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Effects of the ratio of exogenous eicosapentaenoic acid to arachidonic acid on platelet aggregation and serotonin release
THROMBOSIS RESEARCH 34; 439-446, 1984 0049-3848184 $3.00 + .OO Printed in the USA. Copyright (cl 1984 Pergamon Press Ltd. All rights reserved.
EFFECTS OF THE RATIO OF EXOGENOUS EICOSAPENTAENOIC ACID TO ARACHIDONIC ACID ON PLATELET AGGREGATION AND SEROTONIN RELEASE
Yoshiaki Hashimoto, Chikayuki Naito*, Mitsunobu Kawamura* and Hiroshi Oka the First Department of Internal Medicine, Faculty of Medicine, University of Tokyo and the Department of Internal Medicine, The Tokyo Teishin Hospital*, Japan
(Received 14.11.1983; Accepted in revised form 7.3.1984 by Editor M. Matsuda)
ABSTRACT We added arachidonic acid (AA) and eicosapentaenoic acid (EPA) to washed platelet suspensions in the absence of albumin, holding the total amount of the fatty acids constant at 2 uM, and changing the ratio of EPA to AA. Platelet aggregation, serotonin release and the amount of thromboxane (TX) BP, a cyclooxygenase product synthesized from exogenous AA,decreased as the ratio was increased. The decreases were greater than the expected ones from the diminution of the amount of exogenous AA. On the other hand, 12-hydroxyeicosatetraenoic acid (HETE), a lipoxygenase product synthesized from exogenous AA,increased in the presence of EPA. Although EPA was reported to be a poor substrate for platelet cyclooxygenase, the amount of TXBs synthesized from exogenous EPA increased markedly by the simultaneous addition of AA. These results suggest that the EPA / AA ratio-dependent decrease in platelet aggregation and serotonin release is caused at least by both the decrease in the absolute amount of AA and the inhibitory effect of EPA on AAmetabolism via the cyclooxygenase pathway. Further studies on effects of EPA-metabolites via the cyclooxygenase pathway on platelet responses will be needed.
Key Words:
Arachidonic Acid, Eicosapentaenoic Acid, Platelet Aggregation, Serotonin Release. 439
440
EPA AND PLATELET
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INTRODUCTION Epidemiological studies have shown that the incidence of ischemic heart diseases in Greenland Eskimos is very low (1,Z). Dyerberg and Bang have suggested that it could be partly due to the decrease in platelet aggregability which might be caused by the increase in EPA of platelets (3). The mechanisms by which the increase in this fatty acid decreases platelet aggregability have been thought to be the decrease in the absolute amount of AA in platelets, the inhibition of AA-metabolism via cyclooxygenase pathway (3,4) by EPA and/or the increase in cyclic AMP of platelets by EPA- metabolites produced via cyclooxygenase (4). However, it is scarcely known how each of these mechanisms participates in the decrease in platelet aggregability. In this report, we examined the effects of the EPA / AA ratio on platelet aggregation and serotonin release and the possible mechanisms of the decrease in aggregability. MATERIALS AND METHODS Materials [l-14C]AA (52.1 mCi/nanol)and [14C(U)]EPA (212 mCi/mmol) were obtained from New England Nuclear, Boston, MA. ['4C]Serotonin (5_hydroxy[sidechain2-14C]tryptamine creatinine sulphate, 58 mCi/nnnol)was obtained from Amersham Int., England. AA (free form, 99% pure) was purchased from Sigma Chemical Co., St. Louis, MO. EPA (free form, 98% pure), and eicosateraynoic acid (ETYA) were gifts from Nippon Oil and Fats Co., LTD, Tokyo, Japan, and Nippon Roche Co., LTD, Tokyo, Japan, respectively. TKB2 and OKY-1581 were gifts from the Ono Central Research Institute, Osaka, Japan. HETE was purchased from Ran Biochem. Tel Aviv, Israel. Lactate dehydrogenase (LDH) assay kits were obtained from Nippon Shoji Co., LTD, Tokyo, Japan. Silica-gel(60)-precoated plates with a concentration zone were from E. Merk, Darmstadt, Germany. 