THE INFLUENCE OF ISOPROSTANES ON ADP-INDUCED PLATELET AGGREGATION AND CYCLIC AMP-GENERATION IN HUMAN PLATELETS

Thrombosis Research, Vol. 86, No. 4, pp. 337-342, 1997 Copyright @ 1997 Elsevier Science Ltd e Printed in the USA. All rights reserved 0049-3848/97 $17.00 + ,00

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BRIEFCOMMUNICATION

THE INFLUENCE OF ISOPROSTANES ON ADP-INDUCED PLATELET AGGREGATION AND CYCLIC AMP-GENERATION IN HUMAN PLATELETS NorbertLeitinger,IngridBlazek,HelmutSinzinger Departmentof NuclearMedicine,Universityof Vienna,Austria,andWilhelmAuerswald AtherosclerosisResearchGroup(ASF),Vienna,Austria

(Received 11November 1996 by Editor J. Sturzebecher; revised/accepted 21 March 1997)

Isoprostanesare eicosanoidsthatare non-enzymaticproductsof freeradicalcatalyzedperoxidation of arachidonylcontainingphospholipids(l). They are subsequentlyreleased from the site of generationas estersof phospholipid(bound)or throughthe actionof phospholipase(s)Az in free form (2). One F2-isoprostanewhose formationis highlyfavoredis 8-iso-PGFzuwhich has been shown to be a potentpulmonaryand renal vasoconstrictor(3,4). Actions of 8-iso-PGF2mwere demonstratedto be mediated through a receptor related to but probably distinct from the thromboxane(TXAJ / endoperoxide(PGH2) receptor (5). Although 8-epi-PGF2ais a potent agonistof TXA2/PGH2receptorsin vascularsmoothmuscle, interestinglyit acts primarilyas an antagonistof TXA2/PGHzreceptorson both humanand rat platelets(6). Thereis also evidencefor the generationof D- and E-ring isoprostanes(7) and their receptor-mediatedaction on smooth muscle cells (8) and platelets (9). Recent reports support the hypothesis that Ez-isoprostane receptorsare distinctfrom TXA2/PGHzreceptors,suggestingat least different subtypes, one of thesespecificallyrecognizingEz-isoprostanes(9). Isoprostaneshave been suggested to be useful markers for oxidant injury. For example, F2isoprostaneswere significantlyelevatedin plasmaof rats duringreperfusionafter hepaticischemia (10)and in patientswith hepatorenalsyndrome(11). It has been suggestedthat the releaseof F,isoprostanesfromoxidizedLDL in macrophagescouldbe a contributoryfactorin the development of atherosclerosisand at sites of inflammation,locally elevated levels of isoprostanes could contributeto blood cell activation.In this study we investigatepossible pro- or antiaggregatory propertiesof variousF- and E-typeisoprostaneson humanplatelets. MATERIALANDMETHODS Venousbloodwas collectedfromhealthyhumanvolunteers(5 females,2 males,age 23-31 years) not havingtaken medicationsfor at leasttwo weeks.Bloodwas collectedinto syringescontaining 0.38% sodiumcitrate(finalconcentration)as anticoagulant.Plateletrich plasma (PRP) was then Key words:isoprostanes,plateletaggregation,cyclicAMP. Correspondingauthor:Dr. NorbertLeitinger,Departmentof Medicine/ Cardiology,Centerfor the HealthSciences,Room47-123,UCLAMedicalCenter,Los Angeles,CA 90095-1679,USA. Fax: (310)206-5178;E-mail:nleit@pathology .medsch.ucla.edu

