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Effect of 15-hydroperoxy-arachidonic acid on platelet functions
EFFECT OF 15-HYDROPEROXY-ARACHIDONIC ACID ON PLATELET FUNCTIONS E VERICELand M. LAGARDE Labororoire d'H~rnoh~oloflle, Facult~ Alexis Carrel 6V372. l..~n Cedcx 2 and In~erm L' 63. in~titut Pa,~teur, L)'on. France.
INTRODUCTION
Endothelial cells generate a strong inhibitor of platelet aggregation called prostacyclin. T M This compound is produced from arachidonic acid via prostaglandin endoperoxides. '+ Various hydroperoxides of fatty acids and mainly 15-hydroperoxy-arachidonic acid (15-HPETE) have been described as potent inhibitors of prostacyclin synthetase. l+'ts It has been shown that prostaglandins are synthesized during platelet aggregation, to and more recently, that platelets produce thromboxane A2, a very potent aggregating agent, from arachidonic acid. 6 12-Hydroperoxy-arachidonic acid (12-H PETE) produced by platelet lipoxygenase has been found to inhibit platelet thromboxane synthetase. ~ We report here the effect of 15-HPETE on platelet functions and on arachidonic acid metabolism by these cells.
METHODS 15-HPETE was synthesized from arachidonic acid by soybean lipoxidase ~'~ and purified by thin-layer chromatography on silica gel plates (hexane/diethyl ether/acetic acid, 60:40:1, as eluent). After purification, this hydroperoxide was measured by its absorbance at 234 nm and by an enzymic method using oxidation of NADPH." Human platelets were isolated from their plasma as previously described.' 0 15-H PETE in ethanol (less than 1:'200) was added to platelets simultaneously with aggregating agents. Platelet aggregation was performed according to the turbidimetric method of Born.t Metabolism of exogenous arachidonic acid by platelets was studied with a radiochemical technique.t t
RESULTS AND DISCUSSION Platelet aggregation induced by sodium arachidonate was inhibited by 15-H PETE in a dose dependent manner. Figure l shows the aggregation induced by two different concentrations of arachidonate. The effects of either prostaglandin H2 (PGH2) or its 9-methano-analogue on platelets were also counteracted by 15-HPETE (Fig. 2). The inhibition of human platelet aggregation by hydroperoxides was already mentioned by using linoleic acid hydroperoxide, t~ However, these data were obtained with utilization of 10 times more of linoleic acid hydroperoxide to block the aggregation. Results of Figs. 1 and 2 suggest that 15-HPETE did not act against cyclooxygenase activity. Besides, platelet aggregation induced by thrombin, an agent which does not need PGH2 and thromboxane A2 to aggregate platelets, was not inhibited by 15-HPETE (Fig. 3~.
~.v 2o~.+4 ~,
553
554
E. Vcriccl and M. Lagardc
Sodium arachidonate lO-SM
Sodium arachldonate0.25.lO-SM
~
~ ~
0.8. IO-SM 15-HPETE
2.10-SM 15-HPETE
lO-SM 15-RPETE light [ Trans-] missi°nI
~
~
lO-SM 15-HPETE
0
60 sec. FIe_;.I. Effect of 15-HPI~TI~ on arachidonatc-induccd platclcl aggregation. 15-HPETE and arachidonate wcrc added to platclcts at lhc same timc.
PGH2 5.Io-TM
9-methano~analogueof PGH2 2.8.10-~M ~5 - I~ETE 10-SM 15 - h-PETE 0.25,10-SM 15 - HPETE
Ligh~ I Trans- I
0.6.10-SM 15 - HPETE
mi.slo, t
~t o 60 SeCo FIG. 2. Effccl of IS-HPETE
on either P G H 2 or its 9-methano analogue-induced aggregation.
plate|ct
Effect of 15-hydroperoxy-arachidonicacid on platelet functions Thr
0.1 U/ml
555
Thr O.I U/ml
/
°
~
0
2.10-SM 15-HPETE
FIG. 3. Effect of 15-HPETE on thrombin-induced platelet aggregation.
