interestlethergo

REVIEWARTICLEMeasuringantiplateletdrugeffectsinthelaboratoryPaulHarrisona,A.
L.
FrelingerIIIb,d,MarkI.
Furmanb,c,e,AlanD.
Michelsonb,d,e,aOxfordHaemophiliaCentreandThrombosisUnit,ChurchillHospital,Oxford,UnitedKingdombCenterforPlateletFunctionStudies,UniversityofMassachusettsMedicalSchool,Worcester,MA,USAcDivisionofCardiovascularMedicine,UniversityofMassachusettsMedicalSchool,Worcester,MA,USAdDepartmentofPediatrics,UniversityofMassachusettsMedicalSchool,Worcester,MA,USAeDepartmentofMedicine,UniversityofMassachusettsMedicalSchool,Worcester,MA,USAReceived14October2006;receivedinrevisedform14October2006;accepted27November2006Availableonline17January2007AbstractThisreviewdiscussestheadvantagesanddisadvantagesofcurrentlyavailabletestsforthemonitoringofantiplatelettherapy(especiallyaspirinandclopidogrel).
Manytestsofplateletfunctionarenowavailableforclinicaluse,andsomeofthesetestshavebeenshowntopredictclinicaloutcomesafterantiplatelettherapy.
However,inmostofthesestudies,thenumberofmajoradverseclinicaleventswaslow.
Nopublishedstudiesaddresstheclinicaleffectivenessofalteringtherapybasedontheresultsofmonitoringantiplatelettherapy.
Therefore,thecorrecttreatment,ifany,of"resistance"toantiplatelettherapyisunknownand,otherthaninresearchtrials,monitoringofantiplatelettherapyinpatientsisnotgenerallyrecommended.
Aclinicallymeaningfuldefinitionof"resistance"toantiplateletdrugsneedstobedeveloped,basedondatalinkingdrug-dependentlaboratoryteststoclinicaloutcomesinpatients.
2006ElsevierLtd.
Allrightsreserved.
ContentsIntroduction324Historyofplateletfunctiontestingandoverviewofcurrentlyavailabletests324Monitoringofanti-platelettherapy.
328Monitoringofaspirin328Monitoringofclopidogrel.
330MonitoringofGPIIb–IIIaantagonists331intl.
elsevierhealth.
com/journals/threCorrespondingauthor.
CenterforPlateletFunctionStudies,UniversityofMassachusettsMedicalSchool,RoomS5-846,55LakeAvenueNorth,Worcester,MA01655,USA.
Tel.
:+15088560056;fax:+15088564282.
E-mailaddress:michelson@platelets.
org(A.
D.
Michelson).
0049-3848/$-seefrontmatter2006ElsevierLtd.
Allrightsreserved.
doi:10.
1016/j.
thromres.
2006.
11.
012ThrombosisResearch(2007)120,323–336Conclusions332References.
332IntroductionMostplateletfunctiontestshavebeentraditionallyutilizedforthediagnosisandmanagementofpatientspresentingwithbleedingproblemsratherthanthrombosis[1].
However,asplateletsarenowimplicatedinthedevelopmentofatherothrombosis,whichistheleadingcauseofmortalityintheWesternworld,[2,3]newandexistingplateletfunctiontestsareincreasinglybeingusedformonitoringtheefficacyoftheantiplateletdrugsusedtotreattheseconditions.
This,coupledwiththedevelopmentofnew,simplertestsandpoint-of-care(POC)instruments,hasresultedintheincreas-ingtendencyofplateletfunctiontestingtobeperformedawayfromspecializedhemostasisclini-calorresearchlaboratories,wherethemoretraditionalandcomplextestsarestillperformed[4,5].
Table1isasummaryofthecurrentlyavailabletestsforthemonitoringofantiplatelettherapy,includingtheiradvantagesanddisadvantages.
HistoryofplateletfunctiontestingandoverviewofcurrentlyavailabletestsPlateletswerediscoveredinthe1880's[6].
PlateletfunctiontestingbeganwiththeapplicationoftheinvivobleedingtimebyDukein1910[7].
Thebleedingtimewasstillregardedasthemostusefulscreeningtestofplateletfunctionuntiltheearly1990's[1,8,9].
Recently,thewidespreaduseofthebleedingtimehasrapidlydeclinedbecauseitslimitationshavebeenrecognized(seebelow)andother,lessinvasive,screeningtestshavebecomeavailable[10–12].