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prepzuedby centrifugingthe blood at 150 x g for 5 min at 25 W and plateletpoor plasma (PPP) was obtainedby centrifugingPRP at 500 x g for 10 min at 25 ‘C. Plateletcount in PRP was usuallyabout280.000per pl or otherwiseadjustedwith PPP. PRP was used within 2 hours after preparation.Aggregationstudieswere performedaccordingto the methodof Born (12) using a CHRONO-LOG500VS aggregometer(CHRONO-LOGCorporation, FIavertown, PA, USA). Full light transmission was set using PPP. For each set of experiments a threshold ADP (adenosine-5’-diphosphatedisodium salt, Boehringer Mannheim, Germany) concentration sufficientto reach maximalaggregationwas determined.Thisconcentration(2-5@f) was used to preparea controlcurve. Isoprostanes(CaymanChemical,Ann Arbor,MI, USA) were preparedas stocksolutionsin ethanoland the finalconcentrationof ethanoladded to the plasmawas < 0.5%. Controlexperimentsdemonstratedthat ethanolin these concentrationsdid not influenceplatelet aggregation.Isoprostaneswere added to the plateletsuspension 1 minutebefore the additionof ADP. The maximumincrease in light transmission (at 4 minutes after the addition of ADP) expressed as percent of that in the control was taken as a criterion for the degree of platelet aggregation. CyclicAMP levels in plateletswere determinedusing an EnzymeImmunoAssay (EIA, Cayman Chemical,Ann Arbor, MI, USA). SixhundredW1PRP were incubatedfor 15 minuteswith 8.34 wghn.1(final concentration) of various isoprostanes with or without the addition of the thromboxaneantagonistSQ29548 (10 ~ghrd, Cayman Chemical,Ann Arbor, MI, USA). The assaywas performedaccordingto the manufacturer’sinstructions. RESULTS The influenceof differentisoprostanesin concentrationsfrom 10pghnl to 10 Lghnl was tested on ADP-inducedin-vitro plateletaggregation.8-iso-PGFz@ did not show any influence on ADPinducedplateletaggregation,even whentestedat highconcentrations(10 ~ghl). However, slight reversibleaggregationcouldbe observedwhenthiscompoundwas added in concentrationsof 10 and 1 ~ghnl (data not shown). 8-iso-PGF1awas also inactive on ADP-induced platelet aggregation.At 10 Lghnl (but not at lower concentrations)slightreversibleaggregationcouldbe observed, similar to the effect of 8-iso-PGFza(data not shown). The ~-isomers of these two compounds, 8-iso-PGFlp and 8-iso-PGFzP did not show any effect on plateletsand did not influenceADP-inducedplateletaggregationat the tested concentrations,suggestingthat the cxconfigurationis requiredfor interactionwithcertainplateletreceptors.8-iso-15-keto-PGFhand 8iso-13,14-dihydro-15-keto-PGF2@ did not show any influence on ADP-induced platelet aggregation (data not shown). 8-iso-PGF~minhibited ADP-induced platelet aggregation at concentrationsgreater than 1 pghnl (Figure la). ~igure lb demonstratesan example for the inhibitionof ADP-inducedplateletaggregationby 8-iso-PGF~u. From the group of testedE-type isoprostanes, 8-iso-PGEldemonstratedan inhibitionof platelet aggregationin a concentration-dependent mannerstartingat concentrationsgreaterthan 100 nghd (Figure la). The addition of 8-iso-PGE2 led to spontaneous aggregation of platelets. This compoundwas testedat concentrationsof 10, 1 and 0.1 @rd and was able to aggregateplatelets for 31.3, 25.9 and 4.7 %, respectively,comparedto the control curve (mean values of three differentvolunteers). At 10 nghnl and 1 nghnl, 8-iso-PGE2was no longer able to aggregate platelets.Figure Ic demonstratesirreversible aggregationof plateletsinducedby 1 j.@nl 8-isoPGE2. However, a significantpotentiationof ADP-inducedplateletaggregationby 8-iso-PGEz couldnot be observed. To demonstratewhetherthe describedeffectsof isoprostaneson human plateletsare mediatedby cyclicAMP,measurementsof this intracellularmediatorin plateletsafter incubationwith different isoprostanes were performed using a cyclic AMP EIA. Whereas 8-iso-PGF2., 8-iso-13,14dihydro-15-keto-PGF2a,8-iso-PGFzP,8-iso-PGF1a,8-iso-PGFIP,8-iso-PGFqa,and 8-iso-PGE2 didnot influencethe generationof cyclicAMPin humanplatelets,8-iso-PGEl and 8-iso-15-ketoPGFZUseemed to increasecyclic AMP about 1.9 fold. Prostacyclinused as a positive control increasedcyclic AMP about 3.2 fold in these experiments.However, due to large standard deviationsthe effectsof 8-iso-PGE1and 8-iso-15-keto-PGF2@ were not significant(Figure2). The additionof indomethacindid not haveany influenceon theseresults(datanot shown).However,

