Effects of arachidonic acid on the metabolism of eicosapentaenoic acid in washed human platelets

THROMBOSIS RESEARCH 40; 307-317, I985 0049-3848/85 $3.00 t .OO Printed in the USA. Copyright (c) 1985 Pergamon Press Ltd. All rights reserved.

EFFECTS OF ARACHIDONIC ACID ON THE METABOLISM OF EICOSAPENTAENOIC ACID IN WASHED HUMAN PLATELETS Yoshiaki Hashimoto, Chikayuki Naito*, Tamio Teramoto, Hirokazu Kato, Mitsunobu Kawamura*, and Hiroshi Oka the First Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 3-1, Hongo 7-chome, Bunkyo-ku, Tokyo 113, Japan *the Department of Internal Medicine, The Tokyo Teishin Hospital 14-23, Fujimi 2-chome, Chiyoda-ku, Tokyo 102, Japan

(Received 30.5.1985; Accepted in revised form 1.8.1985 by Editor M. Matsuda)

ABSTRACT

We examined effects of arachidonic acid (AA) on eicosapentaenoic acid (EPA) metabolism in washed human Although human platelets had been considerplatelets. ed to metabolize scarcely EPA, a simultaneous addition of EPA and AA to washed platelet suspensions stimulated In addition, the stimulatory markedly EPA metabolism. effect was more potent over the formation of thromboxane (TX) B3 than that of 12-hydroxy-5,8,10,14,17eicosapentaenoic acid (HEPE). The stimulation by AA Indocan be due to AA itself and/or AA metabolites. methacin decreased the stimulatory effect of AA on the

Abbreviations:

AA, arachidonic acid; EPA, eicosapentaenoic acid; PG, prostaglandin; TX, thromboxane; HETE, 12-hydroxy-5,8,10,14-eicosatetraenoic acid; HEPE, 12-hydroxy-5,8,10,14,17-eicosapentaenoic acid; HHTE, 12-hydroxy-5,8,10,14_heptadecatetraenoic acia; HPETE, 12-hydroperoxy-5,8,10, 14-eicosatetraenoic acid; PRP, platelet rich plasma.

Key Words:

Eicosapentaenoic Acid Effect

Acid Metabolism,

307

Arachidonic

EPA METABOLISM BY PLATELETS

308

Vol. 40, No. 3

HEPE formation, suggesting that cyclooxygenase product(s) of AA stimulated the HEPE formation. Among the metabolites of AA investigated, prostaglandin (PG)Gz and 12-hydroperoxy-5,8,10,14_eicosatetraenoic acid had the stimulatory effect on both TXB2 and HEPE formations, whereas PGH2, PGD2, TXB;! and 12-hydroxy-5, 8,10,14-eicosatetraenoic acid were ineffective.

INTRODUCTION An increase in EPA in platelet membrane decreases platelet aggregability (l-3). This was partly explained by the antiaggregating activity of EPA metabolites produced via cyclooxygenase (4,5). However, since EPA was a poor substrate in vitro for platelet cyclooxygenase (4,6,7) some authors have claimed that EPA is scarcely metabolized in platelets and that EPA acts mainly as a competitive inhibitor of the oxidation of AA by cyclooxygenase (7,9). Recently, we (10) and other groups (11,121 have shown that EPA metabolism is promoted in the presence of AA. As EPA always coexisted with AA in platelet membrane lipid, we further examined in detail the stimulatory effect of AA on EPA metabolism and the mechanism of the stimulation in this paper. MATERIALS AND METHODS Materials ['4C(U)IEPA (212 mCi/mmol) and [1-14CIAA (52.1 mCi/mmol) were obtained from New England Nuclear, Boston, MA. AA (99% pure), indomethacin and aspirin were purchased from Sigma Chemical Co., St. Louis., MO. EPA (98% pure) was a gift from Nippon Oil and Fats Co., Tokyo. PGH2, PGD2 and TXB2 were gifts from the Ono Central Research Institute, Osaka. HPETE, HETE and PGG2 were purchased from Ran Biochem., Tel Aviv. Silica-gel (60)-precoated plates with a concentration zone were from E. All fatty acids and aspirin were dissolved in Merk, Darmstadt. ethanol, and indomethacin in dimethyl sulfoxide. 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 one-ninth volume of 3.8% (w/v) trisodium citrate and was centrifuged at 200 x g for 8 min at 22°C. The upper phase, PRP, was recovered and one-sixth volume of ACD (2.2% trisodium citrate, 2.2% glucose, 0.8% citric acid, w/v) was added. The further manipulations were performed at 0-4OC. The PRP-ACD mixture was centrifuged at 600 x g for 12 min. The platelet pellet was washed twice with 15 mM Tris-HCl buffer (pH 6.5) containing 134 mM NaCl, 5 mM glucose and 1 mM EDTA,and then resuspended to 3 xlO*/mR in 15 mM Tris-HCl buffer (pH 7.4) containing 134 mM NaCl and 5 mM glucose.