Results obtained on platelet aggregation were confirmed by study of serotonin release. Table 1 shows that serotonin release from platelets by either arachidonate or collagen (which mainly activates platelets via prostaglandin formation) was completely abolished by 15-HPETE. Whereas, the release induced by thrombin was not modified by the hydroperoxide. Thus, the action of 15-HPETE on platelet functions, as shown above, would seem only to be due to the counteracting of prostaglandin pathway. Moreover, utilization of exogenous arachidonic acid by platelets was not modified by the hydroperoxide, except for a slight decrease of 12-hydroxy-arachidonic acid (12-HETE) formed by the lipoxygenase pathway (Table 2). Therefore, platelet inhibition by 15-HPETE cannot be explained by its action on cyclooxygenase and/or thromboxane synthetase. It has been previously mentioned that 15-HPETE does not inhibit platelet thromboxane synthetase. ~3 The inhibition of the formation of lipoxygenase products could be due to a structural analogy between these products and 15-HPETE itself. Some investigators have shown that inhibition of rat platelet lipoxygenase induces a decrease of aggregation. 2 However, the inhibition of platelet lipoxygenase observed in the presence of 15-HPETE is probably insufficient to provoke the abolition of aggregation. Findings reported above rather suggest that 15-HPETE could counteract the action of PGH2 and/or thromboxane A2. Finally, 15-HPETE was previously shown to inhibit prostacyclin synthetase with an ICso of about 10 -6 M. Our results showed a higher ICso of platelet aggregation. Thus, in the presence of both endothelial cells and platelets, such as the in vivo situation, 15-HPETE should be a more specific inhibitor of prostacyclin formation.
E. Vcric¢l and M. Lagarde
556
T^B~ 1. Serotomn Release from Plat¢lcts Stimulated by Three Different Aggregating Agents. Arachidonate Control
15-H PETE
Collagen
Thrombin
lO s M
2.S/~&:ml
0.I U ml
22.8
36.0
75.2
1.0
[.3
73.4
2.10 s M
Results (n =, 3) were given in percentage of total radioactivity in platelcts prelabelled by [l'=C]scrotonin. T^aL~ 2. Production of prostaglandins and related compounds by human platelets from exogenous arachidonate 10 s M (n = 121. nmoles, 10~ platelets Control 15-HPETE 2.10 -s M
PGF~,
PGEz
0.26 + 0.12 0.38 + 0.18 0.28 + 0.II 0.38 + 0.16 NS. N.S.
TXB=
HHT
12-HETE
3.5 + 2.1 2.5 _+ 1.2 N.S.
2.4 + 2.1 1.9 __. I.I N.S.
4.6 _+.2.6 2.8 + 1.4 P < O.O.S
SUMMARY
15-Hydroperoxy-arachidonic acid (15-HPETE)inhibits human platelet aggregation induced by either sodium arachidonat¢, PGH2, its 9-methano-analogue or collagen but not by thrombin. Serotonin release is also inhibited in the same extent. However, prostaglandin and thromboxane production from exogenous arachidonic acid is not modified whereas lipoxygenase activity is slightly decreased. .4c/,n~,'/rdgrments This
work was supporte~ by grants from I N S E R M A S R 5 and C R L "/9-5-289-5.W e lhank Dr. J. E. Pike of Upjohn Co.. Kalamazoo. MI.. for providing standard prostaglandins and PGHz-analogoc
REFERENCES I. BO~N. G. V. R. Nature 194, 927 929 (1962). 2. DU'ttLH. C. E.. H~,oo~.~4~,.~.E.. J o u w ~ , z . G. H.. T ~ H ~ . (1979).
F. and N u G ~ E ~ . D. H. Lip~+ 14, 241-2~
3. FuN~. M. O.. l~,c. R. and P ~ . N. A. Lip,s II. 113 117 (1976) 4. GRYGI.EWSKI. R.. Bt:NnN(;. S.. M O N C A D ~ S.. FLOWER. R. and VANF, J. Prost~Iondins I~ ~ 5
713 (1976).
