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Influence of anti-platelet drugs on platelet-vessel waif interacticnks
Pmtaglandins, Lcukotriem and Medicine(1987) 30, 133-145 0 Longman Group UK Ltd 1987
Gunk
H. R. I&o
DE%pxbeofLaboratcry~cine~mPathology
univelsityofMhmsutamthmcerrter Box198,vnivsrsity#oslpitalsandcl~~ IChs3ah55455 Wh (mprimt mques+s to G.H.R.Rao)
The influence of in vitro trmtrnent of platelets with antiplateletdrugs on the interactiaaof these cells with the s&xx&WUtanwasstudiedue~ci~tnaaanblood~fraa YlormdLanltxQl~. Bconstitutedblood followhq chug tnshwmt wasdm&ited~aspecidLchaaaberwhitiM~~ ofde+dutkializedrabbitaorta. !&ewaU&earratsumdintIme studieswas800 set-l. srtrfaae~crfplatel&3C#IthslZRWdOthelium m lno@mhdcally evaluahL Am*, Ilxlpaoten, l?mst@andinElandl3 plateletBmztbionexpceedsukmM&d Quin2 andDiltiazem,exdxdeimilar~i~eff~,~ Vm was a poor inhibitor. Aspirintm&mntsignificently erhncd~teletadhesiorlto~~~surf~ se&ylate and SaliqUmide did mt emhatxm platelet adberaace. Cmly Aspirin enhamed the fonnatial of l_genase metabolitesof radiolabeled arz&Mmate. B6ults~thtdrugswhi.chinhibit plaW.let eggrqation and secxetim of granule umkents zxm&xs fomatimofplateletulru&L x%omver,these~nlayar~ynot have a similar influemeanplatelet intezmdmwiththe edhmnce or formation of suberrlbuleliml~to~ aggregates.
133
PROS-
D
Plateletsappearto play a criticalrole in the con@icatiom that associatedwith ischemicheart disease(l-lo). The poesibility platelets may mediate one or xrxe of the pathologicalevents a.rdhloIdershasproqtedmanyclini.cal associatedwith cardiovascul trials with antiplateletdrugs (11-16). Several studies have den-on&ratedthat arachidmate metdbolitesOf Q'clocorygenaSe are potentmediatorsof plateletactivation(8,9,17-19). These firr%iqs pmqtedscmenhqofcyclooxygenaseinhibitorsaimdatreducingthe risk for mmmence of thnxhtic episodes(11-16).F&sults,hmever, have not conclusively provedthebeneficialeffectof such a therapy (16,20-21). Fbcent studies suggestiqa criticalmle for,inositoltriqhosphatesin mbilizing plateletcalciumhave amused mterest in the use of new antiplatelet drugs air& at preventirqactivationat earlier steps (22-33).Sinceintracelluhrcalciumelevaticmmaybeessential for the initialstages of plateletactivation,the use of calcium blockersto preventthis processhas been advocated(25-27). Calcium blockers(Verapmil,Diltiazemand Nifedipine) specifically blockthe voltage-sensitive calciumentry channel,whereasthefluorophor,Quin 2, buffers intracellular calcium (28-30). Wehaveevaluatedthe effectofdrugsthatinterferewiththe~~~cascade,calci~ entry into platelets,or agentsthat kuffer free plateletcytctmlic calcium to determine their influence cm platelet vesEielwall interaction.Wsults suggest that dnlgs that interfti with arachidonatecascade effectivelyreduced the thr&xs formation, whereas,antagoniststhatprevmted calciumentrywerenoteffeotive.
Arachidonicacid as the sodiumsaltwas obtainedfranNuclear Prep,Elysian,M.imesh ardmade up in O.lMTrisbufferat#I 7.4. Radiolabeled arachidonic acid was fm New Eqlard Nuclear(NEC-661), Boston,Maseachsetts. Ibupmfen,Pmstagli?&i.nE~(EGEl)were gifts frm the Upjohn Ccqany, Kalamazoo, Michigan. 13-azap1mst21mic acid (13~APA) was a gift frcaa Guy C. LeBmton,Depa&mtofRIannacology, Universityof Illinois,CC.oago,Illinois. 9mWoxam qnth&me inhibitorOK!+046 was a genemus gift fxan ON0 Phamaoeuticals Conqmy, Ltd., Osaka, Japan. Unlessotherwisestat&,allomer cherc!icalswerefrurftSigma~calccmpanY,St.~,Missauri. Blood was drawn fmm adultvolunteem WhOhadn0ttdkeI-l~ medicationfor at least two weeks prior to -cm and m.ixeJ immediatelywith citrate-citric acid-d&xose (CX!D)anticoagulant (citrate0.1 M, citricacid 7 mM, dextmse 0.14M, pH 6.5) in a ratio Platelet-rich plasma of 9 parts blood to 1 part anticoagu.lant(30)' (PRP)was obtainedbycentrifugirqwholebloodatxwmtenperature for 20 minutesat 100 x g and platelet-poor plasma (PPP)by spinningthe
134
remainingblood at 2000 x g for 20 minutes. PPPwas storedfrozenat -350 C during the plateletwashhq period. WeShlythslwedPPPWlS used for rewn§tiw plateletasEdD.ates (31).
Pramration
of Platelets
Plateletsfrom PRP m washedtwice witi equal volume of CCD 5madeno6ine~3llMlzhw#lyllin8. WashedplMtelets flxmthemstm&lmrtitestxdanthe totheactimofagcd6t8bef~ YforW=ww=e fu%er evalwtim by the Brwaqartner m&hod (32). The caroeartsaticn of platelets was adjusted to 4-5 x10-5 plateletsper ul with Ppp. TM,8 xmcmstiti Pppwas incubataafor 30minuWsat370Cwith varims.i.nhibitom3. Fwfusatescxxls~of~~pRpaanb~wfth a 40% volum of washed red cells. 'he final plateletsin thesew=almys b&men 1.5 - 2.5 x 1 Theperfusalw3mreirmbatdfor30mhutesinawaterbath psru. at37°Ckefore initiatingstudies.
Perfusionswere carrid azt at 37O C in perfusionchimhrs de=lmbym\rmgartsler withaneffectiveatmularwidthof2.2nmaIKI rod lengthof 7.2 QP (32). FlcwwasobtahdbypuQingtherawxlstitut&blcadthruqh a Cole Falmr Mmterflex~withautocl~havrd at 140 mljhin (wallshearrate 800 sacol). (31) Blood tas perfumd DEumded~itabdanindlaortasegmerrts~usedinallthe~iJlmntsinthisstudy.~c~~xllmted CllplEtStiCXUdSt#Uld rinsdwith#xs@ate 3mfferedsalhforlOmimh3. Aftas five minut8sofperfusic4nwiul wererinmdwi~&cephate mite's saline hlffer. suppilsrting=h~~ ehctmllni~ TherestoftEY&mmtuae~W~a graKhdserie80fedxLnol~ti~, cswkIedinJB-4Ps?msyl~),thinsectfcaredfor ~terial(polyscienoes,~ li@t micmsccqy anA stainedwith mthylem blm (31). ??