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Influenceof 8-iso-PGFJa and 8-iso-PGE, on ADP-inducedplatelet aggregation(a). Aggregationstudies were performed as described in methods. The % aggregation compared with the standard curve which was prepared for each set of experiments (withoutisoprostanes)were evaluatedfrom the aggregationcurves. Data are expressed as mean + SD, n=7. Examplefor the inhibitoryeffect of 10 ~ghnl 8-iso-PGFqa(b). Spontaneousplateletaggregationafteradditionof 1 pghnl 8-iso-PGEz(c). whenthe thromboxanereceptorantagonistSQ29548was addedto the plateletsprior to incubation with isoprostanes,8-iso-PGElwas the only compoundwhich significantlyincreasedcyclicAMP generationabout2.4 times.Prostacyclinused as a positivecontrolincreasedcyclicAMP about4.1 fold in these experiments(Figure 2). SQ29548itselfhad no effect on cyclicAMP generationin platelets(datanot shown). DISCUSSION The actionsof regularF- and E-typeprostaglandins(producedthroughenzymaticconversionof freearachidonicacid by cyclooxygenase)on plateletshave been extensivelystudied. PGEZat low concentrationswas shown to potentate plateletaggregationinducedby ADP (13) or arachidonic acid (14). However, when higher concentrationsof PGE2 (0.1-100 @vi)were used, platelet inhibitionwas observed. This biphasic effect displayed by PGE2 is believed to be due to a

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decreasein the cyclicAMP contentof the plateletsby low concentrationsand to an increase by high concentrations(15). PGE1 was shown to inhibitplateletaggregationinduced by various agonistsby increasingcyclicAMP (16).PGF2ahas been shownto antagonizeplateletaggregation induced by either arachidonic acid or an endoperoxideanalog, possibly by acting at the thromboxanereceptor(17). Thiseffecthas also been demonstratedfor 8-iso-PGF,Q(6). The additionaldouble bond at position 17 in PGH~ and TXAJ eliminatesplatelet aggregatory activity,thesetrienoicproductsof eicosapentaenoicacid wereshownto inhibitplateletaggregation by elevatingintracellularcyclicAMP (18). In this study, the effects of isoprostaneson human platelets(cAMP-generationin plateletsand ADP-inducedplateletaggregation)were investigated.Isoprostanesare producedby the actionof free radicals on phospholipidbound arachidonicacid and they possess a different side chain orientation(cis-cis) than enzymaticallyproduced prostaglandins(all-trans). 8-iso-PGFztiand 8iso-PGF1aseemed to act on plateletsby showing slight reversibleaggregation,however, both compoundsdid not influenceADP-inducedplateletaggregation.The influenceof isoprostaneson plateletfunctionhas only been testedfor 8-iso-PGFzaand 8-iso-PGE2so far. Morrowet al. found

+SQ29548

-SQ29548 PGI* iso-PGE2 iso-PGE1 iso-PGF3a iso-15-keto-PGF2a iso-dh-15-k-PGFza iso-PGFzP iso-PGFIP iso-PGF2a iso-PGF1a

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fold increase cAMP FIG. 2. Influenceof isoprostaneson cyclicAMP generationin human platelets.Sixhundred@ PRP were incubatedfor 15 minuteswith 8.34 pghnl (final concentration)of various isoprostaneswith or withoutthe additionof the thromboxaneantagonistSQ29548 (10 ~ghnl).Cyclic AMP assays were performedin triplicateas describedin methods. Data of three independentexperimentswith plateletsobtained from three volunteers are presentedas mean* SD.