Vol. 40, No. 3

EPA METABOLISM BY PLATELETS

0.20-

309

0.08 ;

(A)

z * 0.15>al 'i5 E O.lO-

i 0.06 ; 0.04 ;

iii $

0.050L

0.02

10

0

20

30

40

F

0

AA

He-

_-p--------&,_____

p ---__

-------

a

AA (PM) FIG. 1 Washed platelet susEffect of AA on EPA metabolism. pensions prewarmed at 37°C for 2 min were incubated with 1 ~.IM[14ClEPA and various concentrations of AA. After 15 s, the reaction was terminated by the addition of extraction solvent. HEPE and TXB3 were measured as described under "Experimental Procedures". Results were expressed as the mean? S.E. of the quadruplicate determinations in one experiment. Measurement

of Metabolites

from Added

[14ClEPA and

["CIAA

The washed platelet suspension prewarmed at 37'C for 2 min was incubated with the radiolabeled fatty acid at 37'C. At various times as indicated, the reaction was terminated by

EPA METABOLISM BY PLATELETS

310

Vol. 40, No. 3

AA

AA&M) lo-

ii-

--42

d’-

8-

1

/ i

6B

:

_,____---J

0.5

,’

/

0.5

1 Time

1.5

2 Time

mm

rn~n

FIG. 2 Effect of AA on time courses of HEPE and TXB, formations. Washed platelet suspensions prewarmed at 37'C for 2 min were incubated with 1 UM [14C]EPA and various concentrations of AA. At indicated times, aliquots were transferred into tubes containing extraction solvent. Results were expressed as the mean + S.E. of three separate experiments. (01, AA 0 PM; (x), AA 0.5 PM; (01, AA 1 uM; (A), AA 2 I_IM. mixing with 10 volumes of chloroform/methanol (l:l, v/v) and 2 volumes of 0.85% formic acid (extraction solvent), and the mixture was vortexed and immediately cooled in an ice-water bath. After centrifuqation at 600 x g for 5 min at 4“C, the organic lower layer was taken and evaporated under a dry N2 stream at 3o"c. The radioactivity recovered in the organic layer was The lipids were separatabout 95% of the total radioactivity. ed by thin layer chromatography at 4°C using a solvent consisting of the upper phase of the mixture of ethyl acetate/2,2,4trimethylpentane/acetic acid/water (90:50:20:100, v/v) (13). Metaboiites from ["CIEPA or [I4C]AA were localized by autoradiography and by co-chromatography with unlabeled compounds (HETE, TXB2) which were visualized by iodine vapor. The identification of HEPE or TXB3 was done as described previously (10). The silica-gel in each region was scraped off and the radioactivity was counted in a Pachard Prias liquid scintillation counter (model 400 CL/D). As the conversion of EPA was only a little affected by the concentration of ethanol (Hashimoto, Y., unpublished data), we held its concentration constant at 1% in all experiments. Data Analysis Tests of the statistical significance of differences

