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Potentiation of the antiplatelet action of adenosine in whole blood by dipyridamole or dillazep and the camp phosphodiesterase inhibitor, RA 233
THROMBOSIS RESEARCH 43; 161-175, 1986 0049-3848/86 $3.00t .OO Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.
Doloretta D. Dawicki,KailashC. Agarwaland RobertE. Parks,Jr. Sectionof BiochemicalPharmacology,Division of Biology and Medicine, BrownUniversity,Providence,RI 02912,U.S.A. (Received
15.1.1986; Accepted in revised form 15.4.1986 by Editor J.G. White)
Adenosine (Ado,10 - 50 PM), a potent inhibitorof ADP-inducedhuman plateletaggregationin platelet-rich plasma U?F!P), does not inhibit aggregation in whole blood. However, the Ado analogs, 2-fluoroadenosine, 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine (NECA) which are resistantto deamination(2-fluoroadenosine) or deamination and phosphorylation (2-chloroadenosine and NECA), inhibitaggregation in whole blood with IC50 values of 12 uM, 2.3 uM and 0.26uM, respectively. The inhibitory effectof NBCA (200nM) is potentiated by the platelet cAMP phosphodiesterase(PDE)inhibitorBA 233 (5PM). Inhibition of the erythrocytic nucleosidetransportsystemby dilazep (1uM) or dipyridamole (10 uM), or blockade of Ado metabolism by 2Weoxycoformycin(5 uM) plus 5-iodotubercidin (10PM), evokesthe antiaggregatory actionof Ado in whole blood (IC50= 2 uM). RA 233 (5 PM) potentiates Ado-mediated inhibition about lo-fold when nucleosidetransportor Ado metabolismis blocked. Ado (10LIM or 200 nM) is rapidly metabolized within 1 min in whole blood. When nucleosidetransportis inhibitedby dilazepor dipyridasole, or when Ado metabolismis blockedby 2'-deoxycoformycin and 5-iodotubercidin, 50 - 60 % of the Ado remainsin the plasmaafter5 min. Theseresults show that the failureof Ado to inhibitplateletaggregation in mole blood results from its rapid uptakeandmetabolism byerythrocytes. More importantly,these data emphasizethe key role of nucleoside transportinhibitionin the antiplateletactions of dipyridamole and dilazep.In addition,superiortherapeutic resultsmay be obtainedfrcm the combinationof blockade of the nucleosidetransportsystemwith inhibition of plateletCAMP PDE.
Key words:plateletaggregation, adenosine, dipyriwle, dilazep,PA 233 161
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Althoughdipyridamole (PersantineR) has seen widespreadclinicaluse as a vasodilatorand antiplatelet drug for a quartercentury(1,2), therehas been no generallyaccepted explanation of itsmechanismof action. Sinceelevated intraplateletcAMP levels are associatedwith inhibitionof platelet aggregation (3), the fact that dipyridamole can inhibit platelet CAMP phosphodiesterase (PDE)has led many to assm that this is the targetenzyme for its antithromboticeffect (4,5). It must be noted, however, that publishedinhibitionconstants(Ki)of dipyridamole with CAMP PDE are 12 I.~M and 19pM (6,7), whichare considerably higherthan the plasma levelsof free dipyridamoleusually achieved during therapy (8,9).Thatdipyridamole can block adenosine(Ado)metabolismin intactcells by inhibiting the membraneassociated nucleosidetransportsystemhas also been recognized for manyyears (10,ll). However, the role of nucleoside transport inhibition in the antiplatelet effectsof dipyridamole couldnot be accuratelyassessedbecause of limitations in methodsfor studyingaggregation in the physiological milieu of whole blood. The developmentof impedance aggregometry (12),whichpermitsthe studyof plateletaggregation in whole blood,has made necessarya reevaluation of the antiplateletactionsof dipyridamole and relatedagentssuch as dilazepand nitrobenzylthioinosine. Recentinvestigations in this laboratory(13,14) have shown that dipyridamole, dilazepand nitrobenzylthioinosine whichare potent inhibitors of humanerythrocytic nucleosidetransport(15-18), neitherinhibit alone nor potentiate the antiaggregatory effectof Ado in platelet-rich plasma (PRP). However, these nucleoside transport inhibitors restore the antiplatelet action of Ado in whole blood when administered at pharmacologically relevant doses. l3ycontrast, the cqund RA 233,whichis a closechemicalrelativeof dipyridamole, is essentially devoidof nucleoside transportinhibition, but causes strikingpotentiationof the antiplatelet actionof Ado in PRP due to its relativelypotentCAMP PDH inhibitory activity (Ki= 0.61.1M1 (6). These observationshavebeenexplainedbythe very rapid metabolism of Ado by the erythrocyticenzymes, adenosine deaminaseand adenosinekinase,which,in whole blood,catalysethe disappearance of 10 uM Ado from the plasma in less than 1 min (13,14,19). When the actions of adenos,ine deaminaseand adenosinekinaseare blockedby specificinhibitors, i.e.2- deoxycoforqzin(20)and 5-iodotubercidin (21),respectively, or when nucleosidetransportis inhibited,the rate of disappearanceof Ado from plasma is markedly decreasedand its antiplateletaction is evoked. An impressive body of evidence has accumulated indicatingthat Ado is an important physiological regulator of adenylate cyclase through its interactionswith specific Ado receptors, which either inhibit (Al)or stimulate (AZ)adenylatecyclase dependingon the specificcell or tissue involved. (Forrecent reviews of the regulatoryactionsof Ado see 22,23). For example,plateletspossessA2 receptorsfor Ado. Our earlierfindingsraisedthe questionof whetherthe canbination of a potent cAMP PDE inhibitor with a blocker of nucleoside transportand, therefore, of Ado metabolism, mightenhancethe antiplatelet actionof Ado and perhapsotherof its physiological functions as well. It was proposedthatan
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ideal antiplatelet drug would be one that combinesinhibitoryeffects on nucleosidetransportwith thoseon platelet-specific CAMP PDE. The present studytests this conceptbyexaminingthe antiplateletactionof Ado or Ado analogs,e.g.5'-N-ethylcarboxamidoadenosine (NEW , in combinationwith the nucleosidetransportinhibitors, dilazepor dipyridarrole and/orthe CAMP PDE inhibitor, BA 233. Portionsof this work have been presentedin preliminary form elsewhere(24).
Adenosine 5'-diphosphate(ADP),adenosine (Ado),2-chloroadenosine, 5'-N-ethylcarboxamidoadenosine (NEW, and dipyridamole(PersantineR) were obtainedfromsigma ChemicalCo.,St.Louis,MO. Dilazep (CormelianR) was a gift frcmHoffmann-IaHoche, Inc.,Nutley,NJ. PA 233 was a gift from Dr. Karl ThanaeG&H, Biberachan der Hiss,Germany. 2Weoxycoformycin(Pentostatir?) and 2-fluoroadenosinewere obtained from the Drug Development Branch, Divisionof CancerTreatmentof the NationalCancerInstitute, Bethesda,MD. 5-Iodotubercidin was a gift fromDr.L.B.Townsend,Universityof Michigan, was obtainedfran Moravek Ann Arbor,MI. [8-14C]Ado (sp. act.= 59 mCi/mrole) Biochemicals, Brea, CA. and was >95% pure as shown by thin-layer chranatography on polyethyleneimine-cellulose sheetswith potassiumchloride (100mM) as the eluent. [U-14C]Ado(sp.act. = 300 mCi/mmole)was obtained from ICN Hadiochemicals, Irvine,CA.
Blood was drawnfrom healthydonorswho had not takenantiplatelet drugs for at leastten days. The blood was dispensedinto plastictubescontaining 0.1volume of sodiumcitrate (3.8%)topreventcoagulation. Platelet-rich plasma (PFU?) was obtainedfran the blood by centrifugation at 300 g for 10 min at room temp. Platelet-poorplasma was obtainedfrom the remainingblood constituentsby centrifugationat 1500 g for 10 min at room temp. Platelet aggregation was measuredin PI@ by the turbidimetric methodof Born (25)and in whole blood by the impedancetechnicof Cardinal and Flower (12)using Chrono-Iog PHP (Model440)and Whole Blood (Model500)Aggregcmeters (ChronoLog Corp.,Havertown,PA). Total reactionvolumeswere 1 ml for whole blood and 0.4ml for PHP.