1

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that plateletsexhibiteda modest degree of reversible aggregationwhen incubated with 8-isoPGF2Uat a concentrationof 10”4M (35.5 yg/ml). Irreversibleaggregationdid not occur (6). In addition, they observed a plateletshape change beginningat a concentrationof 10-6M (0.35 I.@@ 8-iso-PGF2w.These observationsare consistentwith the results obtainedin this study. Moreover, Morrow and collegues (6) demonstratedthat plateletshape change and reversible platelet aggregation could be inhibited by addition of a competitiveTXA2/PGHz receptor antagonist,SQ29548 (10-7M).8-iso-PGFzawas also able to inhibithuman plateletaggregation inducedby the two TXAz/PGH2receptor agonists U46619 and IBOP. 8-iso-PGE also was shown to act on plateletsvia the TXA~PGH2receptor. At high concentrations(>103M) 8-isoPGEZwas ableto inducehumanplateletaggregation.Thiseffectwas unaffectedby indomethacin, but couldbe inhibitedby SQ29548.8-iso-PGEzinhibitedthe actionsof the thromboxaneagonists U46619and IBOP and plateletaggregationinducedby arachidonicacid,but not by ADP. Since 8iso-PGEzis a potentagonistof TXA~GH2 receptorson smoothmusclecells, it seems to act as an antagonistfor this receptor on human platelets.It can be concluded that there exist either differentTXA2/PGH2receptorsin plateletsand smooth muscle cells, or a unique isoprostane receptoron vascularsmoothmusclecells(9). The resultsshownin this reportdemonstratethatthe effectof F-isoprostaneson plateletsis similar to the effect of enzymaticallyproduced F-prostaglandins,suggesting that the prostane ring configurationand the structureof the side chainsbut not side chain orientationis responsiblefor receptor-ligandinteraction.The lack of activityof fl-isomersand 15-keto-and 13,14-dihydro-15keto- derivatives of F-isoprostanes supports this conclusion. 8-iso-PGE2 concentrationdependentlystimulatedplateletsresultingin irreversibleaggregation.Althoughconcentrationsup to 10pg/ml were used, an inhibitoryeffect,as shown for enzymaticallyproducedPGE2, was not observed.The inhibitoryactivityof 8-iso-PGE1(unlikethat of 8-iso-PGF~a)seemedto be due to elevationof cyclicAMP levels.Thiseffectwas not influencedby the additionof the TXAz/PGH2 receptorantagonistSQ29548, suggestinginteractionof this compoundwith a differentreceptor, probablywith the PG12receptor,as shownfor enzymaticallyproducedPGEI. REFERENCES 1. MORROW, J.D., HILL, K.E., BURK, R.F., NAMMOUR, T.M., BADR, K.F. and ROBERTS,L.J. A seriesof prostaglandinFz-likecompoundsare producedin vivo in humansby a non-cyclooxygenase,free radicalcatalyzedmechanism.Proc Natl Acad Sci USA 87, 93839387, 1990. 2. MORROW, J.D., HARRIS, T.M. and ROBERTS, L.J. Non-cyclooxygenaseoxidative formationof a series of novel prostaglandins:analyticalramificationsfor the measurementof eicosanoids.AnalytBiochem184, 1-10,1990. 3. BANERJEE, M., KANG, K.H., MORROW,J.D., ROBERTS, L.J. and NEWMAN, J.H. Effectsof a novelprostaglandin,8-epi-PGFza,in rabbitlungin situ.Am J Physiol 26.3,660-663, 1992. 4. TAKAHASHI, K., NAMMOUR, T.M., EBERT, J., MORROW,J.D., ROBERTS, L.J. and BADR, K.F. Glomerularactionsof a free radicalgeneratednovelprostaglandin,8-epi-PGF,., in the rat: evidencefor interactionwith thromboxaneAzreceptors.J Clin Invest90, 136-141,1992. 5. FUKANAGA, M., MAKITA, N,, ROBERTS,L.J., MORROW,J.D., TAKAHASHI, K. and BADR, K.F. Evidence for the existenceof Fz-isoprostanereceptors on rat vascular smooth musclecells. Am J Physiol264, C1619-C1624,1993. 6. MORROW, J.D., MINTON, T.A. and ROBERTS, L.J. The F2-isoprostane, 8-epiprostaglandinFzu, a potentagonistof the vascular thrmbcwnelendoperoxide recwtw iS a plateletthromboxane/endoperoxide receptorantagonist.Prostaglandins44, 155-163,1992. 7. MORROW,J.D., MINTON T.A., MUKUNDAN, C.R., CAMPBELL,M.D., ZACKERT, W.E., DANIEL, V.C., BADR, K.F., BLAIR, I.A. and ROBERTS, L.J. Free radical-induced generationof isoprostanesin vivo.J Biol Chem269,4317-4326, 1994. 8. FUKUNAGA, M., TAKAHASHI, K. and BADR, K.F. Vascularsmoothmuscle actionsand receptor interactions of 8-iso-prostaglandinEz, an Ez-isoprostane.Biochem Biophys Res Commun 295,507-515, 1993.