were

25 I-

311

EPA METABOLISM BY PLATELETS

Vol. 40, No. 3

(*I

40C

AA (,N

T 03

FIG. 3 Effect of AA on the HEPE/ TXB3 ratio. The ratio in the absence (control) and the presence of AA was calculated using the values measured in the experiments described in Fig bated2for(;k ys incu, and the column marked (a) in (B) for 5 min and others in (B) for 2 min. During the incubation time in (B), more than 90% of 1 UM EPA was metabolized. Results were expressed as the mean f S.E. of three separate experiments. b) PcO.05 compared with the control. c) P
AA (PM)

TABLE 1 Effect of Preincubation AA on EPA Metabolism.

Addition

Ethanol AA (1 I-IM)

Preincubation Time (s) 0 0 15 30 60

of Washed Platelet Suspensions with

Percentage of AA remained after preincubation

Percentage conversion HEPE

TXB3

47.2 + 4.7 33.2 + 6.3 15.4? 3.6

2.7 + 0.3 12.4 +1.3 20.3 + 3.9 22.5? 5.1 16.7 f 3.5

0.22 rt0.02 1.61+ 0.17 0.90 f 0.16 0.87 + 0.12 0.58 + 0.69

Washed platelet suspensions prewarmed at 37OC for 2 min were preincubated with 1 PM AA for indicated times, and then 1 I.~M[ 14C]EPA was incubated for 15 s. The further procedures were done as described in the legend to Fig. 1. Results were expressed as the mean+S.E. of three separate experiments.

312

EPA METABOLISM BY PLATELETS

Vol. 40, No. 3

FIG. 4 Effects of AA metabolites on EPA metabolism. Washed platelet suspensions prewarmed at 37OC for 2 min were incubated with 1 PM [14c]~p~ and various concentrations of the AA metabolite for 30 s. The further procedures were done as described in the legend to Fig. 1. Results were expressed as the mean f.S.E. of four separate experiments. performed by using analysis of variance. RESULTS Effects of AA on EPA Metabolism Incubation of EPA with human platelet suspensions leads to the formation of three main compounds, namely HEPE, TXB3 and HHTE (6). The chromatography which we used separated clearly HEPE and TXB3, but not HHTE, from other unknown product(s). Therefore, we assayed HEPE and TXB3. EPA has been considered to be a poor substrate for platelet cyclooxygenase and lipoxygenase (4,7). We also observed that although 53* 4.6% (the mean+ S.E., n=3) of 1 PM AA were metabolized during the incubation with platelets for 15 s, only 4.6 + 0.5% (the mean-I S.E., n=3) of 1 )JM EPA were metabolized in the same condition. However, the addition of AA promoted As shown in markedly the metabolism of EPA (Fig.lA, Fig.2). Fig.lA, less than 5 uM and less than 2 M AA stimulated the formation of HEPE and TXB, dose-dependently, respectively, and the stimulatory effect of AA decreased at concentrations of more than those. Besides, the HEPE/TXBa ratio decreased in the

Vol. 40, No. 3

EPA METABOLISM BY PLATELETS

80

(-)

PGGz

HPETE

1 /tMl

; 1 r(M)

313

c

AA 1 r,M;

Time (s) FIG. 5 Effects of indomethacin on the stimulatory effect of AA, PGG2 or HPETE on the HEPE formation. After pretreatment with 50 uM indomethacin (shaded bars) or dimethyl sulfoxide (open bars) for more than 5 min, washed platelet suspensions prewarmed at 37OC for 2 min were incubated with 1 uM [14C1~pA, and 1 I_IM PGG2, 1 PM HPETE or 1 uM AA for 15 s. The further procedures were done as described in the legend to Fig. 1. Results were expressed as the mean+ S.E. of three separate experiments. The final concentration of dimethyl sulfoxide in platelet suspensions was 0.25% in all experiments.