[ C]Ado upqtake by blood cells was determinedat 37'C in whole blood. sp.act.= 19.4mCi/mmoleor 200 nM, sp. act. = After adding [ C]Ado (10F.IM, 300 mCi/mmole), aliquots(300ul.) were removedat 1 min and 5 min and pipetted on top of an oil mixture (Paraffinand DC 550, 250 ~1, d=1.04)containedin 1.5ml microcentrifugetubes held in an EppendorfMicrocentrifuge(Model 5414). The sampleswere centrifuged(12,000g, lmin), aliquots (100~1) of supernatantplasmawereimmediatelyremovedandaddedwith rapidmixingto ice-cold perchloric acid (25 ul, 20%). After removing the precipitated proteins by centrifugation(12,000g, 4 min, 4'C), aliquots (75 ~1) of supernatewere removed and added to tubes containingpotassiumphosphate
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buffer (50 mM, pH=7.4) and phenol red (0.0005%). Samples were then neutralized with KOH (5N) and the precipitated KC104 removed by centrifugation. To determineAdo and metabolitespresentin plasmaplus cells, aliquots (300I.cl) of whole blood were removedat 1 min and 5 min afteradding [14C]Ado and perchloricacid extractswere made as describedabove. Ado and metabolites(hypoxanthine, inosine, and total nucleotides)were identified by thin-layerchromatography on Silicagel usinga modification of the method of Shimizuet al (26). Silica IB2 thin layer sheets (J.T.Baker Chemical Co.) were developed twice with n-butanol:ethyl acetate:methanol:amnonium hydroxide (7:4:3:4). To study the effectsof variousagents on the uptakeandmetabolism of Ado, blood (0.95ml)was preincubatedat37'C for 3 min with HA 233, dilazep or dipyridamole;for 5 min with 5-iodotubercidin; and 2'-deoxycoformycin (50PM) was presentin the sodiumcitrateat the time of blood collection.
.
*
9
Ado, a potent inhibitorof ADP-inducedplatelet aggregation in PHP, is non-inhibitory in hole blood due to its rapidcellularuptakeand metabolism (13,14).The studiespresentedin Table lexamine Ado and several related analogs which differ biochemically (i.e. resistance to deamination, phosphorylation or both)for theireffectson ADP-induced plateletaggregation in whole blood and PI@. 2-Fluoroadenosinewhich is not deaminatedby adenosinedeaminase but is a good substratefor adenosinekinase (27-29) inhibitsplatelet aggregationin whole blood with an IC56 value, 12 uM. 2-Fluoroadenosine is aboutlo-foldmore potent(IC59= 1.3PM) when adenosine kinase activity is blocked by 5-iodotubercidin (10 PM). NECA and 2-&loroadenosine, which are not substrates for eitheradenosinedeamiMse or adenosinekinase(30),stronglyinhibitADP-inducedplatelet aggregationin both whole blood and PHP, although NECAis about lo-foldmore potent than Recently,NBCA has been shownto be a betteragonistfor 2-chloroadenosine. plateletA2 receptorsthanmost otherAdo analogsincluding2-chloroadenosine (31). The inhibitoryeffectofNECAonplateletaggregation in whole blood can be potentiated by the CAMP PDE inhibitor, HA 233 (Fig.1). It is of interest that Ado analogs that resist metabolism are more inhibitoryto platelet aggregationin whole blood than in PBP. This may resultfrom differences in the aggregation technicsemployed;i.e.,the whole blood aggreganeter measuresthe increasein ixpedance due to plateletadhesion and aggregationon electrodes (12)whereas the PHP aggregometermonitors changesin opticaldensitydue to plateletshapechangeand aggregation (25).
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ANTIPLATELET ACTIONOF ADENOSINE
Inhibition of ADP-Induced Hmn Platelet Aggregationin Whole BloodandPBPbyAdoandAnalogs IC5OoJM) CJholeBlood
4.4 3.2 NJ. 4.8 0.36
>50 12 1.3 2.3 0.26
MO
F-Ado F-Ado+ ITu Cl-Ad0
Whole blood (0.95ml) or PBP (0.35 ml) ms incubated at 3PC with Ado plateletaggregation was or an analog for 5 min. After incubations inducedby ADP (10uM for whole blcod; 2-4 UM for PRP). IC50 values were obtainedfrom log-doseresponseplots such as Fig. 3 (n = 5). N.D. stands for not determined. Abbreviations:Ado, adenosine; F-Ado, 2-fluoroadenosine; ITu, 5-iodotubercidin; Cl-Ado, 2-chloroadenosine; and MKA, 5'-N-ethylcarboxamidoadenosine.
Control
NECA
FIG. 1 Effects of HA 233 and/or NECA on ADP-induced human platelet aggregationin hole blood. Whole blood (0.95ml) was incubatedat 37'C with (A)normal saline for 8 min; (B)HA 233 (5 yM) for 8min; (C)NECA (200nM) for 5 min; and (D)BA 233 (5 PM) for 3 min then MBCA (200nM) for 5 min. After incubations,ADP (5 uM) was added typicalresultsfrun (+) to induceaggregation.Tracings represent one experimentout of three.
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Recently, we have reported that Ado in the presence of a nucleoside transportinhibitor, e.g.dilazep,dipyridamole or nitrobenzylthioinosine acts as a potent inhibitorUC50 = 2 PM) of ADP-inducedplateletaggregationin whole blood (14). The data in Fig. 2 demonstratethat the effect of Ado (l@!)in the presenceof dilazep (1 fl) can be potentiatedif the blood is pretreated with RA 233 (5 vM).
RA 233
FIG.
l
Ado Dilazrp
+ Ado
Dilazrp
+ RA 233
2
Effects of RA 233 and/or dilazep and Ado on ADP-inducedhuman platelet aggregationin whole blood. Whole blood (0.95ml) was incubatedat 37'Cwith (A)normal saline,RA 233 (5pM) or dilazep (1 fl) for 8 min or Ado (1 PM) for 5 mint (B)RA 233 (5uM) for 3 min then Ado (1 $4) for 5 min; (C)dilazep (1 lJM)for 3 min then Ado (1pM) for 5 min; and (D) dilazep (11.rM) and RA 233 (5uM) for 3 min thenAdo (1PM) for 5 min. After incubations,ADP (10pM) was added typicalresultsfran ( t) to induceaggregation.Tracingsrepresent one experiment out of five. Fig. 3 shows a log-doseresponseplot for Ado inhibitioninthepresenceof dilazep (1pM) andRA 233 (5 $l). These studiesdemonstratethat a CAMP PDE inhibitorsuch as RA 233 can enhancethe antiplatelet actionof Ado by about lo-fold (X50 = 180 nM) if combinedwith a potent inhibitorof nucleoside transport. Similar potentiationof Ado inhibitionis seenwhen dilazepis replacedby other nucleosidetransportinhibitors, e.g.dipyridamole(10uM) or by inhibitorsof both adenosinedeaminase(2'-deoxycoformycin, 5 PM) and adenosinekinase (5-iodotubercidin, 10 uM) (Table2).
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ANTIPLATELET ACTIONOF ADENOSINE
3 ‘E 60 0 ._ .‘I n z
40
IfI 20
1.6
2.2
2.6
Log[Ado]
3.0
nM
FIG. 3
Inhibition of ADP-induced humanplateletaggregation in whole blood by Ado in the presenceof dilazepand RA 233. Whole blood (0.95ml) was preincubatedfor 3 min at 37'C with dilazep (11~.M) and RA 233 (5 uM), then Ado (63-1000 nM) was addedand the incubation continued for 5 min. After the incubations,ADP (10 W) was added to induce aggregation.Inhibition of aggregation(%)represents the difference betweenpercentaggregations of the control (100%)and drug-treated samples (n = 5, z +SEM).