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 3.8% (w/v) sodium citrate in the ratio of 1 to 9 volumes of blood. The mixed blood was centrifuged at 250 g for 6 min. The upper phase, platelet rich plasma (PRP), was removed, and then acid-citrate-dextrose (ACD) was added in the ratio of 1 to 6 volumes of PRP. The PRP-ACD mixture was centrifuged at 600 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 OIMEDTA, and then resuspended in 15 mM tris-HCl buffer (pH 7.4) containing 134 mM NaCl and 5 mM glucose to make 1 ul of the suspension contain 3 x10' platelets. Platelet Aggregation and Serotonin Release A washed platelet suspension (3 x10' platelets per ~1) was labeled by incubating with ['4C]serotonin (0.1 uCi/ml platelet suspension) for 1 hr at 30°C. Aggregation of the [ 14C]serotonin-labeled platelets was monitored photometrically at 37°C by use of a Niko bioscience four-channel aggregometer (model PAT-LA). We incubated 200 ul of washed platelet suspension with 1 ul of 200 mM CaCla and 2 ul of the fatty acid(s) dissolved in ethanol. Aggregation and serotonin release were allowed to proceed for 3 min and were terminated by adding 50 1.11 of 0.5% glutaraldehyde and cooling the sample in an ice bath. Serotonin release was calculated according to
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441
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the method of Holmsen et al. (5) with slight modification, i.e. % secreted= (St-Sc)/(T-Sc) x100, where T, St and SC stand for ['4C]radioactivity in uncentrifuged cuvett contents, [!4C]radioactivity in supernatants of cuvett contents incubated for 3 min at 37°C with the fatty acid(s), and ['4C]radioactivity in supernatants of untreated cuvett contents, respectively. Metabolites of [14C]AA and [14c]~p~ The various products formed from exogenous [!"ClAA or ['?IEPA were separated and measured by use of thin layer chromatography. After the incubation of a washed platelet suspension with the fatty acid(s) for 3 min at 37"C, the reaction was terminated by the addition of 10 volumes of chloroform / methanol (1:2, v/v) mixture. The mixture was filtered through a defatted filter paper and the extracts were dried under a Nz stream. Dried lipids and authentic TXB:!and HETE were dissolved in chloroform and applied to a silica-gel(60)-precoated plate. The chromatogram was run as described by Lapetina et al. (6). The localization of the radioactivity on the chromatogram was determined by autoradiography and the spots with radioactivity were scraped off and counted in a Pachard Prias liquid scintillation counter (model 400 CL/D). We regarded the substances located on the chromatogram in the immediate proximal vicinity of the authentic TXB2 and HETE as TXBz and 12-hydroxyeicosapentaenoicacid (HEPE), respectively. Because the TXB2, which we regarded, was stable and its formation was inhibited by 500 uM acetylsalicylic acid (ASA) or 10 uM OKY-1581 (18), and the HEPE was stable and its formation was inhibited not by 500 J+IM ASA, but by 1 PM ETYA (19). The recovery of [14 Clradioactivity was about 60% of the radioactivity added to a washed platelet suspension. The result was expressed in terms of the amount contained in 1 ml of the original platelet suspension. Detection of Platelet Lysis To detect platelet damage, LDH was measured by use of a LDH assay kit. LDH activity recovered in the medium after platelets were stirred in the AAcontaining medium for 3 min at 37°C was calculated as a percentage of LDH activity released from the platelets after 3 times repetition of freezing and thawing. RESULTS EPA/AA 0 l/3 1 3
EPA ('JM)AA 0 0.5 1 1.5
Aggregation %
2 1.5 1 0.5
53 39 32 10 TABLE
(45) (40) (33)
release % 29 12 5 1
(20) (15) (13)
1
Effects of the Ratio of Exogenous EPA to A_4on Platelet Aggregation and Serotonin Release. Each value represents the maximum aggregation and serotonin release rate within 3 min after the addition of the fatty acid(s) to a platelet suspension. The value in parentheses shows AA-induced platelet responses when EPA was not added. Three separate experiments were performed on different platelets from three donors. Though quantitative responses were different in each experiment, the general tendency of responses was the same in three experiments. Therefore, we presented the average value of duplicate determinations of one typical experiment.