Lighttransmissionaggregometry(LTA)wasinventedinthe1960'sandsoonrevolutionizedthediagnosisofprimaryhemostaticdefects[13,14].
LTAisstillregardedasthegoldstandardofplateletfunctiontestingandbyaddingapanelofagoniststostirredplateletsitispossibletoobtainalargeamountofinformationaboutmanydifferentaspectsofplateletfunction[15].
Thistest,oftennowcoupledwiththemeasurementofstoredandreleasableplateletnucleotidecontent,isstillutilizedinmostlaboratoriesforthediagnosisofmanyplateletdefects[16].
Recently,commercialaggregometershavebecomeeasiertousewithmulti-channelcapability,simpleautomaticsettingof100%and0%baselines,andcomputeroperationandstorageofresults.
Forexample,anewfullycomputerized8-channelaggregometerhasjustbecomeavailable(Fig.
1).
Someinstrumentscansimultaneouslymeasureluminescence,tomonitorthereleasereactionofdensegranularnucleotidesduringsecondaryaggregation.
LTAisrelativelynon-physiological,asseparatedplateletsarestirredunderlowshearconditionsandonlyformaggre-gatesafteradditionofagonists,conditionswhichdonotaccuratelymimicplateletadhesion,activationandaggregationuponvesselwalldamage.
Also,conventionalLTAusingafullpanelofagonistsrequiresbothlargebloodvolumesandasignificantexpertisetoperformthetestsandinterpretthetracings.
InresponsetotheproblemswiththebleedingtimeandLTA,anumberofalternativetestshavebeendeveloped,includingimpedancewholebloodaggregometry(WBA),afullyautomatedcartridge-basedinstrument(VerifyNow)thatmea-suresplateletLTAinanticoagulatedwholeblood,andavarietyofteststhatattempttosimulateprimaryhemostasisinvitro(Table1).
WBAprovidesameanstostudyplateletfunctioninanticoagulatedwholeblood[17].
Thetestmeasuresthechangeinresistanceorimpedancebetweentwoelectrodesasplateletsadhereandaggregateinresponsetoclassicalagonists.
Anewfullycomputer-izedtwoorfourchannelinstrumenthasnowbecomeavailable(Fig.
2).
AlthoughthelatterinstrumentcanalsobeusedforLTAofplatelet-richplasma(PRP),WBAhasmanysignificantadvantagesincludingtheuseofsmallersamplevolumesandtheimmediateanalysisofsampleswithoutmanipulation,lossoftimeorpotentiallossofplateletsubpopulationsorplateletactivationduringcentrifugation.
AnewfivechannelcomputerizedWBAinstrument(MultiplePlateletFunctionAnalyzerorMultiplate)nowhasdisposablecuvettes/electrodeswitharangeofdifferentagonistsforbothdiagnosisandmonitoringantiplatelettherapy.
TheVerifyNow(formerlyknownastheUltegraRapidPlateletFunctionAnalyzer[RPFA])instrument(Fig.
3)isafullyautomatedPOCtestthatwasoriginallydevelopedtomonitorglycoprotein(GP)IIb–IIIaantagonistswithinaself-containedcartridge(containingaplateletactivatorandfibrinogen-coatedbeads)[18–20].
Bloodsampletubesarethensimplymixedpriortoinsertionontothecartridgethathasbeenpre-mountedontotheinstrument.
Aggregationinresponsetotheagonistismonitoredbylighttransmissionthroughtwoduplicatereactionchambersineachcartridge.
Otherspecializedcar-tridgesarenowavailableformeasuringplatelet324P.
Harrisonetal.
Table1AnalphabeticallistofcurrentlyavailabletestsforthemonitoringofantiplatelettherapyNameoftestPrincipleAdvantagesDisadvantagesFrequencyofuseAspirinWorksImmunoassayofurinary11-dehydrothromboxaneB2MeasuresstablethromboxanemetaboliteIndirectassayIncreasinguseDependentuponCOX-1activityNotplatelet-specificRenalfunction-dependentBleedingtimeInvivocessationofbloodflowInvivotestInsensitiveDecreasingpopularityPhysiologicalPOCInvasiveScarringHighCVFlowCytometryMeasurementofplateletglycoproteinsandactivationmarkersbyfluorescence(e.
g.