Plateletintmxtimwith subsprdcrtheliLnn~evaluateadinJB-4 sectkmsof3u?Jlm~. %m 6eriesof axialslicesevecy0.5 RIn werecutfrcmtheblock. Sectiam saqle were alway discmA& """"-&iE?&iZ Ad===?= nlicmneter~vided .into100 parts wa!sused forthese waluations. Platelets mtemdzq with eium m way evaluatedaccmdhqtoadmrmdificationbySakaria6mnofthe originallIlemoddescribeclby~ Plateletsthat were attachedwem classifiedas: ocntact (C), a-mnotspreadculthe~i\apt hich Iem spmad m the
-ial
tsurfa:
135
of less than 5 um in height. A furtherstage in which adherent plateletshad formd with multiplelayersbut did not exceed5 um in height,thrmbi (T),platelet aggregates of more than 5 um in height. All of these basic parameterswere expressedas percentageof the totalsurfaceemmined.
Measummtsof
AxachidonicZuzidConversion
For M of arachidonicacid metabolism,each readion mixhxe (1 ml.),contii?i"s1.5 x 109 cells in Hank'sBalancedsalt solution(HESS),was tsturd with 1 ug of labeledarachidonic acid on an am for fiveminutes(34). Attheendoftheexperiment, 1mlofethyla~~tewasiVWFYjtOeachreaction~andacidified with 10 ul of 0.5 M citricacid. AfterthomughIlIixhq,theethyl acetatelayerwasseparatedandthereactimxaixhrewas~~ withanequalvolumofethylacetate. FYacti~oftheorganicphase was pooled,cmncmtmted over nitrogenand platedon a silicagel G of amzhidonicacid plate. The solvent tsystem used for the sepaxation metaboliteswaspetrolemether : ethyl ether: aceticacid (69 : 30 : 1v/v). Ra~~~ivespatswerenrmitoredwithaBertholdradiolabel scannerardquantitati~wasa~~byseparati~oftheradioactive spots~scintillationcountirq.
Studieson PlateletAuareuation Contmlplateletsinplasmaaggregat&inanormlfashionwl‘len stirredwith epimqhrine (5 uM), Wine diphoq$de (ADP,3 UM), thmnbin (0.2q/ml)and arachidonate (450uM). Platelets prepamd for thesestudiesbywashhqtwiceand reconstitutingindonorppP,when tested with various agmists, mqxmded in a normal fashion, indicating full functional capabilities followhq preparative cascade procedures. Agentx that interferedwith the arachidonate pmvWkedaW!hidonateinduced immersible plateletaggregation.The onlynotableexceptionwasthethmkmme synthetase inhibitor, OK!& 046. At the concentmtionstest& (100 ug$l) it did not inhibit arachidonate i_Wucedaggregation.Hmever, it effectively blockedthe g= abilityto corn& 14C-ara&+onateto thr&mme (datanot . Of the calcium antagxmsts, only Quin 2 at 40 uM concentration proved inhibitory. Vempamil (100 uM) ard Diltiazem (1OOuM)didnot inhibitarachidonate imlucedaggregation. PlateletInteractionwithSuberdothelium
Under the cmklitions us& in this study (800set-l,shearrate: tely 60% of the de-mdothelialized 5' perfusiontime),approxima area was covemd with platelets(Table1). 136
contml
(8)
12.3 _+ 1.2
Ibuprofen (10) 16.3 _+1.8 (200 UM)
19.2 _+3.3 31.0 _+3.2
27.7 _+2.9 l5.1_+ 2.1
59.2 f
7.4
62.4 _+ 7.1*
Fghm;9)
33.0 _+2.3** 27.7 _+3.7
8.9 _+2.5** 69.6 _+ 8.5*
13-APA (7) (50 w
16.2 + 2.2
25.9 _+6.0
6.8 ,+2.2** 48.9 _+10.4*
Tug)
22.4 _+4.4** 21.7 _+5.8
3.7 ,+1.2** 47.8 _+11.4*
OK!+046 (5) (100 w/w
6.8 _+2.7
Meanarrlthestmhrd * **
P < 0.1 P < 0.001
17.4 ,+3.8
36.5 f 8.7*
60.7 f l5.1*
error (n)
Not significant. Significantcmpamdtocontmlvalues.
Table 1: Infl~Of~thatinterferedwiti~~tecascade was follow@ on the interaction of platelets with sub@bm&ium. Normal contmlplatelets fomed a significantammt OfthraaJxlson Ibupmfen, Aspirin, Fq and 13-APA the exposed vascular surface. mxunsignifbntly pmmnted the fon&iculofplatelet~. boxamsyW&ase inhMtorOKY-O46hadnosu&inhibibryeffecton platelet reactivity.
ofthmnbifoznmdwas Intheunbeatedamtml~grerup~ (3.8%) the 28% whqzws, Pmstagl~ El significantly decmamd foxmtion of platelet thrcmbi. Aspirin (100 UM), acid, and lkupmfen (200 uM) significantly xmdmxd 13mV2 inhibitor,oKYplateletUx-cmbiformd. TheUmWoxamqMhebse effect cn platelet th?rMms 046 (100 WW, , had no inhaiw fonnaticn. spmadaq andaggregate fozmaticmofplatelets on enwheliulnwassignificarrtlygreaterintheAspirinand~andin El graupS*
137
all Influenceof CalcimAntacmnistsonPlatelet-VesselW Interaction Vexapamil,Diltiazemand C&in 2 were the calciumw employed(Table2).
Surface &nrered 9.8 _+ 1.4
19.0 _+ 2.2
?Lspirin (100uM)
40.7 _+ 5.6*
25.7 t 3.4
8.2 _+ 1.4*
74.6 f 8.6
Quin2 (40w
30.2 _+ 2.8*
17.7 2 1.6
9.9 _+ 1.0*
57.8 _+ 7.4
Diltiazem (100w
27.6 _+ 2.4
26.0 f 2.4
16.6 _+ 2.4
70.2 _+ 8.4
Verapmil (100uM)
14.4 _+ 1.8
23.4 _+ 2.2
29.6 f 4.6
67.4 _+ 9.6
control
Meanard values.
standard
error
(IF3);
*p
39.6 _+ 3.4
< 0.001
culpald
68.4 _+ 6.8
to the
ax-k-ml
Table 2: Influenceof cdlciumantagmistsonplatelet-vesselwall interaction was follcwed. Ccntrclplateletsmactedwiththeexposed surface and form&l Umakus. Aspirin and intracellular calcium chelatorQuin2 effectively r&uced the formation of plateletthrmbi. Verapamilhad nc such inhibitcryeffect cn platelet interaction leadingtothK&U formation.