442
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TXB,
RESPONSES
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7
0.6
1
t
HETE
T
AA (PM) 2
1.51.5
1
1
0.50.5
2
1.51.5
1
I
EPA(PM) 0
00.5
0
1
0 1.5
0
00.5
0
1
0.50.5 0 1.5
FIG. 1 Effects of exogenous EPA on TXB2 and HETE productions from exogenous AA. The data represent mean ?:SD of three values from three separate experiments, each of which was determined duplicately. The t-test for paired comparisons ** : The value was was used. NS: not significant, *: pcO.05, **: pcO.01, significantly greater (pcO.01) than any of the others. m: [14C]AA alone was added. m: Mixture of [14C]AA and cold EPA was added. The maximum aggregation of the platelets suspended in the 1% ethanolcontaining buffer solution was about 85% of that suspended in the 0.2% ethanol-containing buffer solution when 2 uM AA was used as an aggregating agent. But LDH loss and serotonin release from platelets were not observed in the 1% ethanol-containing buffer solution if an aggregating agent was not added. Moreover, the production rate of TXBs and HETE was the same in the 1% ethanol solution as in the 0.2% ethanol one. Therefore, all experiments were performed in making the final concentration of ethanol 1% when the ethanol solution of fatty acids was added to platelet suspensions. When we added EPA and AA to washed platelet suspensions, holding the and changing the EPA / AA ratio, total amount of fatty acids constant at 2 i.lM, platelet aggregation and serotonin release decreased as the ratio was increased. Though platelet responses decreased with the reduction of exogenous AA, the decrease of the responses was greater with the simultaneous addition of EPA (Table 1). The amount of TXB2 synthesized from exogenous AA 3 min after the addition of AA and EPA decreased as the EPA / AA ratio was increased. The decrease was greater than the expected one from the amount of AA added to platelet suspensions. However, on the other hand, the amount of HETE synthesized from exogenous AA increased when EPA
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0.8 I
TX&
RESPONSES
HEPE
443
FIG. 2 Effects of exogenous AA on TXB3 and HEPE productions from exogenous EPA. The data represent mean k SD of three values from three separate experiments, each of which was determined duplicately. The t-test for paired comparisons was used. **: ~~0.01 I':_::.il: [14C]~~~ (1 PM) alone was added m Mixture of [14C]EPA (1 PM) and cold AA (1 PM) was added.
was added simultaneously (Fig. 1). The amount of TXBs synthesized from exogenous EPA increased about 4 times and the amount of HEPE synthesized from exogenous EPA increased slightly by the simultaneous addition of AA (Fig. 2). DISCUSSION When AA or EPA is added to PRP or washed platelet suspensions in the presence of albumin, a part of the acid binds to albumin in the suspension medium (8) and the remainder enters platelets. On the other hand, when suspension mediums do not contain albumin, most of the fatty acid rapidly enters platelets (9,lO). Therefore, we used washed platelet suspensions in the absence of albumin. In these studies, platelet aggregation and serotonin release decreased significantly as the ratio of EPA to AA added to platelet suspensions was increased. Recently, we reported that platelet aggregability did not decrease significantly in spite of the increase in the ratio of EPA to AA in platelet phospholipids by the ingestion of EPA ethylester in human (11). Some reported almost the same results (12-14). Merely insignificant decrease in platelet aggregation even when the EPA / AA ratio of platelet phospholipids increased to 0.34 as reported in the literatures (12) might be caused at least partly by the use of ADP, adrenaline or collagen as an aggregating agent. As all these aggregating agents have both TXAz-dependent and independent platelet-activating actions, the reduction of TXAz production may not always reflect on platelet aggregation. In the present studies, the significant decrease in platelet aggregation was observed even at the EPA / AA ratio of 0.33. This inconsistent result may be explained by the fact that the decrease in TXAz production directly reflects on aggregation because AA-induced platelet aggregation depends on only TXAz.