VASPphosphorylationtomonitorP2Y12inhibition)WholebloodtestSpecializedoperatorWidelyusedSmallbloodvolumesExpensiveWidevarietyoftestsHemoStatusDevicePlateletprocoagulantactivitySimpleInsensitivetoaspirinandGPIbfunctionUsedinsurgeryandcardiologyPOCIchor–PlateletworksPlateletcountingpre-andpost-activationRapidIndirecttestmeasuringcountafteraggregationUsedinsurgeryandcardiologySimplePOCSmallbloodvolumeImpactconeandplate(let)analyzerQuantificationofhighshearplateletadhesion/aggregationontosurfaceSmallbloodvolumerequiredInstrumentnotyetwidelyavailable.
LittlewidespreadexperienceasonlyrecentlycommerciallyavailableHighshearRapidSimpleResearch(variable)andfixedversionsavailablePOCLightTransmissionAggregometry(LTA)Lowshearplatelet-to-plateletaggregationinresponsetoclassicalagonistsHistoricalgoldstandardTimeconsumingWidelyusedinspecializedlabsSamplepreparationExpensivePFA-100HighshearplateletadhesionandaggregationduringformationofaplateletplugWholebloodtestInflexibleWidelyusedHighshearVWF-dependentSmallbloodvolumesHct-dependentSimpleInsensitivetoclopidogrelRapidPOCPlateletReactivityIndexMeasurementofplateletaggregatesinwholeblood(modifiedWuandHoakmethod)SimpleRequiresbloodcounterLittlewidespreadexperienceRapidIndirecttestmeasuringcountafteraggregationInexpensiveSerumThromboxaneB2ImmunoassayDependentuponCOX-1activityPronetoartefactWidespreaduseNotplatelet-specificThromboelastography(TEGorROTEM)MonitoringofrateandqualityofclotformationGlobalwholebloodtestPOCMeasuresclotpropertiesonly;largelyplatelet-independentunlessplateletactivatorsareusedUsedinsurgeryandanesthesiologyVerifyNowFullyautomatedplateletaggregometertomeasureantiplatelettherapySimpleInflexibleIncreasingusePOC3testcartridges(aspirin,P2Y12andGPIIb–IIIa)CartridgescanonlybeusedforsinglepurposeWholeBloodAggregometryMonitorschangesinimpedanceinresponsetoclassicalagonistsWholebloodtestOlderinstrumentsrequireelectrodestobecleanedandrecycledWidelyusedinspecializedlabsalthoughlessthanLTAAbbreviations:COX-1,cyclooxygenase1;CV,coefficientofvariation;GP,glycoprotein;Hct,hematocrit;LTA,lighttransmissionaggregometry;PFA-100,plateletfunctionanalyzer100;POC,point-of-care;VASP,vasodilator-stimulatedphosphoprotein;VWF,vonWillebrandfactor.
325Measuringantiplateletdrugeffectsinthelaboratoryresponsestoeitheraspirin(VerifyNowAspirin)orclopidogrelandotherP2Y12antagonists(VerifyNowP2Y12).
Thisinstrumentisaconsiderableadvance,asthetestisafullyautomatedPOCtestwithouttherequirementsofsampletransport,timedelaysoraspecializedlaboratory,anditcanprovideimmediateinformation.
Itisalsopossibletomonitorplateletaggregometryinwholebloodbyasimpleplateletcountingtech-nique.
Afteradditionofanagonisttoanticoagulated,stirredwholeblood,plateletsaggregateandtheplateletcountdecreaseswhencomparedtoacontroltube[21–23].
ThePlateletworksaggregationkits(HelenaBiosciences)aresimplybaseduponcomparingplateletcountswithinacontrolEDTAtubeandafteraggregationwitheitherADPorcollagenwithincitratedtubes[24-27].
Anumberoftestshavealsobeendevelopedthatattemptedtomimictheprocessesthatoccurduringvesselwalldamage.
Manyofthesetechniqueshaveremainedprimarilyresearchtoolswithinexpertlaboratoriesbecauseoftheirinherentcomplexityandtechnicaldifficulty.
Commerciallyavailableinstrumentsincludetheplateletfunctionanalyzer100(PFA-100,Fig.
4)andtheImpactconeandplate(let)analyzer(Fig.
5).
Bothofthesetestsmeasureplateletadhesionandaggregationunderconditionsofhighshear,inanattempttosimulateprimaryhemostaticmechanismsthatareencoun-teredinvivo.