TpIBIE2 Effectof Ado by RA 233 in the Potentiationof the Antiaggregatory Presence of Inhibitorsof Nucleoside Transportor Ado Metabolism Ado, IC50 (vM1
Dilazep Dipyridamole dCF + ITu
concno 1 10 5 + 10
sAi2xiP 2.2 1.7 1.5
sA2xi 0.18 0.17 0.23
Human whole blood (0.95ml) was preinbuatedat 37'C for 3 min with RA 233 (5 uM) and dilazep or dipyridamole; for 2 min with 5-iodotubercidin (ITu) then for 3 min with RA 233; and 2'-deoxycoformycin (dCF', 50 PM) was presentin the sodiumcitrateat the time of blood collection.Then Ado (0.063 - 1 uM) was addedand the incubationcontinuedfor 5 min. After incubations platelet aggregation was inducedby ADP (10fl). IC50 valueswere obtained from log-dose response plots suchas Fig.3 (n= 5). *IC50 values taken from ref 14.
168
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170
ANTIPLATELET ACTION OF ADENOSINE
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Ihe data in Tables3 and 4 compareAdo metabolismin whole blood at 200 no and 10 uM in the presence of inhibitorsof nucleosidetransportand Ado metaboliSm.Ado in the absenceof a nucleosidetransportinhibitoris rapidly metabolized(< 1 min) by cellular adenosinedeaminaseand adenosinekinase. At 200 nM, Ado is mostly (96%)incorporatedinto nucleotides,whereas,at 10 UM it is only partially(28%)convertedto nucleotides, the rer&nderbeing deaminated and phosphorolyzed to form inosineand hypoxanthine, respectively. Similarresultsare obtainedin the presenceof PA 233 (5- 10 PM) indicating that RA 233 does not block cellular Ado uptake. This correlateswith the previousfindingthat PA 233 (10PM) does not restoreAdo inhibition of AUPinducedplateletaggregation in whole blood (13,14).At high (10PM) and low (200nM) Ado concentrations,both dilazep (1 ~J.M) and dipyridamole(10PM) block the cellularuptakeof Ado, with plasmaAdo levels decreasing by only 37-54% after 5minof incubation.Whennucleosidetransport is inhibited, 15-36%of the Ado entersthe nucleotidepools and 14-17%remainsin the plasma as inosine. Under these conditionsdilazep is somewhatmore potent as a nucleosidetransportinhibitor thandipyridamole whichmay be explainedby the non-specificbinding of dipyridamole to the plasma proteins, al-acid glycoproteinand albumin (9,32).The inosineformedprobablyderives from deamination of Ado by plasmaadenosinedeaminasesince inosineis not present in the cells and the plasmacan deaminateabout15% of 200 nM or 10 PM Ado in 5 min (data not shown). In the presence of 2'-deoxycoformycin and 5-iodotubercidin,inhibitorsof Ado metabolism,most of the Ado (75-96%) remainsunmetabolized in the plasma and cellularfluids. However,if only 5-iodotubercidin is employedto blockadenosinekinase,more than 95% of 10 PM Ado is deaminatedand phosphorolyzedto form inosine and hypoxanthine, respectively. At low Ado concentration (200nM) only about 50% is deaminated indicatingthe low reactivityof adenosinedeaminaseunderthese conditions. The Km value of Ado for erythrocytic adenosinedeaminaseis 25 - 30 PM (27,33) and for adenosinekinase is 0.7 fl (34). On the other hand, if adenosine deaminaseactivityis blockedby 2Weoxycoformycin(5PM), approximately 97% of the Ado is incorporatedinto nucleotide pools. These results are consistentwith our findings on Ado inhibitionof AUP-inducedplatelet aggregation in whole blood and with our earlierproposalthatwhenAdo enters the erythrocyte in relativelyhigh concentrations as duringtissuebreakdown, e.g.> 10 PM, deaminationis favored because of thehigher activity (about lo-fold) of adenosine deaminase than of adenosine kinase in human erythrocytes. On the other hand, Ado entering the erythrocyte at concentrationsbelow the Km of adenosinedeaminase,e.g.< 1 ).IM, as under normal physiological conditions, would be preferentially salvaged by conversionto AMP by adenosinekinase (27,28,35,36).
Thesefindingsconfirmand extendour earlierobservations which indicate that uptakeof Ado via nucleosidetransportand its subseguent metabolismby the enzymesadenosinedeaminaseand adenosinekinaseplay a significant role in the antiplateletactionsof compoundssuch as dipyridamoleand dilazep. Presumably, thesedrugsact by inhibiting nucleosidetransport,thus blocking
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the metabolism of Ado and permittingthe attainmentof higher steady-state levels in plasma. Theseelevatedlevels of Ado in turnwould causeincreased stimulationof adenylate cyclase, thus elevatingthe intraplatelet steadystate levels of CAMP. Significantly,when the CAMP PDE inhibitor,HA 233, which is inactive alone, is combinedwith a potent nucleosidetransport inhibitor,the antiaggregatory actionof Ado is enhancedabout lO-fold;i.e., the IC50 is decreasedfromabout2 uM to about200 nM. Consistent with these findings is the marked decrease in the rate of disappearanceof Ado from plasma when whole blood is incubatedwith a potent nucleosidetransport inhibitor. At both high and low concentrationsof Ado (10 VM and 200 nM), about 10% of the Ado disappearsfrom plasma per min, suggestingthat when nucleosidetransportis blocked Ado influx occurs via simple diffusion,a pseudofirst-order process. It should be noted that the IC50 values reportedabove are strongly influenced by the experimental conditions used. With both PPP and whole blood there is a lag of about 3 min before the plateletinhibitoryactionsof Ado are fully expressed (14,29), presumablyduetothe delay in attaininga new steady-statelevel of intraplateletcAMP and the necessarycAMPdependent the Ado was added 5 min beforeinductionof reactions.In theseexperiments aggregation.Even in the presenceof nucleosidetransportinhibition, 37-48% of the added Ado is metabolized in 5 min. However, the remainingAdo is confined to the plasma, which representsabout 67 % of the total volume (hematocritc-33%),and thereforethe IC50 values presentedin Fig. 3 and Table 2 approximate the concentration of non-metabolized Ado remainingin the extracellular fluidat the end of this incubation period. It is of interest that the IC50 value of NECA alone on platelet aggregation in wholeblood is closeto that determined for Ado in the presence of HA 233 when the metabolism of Ado is blocked (see Tables 1 and 2). Althoughthe antiplatelet effectof NECA is potentiated by HA 233, it is not affectedby dilazep (datanot shown).This suggeststhat NECA does not enter erythrocytesvia the nucleosidetransportsystem. We have not yethadthe opportunity to examinethis pointwith radiolabelled NECA. As seen in these and other studies,nucleosidetransportis not rate limiting in the metabolism of Ado by intact erythrocytes (37). The predcminant metabolicreactioninvolvesthe enzymeadenosinedeaminase,the activity of which is about 0.2 - 0.3 E.U.per ml of erythrocytes(35,38). When one considersthat an adult human has about 2.5 litersof erythrocytes, there is sufficientadenosinedeaminasetoeliminate 8-12 g of Adoper hour assumingoptimal reactionconditions.This calculationconsidersonly the adenosinedeaminaseactivityin human erythrocytes and not the high activity found in other tissues,such as liver, spleen,gastrointestinal mucosa,and vascularendothelium.This high rate of Ado metabolism,especiallyby human erythrocytes,must be taken into account when evaluating the regulatory functionsof Ado in organs and tissues such as the heart, vascular smooth muscle,etc.
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This work was supportedby PHS GRAKCSCA 07340and 09204and funds from the G.D.SearleCo. We are grateful to Ms.Joyce Rose for her assistancein the typing of this manuscript.
1. RIVEY, HS.,B, evaluation. u 2.
J.&&L
H.R andTAYUR, J.W.Dipyridamole: A critical & Uir~ &UL J& 869-880,1984.
BARKER, L.A. and KADATZ, R.A. Mechanism of action of dipyridamole.
Thromb.Res..uU,
39-46,1983.
3.
SMITE, J.B.The influence on plateletaggregation of drugs that affect the accumulation of adenosine 3':5'-cyclic mono@osphatein platelets. E&F&XL L Ul, 185-l%, 1971.
4.
HO-, S. and KORBDT, R Dipyridamoleand other phosphodiesterase inhibitorsact as antithromboticagents by potentiatingendogenous prostacyclin. Lancet.L 1286-1289,1978.
5.
RC. Therapyof ischemiccerebral KBTLBR, J.P.,lUEPBR,A.JiI.andHEROS, vasculardiseasedue to atherothro&osis. L-L&X&L IL Ll& Z& 100-105, 1984.
6.