444
EPA AND PLATELET
RESPONSES
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Needleman et al. reported that the production of both TXB: and HETE synthesized from exogenous AA markedly decreased at the E?A / AA ratio more than 1 (4), but our studies revealed the decrease in TXBz production and the increase in HETE production from exogenous AA by the simultaneous addition of EPA. Moreover, we observed that the production of both TXB2 and HETE 3 min after the simultaneous addition of AA and EPA decreased at the EPA / M ratio of 1 when the total amount of the acids added to platelet suspensions exceeded 20 PM (data not shown). Therefore, it is possible that the difference between the results of Needleman et al. and ours was caused by the difference in the amount of the acids added to platelet suspensions. Although EPA was regarded as a poor substrate for platelet cyclooxygenase (4,15,16), the marked increase in TXBs production was observed by the simultaneous addition of AA. This increase in TXBs production by the addition of AA could not attribute to any contaminations of peroxides in the AA solutions, because even either the AA solutions pretreated with 5 mM sodium diethyldithiocarbamate which was reported to remove peroxides in the AA solutions (20),or the solutions of AA purified by a thin layer chromatography did increase TXBs production as well (data not shown). The production of PGD3, a cyclooxygenase product synthesized from EPA, may also increase by the simultaneous addition of AA. PGD3 is thought to inhibit platelet responses by increasing the level of cyclic AMP in platelets (17). However, we could not find any increase in the amount of cyclic AMP in platelets by the simultaneous addition of AA (1 PM) and EPA (1 uM) (data not shown). As we examined the amount of cyclic AMP in platelets merely 1 min after the addition of the fatty acid(s), the further studies will be needed to reach a definite conclusion. Our studies have suggested that the decrease in platelet responses due to the increase in the EPA / AA ratio of platelet phospholipids is caused at least by the decrease of TXA2 production owing to both the absolute reduction of AA in platelet phospholipids and the inhibition of AA-metabolism via the cyclooxygenase pathway by EPA. Further studies about the effects of EPA-metabolites produced via the cyclooxygenase pathway on platelet responses will be needed. ACKNOWLEDGEMENT This study was supported by a grant from the Ministry of Education (No. 56370038; the chairman: Prof. M. Yamanaka of university of Tokyo.) We thank Drs. T. Teramoto, H. Kato, H. Hayashi and M. Kinoshita of our group for helpful discussions concerning this work. REFERENCES 1.
BANG, H.O., DYERBERG, J., NIELSEN, Aa. B.. Plasma lipid and lipoprotein pattern in Greenlandic west-coast Eskimos. Lancet. 1,1143-1145, 1971.
2.
BANG, H.O., DYERBERG, J.: Plasma lipids and lipoproteins in Greenlandic west coast Eskimos. Acta Med. Stand. =,85-94, 1972.
3.
DYERBERG, J., BANG, H.O.: 1978.
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. USA. &944-948, 1979.
Dietary fat and thrombosis. Lancet.l_,152,
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5.
Evidence that the platelet plasma HOLMSEN, H. and DANGERMAIER, C.A. membrane is impermeable to calcium and magnesium complexes of A23187. J. Biol. Chem. 256,10449-10452, 1981.
6.
Stimulation of phosphatidic acid LAPETINA, E.G. and CUATRECASAS, P. production in platelets procedes the formation of arachidonate and parallels the release of serotonin. Biochim. Biophys. Acta. 52,394402, 1979.
7.
An ultrasensitive method HONMA, M., SATOH, T., TAKEZAWA, J. and UI, M. for the simultaneous determination of cyclic AMP and cyclic GMP in small volume samples from blood and tissue. Biochem. Med. l&257-273, 1977.
a.
HOAK, J.C., WARNER, E.D. and CONNER, W.E. thrombosis. Circ. Res. 20,11-17, 1967.
9.
LAPETINA, E.G. and CUATRECASAS, P. Rapid inactivation of cyclooxygenase activity after stimulation of intact platelets. Proc. Natl. Acad. Sci. USA. &121-125, 1979.
10.
MACLOVF, J., DE LA BAUME, H., LEVY-TOLEDANO, S. and CAEN, J.P. Selective stimulation of human platelet lipoxygenase product 12-hydroxy5,8,10,14-eicosatetraenoic acid by chlorpromazine and 8-(n,n-diethylamino) octyl-3,4,5_trimethoxybenzoate. Biochim. Biophys. Acta. 72, 377-385, 1982.
11.
KAWAMURA, M., NAITO, C., HAYASHI, H., HASHIMOTO, Y., KATO, H. and Effects of 4 weeks' intake of polyunsaturated fatty MATSUSHIMA, T. acid ethylester rich in eicosapentaenoic acid (ethylester) on plasma lipids, plasma and platelet phospholipid fatty acid composition and platelet-aggregation; a double biind study. 3: Jap. Sot: Intern. Med. 1_2,18-24,1983.
12.
SIESS, W., ROTH, P., SCHERER, B., KURZMANN, I., BOHLIG, B. and WEBER, P.C.: Platelet-membrane fatty acids, platelet aggregation, and thromboxane formation during a mackerel diet. Lancet. 1 441-444, 1980.
13.
GOODNIGHT, S.H., HARRIS, W.S. and CONNOR, W.E. The effects of dietary w3 fatty acids on platelet composition and function in man: a prospective, controlled study. B100dz58,aa0-885, 1981.
14.