Aspirinwasusedasoneoftheccntrolstomnitorthesestudies. untreatedconrOls had 39% thmnbi cmpared to 8% for the Aspirin treat4EdgroqL 0fthecalcimantagoniststested,onlytheintracellular calciumchelator,Quin 2, exertedan inhibitoryeffectcn the
138
d0velw of platelet thrcmbi. Aspirin Diltiazemand Quin to the submWA&im, enbncad platelet qxeadbq and aggmgath allpamdtothevalues ~fortheIlnkreated~grarp.
2
Platelet-VesselWall Interactions zspirintreatmentsignificaRtly~theperc#lltcbgeof~i formed (11%) cu@amd to the untreated ccmtmls (38%) (Table 3).
CQlTbA
16.5 ,+ 2.4
38.12
22.3 f 3.8*
35.2 _+ 3.8
11.5 _+ 1.4*
69.0 _+ 7.8
Sal&late (100 UM)
8.6 _+ 1.8
15.6 _+ 2.6
26.1_+ 2.8
50.3 ,+ 6.4
Salicylamiae (100 w
8.0 _+ 2.0
15.4 ,+ 1.8
26.6 _+ 3.0
50.0 ,+ 7.4
A5pM.n (100 w
9.1*
Msanand*P < 0.001 ccqamd
2.4
4.2
63.7 _+ 8.6
error (xF3). to the CcntxQlvalues.
mle 3: Inflw of Aspirin, Salicylate aW Salicylamide on platslet-vesselwallintemcU on was follcmEd. A5pirin s' lwwxdthsformticmofplab.letthmrbi,htmhana3d spmadhg of platelets. Whereas Salicylate and Salicylamide did mt influence inanywayplatelet hteractionwiththevesselwal1.
ELz+z!!Z
Salicylate anA Salicylamiae mre less effective fornmtion of platelet thrabi, ht, unlike Aspirin erhamespmadhganda54regatianof platelets: agentsdidmt
139
Influence of Acetvlsalicvlic Acid,Salicvlate and Saliwlmide on u& axachidonic CmversionbvPlateletEWvmes quantiUntreatednormalcmtrolplateletscomer&d significant ties of radiolaheledsubstrateto cyclooxygenase(HHT, 42%) ard lipoxygenase (HE!TE, 18.7%)metabolites(Table4).
RADIOAc!mnTYRMxlvERED INvAEuCUSFRACrIoNs (*PERXTTOF !NYlY& m) Fhcepholipids Control
38.52 5.4
ASA
14.7_+2.2
HID
41.8_+3.6 1.3 _+0.2*
AA 18.7_+1.2
1.0 LO.2
76.7f 3.8*
7.3 _+1.0
Salicylate 43.1_+4.6
39.9_+3.8** 14.8_+1.4**
2.2 _+0.4
Salicylamide48.5f 6.4
35.12 4.0** 14.9_+1.4**
1.5 _+0.5
*Meanandthestandanl
*P c
0.001,
error (n=3).
significant cmpared to control
values.
**P < 0.1,not significant. Table4: Rildiohbeledarachidonate(AA) transformation to cyclooxygenase(HITI?) and lipoxysenase (HElZ)pmductsbyintactplatelets was follmed. Normalcontrolplateletscommked significant amounts OfAAtoHHTandHEm. AspirinpreventedtheformationofHmand channeledAAconversiontoHEIE. Salicylate arrdSalicylamide did not exertanyinhibitoryeffectoncycloaxygenase andhadverylittleinfluenceonlipoqgemm. Aspirinccmpletelyblockedarachidonicacid conversionvia cyclooxy genasebutenhamedconversionviathelipcorysenase pamy (78%),-icylate and Salicyhmide did not exzrt any inhibitoryeffect on cycl~enaseandhadslightstinnilatoryaCti~onlipoxygenases.
140
l%mlltsoftheprewntirnrestigatianbaveshownthatagents~ch prwent irmvexsibleplateletaggregaticlltisecreticolinvi~, reducedthefomaticnofplateletthrcm\biaathaeqxmed significantly vasaihr surface. Of me variuls drugs tested, the S synthetaseinhibitar,OK!f-O46,andcalcim~V~~ the foxzmtim of platelet thrau. not effective in p?mmthg AspirhsignificanUyreducedths foxmati~ofplateletthmbi,but misilqmmmtwasoffsetby~~,~and aggmgation ofplatalets cmthe vascum~il.ml.~1activates admylate,cyclase. Iharemltingel6watimofcyclicAwpis etib_h$t plamet adhmmoebyblooJ&cytosoliccalcium expressionof the cell adhesionmolecules(35-37). Hcwever,FGE~inthisstudydidnotmduceplateletintem&i~wi~ theeilml. , Hman plateletsnomally wkqo mible qgmgaticm when stlrredwiti~andreleasetbeirgranilecenteorto. Sh aspirininhibi~seuxld~ r@qxmeofplatel~totbeacuonof inwemible aggregaticm,the majority of wandprevents clinicaltrialshaveainmdatinhibitia~ofcyclooxygwm~, zmUm$mwill r+ce ths risk for L!mcmmt~iW j_s&muc heart disease (11-16). a,-fronalr~toryh?lve chmerated that platelets can becme sticky, adhere and aggmgate i.rJmmmibly indarpsnaeaR of pbteB and ths releaseIzeactim(38,39). In aelitial, letsofpatientswi~stomgepooldeficiencyinteractwith~ t&eliumandfomthm&i,al~#eydonotbave~drana thatstoreareleasablepoolofADP(40). Inviewofthsefindingrs, it is reasmable to malx that hh&M.ah of cam ofthebiochmicaleventsassociatedwitiplaWhtactivaticnmaymtc@lakly p~theirparticipaticplintheclMcal~~~~~~atedto ca?xliovasculardi.sorders. a criticalmle !3tM.iesillthelasttWoaarulnahave inplicated for cdl&m in plateletactivaticn(41, 42). Fiaever, the specific role of icmizedCdLciumin the mdulatiar of varims mzphological/ bio&emical/@ysiologicalalterationsisnotclear. Inspite ofthis of calcium in the [email protected] lxlm&MyMbbeeninterest a&agmMs inthepreve&imofplateletrelat&clinicalccs@ications (25-27,43, 44). Vempamil,NifedipineandDil~havebaen fkeqmtlyused.Inthisstudy,verapamilwasineffectivein~~Urefonmtionofplateletthmbi. Diltiazemwashtterthan ofplatel~appearingas verapmilinreducingthepene&qe thrtxbi. In earlierstudies,Diltiazenhas been shmn to parrarrte disscciationof platelet aggmgates amI potenthte the actim of aspirin(45). sinLi.larlythecalciumantagonists, VWd==-6, havebeenskIwnto dhggmgatePAF~t@!dplateletsandrweree phoqhrylation of 40K Md 20K pmteins (46). (&in 2, an irrtrscellcalcium bufferingagent, waseffectiveinreducilqpli?Mlet ular interacti~l~tothef~ti~of~i,and~effective
141