HcEIsdDY, P.A.
and PEILP, RR Relative potenciesof dipyridamoleand relatedagentsas inhibitorsof cyclic nucleotidephosphodiesterases: Possibleexplanationof mechanismof inhibitionof platelet function. hifie& J& 1479-1494,1975.
7.
ASANO, T., OCSIAI, Y.and HIDMA, H. Selective inhibitionof separated formsof humanplateletcyclicnucleotidephosphodiesterase by platelet 400-406,1977. aggregation inhibitors. MoL pharrrracol.J&
8.
R?UAH, S.H.,CRcB7,&I., PENNY, As., AEIHAD,R andWATSON, DAThe effect of dipyridamoleon platelet function:Correlationwith blood levelsinman.BLLClin.Pharmac. 4, 129-133,1977.
9.
IlAEONY, C. WOLFRAH, K.H., COCCHE!C’N), Dipyridamole kinetics. Q&L pharmacol. w
HIIJ.S,D.C.B.and
D.H.
&
and
SJORNSSON, T.D.
330-338,1982.
10. ml R-D.,DOUGUS, C.R. IMAI, S. and BKRNB, RH. Influence of a pyrimidopyrimidine derivative on deaminationof adenosineby blood. Circ.wfi, 83-88,1964. of adenosine uptakein 11. TURUEBIH, IL, PLhNK, B-and KOIMSA, N. Inhibition human erythrocytesby adenosine-5'-carboxamides, xylosyladenine, dipyridamole,hexobendineand p-nitrobenzylthioguanosine. Biochem. m & 2191-2197,1978.
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12. CARDINAL,D.C.and FUHER, R.J.The electronicaggregometer:A novel devicefor assessingplateletbehaviorin blood.1Lm M&ho&.& 135-158,1980. 13. PABKS,B.B.,Jr, MBICKI, D.D.,AGARWAL,KC., CBBB, S.P.and S.S'iUW, J.D.Role of nucleosidetransportin drug action:The adenosinedeaminase inhibitor, deoxycoformycin and the antiplateletdrugs,dipyridamole and dilazep.&UAcad.ti&l, 188-203,1985. KC. and PARKS, R.B.,Jr. Role of adenosine 14. MWICKI, D.D.,m, uptakeand metabolismby blood cells in the antiplateletactions of dipyridamole, dilazepand nitrobenzylthioinosine. Biochem.m 3, 3965-3972,1985. 15. CASS,cB.,G?wDBTYB,TJ1andPATgRsoN, A&P. Mediated transportof nucleosidesin human erythrocytes.Specificbinding of the inhibitor nitrobenzylthioinosine to nucleosidetransportsites in the erythrocyte membrane.Biochim Biochvs.m 3&, l-10,1974. 16. JABVIS, S.H., MCBRIDE, D. and YOUNG, J.D. Erythrocytenucleoside transport:Asyrmnetrical bindingof nitrobenzylthioinosine to nucleoside permeationsites.;L phvsiol.X4, 31-46,1982. A&P., JAKOBS, ES., BAFGBY, E.R, FU, N-U.,ROBINS, H.J.and 17. PATEIRSON, CASS, Cb. Inhibition of nucleosidetransport. In: &gU&Xy Furctions af m. R.M. Berne, T.W. Rall and R. Rubio (Eds.)The Hague: MartinusNijhoff,1983 pp. 203-220. zur henmung des 18. POBL, V.J.and EROCK,N. Vergleichendeuntersuchungen adenosinabbausin vitro durch dilazep.Arzneim-Forsch,2&1901-1905, 1974. 19.
m, R.B.Dipyridaxole inhibition of adenosinemetabolismin human eZ, 21-26, 1983. blood.U5,Pharmacol.
R.P.,SPBClQR,T-and PARKS,RJI.,Jr.Tight-bindinginhibitors 20. m, -IV. Inhibitionof adenosinedeaminasesby variousinhibitors. E&&r& PharIMcol 26, 359-367,1977. 21. B, J.P.,PATBBSOB,AILP., CAukilELL, IX., PADL, a, CBAN, B.C. and LAU, K.P.Inhibitors of nucleosideand nucleotide metabolism. Cancer Chemother.f~~~, 71-85,1972. 22. BERNE, KM., R?u.L,T.W.and BDBIO, R (Eds.)&gUatorv FBenosine . The Hague.MartinusNijhoff.1983.
Functions af
23. PATON, D.B. (Ed.)Methods used in adenosine research. Methods in m 4. Mew York-London. Plenm Press.1985. 24. DAWICKI,D.D.,~, K.C.andPABKS,B.B.,Jr.Antiplatelet actionsof dipyridamole@PM), RA233and dilazep @LZ). Z& Pharmacologist X!, 111, 1985.
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25.
BORN, G.V.R. Quantitative investigations into the aggregation of blood platelets. L phvsiol.l62, 67P-68P,1962.
26.
SHIIIZU, H., LhALY,J.W. and CRJ2VBLlX, CJL A radioisotopicmethod for measuring the formation of adenosine 3',5'-cyclic monophosphatein incubatedslicesof brain.JL NeurochemJ.6,1609-1619,1969.
27.
AGARWAL,R.P.,SAGAR, S.&and PARKS, RJ3.,Jr. Adenosinedeaminasefrom human erythrocytes.Purificationand effects of adenosineanalogs. Biochem BWIBGQL 24, 693-701,1975.
28.
PARKS, RJL, Jr. and BRIlwN,P.R. Incorporationof nucleosidesinto the nucleotide pools of human erythrocytes.Adenosine and its analogs. Biochem J& 3294-3302,1973.
29.
AGAIDAL,K.C.and PARKS, RJL, Jr. Adenosine analogs and humanplatelets. Effects on nucleotide pools and the aggregation phenomenon. E~.QG&L pharrrracol 24, 2239-2248,1975.
30.
MLY, J.W.Adenosinereceptors:Targets for futuredrugs.LWChem. a, 197-207,1982.
31.
D%TJMMN, E., DKSNA, D., LENSCECW,V. and SCAWABB, U. R, adenosine receptors in human platelets. Characterization by 5'-N-ethyl carboxamido[3H]adenosine binding in relation to adenylate cyclase activity.m && BUWUZ& m 226-233,1984.
32.
NIBWIARWSKI, S., LIJlWXEWICX,H.,NM& N. and SHA, A.T. Inhibitionof humanplateletaggregation by dipyridamole and two relatedcarpoundsand its modificationby acid glycoproteinsof human plasma.LUClin. B&L.&, 64-76,1975.
33.
OGDDRNE,W.IU. andSPENcER, N. Partial purificationand propertiesof the common inherited forms of adenosine deaminase from human erythrocytes. Biochem.L l& 117-123,1973.
34.
A.S.Adenosinekinase from human erythrocytes: KYD,J.IkandMGNARA, Determination of the conditions requiredfor assay in crudehemolysates. clin.u m J& 145-153,1980.
35.
PARKS, RJL, Jr., CRABFRB, G.W., KONG, Cd., -.RS.r -, K.C.andSCMOLAR, EJI, Incorporationof analog purine nucleosidesinto
the formed elements of human blood: Erythrocytes,platelets and lymphocytes.Bnn,UAcad.&&ZL 412-434,1975. 36.
HAWKINS,C.F.,KYD,JW.andW4GNARA, A.S.Adenosinemetabolismin human erythrocytes: A studyof sane factorswhichaffectthe metabolicfate of adenosinein intactred cells in vitro.&& &iochem -2Q2, 380387, 1980.
Vol. 43, No. 2 37.
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MdiRWAL, R.P. and PARKS, R.R., Jr.
A possible associationbetween the nucleoside transport system of human erythrocytes and adenosine deaminase.Biochem. pfiarmacol24, 547-550,1975.
38. CHEN, S.F., STOECKLBR, J.D. and PARKS, R.E., Jr. Transport of deoxycoformycin inhumanerythrocytes:Measurementbyadenosinedeaminase titrationand radioisotopeassays.Biochem.Pharmacol.U, 4069-4079, 1984.