THORNGREN, M. and GUSTAFSON, A. Effects of 11-week increase in dietary eicosapentaenoic acid on bleeding time, lipids, and platelet aggregation. Lancet. 2,1190-1193, 1981.
15.
NEEDLEMAN, P., MINKES, M. and RAZ. A. Tromboxanes: selective biosynthesis and distinct biological properties. Science. 193,163-165, -1976.
16.
HAMBERG, M. Transformations of 5,8,11,14,17-eicosapentaenoicacid in human platelets. Biochim. Biophys. Acta. 62,389-398, 1980.
17.
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 PGHs on platelet aggregation. Prostaglandins, 1_8,453478, 1979.
Platelets, fatty acids and
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RESPOIJSES
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OKY-158: a selective inhibitor of S?lITH,J.B. and JUBIZ, W. thromboxane synthesis in vivo and in vitro. Prostaglandins, 2.,353363, 1981.
19.
WILHELM, T.E., SAXARAPPA, S.K., VANROLLINS, 41.and SPRECHER, H. Selective inhibitors of platelet lipoxygenase: 4,7,10,13-icosatetraynoic acid and 5,8,11,14-henicosatetraynoic acid. Prostaglandins, Z&323-332, 1981.
20.
Prostaglandin biosynthesis HEMLER, M.E., COOK, H.W. and LANDS, W.E.M. can be triggered by lipid peroxides. Arch. Biochem. Biophys. w,340345, 1979.
EFFECTS OF THE RATIO OF EXOGENOUS EICOSAPENTAENOIC ACID TO ARACHIDONIC ACID ON PLATELET AGGREGATION AND SEROTONIN RELEASE
Yoshiaki Hashimoto, Chikayuki Naito*, Mitsunobu Kawamura* and Hiroshi Oka the First Department of Internal Medicine, Faculty of Medicine, University of Tokyo and the Department of Internal Medicine, The Tokyo Teishin Hospital*, Japan
(Received 14.11.1983; Accepted in revised form 7.3.1984 by Editor M. Matsuda)
ABSTRACT We added arachidonic acid (AA) and eicosapentaenoic acid (EPA) to washed platelet suspensions in the absence of albumin, holding the total amount of the fatty acids constant at 2 uM, and changing the ratio of EPA to AA. Platelet aggregation, serotonin release and the amount of thromboxane (TX) BP, a cyclooxygenase product synthesized from exogenous AA,decreased as the ratio was increased. The decreases were greater than the expected ones from the diminution of the amount of exogenous AA. On the other hand, 12-hydroxyeicosatetraenoic acid (HETE), a lipoxygenase product synthesized from exogenous AA,increased in the presence of EPA. Although EPA was reported to be a poor substrate for platelet cyclooxygenase, the amount of TXBs synthesized from exogenous EPA increased markedly by the simultaneous addition of AA. These results suggest that the EPA / AA ratio-dependent decrease in platelet aggregation and serotonin release is caused at least by both the decrease in the absolute amount of AA and the inhibitory effect of EPA on AAmetabolism via the cyclooxygenase pathway. Further studies on effects of EPA-metabolites via the cyclooxygenase pathway on platelet responses will be needed.