FIG. 6 Effect of indomethacin on time course of the HEPE formation in the presence of AA. After pretreatment with 50 PM indomethacin (01 or dimethyl sulfoxide (0) for more than 5 min, washed platelet suspensions prewarmed at 37'C for 2 min were incubated with 1 ~.IM [14c]~p~ and 1 uM AA. The further procedures were done as described in the legend to Fig. 2. The final concentration of dimethyl sulfoxide in platelet suspensions was 0.25% in all experiments.

presence of AA (Fig.lB, Fig.31, suggesting that AA promoted the TXB3 formation more strongly than the HEPE formation. One uM of palmitic, stearic or arachidic acid which were saturated fatty acids and 1 uM of oleic, linoleic or linolenic acid which were unsaturated fatty acids did not stimulate the metabolism of 1 uM EPA. The stimulatory effects of AA on EPA metabolism could not

314

EPA METABOLISM BY PLATELETS

Vol. 40, No. 3

attribute to any contaminations in the commercial AA solutions, because even either the AA solutions pretreated with 5 mM sodium diethyldithiocarbamate which was reported to remove peroxides in the AA solutions (141, or the solutions of AA purified by thin layer chromatography did promote EPA metabolism. Effects of preincubation

of platelets with AA on EPA metabolism

Although AA decreased promptly during the preincubation periods, the HEPE formation from EPA was stimulated by the preincubation with AA more strongly than by the simultaneous addition of EPA and AA. On the other hand, although the TXB3 formation was increased in the presence of AA,the effect was most prominent by the simultaneous addition of EPA and AA (Table I). Effects of AA metabolites

on EPA metabolism

We examined effects of PGG2, PGHp, HPETE, and HETE on EPA metabolism (Fig. 4). PGG2 or HPETE promoted both TXB3 and HEPE formations dose-dependently, whereas PGH2 or HETE did not show any effects. PGD2 or 2 ~JM TXB2 also did not Moreover, 2 ~.IM stimulate the metabolism of 1 uM EPA. Although the addition of AA to platelets decreased the HEPE/TXBs ratio (Fig. lB, Fig. 31, PGG2 or HPETE did not affect the ratio (Fig. 4). Effects of indomethacin-pretreatment latory effect of AA, PGG2 or HPETE

of platelets on the stimu-

When EPA and AA were simultaneously added to washed platelet suspensions, the HEPE formation was delayed by the preincubation of platelets with indomethacin (Fig. 5, Fig. 6). On the other hand, when EPA alone, EPA and PGG2, or EPA and HPETE were added, the HEPE formation was promoted by the preincubation of platelets with indomethacin (Fig. 5). Almost the same results were observed using 500 uM aspirin in place of 50 UM indomethacin. DISCUSSION We showed that AA stimulated markedly EPA metabolism and the stimulatory effect was more potent over the formation of TXB, than that of HEPE. The stimulatory effect of AA on EPA metabolism can be caused by AA itself and/or AA metabolites. Indomethacin, a specific inhibitor of cyclooxygenase, decreased the stimulatory effect of AA on the HEPE formation (Fig. 5, Fig. 61, suggesting that cyclooxygenase product(s) of AA stimulated the HEPE forAmong the metabolites investigated, PGG2 and HPETE mation. stimulated EPA metabolism (Fig.4). The mechanism which PGG2 and HPETE stimulate EPA metabolism in washed human platelets is not clear. As the cyclooxygenase purified from sheep seminal vesicle (9) or the lipoxygenase purified from bovine platelets (15) metabolizes EPA about half as fast as AA, it may be reasonable to consider that AA metabolites promote the trans-

