) from the abdominal aorta of male rabbits (2-2.5 kg) under sodium pentobarbital anaesthesia. Platelets were then collected by differential centrifugation. Whole blood was centrifuged for 10 min at 200 × g at room temperature and the platelet-rich plasma was withdrawn from above the pelleted erythrocytes. Alter addition of EDTA (to a final concentration of 1 raM), the plateletrich plasma was cooled to 0°C and centrifuged at 2000 X g for 10 min. The platelet pellet was washed twice with 134 mM NaCI, 5 mM glucose, 15 mM Tris-HCl buffer (pH 7.4; buffer A) containing 1 mM EDTA, and then resuspended in buffer A.

2.3. Incubation of platelets The washed platelet suspension (3 × l0 s platelets) was preincubated for 5 rain at 37°C in 1 ml of buffer A with or without the indicated concentrations of EPA, 15-HPEPE or

The data shown are means _+ S.E. Statistical analyses were performed with Student's t-test and P < 0.05 was considered as significant.

3. Results and discussion

Fig. 1 illustrates the effect of various concentrations of 15-HPEPE on the formation of 12-HETE, TXB 2 and HHT from exogenous AA in rabbit platelets. By being preincubated with platelets for 5 rain before an addition of AA, 15-HPEPE inhibited the formation of 12-HETE, TXB~ and HHT at concentrations ranging from 2 to 8 /xM. The effect of 15-HPEPE was concentration-dependent (12-HETE, 16.0-82.9% inhibition; TXB2, 16.7-57.2% inhibition; HHT, 4.6-52.0% inhibition). In addition, the amount of AA remaining after incubation was quantified by HPLC. In the control experiment, when the platelets were preincu-

M. Tsunomori et al. / Biochimica i0

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Biophysica Acta 1300 (1996) 171-176

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Fig. I. Effect of 15-HPEPE on the formation of 12-HETE, TXB~ and HHT in washed rabbit platelets. Platelets (3 × 1 0 a / m l ) were preincubated with various concentrations of 15-HPEPE for 5 rain at 37°C prior to the incubation with arachidonic acid (40 /xM) for 5 min. Each point indicates the mean of four experiments; vertical lines show S.E. * P < 0.05, • P < 0 . 0 1 ; compared to corresponding value in the absence of 15HPEPE.

bated in the absence of 15-HPEPE followed by the addition of A A (40 /xM), 13.8 _+ 1.0 nmol A A was detected (n = 4). Raising the 15-HPEPE concentration from 0 to 8 /xM increased the amount of AA from 13.8 4-1.0 to 31,5 _+ 3.2 nmol (n = 4). This result lessens the possibility that 15-HPEPE-induced reduction of 12-HETE, TXB 2 and HHT formation can be ascribed to decreased availability of AA by its peroxidative action. Further, we investigated the effect of 15-HPEPE on the platelet 12-1ipoxygenase and cyclo-oxygenase activities using the broken cell preparation (washed rabbit platelets were lysed by freeze-thawing three times). 15-HPEPE inhibited the formation of 12HETE, TXB 2 and HHT from exogenous AA (40 /xM) at concentrations ranging from 2 to 8 /xM (12-HETE, 2 8 . 6 81.4% inhibition; TXB 2, 13.1-54.8% inhibition; HHT, 15.0-42.5% inhibition; n = 3) (data not shown). These results show that the 12-1ipoxygenase and cyclo-oxygenase activities in platelets can be inhibited by 15-HPEPE. As shown in Fig. 2, the inhibitory effects of 15-HPEPE (6 /xM) on 12-HETE, TXB 2 and HHT formation were apparent within 30 s after addition to the preincubation mixture. 12-HETE formation was decreased depending on the duration of preincubation time. On the other hand, 75-78% inhibition of TXB 2 and HHT formation was observed at 30 s. EPA, the native fatty acid of 15-HPEPE, has been reported to inhibit the A A metabolism in platelets. The chemical structure of EPA resembles that of A A with only one additional double bond. This makes EPA a fairly good candidate for competing the cell metabolism of A A [10]. Needleman et al. [11] have also reported that this polyunsaturated fatty acid can inhibit the platelet cyclooxygenase directly. To compare the effect of 15-HPEPE on the formation of 12-HETE, TXB 2 and HHT with the