than calciumentry blockersin prevent- the formationof platelet thmmbi. Aspirinis themstwidelyused of all the antiplateletdrugs in frcm ourlabomkory as well as clinicaltrials (11-16). studies othershave demnstmted that this drug is effectivein r&u&q the However,italsoseerrrs adhmncetoer&thelium.Inaseparatestudy we have shumthataspirintreatnmtaltersnmWanedefomability of plateletsinaependent of its effecton cyclooxygenase Mzymes (47). Ofthevariouscycl~ *, Aspirinsemstohavesignificxnt influenceinchaMeling~~cacidconversionvialipcacygenase. Salicylateand Salicylamide used in this study did not exert any influenceon platelet interactionwith Vium. studies wiul specific lipcorysenase criticallyevaluatethe role, if any, of lipoxygenase n&abolites of arachidomtein the mdulation of plateletinteractionwithsuberhfKtnnthis laboratoq demon&mthat thelium. Prwicus stl.xIies lipoxygemsemtabolitesof arachidonate do nut have any criticalrole i.nagonistimhc& immersible aggregation of platelets(48). In conclusion,results of our study demnstmte that agents capableofpreventiqthmhmne synthesisor~easeofgranule amtfhs significantly reducethe formationof plateletthrmbi upon activationin the extracorpo& circulation.The majorityof these drugs semtoke ineffective inmiqplatelet interactionwith subendathelium leading to spreading adhesionand aggregationof platelets. Theseresultsfwkher'supportourBarlierfhdhgs suggesting that elevationof cyhslie calcim, formationof thmnboxanes and releaseof granulecontentsare not essentialfor platelet shape change, spreading adhemnce or irzwersible aggregation, alkhoqhtheypotentiateheseevents follmingplateletactivation.
TheauthorwishestothankMrs.JulieLeeatxIMrs.DeborahC. JohnsonfOrtheirtecbnimlhelpamIMs.SusanSchwarzeforherhelp with the manuscript. m by USPiiSgrants-11880, CA-21737, Hk30217, a grant fmn the Mar& of Dims (BBBDl-886), Clinical Reseamh C!enta (ES 400) and the Mhmsota MedicalFoundation(HRDF 38-85ti HRF 47-87). REmRENcEs
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Gmqx Aepirininc%mmary 14.meCorwary~prpject~ heart disease. Cimulation 62 (Sqpl V): V-59, 1980. 15. Brsddin K, L D, Iechmr K, Dberla K, Walter E: seoordary Cmparkof~lealiprevmtion of myocadminfdm cylic acid, placebo and mbmwof qmadial infarction. Circulation62 (wIlV):V-63, 1980. 16. B ER: sxmmaryof~~clMcaltrialsofpla&latactivedrugsincardi~ar~ (%mhtbm62(Suppl IT):V-106, 1980.
143
17. Robabon PM, Rob&son D, m IJ, Maas I&, FitzgeraldGA, Fkiesiqer GC, Oates JA: v A2 in vamtonicangina pectoris.New ErqlandJamml of Medicine304:998, 1981. 18. Lewy RI, Wiener L, WalinskyP, Lefer AM, SilverMT, Smith JB: Thr&mme release durw paciq-w am&ml pectoris: Possiblevasocmstrictioninfluenceon the ccmmry vascdatum. Cimulatim 61: 1165,1979. SobelM, SalzmanEW,DaviesGL, Ha&in=, Sweeney J, IloetzRN, 19. Kkload G: Circulatirqplateletpmducts in unstableax&m 63: 300, 1981. pectoris. Circulation 20. Clinical. trialsof platelet-active drugs in : Sumnaryofdesignfeatures. Circulation ZrnCyL 62 @.I@): V-49, 1980. of plateletlnprpressant 21. Gmton E: A perspective dnlgtreatment onco?mAaryarteryandcxmb mvasallardisease. Cimxlation62 (SupplV): V-111,1980. 22. NishizukaY: Turnover of inositolFhospholipids ard signal Wm. Science225: 1365,1984. 23. Authi KS, Crawford N: Inositol 1,4,5-triphosphate-i releaseof m Ca2+ frcmhigblypurifiedhmmn platelet intracellularxlmnbranes. Bio&emicalJanmal 230:247,1985. 24. chapH, Simm MF, F'auvelJ, Plantavid M, MaucoG, Douste-BlazyL: Relationshipbetweenphospholipid metabolismti intracellular calciummobilization du?&g plateletactivation.Nmvelle Revue Franchise d%exaatilcgie 27: 229, 1985. 25. Burns ER, FrishmanWH: The anti-platelet effectsof calcium channelblockersadditionalto their anti-am&al properties. Internatiti Jmmal of (Xdiology4: 372, 1983. 26. m ~t~~,o~~~_~~ ~~XS~XV JH, vhksb3 m, action of calcium thmbutic antagon&drugswithdypyridamleindogs. AmericanJcurnalof Qrdiology51: 592, 1983. 27. onoda JM, Sloane BF, Honn KF: Antithmnbogmic effects of calcium channel blockers: Synergismwith pmtacyclin and Uum&mne synthetaseinhibitors.lbxmbosisResearch34: 367, 1984. 28. He.nry PD: cUpara+ive pharmacx3logy of cdlcim antigonists: nifedipine,venqlmil ard diltiazm. American Joumal of Cardiology 46: 1047,1980. 29. Bmmwald E: Mechanismof action of calcim charnel-blocking agents. New E@?md Jaxnal of Medicine307: 1618,1982. 30. RaoGHR, FellerJD, SmbaCP, WhiteJG: Influenceofthetxlcium sensitivefluorophor Quin 2 cm plateletfmction. Blood 67:354, 1986. 31. Rao GHR, Ekzcolar G, White JG: Epinephrinexwemestheinhibitory influenceof aspirinan platelet-vessel wall interactions. mrc&osisResearch 44: 65, 1986. 144
32. Bamqartmr RR: Effects of acetylsalicylicacid, sulfinpymwm and dipyrmle cnplatelet Mhesicnatd aqpxqation in flowhq nativeanticoagulatedblood. I%mm&~is 8: 340, 1979. 33. 8akariassenKS,BolhuisRA,Blc&dcM,lhorellL,BlaubackB: Associaticxlbetween bleeding time ard platelet adtmmme to 52: 144, artery subendothelilml. T?lm&msisand Baemshsis 1984. 34. RAO QSR, Johnson GJ, Reddy XR, White JG: mpidretunlof cycl~activeplateletsindcgsafterasixyleoraldcse of aspirin. Pnmtaglandins 22: 761, 1981. 35. carlsonIA,Er~1: l%lmral-artexyinfusim of pzmtaglandin El in severe peripheralvasculardisease. Lamet 1: 155, 1973. 36. tarlson IA, Olsson Al;: ~pZlt3hglendhE+lasVere peripheralvasmlar dimase. Iamet 2: 810, 1976. 37. Packham MA: Platelet furnztioninhibitors. lhrcmsxlsisard Haenustasis50: 610, 1983. 38. F&o c;HR,Jchsm GJ, Whih JG: Infl~ofep~inemtbe of aspirh-txm~ platelets. aggregation v glandinsand Medicine 5: 45, 1980.