5. H A ~ R G . M. a ~ SAML'E~N, B. J. biol. Chem. ~ 5 3 ~ 5 3 3 5 (1~7~ 6. HA~aERG. M., S ~ N ~ N , J. and S A ~ L ' E ~ . B. Pr~. Na~ ~ c ~ . Sci. ~ 2 ~ 8 (19~5). 7. H ~ M M ~ O M , S. and F A L ~ U , P. P r ~ N a t / R c ~ . ~ . 7~ ~ 9 1 - ~ 9 5 (1977). 8. H ~ H , R. a ~ T ~ P ~ A. ~nal. B i ~ m . ~6. 18~i9~ [1976). 9. J ~ N ~ s . R. A., MO~7ON. D. R. K ~ s ~ . J. H. ~ M ~ N . R R., M ( ~ u ~ , J. C. and SL's. F. F. Prosta@la~ms I ~ 915-928 (1978). 10. L A G ~ M., B~YOS, P. A., GL'~CH~RDAST, M. and ~('H~VASNL M. T h r o ~ s . Res. 17, 581-588 (19~). I I. L A ~ G ~ M., G H ~ A. and ~ C H A V ~ S ~ M. C/in. Chim. Acfa ~ . 255 259 (1977). 12. Mo~c~D~, S.. G~YG~wsKt. R, BL'S~ls6. S. and VAst. J. Nalure ~3+ ~ 3 ~ 5 [~976). 13. MoNcAo~. S. G~GLEWSKL R., B U ~ G . S. and V~NE, J. Prostaglandins I ~ 715-737 (1976). 14. Mosc'~o~. S.. V~S~ J. and H I ~ E. A. Lance~ i, 1 8 - ~ (1977). 15. S ~ L ~ . 1.. S m ~ . D., FLOW~ R, MOSCAD~ S. and V ~ ~. Bi~him. Biophrs..4era ~ , 2 ~ 2 6 2 (19781. 16 SM~(. J. B., and W~LL~ A. L. Nature N~" Biol. ~ 1 . 235 237 (1971). 17. T A ~ t ~ L 1., S ~ I ~ , M. a ~ B~LD~SL M. ~. ~ . Clin. Med. 81. 5 8 7 ~ 2 (1973).
INTRODUCTION
Endothelial cells generate a strong inhibitor of platelet aggregation called prostacyclin. T M This compound is produced from arachidonic acid via prostaglandin endoperoxides. '+ Various hydroperoxides of fatty acids and mainly 15-hydroperoxy-arachidonic acid (15-HPETE) have been described as potent inhibitors of prostacyclin synthetase. l+'ts It has been shown that prostaglandins are synthesized during platelet aggregation, to and more recently, that platelets produce thromboxane A2, a very potent aggregating agent, from arachidonic acid. 6 12-Hydroperoxy-arachidonic acid (12-H PETE) produced by platelet lipoxygenase has been found to inhibit platelet thromboxane synthetase. ~ We report here the effect of 15-HPETE on platelet functions and on arachidonic acid metabolism by these cells.
METHODS 15-HPETE was synthesized from arachidonic acid by soybean lipoxidase ~'~ and purified by thin-layer chromatography on silica gel plates (hexane/diethyl ether/acetic acid, 60:40:1, as eluent). After purification, this hydroperoxide was measured by its absorbance at 234 nm and by an enzymic method using oxidation of NADPH." Human platelets were isolated from their plasma as previously described.' 0 15-H PETE in ethanol (less than 1:'200) was added to platelets simultaneously with aggregating agents. Platelet aggregation was performed according to the turbidimetric method of Born.t Metabolism of exogenous arachidonic acid by platelets was studied with a radiochemical technique.t t
RESULTS AND DISCUSSION Platelet aggregation induced by sodium arachidonate was inhibited by 15-H PETE in a dose dependent manner. Figure l shows the aggregation induced by two different concentrations of arachidonate. The effects of either prostaglandin H2 (PGH2) or its 9-methano-analogue on platelets were also counteracted by 15-HPETE (Fig. 2). The inhibition of human platelet aggregation by hydroperoxides was already mentioned by using linoleic acid hydroperoxide, t~ However, these data were obtained with utilization of 10 times more of linoleic acid hydroperoxide to block the aggregation. Results of Figs. 1 and 2 suggest that 15-HPETE did not act against cyclooxygenase activity. Besides, platelet aggregation induced by thrombin, an agent which does not need PGH2 and thromboxane A2 to aggregate platelets, was not inhibited by 15-HPETE (Fig. 3~.