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Fig. 2. Effect of the length of preincubation of platelets with I5-HPEPE on the formation of 12-HETE, TXB 2 and HHT in washed rabbit platelets. Platelets (3 × 1 0 ~ / m l ) were preincubated with ethanol (control) or 6 /xM 15-HPEPE at 37°C. At various times as indicated, arachidonic acid (40 /xM) was added to the mixture. After 5 min, the reaction was terminated by an addition of the extraction solvent. Each point indicates the mean of three experiments.

native fatty acid, EPA was subjected to the same experimental protocols as applied to 15-HPEPE (Fig. 3). EPA showed no significant effect on the formation of 12-HETE, TXB 2 and HHT at concentrations of 20 and 40 p~M. At a concentration of 100 /xM, it inhibited the formation of TXB 2 and HHT, and showed an inhibitory tendency on the formation of 12-HETE. This finding shows that EPA inhibited the generation of cyclo-oxygenase and 12-1ipoxygenase metabolites at concentrations over 10-times higher than 15-HPEPE. In platelets, EPA is known to be transformed into various hydroperoxy adducts by lipoxygenases or cyclo-oxygenase [8,15-17]. However, Morita et al. [ 15] 2

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Fig. 3. Effect of EPA on the formation of 12-HETE, TXB 2 and HHT in washed rabbit platelets. Platelets (3;,< 1 0 S / m l ) were preincubated with various concentrations of EPA for 5 min at 37°C prior to the incubation with arachidonic acid (40 /xM) for 5 min. Each point indicates the mean of three experiments; vertical lines show S.E. * P < 0.05; compared to corresponding value in the absence of EPA.

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Table 1 Effect of 15-HEPE on the formation of 12-HETE, TXB2 and HHT in washed rabbit platelets Pretreatment

12-HETE

TXB,

HHT

1.61 +0.14 1.88 ± 0.05 1.96±0i2 2.17±0.11 2.1l_+0.06

1.26_+0.08 1.36 ±0.03 1.42±0.04 1.52±0.02 1.49±0.05

(nmol) Control 15-HEPE 2 ,aM 4 ,aM 6,aM 8 ,aM

6.61 ±0.87 6.34±0.11 5.99 ± 0.08 6.11±0.39 5.80±0.21

Platelets (3 x 10S/ml) were preincubatedwith various concentrationsof 15-HEPE for 5 min at 37°C prior to the incubationwith arachidonic acid (40 ,aM) for 5 min. Values are meansiS.E. (n = 3).

have shown that when platelets are incubated with EPA alone, very few cyclo-oxygenase and lipoxygenase products are formed, but those products appear when AA is co-incubated with EPA. In the present study, washed rabbit platelets were pretreated with EPA, and after 5 min AA was added. This means that, in this study, during preincubation time, EPA is not converted into lipoxygenase and cyclo-oxygenase metabolites. So, EPA in itself may exhibit much weaker inhibitory effect on the cyclooxygenase and lipoxygenase activities in platelets than does 15-HPEPE. Recently, some interesting results were shown concerning the physiological and pathological roles of the hydroxy adducts of polyunsaturated fatty acids. It has been reported that the hydroxy form of docosahexaenoic acid activates 5-1ipoxygenase activity in guinea pig lung parenchymal strip [29]. Also it has been shown that the hydroxy adduct of linoleic acid, 13-hydroxy-9,1 l-octadecadienoic acid, inhibits the adhesion of platelets to endothelial cells [30], and modulates TXA 2 and 12-HETE formation in platelets [31]. We observed, in the present experiments, the rapid conversion of 15-HPEPE to 15-HEPE (platelets converted about 90% of added 15-HPEPE to 15-HEPE after a 30 s incubation at 37°C). Therefore, we examined the effect of 15HEPE on the formation of 12-HETE, TXB 2 and HHT in washed rabbit platelets (Table 1). In contrast to the effect of 15-HPEPE on the formation of 12-HETE, TXB 2 and