Pm&a-
39. Rao c;HR,Gemaxd JM, WitJmp CT, White JG: Platslet a5Fgregatioa mepemntofADPreleaseor~andin~isinpatie!its ~~5heg~=m==* prrretchglandin~~~ :
40. RaoQIR,
I
.
WhiteJG:
Uqublishedohmvation.
41. Massini P, KasercGlanzmaxmR, IuscMr EF: i~andtheirroleinthea~~~~ofplatel~. Haenmtasis 40:2X?, 1978.
r4uvmmtofcdlcium Thmdmsis
42. FeinsteinMB: The role of calcium in platelet fun&m. calcium in Dmq Action (Weiss GB, ed), Plenum Rlblhhem New York, p. 197, 1978. 43. Weiss GB: *"Lx DrugDevelom=nt
In: IrC.,
themxcl&cdnrgs. 1:5pntia
44. Betze E, Hanmerle H, Viele D: Ca2+-entryblcckers and atherosclesosis. InternationalAtyiogra@y 3: 33, 1984. 45. Rirq ME, 0mrigan JJ, F'endm? PE: Effects of oral Diltiazemcn platelet fur&ion alone and in cmbination with 'luw dose' 44: 391, 1986, aspirin. T!lrdmsism 46. Khan SN, Lane PA, Smith AD: DeSqmgatiCxl of PAP-acetm? withmvemal of aggregated platelets byvesMpermil and-8 phqhorylation of 40k arxl20k pmteins. EumpeanJaxnalof Pharmacology107: 189, 1985. 47. Burris BM, smith CM, Rae GHR, White JG: Aspirin tzmahmt reducesplateletmd&anceto deformation.Arterio6cl~is(in ~BSS), 1987. Imevemibleplatelet~ 48. PAOGHR, Radha E,WhiteJG: llM3t&OliteC3. doesiIXltdependoll1~ BiaphysiCdlReseamh &mnunications l3:50, 1985. 145
Gunk
H. R. I&o
DE%pxbeofLaboratcry~cine~mPathology
univelsityofMhmsutamthmcerrter Box198,vnivsrsity#oslpitalsandcl~~ IChs3ah55455 Wh (mprimt mques+s to G.H.R.Rao)
The influence of in vitro trmtrnent of platelets with antiplateletdrugs on the interactiaaof these cells with the s&xx&WUtanwasstudiedue~ci~tnaaanblood~fraa YlormdLanltxQl~. Bconstitutedblood followhq chug tnshwmt wasdm&ited~aspecidLchaaaberwhitiM~~ ofde+dutkializedrabbitaorta. !&ewaU&earratsumdintIme studieswas800 set-l. srtrfaae~crfplatel&3C#IthslZRWdOthelium m lno@mhdcally evaluahL Am*, Ilxlpaoten, l?mst@andinElandl3 plateletBmztbionexpceedsukmM&d Quin2 andDiltiazem,exdxdeimilar~i~eff~,~ Vm was a poor inhibitor. Aspirintm&mntsignificently erhncd~teletadhesiorlto~~~surf~ se&ylate and SaliqUmide did mt emhatxm platelet adberaace. Cmly Aspirin enhamed the fonnatial of l_genase metabolitesof radiolabeled arz&Mmate. B6ults~thtdrugswhi.chinhibit plaW.let eggrqation and secxetim of granule umkents zxm&xs fomatimofplateletulru&L x%omver,these~nlayar~ynot have a similar influemeanplatelet intezmdmwiththe edhmnce or formation of suberrlbuleliml~to~ aggregates.
133
PROS-
D
Plateletsappearto play a criticalrole in the con@icatiom that associatedwith ischemicheart disease(l-lo). The poesibility platelets may mediate one or xrxe of the pathologicalevents a.rdhloIdershasproqtedmanyclini.cal associatedwith cardiovascul trials with antiplateletdrugs (11-16). Several studies have den-on&ratedthat arachidmate metdbolitesOf Q'clocorygenaSe are potentmediatorsof plateletactivation(8,9,17-19). These firr%iqs pmqtedscmenhqofcyclooxygenaseinhibitorsaimdatreducingthe risk for mmmence of thnxhtic episodes(11-16).F&sults,hmever, have not conclusively provedthebeneficialeffectof such a therapy (16,20-21). Fbcent studies suggestiqa criticalmle for,inositoltriqhosphatesin mbilizing plateletcalciumhave amused mterest in the use of new antiplatelet drugs air& at preventirqactivationat earlier steps (22-33).Sinceintracelluhrcalciumelevaticmmaybeessential for the initialstages of plateletactivation,the use of calcium blockersto preventthis processhas been advocated(25-27). Calcium blockers(Verapmil,Diltiazemand Nifedipine) specifically blockthe voltage-sensitive calciumentry channel,whereasthefluorophor,Quin 2, buffers intracellular calcium (28-30). Wehaveevaluatedthe effectofdrugsthatinterferewiththe~~~cascade,calci~ entry into platelets,or agentsthat kuffer free plateletcytctmlic calcium to determine their influence cm platelet vesEielwall interaction.Wsults suggest that dnlgs that interfti with arachidonatecascade effectivelyreduced the thr&xs formation, whereas,antagoniststhatprevmted calciumentrywerenoteffeotive.