~.v 2o~.+4 ~,
553
554
E. Vcriccl and M. Lagardc
Sodium arachidonate lO-SM
Sodium arachldonate0.25.lO-SM
~
~ ~
0.8. IO-SM 15-HPETE
2.10-SM 15-HPETE
lO-SM 15-RPETE light [ Trans-] missi°nI
~
~
lO-SM 15-HPETE
0
60 sec. FIe_;.I. Effect of 15-HPI~TI~ on arachidonatc-induccd platclcl aggregation. 15-HPETE and arachidonate wcrc added to platclcts at lhc same timc.
PGH2 5.Io-TM
9-methano~analogueof PGH2 2.8.10-~M ~5 - I~ETE 10-SM 15 - h-PETE 0.25,10-SM 15 - HPETE
Ligh~ I Trans- I
0.6.10-SM 15 - HPETE
mi.slo, t
~t o 60 SeCo FIG. 2. Effccl of IS-HPETE
on either P G H 2 or its 9-methano analogue-induced aggregation.
plate|ct
Effect of 15-hydroperoxy-arachidonicacid on platelet functions Thr
0.1 U/ml
555
Thr O.I U/ml
/
°
~
0
2.10-SM 15-HPETE
FIG. 3. Effect of 15-HPETE on thrombin-induced platelet aggregation.
Results obtained on platelet aggregation were confirmed by study of serotonin release. Table 1 shows that serotonin release from platelets by either arachidonate or collagen (which mainly activates platelets via prostaglandin formation) was completely abolished by 15-HPETE. Whereas, the release induced by thrombin was not modified by the hydroperoxide. Thus, the action of 15-HPETE on platelet functions, as shown above, would seem only to be due to the counteracting of prostaglandin pathway. Moreover, utilization of exogenous arachidonic acid by platelets was not modified by the hydroperoxide, except for a slight decrease of 12-hydroxy-arachidonic acid (12-HETE) formed by the lipoxygenase pathway (Table 2). Therefore, platelet inhibition by 15-HPETE cannot be explained by its action on cyclooxygenase and/or thromboxane synthetase. It has been previously mentioned that 15-HPETE does not inhibit platelet thromboxane synthetase. ~3 The inhibition of the formation of lipoxygenase products could be due to a structural analogy between these products and 15-HPETE itself. Some investigators have shown that inhibition of rat platelet lipoxygenase induces a decrease of aggregation. 2 However, the inhibition of platelet lipoxygenase observed in the presence of 15-HPETE is probably insufficient to provoke the abolition of aggregation. Findings reported above rather suggest that 15-HPETE could counteract the action of PGH2 and/or thromboxane A2. Finally, 15-HPETE was previously shown to inhibit prostacyclin synthetase with an ICso of about 10 -6 M. Our results showed a higher ICso of platelet aggregation. Thus, in the presence of both endothelial cells and platelets, such as the in vivo situation, 15-HPETE should be a more specific inhibitor of prostacyclin formation.
E. Vcric¢l and M. Lagarde
556
T^B~ 1. Serotomn Release from Plat¢lcts Stimulated by Three Different Aggregating Agents. Arachidonate Control
15-H PETE
Collagen
Thrombin
lO s M
2.S/~&:ml
0.I U ml
22.8
36.0
75.2
1.0
[.3
73.4
2.10 s M
Results (n =, 3) were given in percentage of total radioactivity in platelcts prelabelled by [l'=C]scrotonin. T^aL~ 2. Production of prostaglandins and related compounds by human platelets from exogenous arachidonate 10 s M (n = 121. nmoles, 10~ platelets Control 15-HPETE 2.10 -s M
PGF~,
PGEz
0.26 + 0.12 0.38 + 0.18 0.28 + 0.II 0.38 + 0.16 NS. N.S.