Vol. 40, No. 3

EPA METABO,L.ISM BY PLATELETS

location of EPA across the platelet membrane (11). However, as the purified cyclooxygenase is activated by lipid peroxide (8) and inhibited by the addition of phenol or glutathione peroxidase (91, PGGz and HPETE might activate cyclooxygenase and lipoxygenase directly. While exogenous PGG2 and HPETE scarcely affected the HEPE/ TXBJ ratio (Fig. 41, AA decreased the ratio (Fig. lB, Fig. 3). Because AA is metabolized by cyclooxygenase more rapidly than by lipoxygenase (16,171 and PGGz produced is expected to exist more closely to cyclooxygenase than lipoxygenase, AA might activate cyclooxygenase more promptly than lipoxygenase. On the other hand, exogenous PGGz or HPETE might activate both enzymes at almost the same time and the same extent. The preincubation of AA with platelets decreased the stimulatory effect of AA on the TXBB formation (Table I). The results suggested that AA itself also might stimulate the TXB3 formation or AA metabolite(s) produced during the preincubation period might inhibit the cyclooxygenase pathway. HPETE is reported to inhibit the cyclooxygenase pathway (18,191. We examined effects of preincubation of washed platelet suspensions with 1 ~,IM AA on the metabolism of 1 yM ["'CIAA. The ratios of the amount of TXB2 produced when the preincubation times were 15, 30, and 60 s to that when the preincubation time was 0s were 80, 83 and 95% (the mean of the duplicate determinations in one experiment) (Hashimoto, Y., unpublished data), respectively, suggesting that the cyclooxygenase pathway was not significantly suppressed by the preincubation of 1 I_IM AA These results imply that AA under our experimental conditions. However, further itself might stimulate the TXBJ formation. studies will be needed. Our studies suggest that EPA might be easily metabolized in vivo through the above-mentioned mutual effects, because EPA and AA always coexist in human platelets and tissues. In fact, recently, Fischer et al. showed the direct evidence for ex vivo formation of TXAJ in human platelets (20) and for in vivo Studies on the biological formation of PGI3 in man (21). significance of EPA metabolites will be necessary. ACKNOWLEDGMENTS We thank Dr. Makoto Kinoshita of our department, Dr. Hiroshi Hayashi of The Tokyo Teishin Hospital, Dr. Ushio Sankawa of Faculty of Pharmaceutical Sciences, University of Tokyo, and Dr. Takao Shimizu of Department of Physiological Chemistry and Nutrition, Faculty of Medicine, University of Tokyo, for helpful discussions, and Miss Asami Tajima for her excellent technical assistance. This work has been supported in part by a Grant-in-Aid for Co-operative Research (No. 56370038). REFERENCES 1.

GOODNIGHT, S.H., HARRIS, W.S. and CONNER, W.E. The effects of dietary w3 fatty acids on platelet composition and function in man : a prospective, controlled study. Blood, 58, 880-885, 1981.

315

316

EPA METABOLISM BY PLATELETS

Vol. 40, No. 3

2.

THORNGREN, M. and GUSTAFSON, A. Effects of ll-week increase in dietary eicosapentaenoic acid on bleedinq time, lipids, and platelet aggregation. Lancet, ii, 1190~1193,~ 1981.

3.

KAWAMURA, M., NAITO, C., HAYASHI, H., HASHIMOTO, Y., KATO, H. and MATSUSHIMA, T. Effects of 4 weeks' intake of polyunsaturated fatty acid ethylester rich in eicosapentaenoic acid (ethylester) on plasma lipids, plasma and platelet phospholipid fatty acid composition and platelet aggregation; a double blind study. J. Jap. Sot. Intern. Med. 72, 18-24, 1983. -

4.

NEEDLEMAN, P., RAZ, A., MINKES, M.S., FERRENDELLI, J.A. and SPRECHER, H. Triene prostaglandins : Prostacyclin and thromboxane biosynthesis and unique biological properties. Proc. Natl. Acad. Sci. U.S.A. -' 76 944-948, 1979.

5.

GRYGLEWSKI, R.J., SALMON, J.A., UBATUBA, F.B., WEATHERLY, B.C., MONCADA, S. and VANE, J.R. Effects of all cis-5,8, 11,14,17 eicosapentaenoic acid and PGH3 on platelet aggregation. Prostaglandins, l8_, 453-478, 1979.