Arachidonicacid as the sodiumsaltwas obtainedfranNuclear Prep,Elysian,M.imesh ardmade up in O.lMTrisbufferat#I 7.4. Radiolabeled arachidonic acid was fm New Eqlard Nuclear(NEC-661), Boston,Maseachsetts. Ibupmfen,Pmstagli?&i.nE~(EGEl)were gifts frm the Upjohn Ccqany, Kalamazoo, Michigan. 13-azap1mst21mic acid (13~APA) was a gift frcaa Guy C. LeBmton,Depa&mtofRIannacology, Universityof Illinois,CC.oago,Illinois. 9mWoxam qnth&me inhibitorOK!+046 was a genemus gift fxan ON0 Phamaoeuticals Conqmy, Ltd., Osaka, Japan. Unlessotherwisestat&,allomer cherc!icalswerefrurftSigma~calccmpanY,St.~,Missauri. Blood was drawn fmm adultvolunteem WhOhadn0ttdkeI-l~ medicationfor at least two weeks prior to -cm and m.ixeJ immediatelywith citrate-citric acid-d&xose (CX!D)anticoagulant (citrate0.1 M, citricacid 7 mM, dextmse 0.14M, pH 6.5) in a ratio Platelet-rich plasma of 9 parts blood to 1 part anticoagu.lant(30)' (PRP)was obtainedbycentrifugirqwholebloodatxwmtenperature for 20 minutesat 100 x g and platelet-poor plasma (PPP)by spinningthe
134
remainingblood at 2000 x g for 20 minutes. PPPwas storedfrozenat -350 C during the plateletwashhq period. WeShlythslwedPPPWlS used for rewn§tiw plateletasEdD.ates (31).
Pramration
of Platelets
Plateletsfrom PRP m washedtwice witi equal volume of CCD 5madeno6ine~3llMlzhw#lyllin8. WashedplMtelets flxmthemstm&lmrtitestxdanthe totheactimofagcd6t8bef~ YforW=ww=e fu%er evalwtim by the Brwaqartner m&hod (32). The caroeartsaticn of platelets was adjusted to 4-5 x10-5 plateletsper ul with Ppp. TM,8 xmcmstiti Pppwas incubataafor 30minuWsat370Cwith varims.i.nhibitom3. Fwfusatescxxls~of~~pRpaanb~wfth a 40% volum of washed red cells. 'he final plateletsin thesew=almys b&men 1.5 - 2.5 x 1 Theperfusalw3mreirmbatdfor30mhutesinawaterbath psru. at37°Ckefore initiatingstudies.
Perfusionswere carrid azt at 37O C in perfusionchimhrs de=lmbym\rmgartsler withaneffectiveatmularwidthof2.2nmaIKI rod lengthof 7.2 QP (32). FlcwwasobtahdbypuQingtherawxlstitut&blcadthruqh a Cole Falmr Mmterflex~withautocl~havrd at 140 mljhin (wallshearrate 800 sacol). (31) Blood tas perfumd DEumded~itabdanindlaortasegmerrts~usedinallthe~iJlmntsinthisstudy.~c~~xllmted CllplEtStiCXUdSt#Uld rinsdwith#xs@ate 3mfferedsalhforlOmimh3. Aftas five minut8sofperfusic4nwiul wererinmdwi~&cephate mite's saline hlffer. suppilsrting=h~~ ehctmllni~ TherestoftEY&mmtuae~W~a graKhdserie80fedxLnol~ti~, cswkIedinJB-4Ps?msyl~),thinsectfcaredfor ~terial(polyscienoes,~ li@t micmsccqy anA stainedwith mthylem blm (31). ??
Plateletintmxtimwith subsprdcrtheliLnn~evaluateadinJB-4 sectkmsof3u?Jlm~. %m 6eriesof axialslicesevecy0.5 RIn werecutfrcmtheblock. Sectiam saqle were alway discmA& """"-&iE?&iZ Ad===?= nlicmneter~vided .into100 parts wa!sused forthese waluations. Platelets mtemdzq with eium m way evaluatedaccmdhqtoadmrmdificationbySakaria6mnofthe originallIlemoddescribeclby~ Plateletsthat were attachedwem classifiedas: ocntact (C), a-mnotspreadculthe~i\apt hich Iem spmad m the
-ial
tsurfa:
135
of less than 5 um in height. A furtherstage in which adherent plateletshad formd with multiplelayersbut did not exceed5 um in height,thrmbi (T),platelet aggregates of more than 5 um in height. All of these basic parameterswere expressedas percentageof the totalsurfaceemmined.
Measummtsof
AxachidonicZuzidConversion
For M of arachidonicacid metabolism,each readion mixhxe (1 ml.),contii?i"s1.5 x 109 cells in Hank'sBalancedsalt solution(HESS),was tsturd with 1 ug of labeledarachidonic acid on an am for fiveminutes(34). Attheendoftheexperiment, 1mlofethyla~~tewasiVWFYjtOeachreaction~andacidified with 10 ul of 0.5 M citricacid. AfterthomughIlIixhq,theethyl acetatelayerwasseparatedandthereactimxaixhrewas~~ withanequalvolumofethylacetate. FYacti~oftheorganicphase was pooled,cmncmtmted over nitrogenand platedon a silicagel G of amzhidonicacid plate. The solvent tsystem used for the sepaxation metaboliteswaspetrolemether : ethyl ether: aceticacid (69 : 30 : 1v/v). Ra~~~ivespatswerenrmitoredwithaBertholdradiolabel scannerardquantitati~wasa~~byseparati~oftheradioactive spots~scintillationcountirq.