Theconeandplate(let)analyzer,originallydevelopedbyVaronandSavion,monitorsplateletadhesionandaggregationtoaplatecoatedwithcollagenorextracellularmatrix(ECM)underhighshearconditionsof1800s1[28–30].
Inthecommercialversionofthedevice,theImpact(DiaMed),aplasticplateisutilizedinsteadofacollagenoranECM-coatedsurface.
Thetestisnowfullyautomated,simpletooperate,usesasmallquantityofblood(0.
12mL)anddisplaysresultsin6min.
Theinstrumentcontainsamicroscopeandperformsstainingandimageanalysisoftheplate-letsthathaveadheredandaggregatedontheplate.
Preliminarydatasuggestthetestcanbeusedinthediagnosisofplateletdefectsandmonitoringanti-platelettherapy.
Becausethetesthasonlyjustbecomecommerciallyavailable,widespreadexpe-rienceislimited.
ThePFA-100device(Dade-Behring)hasbeenavailableforanumberofyearsandisnowinwidespreadusewithinmanylaboratories,withoverFigure1Anexampleofamodern8-channelplateletaggregometer.
ThemodelshownistheBiodataPAP-8E.
ReproducedwithpermissionfromBiodataandBiodis.
Figure2TheChrono-logModel700wholeblood/optical2channellumiaggregometer.
ReproducedwithpermissionfromChrono-log.
Figure3TheVerifyNowsystem.
Reproducedwithpermis-sionfromAccumetrics.
326P.
Harrisonetal.
200paperspublishedonvariousclinicalapplications[31,32].
ThetestwasoriginallydevelopedasaprototypeinstrumentcalledtheThrombostat4000byKratzerandBorn[33,34].
ThePFA-100mea-suresthefallinflowrateasplateletswithincitratedwholebloodareaspiratedthroughacapillaryandbegintoseala150μmaperturewithinacollagen-coatedmembrane.
Thisreactiontakesplacecontainedwithinoneoftwotypesofdisposablecartridge(collagen–epinephrineorcollagen–ADP).
Theinstrumentrecordsthetime(closuretimeorCT)ittakestooccludetheaperture,alongwiththetotalvolumeofbloodusedduringthetest.
Plateletscontributesignificantlytothegenera-tionofthrombinandthedynamicsofbloodclottingincludingclotformation,clotretractionandlysis.
ClotretractioncanbeeasilymeasuredinwholebloodorPRPwithinglasstubesaftertheadditionofcalcium.
TheroleofplateletsinclotretractionwasfirstdescribedbyHayeminthelate19thcenturyandGlanzmannfamouslydescribedpatientswithpoorclotretractionorthrombastheniain1918,whoweresubsequentlyshowntobedefectiveinGPIIb–IIIa[35].
Moderntestsareavailablethatcanstudyboththeroleofplateletsinthrombingeneration,clotformationandclotretraction.
Forexample,throm-bingenerationtestscanbeusedtomeasurethrombingenerationinPRPandwholeblood[36–38].
TheHemoStatustest(MedtronicBloodMan-agement,Parker,CO,USA)canbeusedtodetecttheeffectsofGPIIb–IIIaantagonists[39–41].
Therearealsoanumberofinstrumentsthatmeasurethephysicalpropertiesofclotformation.
Thromboelas-tography(TEG)wasdevelopedmorethan50yearsago[42–44].
Anticoagulatedwholebloodisincu-batedinaheatedsamplecupinwhichapinissuspendedthatisconnectedtoachartrecorderorcomputer.
Thecuposcillates5°ineachdirection.
Innormalanticoagulatedbloodthepinisunaffected,butasthebloodclots,themotionofthecupistransmittedtothepin.
Wholebloodorre-calcifiedplasmacanbeused,withorwithoutactivatorsofthetissuefactororcontactfactorpathways.
TheTEGprovidesarelativelyrapidresult(b30min),canbeconductedinaPOCfashionandprovidesvariousdatarelatingtoclotformationandlysis(thelagtimebeforetheclotstartstoform,therateatwhichclottingoccurs,themaximalamplitudeofthetraceandtheextentandrateofamplitudereduction).
RotationalTEG(ROTEGorROTEM)isanadapta-tionoftheTEGinwhichthecupisstationaryandthepinoscillates[42,45].
Unlikeplateletfunctiontests,TEGinstrumentshavebeentraditionallyutilizedwithinsurgicalandanesthesiologydepartmentsasPOCtestsfordeterminingtheriskofbleedingandasaguidetotransfusionrequirements.