Doloretta D. Dawicki,KailashC. Agarwaland RobertE. Parks,Jr. Sectionof BiochemicalPharmacology,Division of Biology and Medicine, BrownUniversity,Providence,RI 02912,U.S.A. (Received
15.1.1986; Accepted in revised form 15.4.1986 by Editor J.G. White)
Adenosine (Ado,10 - 50 PM), a potent inhibitorof ADP-inducedhuman plateletaggregationin platelet-rich plasma U?F!P), does not inhibit aggregation in whole blood. However, the Ado analogs, 2-fluoroadenosine, 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine (NECA) which are resistantto deamination(2-fluoroadenosine) or deamination and phosphorylation (2-chloroadenosine and NECA), inhibitaggregation in whole blood with IC50 values of 12 uM, 2.3 uM and 0.26uM, respectively. The inhibitory effectof NBCA (200nM) is potentiated by the platelet cAMP phosphodiesterase(PDE)inhibitorBA 233 (5PM). Inhibition of the erythrocytic nucleosidetransportsystemby dilazep (1uM) or dipyridamole (10 uM), or blockade of Ado metabolism by 2Weoxycoformycin(5 uM) plus 5-iodotubercidin (10PM), evokesthe antiaggregatory actionof Ado in whole blood (IC50= 2 uM). RA 233 (5 PM) potentiates Ado-mediated inhibition about lo-fold when nucleosidetransportor Ado metabolismis blocked. Ado (10LIM or 200 nM) is rapidly metabolized within 1 min in whole blood. When nucleosidetransportis inhibitedby dilazepor dipyridasole, or when Ado metabolismis blockedby 2'-deoxycoformycin and 5-iodotubercidin, 50 - 60 % of the Ado remainsin the plasmaafter5 min. Theseresults show that the failureof Ado to inhibitplateletaggregation in mole blood results from its rapid uptakeandmetabolism byerythrocytes. More importantly,these data emphasizethe key role of nucleoside transportinhibitionin the antiplateletactions of dipyridamole and dilazep.In addition,superiortherapeutic resultsmay be obtainedfrcm the combinationof blockade of the nucleosidetransportsystemwith inhibition of plateletCAMP PDE.
Key words:plateletaggregation, adenosine, dipyriwle, dilazep,PA 233 161
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Althoughdipyridamole (PersantineR) has seen widespreadclinicaluse as a vasodilatorand antiplatelet drug for a quartercentury(1,2), therehas been no generallyaccepted explanation of itsmechanismof action. Sinceelevated intraplateletcAMP levels are associatedwith inhibitionof platelet aggregation (3), the fact that dipyridamole can inhibit platelet CAMP phosphodiesterase (PDE)has led many to assm that this is the targetenzyme for its antithromboticeffect (4,5). It must be noted, however, that publishedinhibitionconstants(Ki)of dipyridamole with CAMP PDE are 12 I.~M and 19pM (6,7), whichare considerably higherthan the plasma levelsof free dipyridamoleusually achieved during therapy (8,9).Thatdipyridamole can block adenosine(Ado)metabolismin intactcells by inhibiting the membraneassociated nucleosidetransportsystemhas also been recognized for manyyears (10,ll). However, the role of nucleoside transport inhibition in the antiplatelet effectsof dipyridamole couldnot be accuratelyassessedbecause of limitations in methodsfor studyingaggregation in the physiological milieu of whole blood. The developmentof impedance aggregometry (12),whichpermitsthe studyof plateletaggregation in whole blood,has made necessarya reevaluation of the antiplateletactionsof dipyridamole and relatedagentssuch as dilazepand nitrobenzylthioinosine. Recentinvestigations in this laboratory(13,14) have shown that dipyridamole, dilazepand nitrobenzylthioinosine whichare potent inhibitors of humanerythrocytic nucleosidetransport(15-18), neitherinhibit alone nor potentiate the antiaggregatory effectof Ado in platelet-rich plasma (PRP). However, these nucleoside transport inhibitors restore the antiplatelet action of Ado in whole blood when administered at pharmacologically relevant doses. l3ycontrast, the cqund RA 233,whichis a closechemicalrelativeof dipyridamole, is essentially devoidof nucleoside transportinhibition, but causes strikingpotentiationof the antiplatelet actionof Ado in PRP due to its relativelypotentCAMP PDH inhibitory activity (Ki= 0.61.1M1 (6). These observationshavebeenexplainedbythe very rapid metabolism of Ado by the erythrocyticenzymes, adenosine deaminaseand adenosinekinase,which,in whole blood,catalysethe disappearance of 10 uM Ado from the plasma in less than 1 min (13,14,19). When the actions of adenos,ine deaminaseand adenosinekinaseare blockedby specificinhibitors, i.e.2- deoxycoforqzin(20)and 5-iodotubercidin (21),respectively, or when nucleosidetransportis inhibited,the rate of disappearanceof Ado from plasma is markedly decreasedand its antiplateletaction is evoked. An impressive body of evidence has accumulated indicatingthat Ado is an important physiological regulator of adenylate cyclase through its interactionswith specific Ado receptors, which either inhibit (Al)or stimulate (AZ)adenylatecyclase dependingon the specificcell or tissue involved. (Forrecent reviews of the regulatoryactionsof Ado see 22,23). For example,plateletspossessA2 receptorsfor Ado. Our earlierfindingsraisedthe questionof whetherthe canbination of a potent cAMP PDE inhibitor with a blocker of nucleoside transportand, therefore, of Ado metabolism, mightenhancethe antiplatelet actionof Ado and perhapsotherof its physiological functions as well. It was proposedthatan
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ANTIPLATELET ACTION OF ADENOSINE
163
ideal antiplatelet drug would be one that combinesinhibitoryeffects on nucleosidetransportwith thoseon platelet-specific CAMP PDE. The present studytests this conceptbyexaminingthe antiplateletactionof Ado or Ado analogs,e.g.5'-N-ethylcarboxamidoadenosine (NEW , in combinationwith the nucleosidetransportinhibitors, dilazepor dipyridarrole and/orthe CAMP PDE inhibitor, BA 233. Portionsof this work have been presentedin preliminary form elsewhere(24).
Adenosine 5'-diphosphate(ADP),adenosine (Ado),2-chloroadenosine, 5'-N-ethylcarboxamidoadenosine (NEW, and dipyridamole(PersantineR) were obtainedfromsigma ChemicalCo.,St.Louis,MO. Dilazep (CormelianR) was a gift frcmHoffmann-IaHoche, Inc.,Nutley,NJ. PA 233 was a gift from Dr. Karl ThanaeG&H, Biberachan der Hiss,Germany. 2Weoxycoformycin(Pentostatir?) and 2-fluoroadenosinewere obtained from the Drug Development Branch, Divisionof CancerTreatmentof the NationalCancerInstitute, Bethesda,MD. 5-Iodotubercidin was a gift fromDr.L.B.Townsend,Universityof Michigan, was obtainedfran Moravek Ann Arbor,MI. [8-14C]Ado (sp. act.= 59 mCi/mrole) Biochemicals, Brea, CA. and was >95% pure as shown by thin-layer chranatography on polyethyleneimine-cellulose sheetswith potassiumchloride (100mM) as the eluent. [U-14C]Ado(sp.act. = 300 mCi/mmole)was obtained from ICN Hadiochemicals, Irvine,CA.
Blood was drawnfrom healthydonorswho had not takenantiplatelet drugs for at leastten days. The blood was dispensedinto plastictubescontaining 0.1volume of sodiumcitrate (3.8%)topreventcoagulation. Platelet-rich plasma (PFU?) was obtainedfran the blood by centrifugation at 300 g for 10 min at room temp. Platelet-poorplasma was obtainedfrom the remainingblood constituentsby centrifugationat 1500 g for 10 min at room temp. Platelet aggregation was measuredin PI@ by the turbidimetric methodof Born (25)and in whole blood by the impedancetechnicof Cardinal and Flower (12)using Chrono-Iog PHP (Model440)and Whole Blood (Model500)Aggregcmeters (ChronoLog Corp.,Havertown,PA). Total reactionvolumeswere 1 ml for whole blood and 0.4ml for PHP.