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9. LONGMIRE, A.W., ROBERTS,L.J. and MORROW,J.D. Actionsof the E2-isoprostane,8-

iso-PGE2 on the platelet thromboxane/endoperoxidereceptor in humans and rats: Additional evidencefor the existenceof a uniqueisoprostanereceptor.Prostaglandins48,247-257, 1994. 10.MATHEWS, W.R., GUIDO, D.M., FISHER, M.A. and JAESCHKE,H. Lipid peroxidation as molecularmechanismof livercellinjuryduringreperfusionafter ischemia.Free Rad Biol Med 16,763-770, 1994. 11. MORROW,J.D., MOORE, K.P., AWAD, J.A., RAVENSCRAFT,M.D., MARINI, G., BADR, K.F., WILLIAMS, R. and ROBERTS, L.J. Marked overproduction of noncyclooxygenasederived prostanoids (Fz-isoprostanes)in the hepatorenal syndrome. J Lipid Mediat 6,417-420, 1993. 12. BORN, G.C.R. Aggregationof blood plateletsby adenosinediphosphateand its reversal. Nature (London)194,927-929, 1962. 13. SHIO, H. and RAMWELL,P.W. Effectof prostaglandinE2 and aspirin on the secondary aggregationof humanplatelets.NatureNew Biology236,45-46, 1972. 14.VARGAFTIG, B.B. and CHIGNARD, M. Substancesthat increasethe cyclicAMP content prevent plateletaggregationand the concurrentrelease of pharmacologicallyactive substances evokedby arachidonicacid. AgentsActions5, 137-144,1975. 15. SALZMAN, E.W., KENSLER, P.C. and LEVINE, L. Cyclic 3’-5’-adenosine monophosphatein humanbloodplatelets.Ann NY Acad Sci 201, 61-71, 1972. 16. MCDONALD, J.W.D. and STUART, R.K. Interaction of prostaglandin El and Ez in regulation of cyclic AMP and aggregation in human platelets: evidence for a common prostaglandinreceptor.J Lab Clin Med 84, 111-121,1974. 17. HUNG, S.C., GHALI, N.I., VENTON, D.L. and LEBRETON, G.C. ProstagkmdinFza antagonizesthromboxaneA2-induced human plateletaggregation.Prostaglandins24, 195-206, 1982. 18.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 76,944-948, 1979.