Morerecentdevelopmentsincludeanexpansionintherangeofactivatorstoinitiateaggregationratherthancoag-ulatione.
g.
theplateletmappingsystemusingADPandarachidonicacid[46,47].
TheHaemostasisAnalysisSystem(HAS)byHemodyneisbasedupontheoriginaltechniquedevelopedbyCarr[48–51].
TheHASmeasuresanumberofparametersinclottingbloodincludingplateletcontractileforce(PCF),clotelasticmodulusandthrombingenerationtime(TGT)inasmallsample(700μL)ofwholeblood.
Figure5TheImpactconeandplate(let)analyzer.
Repro-ducedwithpermissionfromDiaMed.
Figure4ThePFA-100instrument.
Reproducedwithper-missionfromDade-Behring.
327MeasuringantiplateletdrugeffectsinthelaboratoryInthelast20years,flowcytometricanalysisofplateletshasalsodevelopedintoapopularmeanstostudymanyaspectsofplateletbiologyandfunction.
Preferredmodernmethodsnowutilizedilutedanticoagulatedwholebloodincubatedwithavarietyofreagentsincludingantibodiesanddyesthatbindspecificallytoindividualplateletproteins,granulesandlipidmembranes[52–54].
Flowcyto-metricanalysisofplateletfunctionisdiscussedindetailinRefs.
[52–54].
Monitoringofanti-platelettherapyMostplateletfunctiontestshavebeentraditionallyutilizedtoeitherscreenforordiagnoseplateletdefects.
Mosttraditionaltestsarenotonlydifficulttoperformbutareexpensive,timeconsuming,andrequirerelativelylargevolumesoffreshblood.
Theyarethereforeusuallyperformedwithinspe-cializedhemostasislaboratories,oftenincloseproximitytoassociatedclinics.
Manyofthesetestsarelimitedintheircapacitytopredictbleedingorthrombosis.
Theselimitationshavelargelyrestrict-edtheirwidespreadclinicalusewithinotherdisciplines(e.
g.
,cardiology,strokeandsurgery).
However,thisisnowbeginningtochangeassimplertestsofplateletfunctionbecomeavailablethatcanpotentiallybeutilizedasPOCtestsoratleastwithinnon-specializedlaboratories.
Withtheincreasingdevelopmentofnewclassesofantiplateletdrugsandtheknownheterogeneityintheirbiologicaleffectsbetweenpatients,itmaybecomeusefultomonitoranindividual'sresponsetoantiplatelettherapysothateitherthedosageand/orthetypeofdrug(s)administeredcanbetitratedoroptimizedwithinindividualpatientstohelpcontrolandminimizetheriskofeitherthrombosisorbleeding.
Theantiplateletdrugaspirinhastraditionallybeenadministeredatastandarddosewithnomonitoringofeffect,ontheassumptionthatusualdosesaretwotothreetimesthoserequiredtoinhibitallcyclooxygenase-1(COX-1)activity.
How-ever,thelackofasimple,convenient,reliableandclinicallyrelevanttestofplateletfunctionhasmeantthatlackofeffectinindividualpatientshasgoneundetected.
Withtheavailabilityofotherclassesofantiplateletdrugs(e.
g.
thienopyridines,newP2Y12antagonistsandGPIIb–IIIaantagonists)thereisrenewedinterestinthepotentialutilityofplateletfunctionteststomonitortheefficacyofplateletinhibition.
ThedevelopmentofGPIIb–IIIaantagonistsinparticularresultedinthedevelop-mentofanumberofnewassaystomonitorapatient'sresponse(e.
g.
,VerifyNowIIb/IIIa)mainlybecauseoftheirnarrowtherapeuticwindowwithassociatedincreasedriskofbleeding.
Thestillpoorly-definedphenomenonof"drugresistance"hasledtoanexplosionofinterest,researchandavailabilityofavarietyofteststhatcanpotentiallymonitoranindividual'sresponsetoantiplatelettherapy[55].
Thequestionremainsastowhetherthesetestswillbeclinicallyusefulinthepredictionofbleedingand/orthrombosis.
Patientnon-compli-ancetotheirtherapyisalsoanimportantbutrelativelycommonconfoundingprobleminmanystudies[55].
Itiswellknownthatthereisconsiderablevariationintheresponseofindividuals(eitherpatientsornormalcontrols)toaspirin,clopidogrelandGPIIb–IIIaantagonistsasmeasuredbyvariousplateletfunctiontests.