Studieson PlateletAuareuation Contmlplateletsinplasmaaggregat&inanormlfashionwl‘len stirredwith epimqhrine (5 uM), Wine diphoq$de (ADP,3 UM), thmnbin (0.2q/ml)and arachidonate (450uM). Platelets prepamd for thesestudiesbywashhqtwiceand reconstitutingindonorppP,when tested with various agmists, mqxmded in a normal fashion, indicating full functional capabilities followhq preparative cascade procedures. Agentx that interferedwith the arachidonate pmvWkedaW!hidonateinduced immersible plateletaggregation.The onlynotableexceptionwasthethmkmme synthetase inhibitor, OK!& 046. At the concentmtionstest& (100 ug$l) it did not inhibit arachidonate i_Wucedaggregation.Hmever, it effectively blockedthe g= abilityto corn& 14C-ara&+onateto thr&mme (datanot . Of the calcium antagxmsts, only Quin 2 at 40 uM concentration proved inhibitory. Vempamil (100 uM) ard Diltiazem (1OOuM)didnot inhibitarachidonate imlucedaggregation. PlateletInteractionwithSuberdothelium
Under the cmklitions us& in this study (800set-l,shearrate: tely 60% of the de-mdothelialized 5' perfusiontime),approxima area was covemd with platelets(Table1). 136
contml
(8)
12.3 _+ 1.2
Ibuprofen (10) 16.3 _+1.8 (200 UM)
19.2 _+3.3 31.0 _+3.2
27.7 _+2.9 l5.1_+ 2.1
59.2 f
7.4
62.4 _+ 7.1*
Fghm;9)
33.0 _+2.3** 27.7 _+3.7
8.9 _+2.5** 69.6 _+ 8.5*
13-APA (7) (50 w
16.2 + 2.2
25.9 _+6.0
6.8 ,+2.2** 48.9 _+10.4*
Tug)
22.4 _+4.4** 21.7 _+5.8
3.7 ,+1.2** 47.8 _+11.4*
OK!+046 (5) (100 w/w
6.8 _+2.7
Meanarrlthestmhrd * **
P < 0.1 P < 0.001
17.4 ,+3.8
36.5 f 8.7*
60.7 f l5.1*
error (n)
Not significant. Significantcmpamdtocontmlvalues.
Table 1: Infl~Of~thatinterferedwiti~~tecascade was follow@ on the interaction of platelets with sub@bm&ium. Normal contmlplatelets fomed a significantammt OfthraaJxlson Ibupmfen, Aspirin, Fq and 13-APA the exposed vascular surface. mxunsignifbntly pmmnted the fon&iculofplatelet~. boxamsyW&ase inhMtorOKY-O46hadnosu&inhibibryeffecton platelet reactivity.
ofthmnbifoznmdwas Intheunbeatedamtml~grerup~ (3.8%) the 28% whqzws, Pmstagl~ El significantly decmamd foxmtion of platelet thrcmbi. Aspirin (100 UM), acid, and lkupmfen (200 uM) significantly xmdmxd 13mV2 inhibitor,oKYplateletUx-cmbiformd. TheUmWoxamqMhebse effect cn platelet th?rMms 046 (100 WW, , had no inhaiw fonnaticn. spmadaq andaggregate fozmaticmofplatelets on enwheliulnwassignificarrtlygreaterintheAspirinand~andin El graupS*
137
all Influenceof CalcimAntacmnistsonPlatelet-VesselW Interaction Vexapamil,Diltiazemand C&in 2 were the calciumw employed(Table2).
Surface &nrered 9.8 _+ 1.4
19.0 _+ 2.2
?Lspirin (100uM)
40.7 _+ 5.6*
25.7 t 3.4
8.2 _+ 1.4*
74.6 f 8.6
Quin2 (40w
30.2 _+ 2.8*
17.7 2 1.6
9.9 _+ 1.0*
57.8 _+ 7.4
Diltiazem (100w
27.6 _+ 2.4
26.0 f 2.4
16.6 _+ 2.4
70.2 _+ 8.4
Verapmil (100uM)
14.4 _+ 1.8
23.4 _+ 2.2
29.6 f 4.6
67.4 _+ 9.6
control
Meanard values.
standard
error
(IF3);
*p
39.6 _+ 3.4
< 0.001
culpald
68.4 _+ 6.8
to the
ax-k-ml
Table 2: Influenceof cdlciumantagmistsonplatelet-vesselwall interaction was follcwed. Ccntrclplateletsmactedwiththeexposed surface and form&l Umakus. Aspirin and intracellular calcium chelatorQuin2 effectively r&uced the formation of plateletthrmbi. Verapamilhad nc such inhibitcryeffect cn platelet interaction leadingtothK&U formation.
Aspirinwasusedasoneoftheccntrolstomnitorthesestudies. untreatedconrOls had 39% thmnbi cmpared to 8% for the Aspirin treat4EdgroqL 0fthecalcimantagoniststested,onlytheintracellular calciumchelator,Quin 2, exertedan inhibitoryeffectcn the
138
d0velw of platelet thrcmbi. Aspirin Diltiazemand Quin to the submWA&im, enbncad platelet qxeadbq and aggmgath allpamdtothevalues ~fortheIlnkreated~grarp.
2
Platelet-VesselWall Interactions zspirintreatmentsignificaRtly~theperc#lltcbgeof~i formed (11%) cu@amd to the untreated ccmtmls (38%) (Table 3).
CQlTbA
16.5 ,+ 2.4
38.12
22.3 f 3.8*
35.2 _+ 3.8
11.5 _+ 1.4*
69.0 _+ 7.8
Sal&late (100 UM)
8.6 _+ 1.8
15.6 _+ 2.6
26.1_+ 2.8
50.3 ,+ 6.4
Salicylamiae (100 w
8.0 _+ 2.0
15.4 ,+ 1.8
26.6 _+ 3.0
50.0 ,+ 7.4
A5pM.n (100 w
9.1*
Msanand*P < 0.001 ccqamd
2.4
4.2
63.7 _+ 8.6
error (xF3). to the CcntxQlvalues.
mle 3: Inflw of Aspirin, Salicylate aW Salicylamide on platslet-vesselwallintemcU on was follcmEd. A5pirin s' lwwxdthsformticmofplab.letthmrbi,htmhana3d spmadhg of platelets. Whereas Salicylate and Salicylamide did mt influence inanywayplatelet hteractionwiththevesselwal1.
ELz+z!!Z
Salicylate anA Salicylamiae mre less effective fornmtion of platelet thrabi, ht, unlike Aspirin erhamespmadhganda54regatianof platelets: agentsdidmt
139
Influence of Acetvlsalicvlic Acid,Salicvlate and Saliwlmide on u& axachidonic CmversionbvPlateletEWvmes quantiUntreatednormalcmtrolplateletscomer&d significant ties of radiolaheledsubstrateto cyclooxygenase(HHT, 42%) ard lipoxygenase (HE!TE, 18.7%)metabolites(Table4).
RADIOAc!mnTYRMxlvERED INvAEuCUSFRACrIoNs (*PERXTTOF !NYlY& m) Fhcepholipids Control
38.52 5.4
ASA
14.7_+2.2
HID
41.8_+3.6 1.3 _+0.2*
AA 18.7_+1.2
1.0 LO.2
76.7f 3.8*
7.3 _+1.0
Salicylate 43.1_+4.6
39.9_+3.8** 14.8_+1.4**
2.2 _+0.4
Salicylamide48.5f 6.4
35.12 4.0** 14.9_+1.4**
1.5 _+0.5
*Meanandthestandanl
*P c
0.001,
error (n=3).
significant cmpared to control
values.