HHT, 15-HEPE showed no inhibitory effect on the formation of 12-1ipoxygenase and cyclo-oxygenase products at concentrations ranging from 2 to 8 /zM. This finding implies the requirement of the hydroperoxy moiety for the inhibitory effect of 15-HPEPE on the 12-HETE, TXB 2 and HHT formation. Egan et al. [32] have reported that PG cyclo-oxygenase is irreversibly self-deactivated due to the natural reduction of the hydroperoxide at carbon 15 of PGG 2 to the hydroxyl on PGH 2, and during this reduction, radicals, possibly hydroxyl radicals, are formed which could be oxidizing the enzyme. Ham et al. [33] and Weiss et al. [34] have shown that 12- and 15-HPETEs are, like PGG2, substrates for the peroxidase component of PG synthetase, thereby yielding hydroxyl radicals. Furthermore, they [33,34] found in the same experiments that a reactive radical detected during the reduction of the hydroperoxides was hydroxyl radical by using various hydroxyl radical scavengers. There may be a possibility, therefore, that 15-HPEPE is reduced to 15-HEPE by the same mechanism as 12- or 15-HPETE, and hydroxyl radicals being formed during this reduction. Thus, the inhibitory action of 15-HPEPE on platelet cyclo-oxygenase and lipoxygenase activity could be achieved either through a direct effect or via its participation in the formation of hydroxyl radicals. To clarify this point, we determined the effect of 15-HPEPE in the presence of mannitol or DMSO, a hydroxyl radical scavenger, on the production of 12-HETE, TXB 2 and HHT in washed platelets (Table 2). Mannitol (10 raM) or DMSO (10 raM) alone had no effect on the formation of 12-HETE, TXB 2 and HHT. They also did not alter the effect of 15-HPEPE (6 /xM) in suppressing 12-HETE, TXB 2 and HHT production. So, it appears that the action of 15-HPEPE on the cyclo-oxygenase and 12-1ipoxygenase cannot be explained by hydroxyl radicals which may be formed from 15HPEPE, suggesting that the inhibition of 15-HPEPE on platelet cyclo-oxygenase and 12-1ipoxygenase is due to its direct effect on these enzyme activities. This speculation may be supported, in part, by the previous our findings that tert-butyl hydroperoxide and 13-HPODE can modulate the platelet cyclo-oxygenase a n d / o r 12-1ipoxygenase in the form of the hydroperoxide [14,28].

Table 2 Effect of 15-HPEPE in the presence of hydroxyl radical scavengers on the formationof 12-HETE, TXB2 and HHT in washed rabbit platelets Pretreatment

12-HETE

TXB2

HHT

1.59 ± 0.23 1.85 _+0.16 1.67 ± 0.09 0.42 4- 0.07 0.42 ± 0.07 0.41 ± 0.02

1.27 ± 0.17 1.38 ± 0.07 1.30 ± 0.04 0.36 ± 0.06 0.31 + 0.01 0.33 ± 0.01

(nmol) Control Mannitol(10 mM) DMSO (10 mM) 15-HPEPE (6 ,aM) 15-HPEPE (6 ,aM) + Mannitol(10 mM) 15-HPEPE (6 ,aM) + DMSO (10 raM)

5.46 _ 0.84 5.80 ± 0.11 5.62 ___0.08 3.76 :f 0.39 3.50 ± 0.21 3.61 ± 0.23

Platelets (3 x 108/ml) were preincubatedwith or without mannitol(10 mM) or DMSO (10 mM) for 5 min at 37°C prior to the incubationwith arachidonic acid (40 ,aM) for 5 min. 15-HPEPE(6 /xM) was added at t = 4 min to the preincubations.Values are means ± S.E. (n = 3).