Thoseindividualswhorespondpoorlytoagivendrugarethereforetermed"resistant".
However,thisisapoorly-definedphenomenonandaprecisedefinitionofresistanceshouldonlyrelatetotheactionofaspecificdrugtoinhibititsbiochemicaltarget[56].
Manyplateletfunctiontestsarenon-specificanddonotmeetthiscriterion.
Resistancemaysimplyrepresentnaturalbiologicalvariationinagivendrugresponseormaybeduetospecificormorecomplicatedmechanisms[57].
Isresistancespecifictoanindividualclassofdrugrelatedtoitsmechanismofaction,oraretherecommoninheritedand/oracquiredmechanismsthatmayinfluenceanindividual'sresponsetonotjustonebutpotentiallyallantiplateletdrugs[57]Recentdataindeedsuggeststhataspirinresistantpatientsasagroupalsohaveareducedresponsetoclopidogrel[58].
Whateverthemechanisms,thekeyquestioniswhetheralaboratorytestthatdetectsresistance,ornon-response,predictsfutureclinicalevents—andwhetherchangingtherapybasedonthetestisbeneficialtothepatient.
Untiltheselinksarefirmlyprovenwithinlargetrials,resistanceinthelaboratorycannotnecessarilybeascribedasacauseofthrombosis.
Therefore,exceptinresearchtrials,itisstillnotyetclinicallyusefultotestforresistanceandchangeapatient'stherapyonthebasisofalaboratorytest[55,57,59].
Thefollowingsectionsdiscussthespecificlaboratorytestsforthethreemainantiplateletdrugs.
MonitoringofaspirinAspirinirreversiblyinhibitsCOX-1resultingintheinhibitionofthromboxane(TX)A2generationfortheentirelifespanoftheplatelet[60].
Aspirinisaneffectiveantiplateletagentbecauseitreducestherelativeriskofmajorvasculareventsandvasculardeathbyabout25%afterischemicstrokeandacutecoronarysyndrome[61].
Regularlowdosesofaspirin(e.
g.
,81mg/day)willusuallyresultinN95%inhibitionofthromboxanegeneration.
328P.
Harrisonetal.
Therapeuticmonitoringwasthereforeconsideredtobeunnecessary.
However,theantiplateletpropertiesofaspirinhavebeenshowntovarybetweenindividualsandrecurrenteventsinsomepatientscouldbedueto"aspirinresistance"oraspirinnon-responsiveness[56,57].
Thereportedincidenceofaspirinnon-responsivenessvarieswidely(between5–60%).
Therearealsomanypossiblemechanismsforaspirinresistancewhichhavebeendiscussedindetailelsewhere[57,62].
Recentlyithasbeenproposedthattheterm"aspirinresistance"shouldonlybeutilizedasadescriptionofthefailureofaspirintoinhibitTXA2production,irrespectiveofanon-specifictestofplateletfunction[56].
ThisisbecausetherearemanyotherbiochemicalpathwaysthatcanpotentiallybypassCOX-1evenifthisenzymeisinhibited.
Dependinguponthetestsystememployed,"aspirinresistance"maythereforebedetectedevenifCOX-1isfullyblocked[56].
Recentstudiesalsosuggestthat,incompliantpatients,theincidenceofaspirinresistanceisrareusingmethodsdependentonCOX-1activity[46,63,64].
Additionofinvitroaspirintosamplesfollowedbyretestingshouldalsobeanimportantconsiderationfortestingcompliance[64,65].
Manytestshavebeenusedtoassesstheinfluenceofaspirinonplateletsandaspirinresistance,includingarachidonicacid-andADP-inducedLTA,ADP-andcollagen-inducedimpedanceaggregation,VerifyNowAspirin,PFA-100,Thromboelastogra-phy(TEG—plateletmappingsystem),flowcytometryusingarachidonicacidstimulation,Im-pactconeandplate(let)analyzer,andserumandurinarythromboxane[55].
Testsshouldideallybeperformedpre-andpost-drug.
Someofthetestshavebeenreportedtobepredictiveofadverseclinicalevents[55].
However,thelargemajorityofthesestudiesaresmallandoftenstatisticallyunderpoweredtocompletelyanswerwhethereachtestcanreliablypredictthesmallnumberofadverseoutcomesthatwereobserved[57,62].