[ C]Ado upqtake by blood cells was determinedat 37'C in whole blood. sp.act.= 19.4mCi/mmoleor 200 nM, sp. act. = After adding [ C]Ado (10F.IM, 300 mCi/mmole), aliquots(300ul.) were removedat 1 min and 5 min and pipetted on top of an oil mixture (Paraffinand DC 550, 250 ~1, d=1.04)containedin 1.5ml microcentrifugetubes held in an EppendorfMicrocentrifuge(Model 5414). The sampleswere centrifuged(12,000g, lmin), aliquots (100~1) of supernatantplasmawereimmediatelyremovedandaddedwith rapidmixingto ice-cold perchloric acid (25 ul, 20%). After removing the precipitated proteins by centrifugation(12,000g, 4 min, 4'C), aliquots (75 ~1) of supernatewere removed and added to tubes containingpotassiumphosphate
ANTIPLATELET ACTION OF ADENOSINE
164
Vol. 43, No. 2
buffer (50 mM, pH=7.4) and phenol red (0.0005%). Samples were then neutralized with KOH (5N) and the precipitated KC104 removed by centrifugation. To determineAdo and metabolitespresentin plasmaplus cells, aliquots (300I.cl) of whole blood were removedat 1 min and 5 min afteradding [14C]Ado and perchloricacid extractswere made as describedabove. Ado and metabolites(hypoxanthine, inosine, and total nucleotides)were identified by thin-layerchromatography on Silicagel usinga modification of the method of Shimizuet al (26). Silica IB2 thin layer sheets (J.T.Baker Chemical Co.) were developed twice with n-butanol:ethyl acetate:methanol:amnonium hydroxide (7:4:3:4). To study the effectsof variousagents on the uptakeandmetabolism of Ado, blood (0.95ml)was preincubatedat37'C for 3 min with HA 233, dilazep or dipyridamole;for 5 min with 5-iodotubercidin; and 2'-deoxycoformycin (50PM) was presentin the sodiumcitrateat the time of blood collection.
.
*
9
Ado, a potent inhibitorof ADP-inducedplatelet aggregation in PHP, is non-inhibitory in hole blood due to its rapidcellularuptakeand metabolism (13,14).The studiespresentedin Table lexamine Ado and several related analogs which differ biochemically (i.e. resistance to deamination, phosphorylation or both)for theireffectson ADP-induced plateletaggregation in whole blood and PI@. 2-Fluoroadenosinewhich is not deaminatedby adenosinedeaminase but is a good substratefor adenosinekinase (27-29) inhibitsplatelet aggregationin whole blood with an IC56 value, 12 uM. 2-Fluoroadenosine is aboutlo-foldmore potent(IC59= 1.3PM) when adenosine kinase activity is blocked by 5-iodotubercidin (10 PM). NECA and 2-&loroadenosine, which are not substrates for eitheradenosinedeamiMse or adenosinekinase(30),stronglyinhibitADP-inducedplatelet aggregationin both whole blood and PHP, although NECAis about lo-foldmore potent than Recently,NBCA has been shownto be a betteragonistfor 2-chloroadenosine. plateletA2 receptorsthanmost otherAdo analogsincluding2-chloroadenosine (31). The inhibitoryeffectofNECAonplateletaggregation in whole blood can be potentiated by the CAMP PDE inhibitor, HA 233 (Fig.1). It is of interest that Ado analogs that resist metabolism are more inhibitoryto platelet aggregationin whole blood than in PBP. This may resultfrom differences in the aggregation technicsemployed;i.e.,the whole blood aggreganeter measuresthe increasein ixpedance due to plateletadhesion and aggregationon electrodes (12)whereas the PHP aggregometermonitors changesin opticaldensitydue to plateletshapechangeand aggregation (25).
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165
ANTIPLATELET ACTIONOF ADENOSINE
Inhibition of ADP-Induced Hmn Platelet Aggregationin Whole BloodandPBPbyAdoandAnalogs IC5OoJM) CJholeBlood
4.4 3.2 NJ. 4.8 0.36
>50 12 1.3 2.3 0.26
MO
F-Ado F-Ado+ ITu Cl-Ad0
Whole blood (0.95ml) or PBP (0.35 ml) ms incubated at 3PC with Ado plateletaggregation was or an analog for 5 min. After incubations inducedby ADP (10uM for whole blcod; 2-4 UM for PRP). IC50 values were obtainedfrom log-doseresponseplots such as Fig. 3 (n = 5). N.D. stands for not determined. Abbreviations:Ado, adenosine; F-Ado, 2-fluoroadenosine; ITu, 5-iodotubercidin; Cl-Ado, 2-chloroadenosine; and MKA, 5'-N-ethylcarboxamidoadenosine.
Control
NECA
FIG. 1 Effects of HA 233 and/or NECA on ADP-induced human platelet aggregationin hole blood. Whole blood (0.95ml) was incubatedat 37'C with (A)normal saline for 8 min; (B)HA 233 (5 yM) for 8min; (C)NECA (200nM) for 5 min; and (D)BA 233 (5 PM) for 3 min then MBCA (200nM) for 5 min. After incubations,ADP (5 uM) was added typicalresultsfrun (+) to induceaggregation.Tracings represent one experimentout of three.
166
ANTIPLATELET ACTION OF ADENOSINE
Vol. 43, No. 2
Recently, we have reported that Ado in the presence of a nucleoside transportinhibitor, e.g.dilazep,dipyridamole or nitrobenzylthioinosine acts as a potent inhibitorUC50 = 2 PM) of ADP-inducedplateletaggregationin whole blood (14). The data in Fig. 2 demonstratethat the effect of Ado (l@!)in the presenceof dilazep (1 fl) can be potentiatedif the blood is pretreated with RA 233 (5 vM).
RA 233
FIG.
l
Ado Dilazrp
+ Ado
Dilazrp
+ RA 233
2
Effects of RA 233 and/or dilazep and Ado on ADP-inducedhuman platelet aggregationin whole blood. Whole blood (0.95ml) was incubatedat 37'Cwith (A)normal saline,RA 233 (5pM) or dilazep (1 fl) for 8 min or Ado (1 PM) for 5 mint (B)RA 233 (5uM) for 3 min then Ado (1 $4) for 5 min; (C)dilazep (1 lJM)for 3 min then Ado (1pM) for 5 min; and (D) dilazep (11.rM) and RA 233 (5uM) for 3 min thenAdo (1PM) for 5 min. After incubations,ADP (10pM) was added typicalresultsfran ( t) to induceaggregation.Tracingsrepresent one experiment out of five. Fig. 3 shows a log-doseresponseplot for Ado inhibitioninthepresenceof dilazep (1pM) andRA 233 (5 $l). These studiesdemonstratethat a CAMP PDE inhibitorsuch as RA 233 can enhancethe antiplatelet actionof Ado by about lo-fold (X50 = 180 nM) if combinedwith a potent inhibitorof nucleoside transport. Similar potentiationof Ado inhibitionis seenwhen dilazepis replacedby other nucleosidetransportinhibitors, e.g.dipyridamole(10uM) or by inhibitorsof both adenosinedeaminase(2'-deoxycoformycin, 5 PM) and adenosinekinase (5-iodotubercidin, 10 uM) (Table2).
Vol. 43, No. 2
167
ANTIPLATELET ACTIONOF ADENOSINE
3 ‘E 60 0 ._ .‘I n z
40
IfI 20
1.6
2.2
2.6
Log[Ado]
3.0
nM
FIG. 3
Inhibition of ADP-induced humanplateletaggregation in whole blood by Ado in the presenceof dilazepand RA 233. Whole blood (0.95ml) was preincubatedfor 3 min at 37'C with dilazep (11~.M) and RA 233 (5 uM), then Ado (63-1000 nM) was addedand the incubation continued for 5 min. After the incubations,ADP (10 W) was added to induce aggregation.Inhibition of aggregation(%)represents the difference betweenpercentaggregations of the control (100%)and drug-treated samples (n = 5, z +SEM).
TpIBIE2 Effectof Ado by RA 233 in the Potentiationof the Antiaggregatory Presence of Inhibitorsof Nucleoside Transportor Ado Metabolism Ado, IC50 (vM1
Dilazep Dipyridamole dCF + ITu
concno 1 10 5 + 10
sAi2xiP 2.2 1.7 1.5
sA2xi 0.18 0.17 0.23
Human whole blood (0.95ml) was preinbuatedat 37'C for 3 min with RA 233 (5 uM) and dilazep or dipyridamole; for 2 min with 5-iodotubercidin (ITu) then for 3 min with RA 233; and 2'-deoxycoformycin (dCF', 50 PM) was presentin the sodiumcitrateat the time of blood collection.Then Ado (0.063 - 1 uM) was addedand the incubationcontinuedfor 5 min. After incubations platelet aggregation was inducedby ADP (10fl). IC50 valueswere obtained from log-dose response plots suchas Fig.3 (n= 5). *IC50 values taken from ref 14.