6.

HAMBERG, M. Transformations of 5,8,11,14,17-eicosapenta-. enoic acid in human platelets. Biochim. Biophys. Acta, 618, 389-398, 1980.

7.

MORITA, I., SAITO, Y., CHANG. W.C. and MUROTA, S. Effects of purified eicosapentaenoic acid on arachidonic acid metabolism in cultured murine aortic smooth muscle cells, vessel walls and platelets. Lipids, 18, 42-49, 1983.

8.

LANDS, W.E.M. and BYRNES, M.J. The influence of ambient peroxides on the conversion of 5,8,11,14,17-eicosapentaenoic acid to prostaglandins. Lipid Res. 20, 287-290, 1981.

9.

CULP, B.R., TITUS, B.G. and LANDS, W.E.M. Inhibition of prostaglandin biosynthesis by eicosapentaenoic acid. Prostaglandins and Medicine, 3, 269-278, 1979.

10.

HASHIMOTO, Y., NAITO, C., KAWAMURA, M. and OKA, H. Effects of the ratio of exogenous eicosapentaenoic acid to arachidonic acid on platelet aggregation and serotonin release. Thromb. Res. 34, 439-446, 1984.

11.

MORITA, I., TAKAHASHI, R., SAITO, Y. and MUROTA, S. Stimulation of eicosapentaenoic acid metabolism in washed human platelets by 12-hydroperoxyeicosatetraenoic acid. (1983) J. - Biol. Chem. 258, 10197-10199, 1983.

12.

BOUKHCHACHE, D. and LAGARDE, M. Interactions between prostaglandin precursors during their oxygenation by human platelets. Biochim. Biophys. Acta, 713, 386-392, 1982.

13.

BILLAH, M.M., LAPETINA, E.G. and CUATRECASAS, P. Phospholipase AZ and phospholipase C activities of platelets. J. Biol. Chem. 255, 10227-10231, 1980.

Vol. 40, No. 3

EPA METABQLJSM BY PLATELETS

317

14.

Prostaglandin HEMLER, M.E., COOK, H.W. and LANDS, W.E.M. biosynthesis can be triggered by lipid peroxides. Arch. Biochem. Biophys. 193, 340-345, 1979.

15.

NUGTEREN, D.H. Arachidonate lipoxygenase in blood platelets. Biochim. Biophys. Acta, 380, 299-307, 1975.

16.

DUTILH, C.E., HADDEMAN, E., JOUVENAZ, G.H., HOOR, F.T. and NUGTEREN, D.H. Study of the two pathways for arachidonate oxygenation in blood platelets. Lipids, 14, 241-246, 1979.

17.

LAPETINA, E.G. and CUATRECASAS, P. Rapid inactivation of cyclooxygenase activity after stimulation of intact platelets. Proc. Natl. Acad. Sci. U.S.A. 76, 121-125, 1979.

18.

HAMMARSTReM, S. and FALARDEAU, P. Resolution of prostaglandin endoperoxide synthase and thromboxane synthase of human platelets. Proc. Natl. Acad. Sci. U.S.A. 74, 36913695, 1977.

19.

SIEGEL, M.I., MCCONNELL, R.T., ABRAHAMS, S.L., PORTER, N.A. and CUATRECASAS, P. Regulation of arachidonate metabolism via lipoxygenase and cycle-oxygenase by 12HPETE, the product of human platelet lipoxygenase. Biochem. Biophys. Res. Commun. 89, 1273-1280, 1979.

20.

FISCHER, S. and WEBER, P.C. Thromboxane AB (TKAB) is formed in human platelets after dietary eicosapentaenoic acid (C20:5w3). Biochem. Biophys. Res. Commun. 116, 10911099, 1983.

21.

FISCHER, S. and WEBER, P.C. Prostaglandin vivo in man after dietary eicosapentaenoic 307, 165-168, 1984.

13 is formed in acid. Nature,