Althoughpreliminaryresultsfromsomestudiescouldsuggestthatresponsestoaspirinshouldbemonitored,thereareadditionalproblemsinthatLTAistime-consuming,difficultandcannotrealis-ticallybeperformedonlargenumbersofpatientsinroutinepractice.
However,thesimplertestsofplateletfunction(e.
g.
PFA-100,VerifyNowAspi-rin,TEGplateletmapping,Impact,andurinarythromboxane)couldofferthepossibilityofrapidandreliableidentificationofaspirinnon-responsivepatients.
ThePFA-100usuallygivesaprolongationintheCollagen/Epinephrine(CEPI)CTinresponsetoaspirin,withtheCollagen/ADP(CADP)CTusuallyremainingwithinthenormalrange[66,67].
Anumberofstudieshaveobservedthatanapprecia-blenumberofbothnormalsandpatientsfailtorespondintermsofprolongationoftheirCEPICTinresponsetoaspirin[68–75].
BecausethePFA-100isaglobalhigh-sheartestofplateletfunction,manyvariableshavebeenshowntoinfluencetheCTincludingVWFlevels,plateletcountandhematocrit[32].
Inpatientsidentifiedwith"aspirinresistance"bythePFA-100,anumberofstudieshaveshownthatVWFlevelsareelevatedinnon-responders[73,74,76].
AstheCEPICTishighlydependentuponVWFandothervariables,pre-andpost-aspirinCTsshouldideallythereforebedetermined,becausethetrueaspirinresponsemaybemaskedbeforethedrugisgiven[56].
Also,CADPCTsarelowerinthesepatients,whichmaybecausedbyacombinationofhighVWFbutalsoincreasedsensitivitytocollagenandADP[73,77,78].
Itisthereforenotsurprisingthattheincidenceofaspirinnon-respondersisreportedlymuchhigherwiththePFA-100thanwithothertests[79,80].
Thequestionremainswhetherornotthisgroupofpatientsareatincreasedriskforthrombosis.
PreliminarydatasuggeststhatPFA-100CEPICTswerenon-informa-tiveinpatientswithstablecoronaryarterydisease,incontrasttoLTA[81–84].
However,anotherstudysuggeststhatthePFA-100couldbeinformative,[85]andthatshortenedCTswiththeCADPcartridge(whichisnotaffectedbyaspirin)mayalsobepredictive[77,86-88].
TheVerifyNowAspirinassayoffersthepossibil-ityofrapidandreliableidentificationofaspirinresistanceornon-responsivenesswithoutthere-quirementofaspecializedlaboratory.
Indeed,thetesthasUnitedStatesFoodandDrugAdministration(FDA)approvalformonitoringaspirintherapyandisbeingusedbysomecardiologistsandgeneralpractitionersintheUSA.
TheoriginalVerifyNowAspirincartridgecontainsfibrinogen-coatedbeadsandaplateletactivator(metalliccationsandpropylgallate)tostimulatetheCOX-1pathwayandactivateplatelets[89].
Ideally,thetestshouldproducesimilarresultstothoseobtainedbyarachidonicacid-inducedLTA.
Onestudyshowedan87%agreementwithepinephrine-inducedLTA[90].
Previousdatacomparingpropylgallateandotheragonistsbyplateletaggregometrysuggestthatthisagonistdetectsalowernumberofrespondersinvolunteersreceivingeither100or400mgofaspirin[89].
AmorerecentstudycomparedLTAwithVerifyNowAspirinandPFA-100anddemonstratedthataspirinnon-respon-sivenesswasnotonlyhigherinbothPOCtestsbutthatagreementbetweenthetestswaspoorandfewpatientswerenon-responsivebyall3tests[79].
Nevertheless,theVerifyNowAspirintestcan329Measuringantiplateletdrugeffectsinthelaboratorypotentiallyidentifyacorrelationbetweenaspirinnon-responders,adverseclinicaloutcomesandaspirindose[91–94].
Sincetheendof2004theVerifyNowAspirincartridgehasbeenmodifiedandarachidonicacidhasreplacedpropylgallateastheprincipleagonist.
FurtherstudiesarethereforewarrantedtorelateadverseclinicaloutcomestothenewVerifyNowAspirinassayandtoseewhetherchangingtherapybasedupontheresultcanalsoimproveoutcomes.
Arecentstudysuggeststhataspirinresistantpatients,asdefinedusing=2outof3tests(VerifyNowAspirinAssay,LTAwithADP,LTAwitharachidonicacid),alsoexhibitreducedresponsivenesstoclopidogrel,suggestingthatalternativeantiplateletdrugsmaynotbeeffectiveinaspirinresistantpatients[58].