168
Vol. 43, No. 2
ANTIPLATELET ACTION OF AOENOSINE
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Adenosine (10pM) Metabolismin Whole Blood in the Presenceof Inhibitorsof Transportor Metabolism
170
ANTIPLATELET ACTION OF ADENOSINE
Vol. 43, No. 2
Ihe data in Tables3 and 4 compareAdo metabolismin whole blood at 200 no and 10 uM in the presence of inhibitorsof nucleosidetransportand Ado metaboliSm.Ado in the absenceof a nucleosidetransportinhibitoris rapidly metabolized(< 1 min) by cellular adenosinedeaminaseand adenosinekinase. At 200 nM, Ado is mostly (96%)incorporatedinto nucleotides,whereas,at 10 UM it is only partially(28%)convertedto nucleotides, the rer&nderbeing deaminated and phosphorolyzed to form inosineand hypoxanthine, respectively. Similarresultsare obtainedin the presenceof PA 233 (5- 10 PM) indicating that RA 233 does not block cellular Ado uptake. This correlateswith the previousfindingthat PA 233 (10PM) does not restoreAdo inhibition of AUPinducedplateletaggregation in whole blood (13,14).At high (10PM) and low (200nM) Ado concentrations,both dilazep (1 ~J.M) and dipyridamole(10PM) block the cellularuptakeof Ado, with plasmaAdo levels decreasing by only 37-54% after 5minof incubation.Whennucleosidetransport is inhibited, 15-36%of the Ado entersthe nucleotidepools and 14-17%remainsin the plasma as inosine. Under these conditionsdilazep is somewhatmore potent as a nucleosidetransportinhibitor thandipyridamole whichmay be explainedby the non-specificbinding of dipyridamole to the plasma proteins, al-acid glycoproteinand albumin (9,32).The inosineformedprobablyderives from deamination of Ado by plasmaadenosinedeaminasesince inosineis not present in the cells and the plasmacan deaminateabout15% of 200 nM or 10 PM Ado in 5 min (data not shown). In the presence of 2'-deoxycoformycin and 5-iodotubercidin,inhibitorsof Ado metabolism,most of the Ado (75-96%) remainsunmetabolized in the plasma and cellularfluids. However,if only 5-iodotubercidin is employedto blockadenosinekinase,more than 95% of 10 PM Ado is deaminatedand phosphorolyzedto form inosine and hypoxanthine, respectively. At low Ado concentration (200nM) only about 50% is deaminated indicatingthe low reactivityof adenosinedeaminaseunderthese conditions. The Km value of Ado for erythrocytic adenosinedeaminaseis 25 - 30 PM (27,33) and for adenosinekinase is 0.7 fl (34). On the other hand, if adenosine deaminaseactivityis blockedby 2Weoxycoformycin(5PM), approximately 97% of the Ado is incorporatedinto nucleotide pools. These results are consistentwith our findings on Ado inhibitionof AUP-inducedplatelet aggregation in whole blood and with our earlierproposalthatwhenAdo enters the erythrocyte in relativelyhigh concentrations as duringtissuebreakdown, e.g.> 10 PM, deaminationis favored because of thehigher activity (about lo-fold) of adenosine deaminase than of adenosine kinase in human erythrocytes. On the other hand, Ado entering the erythrocyte at concentrationsbelow the Km of adenosinedeaminase,e.g.< 1 ).IM, as under normal physiological conditions, would be preferentially salvaged by conversionto AMP by adenosinekinase (27,28,35,36).
Thesefindingsconfirmand extendour earlierobservations which indicate that uptakeof Ado via nucleosidetransportand its subseguent metabolismby the enzymesadenosinedeaminaseand adenosinekinaseplay a significant role in the antiplateletactionsof compoundssuch as dipyridamoleand dilazep. Presumably, thesedrugsact by inhibiting nucleosidetransport,thus blocking
Vol. 43, No. 2
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the metabolism of Ado and permittingthe attainmentof higher steady-state levels in plasma. Theseelevatedlevels of Ado in turnwould causeincreased stimulationof adenylate cyclase, thus elevatingthe intraplatelet steadystate levels of CAMP. Significantly,when the CAMP PDE inhibitor,HA 233, which is inactive alone, is combinedwith a potent nucleosidetransport inhibitor,the antiaggregatory actionof Ado is enhancedabout lO-fold;i.e., the IC50 is decreasedfromabout2 uM to about200 nM. Consistent with these findings is the marked decrease in the rate of disappearanceof Ado from plasma when whole blood is incubatedwith a potent nucleosidetransport inhibitor. At both high and low concentrationsof Ado (10 VM and 200 nM), about 10% of the Ado disappearsfrom plasma per min, suggestingthat when nucleosidetransportis blocked Ado influx occurs via simple diffusion,a pseudofirst-order process. It should be noted that the IC50 values reportedabove are strongly influenced by the experimental conditions used. With both PPP and whole blood there is a lag of about 3 min before the plateletinhibitoryactionsof Ado are fully expressed (14,29), presumablyduetothe delay in attaininga new steady-statelevel of intraplateletcAMP and the necessarycAMPdependent the Ado was added 5 min beforeinductionof reactions.In theseexperiments aggregation.Even in the presenceof nucleosidetransportinhibition, 37-48% of the added Ado is metabolized in 5 min. However, the remainingAdo is confined to the plasma, which representsabout 67 % of the total volume (hematocritc-33%),and thereforethe IC50 values presentedin Fig. 3 and Table 2 approximate the concentration of non-metabolized Ado remainingin the extracellular fluidat the end of this incubation period. It is of interest that the IC50 value of NECA alone on platelet aggregation in wholeblood is closeto that determined for Ado in the presence of HA 233 when the metabolism of Ado is blocked (see Tables 1 and 2). Althoughthe antiplatelet effectof NECA is potentiated by HA 233, it is not affectedby dilazep (datanot shown).This suggeststhat NECA does not enter erythrocytesvia the nucleosidetransportsystem. We have not yethadthe opportunity to examinethis pointwith radiolabelled NECA. As seen in these and other studies,nucleosidetransportis not rate limiting in the metabolism of Ado by intact erythrocytes (37). The predcminant metabolicreactioninvolvesthe enzymeadenosinedeaminase,the activity of which is about 0.2 - 0.3 E.U.per ml of erythrocytes(35,38). When one considersthat an adult human has about 2.5 litersof erythrocytes, there is sufficientadenosinedeaminasetoeliminate 8-12 g of Adoper hour assumingoptimal reactionconditions.This calculationconsidersonly the adenosinedeaminaseactivityin human erythrocytes and not the high activity found in other tissues,such as liver, spleen,gastrointestinal mucosa,and vascularendothelium.This high rate of Ado metabolism,especiallyby human erythrocytes,must be taken into account when evaluating the regulatory functionsof Ado in organs and tissues such as the heart, vascular smooth muscle,etc.
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This work was supportedby PHS GRAKCSCA 07340and 09204and funds from the G.D.SearleCo. We are grateful to Ms.Joyce Rose for her assistancein the typing of this manuscript.
1. RIVEY, HS.,B, evaluation. u 2.
J.&&L
H.R andTAYUR, J.W.Dipyridamole: A critical & Uir~ &UL J& 869-880,1984.
BARKER, L.A. and KADATZ, R.A. Mechanism of action of dipyridamole.
Thromb.Res..uU,
39-46,1983.
3.
SMITE, J.B.The influence on plateletaggregation of drugs that affect the accumulation of adenosine 3':5'-cyclic mono@osphatein platelets. E&F&XL L Ul, 185-l%, 1971.
4.
HO-, S. and KORBDT, R Dipyridamoleand other phosphodiesterase inhibitorsact as antithromboticagents by potentiatingendogenous prostacyclin. Lancet.L 1286-1289,1978.
5.
RC. Therapyof ischemiccerebral KBTLBR, J.P.,lUEPBR,A.JiI.andHEROS, vasculardiseasedue to atherothro&osis. L-L&X&L IL Ll& Z& 100-105, 1984.