Key Words:
Arachidonic Acid, Eicosapentaenoic Acid, Platelet Aggregation, Serotonin Release. 439
440
EPA AND PLATELET
RESPONSES
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INTRODUCTION Epidemiological studies have shown that the incidence of ischemic heart diseases in Greenland Eskimos is very low (1,Z). Dyerberg and Bang have suggested that it could be partly due to the decrease in platelet aggregability which might be caused by the increase in EPA of platelets (3). The mechanisms by which the increase in this fatty acid decreases platelet aggregability have been thought to be the decrease in the absolute amount of AA in platelets, the inhibition of AA-metabolism via cyclooxygenase pathway (3,4) by EPA and/or the increase in cyclic AMP of platelets by EPA- metabolites produced via cyclooxygenase (4). However, it is scarcely known how each of these mechanisms participates in the decrease in platelet aggregability. In this report, we examined the effects of the EPA / AA ratio on platelet aggregation and serotonin release and the possible mechanisms of the decrease in aggregability. MATERIALS AND METHODS Materials [l-14C]AA (52.1 mCi/nanol)and [14C(U)]EPA (212 mCi/mmol) were obtained from New England Nuclear, Boston, MA. ['4C]Serotonin (5_hydroxy[sidechain2-14C]tryptamine creatinine sulphate, 58 mCi/nnnol)was obtained from Amersham Int., England. AA (free form, 99% pure) was purchased from Sigma Chemical Co., St. Louis, MO. EPA (free form, 98% pure), and eicosateraynoic acid (ETYA) were gifts from Nippon Oil and Fats Co., LTD, Tokyo, Japan, and Nippon Roche Co., LTD, Tokyo, Japan, respectively. TKB2 and OKY-1581 were gifts from the Ono Central Research Institute, Osaka, Japan. HETE was purchased from Ran Biochem. Tel Aviv, Israel. Lactate dehydrogenase (LDH) assay kits were obtained from Nippon Shoji Co., LTD, Tokyo, Japan. Silica-gel(60)-precoated plates with a concentration zone were from E. Merk, Darmstadt, Germany. 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 3.8% (w/v) sodium citrate in the ratio of 1 to 9 volumes of blood. The mixed blood was centrifuged at 250 g for 6 min. The upper phase, platelet rich plasma (PRP), was removed, and then acid-citrate-dextrose (ACD) was added in the ratio of 1 to 6 volumes of PRP. The PRP-ACD mixture was centrifuged at 600 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 OIMEDTA, and then resuspended in 15 mM tris-HCl buffer (pH 7.4) containing 134 mM NaCl and 5 mM glucose to make 1 ul of the suspension contain 3 x10' platelets. Platelet Aggregation and Serotonin Release A washed platelet suspension (3 x10' platelets per ~1) was labeled by incubating with ['4C]serotonin (0.1 uCi/ml platelet suspension) for 1 hr at 30°C. Aggregation of the [ 14C]serotonin-labeled platelets was monitored photometrically at 37°C by use of a Niko bioscience four-channel aggregometer (model PAT-LA). We incubated 200 ul of washed platelet suspension with 1 ul of 200 mM CaCla and 2 ul of the fatty acid(s) dissolved in ethanol. Aggregation and serotonin release were allowed to proceed for 3 min and were terminated by adding 50 1.11 of 0.5% glutaraldehyde and cooling the sample in an ice bath. Serotonin release was calculated according to
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441
RESPONSES
the method of Holmsen et al. (5) with slight modification, i.e. % secreted= (St-Sc)/(T-Sc) x100, where T, St and SC stand for ['4C]radioactivity in uncentrifuged cuvett contents, [!4C]radioactivity in supernatants of cuvett contents incubated for 3 min at 37°C with the fatty acid(s), and ['4C]radioactivity in supernatants of untreated cuvett contents, respectively. Metabolites of [14C]AA and [14c]~p~ The various products formed from exogenous [!"ClAA or ['?IEPA were separated and measured by use of thin layer chromatography. After the incubation of a washed platelet suspension with the fatty acid(s) for 3 min at 37"C, the reaction was terminated by the addition of 10 volumes of chloroform / methanol (1:2, v/v) mixture. The mixture was filtered through a defatted filter paper and the extracts were dried under a Nz stream. Dried lipids and authentic TXB:!and HETE were dissolved in chloroform and applied to a silica-gel(60)-precoated plate. The chromatogram was run as described by Lapetina et al. (6). The localization of the radioactivity on the chromatogram was determined by autoradiography and the spots with radioactivity were scraped off and counted in a Pachard Prias liquid scintillation counter (model 400 CL/D). We regarded the substances located on the chromatogram in the immediate proximal vicinity of the authentic TXB2 and HETE as TXBz and 12-hydroxyeicosapentaenoicacid (HEPE), respectively. Because the TXB2, which we regarded, was stable and its formation was inhibited by 500 uM acetylsalicylic acid (ASA) or 10 uM OKY-1581 (18), and the HEPE was stable and its formation was inhibited not by 500 J+IM ASA, but by 1 PM ETYA (19). The recovery of [14 Clradioactivity was about 60% of the radioactivity added to a washed platelet suspension. The result was expressed in terms of the amount contained in 1 ml of the original platelet suspension. Detection of Platelet Lysis To detect platelet damage, LDH was measured by use of a LDH assay kit. LDH activity recovered in the medium after platelets were stirred in the AAcontaining medium for 3 min at 37°C was calculated as a percentage of LDH activity released from the platelets after 3 times repetition of freezing and thawing. RESULTS EPA/AA 0 l/3 1 3
EPA ('JM)AA 0 0.5 1 1.5
Aggregation %
2 1.5 1 0.5
53 39 32 10 TABLE
(45) (40) (33)
release % 29 12 5 1
(20) (15) (13)
1
Effects of the Ratio of Exogenous EPA to A_4on Platelet Aggregation and Serotonin Release. Each value represents the maximum aggregation and serotonin release rate within 3 min after the addition of the fatty acid(s) to a platelet suspension. The value in parentheses shows AA-induced platelet responses when EPA was not added. Three separate experiments were performed on different platelets from three donors. Though quantitative responses were different in each experiment, the general tendency of responses was the same in three experiments. Therefore, we presented the average value of duplicate determinations of one typical experiment.