**P < 0.1,not significant. Table4: Rildiohbeledarachidonate(AA) transformation to cyclooxygenase(HITI?) and lipoxysenase (HElZ)pmductsbyintactplatelets was follmed. Normalcontrolplateletscommked significant amounts OfAAtoHHTandHEm. AspirinpreventedtheformationofHmand channeledAAconversiontoHEIE. Salicylate arrdSalicylamide did not exertanyinhibitoryeffectoncycloaxygenase andhadverylittleinfluenceonlipoqgemm. Aspirinccmpletelyblockedarachidonicacid conversionvia cyclooxy genasebutenhamedconversionviathelipcorysenase pamy (78%),-icylate and Salicyhmide did not exzrt any inhibitoryeffect on cycl~enaseandhadslightstinnilatoryaCti~onlipoxygenases.
140
l%mlltsoftheprewntirnrestigatianbaveshownthatagents~ch prwent irmvexsibleplateletaggregaticlltisecreticolinvi~, reducedthefomaticnofplateletthrcm\biaathaeqxmed significantly vasaihr surface. Of me variuls drugs tested, the S synthetaseinhibitar,OK!f-O46,andcalcim~V~~ the foxzmtim of platelet thrau. not effective in p?mmthg AspirhsignificanUyreducedths foxmati~ofplateletthmbi,but misilqmmmtwasoffsetby~~,~and aggmgation ofplatalets cmthe vascum~il.ml.~1activates admylate,cyclase. Iharemltingel6watimofcyclicAwpis etib_h$t plamet adhmmoebyblooJ&cytosoliccalcium expressionof the cell adhesionmolecules(35-37). Hcwever,FGE~inthisstudydidnotmduceplateletintem&i~wi~ theeilml. , Hman plateletsnomally wkqo mible qgmgaticm when stlrredwiti~andreleasetbeirgranilecenteorto. Sh aspirininhibi~seuxld~ r@qxmeofplatel~totbeacuonof inwemible aggregaticm,the majority of wandprevents clinicaltrialshaveainmdatinhibitia~ofcyclooxygwm~, zmUm$mwill r+ce ths risk for L!mcmmt~iW j_s&muc heart disease (11-16). a,-fronalr~toryh?lve chmerated that platelets can becme sticky, adhere and aggmgate i.rJmmmibly indarpsnaeaR of pbteB and ths releaseIzeactim(38,39). In aelitial, letsofpatientswi~stomgepooldeficiencyinteractwith~ t&eliumandfomthm&i,al~#eydonotbave~drana thatstoreareleasablepoolofADP(40). Inviewofthsefindingrs, it is reasmable to malx that hh&M.ah of cam ofthebiochmicaleventsassociatedwitiplaWhtactivaticnmaymtc@lakly p~theirparticipaticplintheclMcal~~~~~~atedto ca?xliovasculardi.sorders. a criticalmle !3tM.iesillthelasttWoaarulnahave inplicated for cdl&m in plateletactivaticn(41, 42). Fiaever, the specific role of icmizedCdLciumin the mdulatiar of varims mzphological/ bio&emical/@ysiologicalalterationsisnotclear. Inspite ofthis of calcium in the [email protected] lxlm&MyMbbeeninterest a&agmMs inthepreve&imofplateletrelat&clinicalccs@ications (25-27,43, 44). Vempamil,NifedipineandDil~havebaen fkeqmtlyused.Inthisstudy,verapamilwasineffectivein~~Urefonmtionofplateletthmbi. Diltiazemwashtterthan ofplatel~appearingas verapmilinreducingthepene&qe thrtxbi. In earlierstudies,Diltiazenhas been shmn to parrarrte disscciationof platelet aggmgates amI potenthte the actim of aspirin(45). sinLi.larlythecalciumantagonists, VWd==-6, havebeenskIwnto dhggmgatePAF~t@!dplateletsandrweree phoqhrylation of 40K Md 20K pmteins (46). (&in 2, an irrtrscellcalcium bufferingagent, waseffectiveinreducilqpli?Mlet ular interacti~l~tothef~ti~of~i,and~effective
141
than calciumentry blockersin prevent- the formationof platelet thmmbi. Aspirinis themstwidelyused of all the antiplateletdrugs in frcm ourlabomkory as well as clinicaltrials (11-16). studies othershave demnstmted that this drug is effectivein r&u&q the However,italsoseerrrs adhmncetoer&thelium.Inaseparatestudy we have shumthataspirintreatnmtaltersnmWanedefomability of plateletsinaependent of its effecton cyclooxygenase Mzymes (47). Ofthevariouscycl~ *, Aspirinsemstohavesignificxnt influenceinchaMeling~~cacidconversionvialipcacygenase. Salicylateand Salicylamide used in this study did not exert any influenceon platelet interactionwith Vium. studies wiul specific lipcorysenase criticallyevaluatethe role, if any, of lipoxygenase n&abolites of arachidomtein the mdulation of plateletinteractionwithsuberhfKtnnthis laboratoq demon&mthat thelium. Prwicus stl.xIies lipoxygemsemtabolitesof arachidonate do nut have any criticalrole i.nagonistimhc& immersible aggregation of platelets(48). In conclusion,results of our study demnstmte that agents capableofpreventiqthmhmne synthesisor~easeofgranule amtfhs significantly reducethe formationof plateletthrmbi upon activationin the extracorpo& circulation.The majorityof these drugs semtoke ineffective inmiqplatelet interactionwith subendathelium leading to spreading adhesionand aggregationof platelets. Theseresultsfwkher'supportourBarlierfhdhgs suggesting that elevationof cyhslie calcim, formationof thmnboxanes and releaseof granulecontentsare not essentialfor platelet shape change, spreading adhemnce or irzwersible aggregation, alkhoqhtheypotentiateheseevents follmingplateletactivation.
TheauthorwishestothankMrs.JulieLeeatxIMrs.DeborahC. JohnsonfOrtheirtecbnimlhelpamIMs.SusanSchwarzeforherhelp with the manuscript. m by USPiiSgrants-11880, CA-21737, Hk30217, a grant fmn the Mar& of Dims (BBBDl-886), Clinical Reseamh C!enta (ES 400) and the Mhmsota MedicalFoundation(HRDF 38-85ti HRF 47-87). REmRENcEs
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