6.
HcEIsdDY, P.A.
and PEILP, RR Relative potenciesof dipyridamoleand relatedagentsas inhibitorsof cyclic nucleotidephosphodiesterases: Possibleexplanationof mechanismof inhibitionof platelet function. hifie& J& 1479-1494,1975.
7.
ASANO, T., OCSIAI, Y.and HIDMA, H. Selective inhibitionof separated formsof humanplateletcyclicnucleotidephosphodiesterase by platelet 400-406,1977. aggregation inhibitors. MoL pharrrracol.J&
8.
R?UAH, S.H.,CRcB7,&I., PENNY, As., AEIHAD,R andWATSON, DAThe effect of dipyridamoleon platelet function:Correlationwith blood levelsinman.BLLClin.Pharmac. 4, 129-133,1977.
9.
IlAEONY, C. WOLFRAH, K.H., COCCHE!C’N), Dipyridamole kinetics. Q&L pharmacol. w
HIIJ.S,D.C.B.and
D.H.
&
and
SJORNSSON, T.D.
330-338,1982.
10. ml R-D.,DOUGUS, C.R. IMAI, S. and BKRNB, RH. Influence of a pyrimidopyrimidine derivative on deaminationof adenosineby blood. Circ.wfi, 83-88,1964. of adenosine uptakein 11. TURUEBIH, IL, PLhNK, B-and KOIMSA, N. Inhibition human erythrocytesby adenosine-5'-carboxamides, xylosyladenine, dipyridamole,hexobendineand p-nitrobenzylthioguanosine. Biochem. m & 2191-2197,1978.
Vol. 43, No. 2
ANTIPLATELET ACTIONOF ADENOSINE
173
12. CARDINAL,D.C.and FUHER, R.J.The electronicaggregometer:A novel devicefor assessingplateletbehaviorin blood.1Lm M&ho&.& 135-158,1980. 13. PABKS,B.B.,Jr, MBICKI, D.D.,AGARWAL,KC., CBBB, S.P.and S.S'iUW, J.D.Role of nucleosidetransportin drug action:The adenosinedeaminase inhibitor, deoxycoformycin and the antiplateletdrugs,dipyridamole and dilazep.&UAcad.ti&l, 188-203,1985. KC. and PARKS, R.B.,Jr. Role of adenosine 14. MWICKI, D.D.,m, uptakeand metabolismby blood cells in the antiplateletactions of dipyridamole, dilazepand nitrobenzylthioinosine. Biochem.m 3, 3965-3972,1985. 15. CASS,cB.,G?wDBTYB,TJ1andPATgRsoN, A&P. Mediated transportof nucleosidesin human erythrocytes.Specificbinding of the inhibitor nitrobenzylthioinosine to nucleosidetransportsites in the erythrocyte membrane.Biochim Biochvs.m 3&, l-10,1974. 16. JABVIS, S.H., MCBRIDE, D. and YOUNG, J.D. Erythrocytenucleoside transport:Asyrmnetrical bindingof nitrobenzylthioinosine to nucleoside permeationsites.;L phvsiol.X4, 31-46,1982. A&P., JAKOBS, ES., BAFGBY, E.R, FU, N-U.,ROBINS, H.J.and 17. PATEIRSON, CASS, Cb. Inhibition of nucleosidetransport. In: &gU&Xy Furctions af m. R.M. Berne, T.W. Rall and R. Rubio (Eds.)The Hague: MartinusNijhoff,1983 pp. 203-220. zur henmung des 18. POBL, V.J.and EROCK,N. Vergleichendeuntersuchungen adenosinabbausin vitro durch dilazep.Arzneim-Forsch,2&1901-1905, 1974. 19.
m, R.B.Dipyridaxole inhibition of adenosinemetabolismin human eZ, 21-26, 1983. blood.U5,Pharmacol.
R.P.,SPBClQR,T-and PARKS,RJI.,Jr.Tight-bindinginhibitors 20. m, -IV. Inhibitionof adenosinedeaminasesby variousinhibitors. E&&r& PharIMcol 26, 359-367,1977. 21. B, J.P.,PATBBSOB,AILP., CAukilELL, IX., PADL, a, CBAN, B.C. and LAU, K.P.Inhibitors of nucleosideand nucleotide metabolism. Cancer Chemother.f~~~, 71-85,1972. 22. BERNE, KM., R?u.L,T.W.and BDBIO, R (Eds.)&gUatorv FBenosine . The Hague.MartinusNijhoff.1983.
Functions af
23. PATON, D.B. (Ed.)Methods used in adenosine research. Methods in m 4. Mew York-London. Plenm Press.1985. 24. DAWICKI,D.D.,~, K.C.andPABKS,B.B.,Jr.Antiplatelet actionsof dipyridamole@PM), RA233and dilazep @LZ). Z& Pharmacologist X!, 111, 1985.
174
ANTIPLATELET ACTION OF ADENOSINE
Vol. 43, No. 2
25.
BORN, G.V.R. Quantitative investigations into the aggregation of blood platelets. L phvsiol.l62, 67P-68P,1962.
26.
SHIIIZU, H., LhALY,J.W. and CRJ2VBLlX, CJL A radioisotopicmethod for measuring the formation of adenosine 3',5'-cyclic monophosphatein incubatedslicesof brain.JL NeurochemJ.6,1609-1619,1969.
27.
AGARWAL,R.P.,SAGAR, S.&and PARKS, RJ3.,Jr. Adenosinedeaminasefrom human erythrocytes.Purificationand effects of adenosineanalogs. Biochem BWIBGQL 24, 693-701,1975.
28.
PARKS, RJL, Jr. and BRIlwN,P.R. Incorporationof nucleosidesinto the nucleotide pools of human erythrocytes.Adenosine and its analogs. Biochem J& 3294-3302,1973.
29.
AGAIDAL,K.C.and PARKS, RJL, Jr. Adenosine analogs and humanplatelets. Effects on nucleotide pools and the aggregation phenomenon. E~.QG&L pharrrracol 24, 2239-2248,1975.
30.
MLY, J.W.Adenosinereceptors:Targets for futuredrugs.LWChem. a, 197-207,1982.
31.
D%TJMMN, E., DKSNA, D., LENSCECW,V. and SCAWABB, U. R, adenosine receptors in human platelets. Characterization by 5'-N-ethyl carboxamido[3H]adenosine binding in relation to adenylate cyclase activity.m && BUWUZ& m 226-233,1984.
32.
NIBWIARWSKI, S., LIJlWXEWICX,H.,NM& N. and SHA, A.T. Inhibitionof humanplateletaggregation by dipyridamole and two relatedcarpoundsand its modificationby acid glycoproteinsof human plasma.LUClin. B&L.&, 64-76,1975.
33.
OGDDRNE,W.IU. andSPENcER, N. Partial purificationand propertiesof the common inherited forms of adenosine deaminase from human erythrocytes. Biochem.L l& 117-123,1973.
34.
A.S.Adenosinekinase from human erythrocytes: KYD,J.IkandMGNARA, Determination of the conditions requiredfor assay in crudehemolysates. clin.u m J& 145-153,1980.
35.
PARKS, RJL, Jr., CRABFRB, G.W., KONG, Cd., -.RS.r -, K.C.andSCMOLAR, EJI, Incorporationof analog purine nucleosidesinto
the formed elements of human blood: Erythrocytes,platelets and lymphocytes.Bnn,UAcad.&&ZL 412-434,1975. 36.
HAWKINS,C.F.,KYD,JW.andW4GNARA, A.S.Adenosinemetabolismin human erythrocytes: A studyof sane factorswhichaffectthe metabolicfate of adenosinein intactred cells in vitro.&& &iochem -2Q2, 380387, 1980.
Vol. 43, No. 2 37.
ANTIPLATELET ACTION OF ADENOSINE
175
MdiRWAL, R.P. and PARKS, R.R., Jr.
A possible associationbetween the nucleoside transport system of human erythrocytes and adenosine deaminase.Biochem. pfiarmacol24, 547-550,1975.
38. CHEN, S.F., STOECKLBR, J.D. and PARKS, R.E., Jr. Transport of deoxycoformycin inhumanerythrocytes:Measurementbyadenosinedeaminase titrationand radioisotopeassays.Biochem.Pharmacol.U, 4069-4079, 1984.