442
EPA AND PLATELET
TXB,
RESPONSES
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7
0.6
1
t
HETE
T
AA (PM) 2
1.51.5
1
1
0.50.5
2
1.51.5
1
I
EPA(PM) 0
00.5
0
1
0 1.5
0
00.5
0
1
0.50.5 0 1.5
FIG. 1 Effects of exogenous EPA on TXB2 and HETE productions from exogenous AA. The data represent mean ?:SD of three values from three separate experiments, each of which was determined duplicately. The t-test for paired comparisons ** : The value was was used. NS: not significant, *: pcO.05, **: pcO.01, significantly greater (pcO.01) than any of the others. m: [14C]AA alone was added. m: Mixture of [14C]AA and cold EPA was added. The maximum aggregation of the platelets suspended in the 1% ethanolcontaining buffer solution was about 85% of that suspended in the 0.2% ethanol-containing buffer solution when 2 uM AA was used as an aggregating agent. But LDH loss and serotonin release from platelets were not observed in the 1% ethanol-containing buffer solution if an aggregating agent was not added. Moreover, the production rate of TXBs and HETE was the same in the 1% ethanol solution as in the 0.2% ethanol one. Therefore, all experiments were performed in making the final concentration of ethanol 1% when the ethanol solution of fatty acids was added to platelet suspensions. When we added EPA and AA to washed platelet suspensions, holding the and changing the EPA / AA ratio, total amount of fatty acids constant at 2 i.lM, platelet aggregation and serotonin release decreased as the ratio was increased. Though platelet responses decreased with the reduction of exogenous AA, the decrease of the responses was greater with the simultaneous addition of EPA (Table 1). The amount of TXB2 synthesized from exogenous AA 3 min after the addition of AA and EPA decreased as the EPA / AA ratio was increased. The decrease was greater than the expected one from the amount of AA added to platelet suspensions. However, on the other hand, the amount of HETE synthesized from exogenous AA increased when EPA
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0.8 I
TX&
RESPONSES
HEPE
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FIG. 2 Effects of exogenous AA on TXB3 and HEPE productions from exogenous EPA. The data represent mean k SD of three values from three separate experiments, each of which was determined duplicately. The t-test for paired comparisons was used. **: ~~0.01 I':_::.il: [14C]~~~ (1 PM) alone was added m Mixture of [14C]EPA (1 PM) and cold AA (1 PM) was added.
was added simultaneously (Fig. 1). The amount of TXBs synthesized from exogenous EPA increased about 4 times and the amount of HEPE synthesized from exogenous EPA increased slightly by the simultaneous addition of AA (Fig. 2). DISCUSSION When AA or EPA is added to PRP or washed platelet suspensions in the presence of albumin, a part of the acid binds to albumin in the suspension medium (8) and the remainder enters platelets. On the other hand, when suspension mediums do not contain albumin, most of the fatty acid rapidly enters platelets (9,lO). Therefore, we used washed platelet suspensions in the absence of albumin. In these studies, platelet aggregation and serotonin release decreased significantly as the ratio of EPA to AA added to platelet suspensions was increased. Recently, we reported that platelet aggregability did not decrease significantly in spite of the increase in the ratio of EPA to AA in platelet phospholipids by the ingestion of EPA ethylester in human (11). Some reported almost the same results (12-14). Merely insignificant decrease in platelet aggregation even when the EPA / AA ratio of platelet phospholipids increased to 0.34 as reported in the literatures (12) might be caused at least partly by the use of ADP, adrenaline or collagen as an aggregating agent. As all these aggregating agents have both TXAz-dependent and independent platelet-activating actions, the reduction of TXAz production may not always reflect on platelet aggregation. In the present studies, the significant decrease in platelet aggregation was observed even at the EPA / AA ratio of 0.33. This inconsistent result may be explained by the fact that the decrease in TXAz production directly reflects on aggregation because AA-induced platelet aggregation depends on only TXAz.