M. Tsunomori et al. / Biochimica et Biophysica Acta 1300 (1996) 171-176

T X A 2 is involved in the initiation and propagation of thrombotic and atherosclerotic disorders. The lipoxygenase metabolites as well as the cyclo-oxygenase metabolites have also been focused as mediators of thrombosis and atherosclerosis. A 12-1ipoxygenase product, 12-HETE, has been reported to amplify thrombin-induced aggregation and counteract the inhibitory effect of a c A M P elevator, PGE~ [2,3]. 12-HETE has also been reported to induce migration of smooth muscle cells from media to intima [5], which event is a significant process in the genesis of atherosclerosis. These reports imply that dual inhibition of the cyclo-oxygenase and 12-1ipoxygenase pathways in platelets may be beneficial to vascular diseases such as thrombosis and atherosclerosis. Populations like Eskimo whose diet contains increased amounts of the n - 3 fatty acid, EPA, are characterized by a low incidence of thrombosis and atherosclerosis [6-10]. This observation has led researchers to investigate the mechanism responsible for the beneficial effect of this fatty acid. Dyerberg and Bang [8] have shown that EPA is transformed into the less vasoconstrictive and less aggregative T X A 3 in platelets, in addition to the formation of the vasodilative and anti-aggregative PGI 3 in blood vessel wall. Several investigators have also made it clear that EPA inhibits the A A metabolism in platelets and suggested that this function may be elicited by the following manner; (a) incorporation of EPA into phospholipids in place of A A [9], (b) competition with A A for the platelet cyclo-oxygenase [10] and (c) inhibition of the platelet cyclo-oxygenase [11]. On the other hand, under physiological conditions, EPA exists in the tissues including platelets. It is known that most of EPAs are firstly incorporated into 2-position of membrane phospholipids in the cells, as well as A A , implying that, upon stimulation, A A and EPA are simultaneously released from the phospholipids by phospholipase A2. It has been reported that human platelets contain 15lipoxygenase [19,20]. Furthermore, human platelet 12lipoxygenase has been shown to have the capacity to act as 15-1ipoxygenase [21,22]. Rabbit peritoneal polymorphonuclear leukocytes [35] and human umbilical vein endothelial cells [36] have also been found to contain 15-1ipoxygenase, or 15-1ipoxygenase like activity. It has become obvious that interactions between the various lipoxygenases existing in platelets, endothelial cells and leukocytes occur and may have important biological significance [21,37-40]. For example, Serhan and Sheppard [21] have shown that co-incubation of human neutrophils and platelets leads to the formation of lipoxins from endogenous sources. Marcus et al. [37,38] have demonstrated that platelet-derived precursors (PG endoperoxides or 12-HETE) are utilized by endothelial cells or neutrophils in the formation of prostacyclin or dihydroxyeicosatetraenoic acid, respectively. Thus, it may be possible that 15- or 12-1ipoxygenase in platelets generates small amounts of 15-HPEPE from endogenous or exogenous EPA, and that leukocytes and endothelial cells can supply 15-HPEPE to platelets.

175

In the present study, we observed that 15-HPEPE inhibited the formation of platelet cyclo-oxygenase and 12lipoxygenase products more potently than did EPA. This finding serves to emphasize that 15-HPEPE can be a potent dual inhibitor of the cyclo-oxygenase and 12lipoxygenase in platelets. Therefore, it may be possible that in vivo 15-HPEPE produced from EPA in the presence of A A can be the potent endogenous regulator of the cyclo-oxygenase and 12-1ipoxygenase activities in platelets. Furthermore, it appears likely that the inhibitory action of EPA ingested on the A A metabolism in platelets is due, in part, to 15-HPEPE which is expected to be formed from the native fatty acid, and that this may be one convincing mechanism for the anti-thrombotic and anti-atherosclerotic actions of EPA. Further studies are needed to clarify the mechanism of modulation; however, we have provided the first direct evidence that 15-HPEPE has the potential to modulate platelet 12-1ipoxygenase and cyclo-oxygenase activities. These observations provide new insight into factors controlling the production of the metabolites derived from A A in platelets.

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