TXB=
HHT
12-HETE
3.5 + 2.1 2.5 _+ 1.2 N.S.
2.4 + 2.1 1.9 __. I.I N.S.
4.6 _+.2.6 2.8 + 1.4 P < O.O.S
SUMMARY
15-Hydroperoxy-arachidonic acid (15-HPETE)inhibits human platelet aggregation induced by either sodium arachidonat¢, PGH2, its 9-methano-analogue or collagen but not by thrombin. Serotonin release is also inhibited in the same extent. However, prostaglandin and thromboxane production from exogenous arachidonic acid is not modified whereas lipoxygenase activity is slightly decreased. .4c/,n~,'/rdgrments This
work was supporte~ by grants from I N S E R M A S R 5 and C R L "/9-5-289-5.W e lhank Dr. J. E. Pike of Upjohn Co.. Kalamazoo. MI.. for providing standard prostaglandins and PGHz-analogoc
REFERENCES I. BO~N. G. V. R. Nature 194, 927 929 (1962). 2. DU'ttLH. C. E.. H~,oo~.~4~,.~.E.. J o u w ~ , z . G. H.. T ~ H ~ . (1979).
F. and N u G ~ E ~ . D. H. Lip~+ 14, 241-2~
3. FuN~. M. O.. l~,c. R. and P ~ . N. A. Lip,s II. 113 117 (1976) 4. GRYGI.EWSKI. R.. Bt:NnN(;. S.. M O N C A D ~ S.. FLOWER. R. and VANF, J. Prost~Iondins I~ ~ 5
713 (1976).
5. H A ~ R G . M. a ~ SAML'E~N, B. J. biol. Chem. ~ 5 3 ~ 5 3 3 5 (1~7~ 6. HA~aERG. M., S ~ N ~ N , J. and S A ~ L ' E ~ . B. Pr~. Na~ ~ c ~ . Sci. ~ 2 ~ 8 (19~5). 7. H ~ M M ~ O M , S. and F A L ~ U , P. P r ~ N a t / R c ~ . ~ . 7~ ~ 9 1 - ~ 9 5 (1977). 8. H ~ H , R. a ~ T ~ P ~ A. ~nal. B i ~ m . ~6. 18~i9~ [1976). 9. J ~ N ~ s . R. A., MO~7ON. D. R. K ~ s ~ . J. H. ~ M ~ N . R R., M ( ~ u ~ , J. C. and SL's. F. F. Prosta@la~ms I ~ 915-928 (1978). 10. L A G ~ M., B~YOS, P. A., GL'~CH~RDAST, M. and ~('H~VASNL M. T h r o ~ s . Res. 17, 581-588 (19~). I I. L A ~ G ~ M., G H ~ A. and ~ C H A V ~ S ~ M. C/in. Chim. Acfa ~ . 255 259 (1977). 12. Mo~c~D~, S.. G~YG~wsKt. R, BL'S~ls6. S. and VAst. J. Nalure ~3+ ~ 3 ~ 5 [~976). 13. MoNcAo~. S. G~GLEWSKL R., B U ~ G . S. and V~NE, J. Prostaglandins I ~ 715-737 (1976). 14. Mosc'~o~. S.. V~S~ J. and H I ~ E. A. Lance~ i, 1 8 - ~ (1977). 15. S ~ L ~ . 1.. S m ~ . D., FLOW~ R, MOSCAD~ S. and V ~ ~. Bi~him. Biophrs..4era ~ , 2 ~ 2 6 2 (19781. 16 SM~(. J. B., and W~LL~ A. L. Nature N~" Biol. ~ 1 . 235 237 (1971). 17. T A ~ t ~ L 1., S ~ I ~ , M. a ~ B~LD~SL M. ~. ~ . Clin. Med. 81. 5 8 7 ~ 2 (1973).