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Needleman et al. reported that the production of both TXB: and HETE synthesized from exogenous AA markedly decreased at the E?A / AA ratio more than 1 (4), but our studies revealed the decrease in TXBz production and the increase in HETE production from exogenous AA by the simultaneous addition of EPA. Moreover, we observed that the production of both TXB2 and HETE 3 min after the simultaneous addition of AA and EPA decreased at the EPA / M ratio of 1 when the total amount of the acids added to platelet suspensions exceeded 20 PM (data not shown). Therefore, it is possible that the difference between the results of Needleman et al. and ours was caused by the difference in the amount of the acids added to platelet suspensions. Although EPA was regarded as a poor substrate for platelet cyclooxygenase (4,15,16), the marked increase in TXBs production was observed by the simultaneous addition of AA. This increase in TXBs production by the addition of AA could not attribute to any contaminations of peroxides in the AA solutions, because even either the AA solutions pretreated with 5 mM sodium diethyldithiocarbamate which was reported to remove peroxides in the AA solutions (20),or the solutions of AA purified by a thin layer chromatography did increase TXBs production as well (data not shown). The production of PGD3, a cyclooxygenase product synthesized from EPA, may also increase by the simultaneous addition of AA. PGD3 is thought to inhibit platelet responses by increasing the level of cyclic AMP in platelets (17). However, we could not find any increase in the amount of cyclic AMP in platelets by the simultaneous addition of AA (1 PM) and EPA (1 uM) (data not shown). As we examined the amount of cyclic AMP in platelets merely 1 min after the addition of the fatty acid(s), the further studies will be needed to reach a definite conclusion. Our studies have suggested that the decrease in platelet responses due to the increase in the EPA / AA ratio of platelet phospholipids is caused at least by the decrease of TXA2 production owing to both the absolute reduction of AA in platelet phospholipids and the inhibition of AA-metabolism via the cyclooxygenase pathway by EPA. Further studies about the effects of EPA-metabolites produced via the cyclooxygenase pathway on platelet responses will be needed. ACKNOWLEDGEMENT This study was supported by a grant from the Ministry of Education (No. 56370038; the chairman: Prof. M. Yamanaka of university of Tokyo.) We thank Drs. T. Teramoto, H. Kato, H. Hayashi and M. Kinoshita of our group for helpful discussions concerning this work. REFERENCES 1.
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KAWAMURA, M., NAITO, C., HAYASHI, H., HASHIMOTO, Y., KATO, H. and Effects of 4 weeks' intake of polyunsaturated fatty MATSUSHIMA, T. acid ethylester rich in eicosapentaenoic acid (ethylester) on plasma lipids, plasma and platelet phospholipid fatty acid composition and platelet-aggregation; a double biind study. 3: Jap. Sot: Intern. Med. 1_2,18-24,1983.
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SIESS, W., ROTH, P., SCHERER, B., KURZMANN, I., BOHLIG, B. and WEBER, P.C.: Platelet-membrane fatty acids, platelet aggregation, and thromboxane formation during a mackerel diet. Lancet. 1 441-444, 1980.
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GOODNIGHT, S.H., HARRIS, W.S. and CONNOR, W.E. The effects of dietary w3 fatty acids on platelet composition and function in man: a prospective, controlled study. B100dz58,aa0-885, 1981.
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THORNGREN, M. and GUSTAFSON, A. Effects of 11-week increase in dietary eicosapentaenoic acid on bleeding time, lipids, and platelet aggregation. Lancet. 2,1190-1193, 1981.
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HAMBERG, M. Transformations of 5,8,11,14,17-eicosapentaenoicacid in human platelets. Biochim. Biophys. Acta. 62,389-398, 1980.
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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 PGHs on platelet aggregation. Prostaglandins, 1_8,453478, 1979.
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WILHELM, T.E., SAXARAPPA, S.K., VANROLLINS, 41.and SPRECHER, H. Selective inhibitors of platelet lipoxygenase: 4,7,10,13-icosatetraynoic acid and 5,8,11,14-henicosatetraynoic acid. Prostaglandins, Z&323-332, 1981.
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Prostaglandin biosynthesis HEMLER, M.E., COOK, H.W. and LANDS, W.E.M. can be triggered by lipid peroxides. Arch. Biochem. Biophys. w,340345, 1979.