Inapparentcontrast,wehaverecentlyreportedthatclopidogrel,inadditiontoitsdirectantiplateleteffectviatheP2Y12ADPreceptor,maydecreaseaspirinresistance(asdefinedbyaresidualarachi-donicacid-inducedincreaseinplateletsurfaceP-selectin)inboththepresenceandabsenceofcoronaryarterydisease[64].
BecauseaspirininhibitsCOX-1,measurementofTXA2anditsmetaboliteseitherwithinserumorurineprovidesarelativelysimplepotentialwaytomonitoraspirintherapy.
Invivo,TXA2israpidlyconvertedintothemorestableandinertmetaboliteTXB2whichisfurthermetabolizedto11-dehydroTXB2,themajorproductfoundinurine.
Measure-mentofTXB2byvariousimmunoassayscanfacilitateanindirectassessmentofthecapacityofplateletstoformTXA2.
AssayscanbestandardisedsothatTXB2ismeasuredeitherwithinserumderivedfromwholebloodclottedfor30minat37°CorinsupernatantsderivedfromPRPorpurifiedplatelets(withstandardizedplateletcounts)activatedbyagoniststostimulateCOX-1activity.
Themetabolite11-dehydroTXB2canalsobemeasuredwithinurinesamplesandtheassayisalsocommerciallyavailableastheAspirinWorkstest.
Thisassayhastheadvantagethatitisnon-invasiveandonelargestudysuggestedthathighlevelsofurinary11-dehydroTXB2areassociatedwithadverseclinicalevents[95].
MonitoringofclopidogrelClopidogrel(Plavix)isaprodrugthatismetabolizedbycytochromeP450inthelivertoanactivemetabolitethatspecificallyandirreversiblyblockstheplateletADPP2Y12receptor[96].
Plateletinhibitionbyclopidogrelisbothdose-andtime-dependentandpatientsareusuallygivealoadingdoseof300–600mgandthenmaintainedon75mg/day.
TheCAPRIEtrialshowedthatclopidogrelpreventedmorethromboticvasculareventsthanaspirin(RRR8.
7%)inpatientswithknownathero-sclerosis[97].
TheCUREtrialshowedaspirinplusclopidogrelwas20%moreeffectivethanaspirinaloneinacutecoronarysyndromes,[98]buttheMATCHstudyshowedthataddingaspirintoclopido-grelinhigh-riskpatientswithrecentischemicstrokeortransientischemicattack(TIA)wasassociatedwithanon-significantdifferenceinreducingmajorvascularevents[99].
Combinationtherapyisregardedasthegoldstandardduringpercutaneouscoronaryintervention(PCI)[100].
However,inter-individualvariabilityinplateletresponsetoclopidogrelhasbeenobserved,[101]and5–10%ofpatientsstillexperienceacuteorsubacutethrombosisaftercoronarystentimplanta-tion[56,102–104].
Thephenomenonof"clopidogrelresistance"hasbeenestimatedtobebetween4%and30%.
ThedefinitionofclopidogrelresistanceisevenmorecomplexthanaspirinresistancebecausethephysiologicaldegreeofinhibitiondetectedbyADP-inducedLTAcanvarywidelybetweenindivi-duals,especiallyasADPcanalsoactivateplateletsviaasecondreceptor,P2Y1,andthereisinter-individualvariabilityofcytochromeP450activity[96].
ThereisaninversecorrelationbetweenP4503A4activityandplateletaggregation,andotherdrugscaneitherpromoteorinhibitmetabolismtoacertaindegree[105].
Pre-existingvariabilityinADPresponsivenessisalsoanimportantvariableandmayprovideanexplanationforresponsevariability[106].
Manymechanismsofclopidogrelresistancehavebeenproposed[55].
LaboratoryresponsestoP2Y12inhibitorsarelargelybaseduponmonitoringADP-stimulatedresponses[107].
PlateletsarestimulatedwithADPandresponsesaremonitoredusingeitherLTA,theVerifyNowP2Y12assay,TEGplateletmappingsystem,Impact,Plateletworksflowcytometricanalysisofactivation-dependentmarkers(e.
g.
,P-selectin,PAC-1),orflowcytometricanalysisofintracellularsignalingbymonitoringthephosphor-ylationofvasodilator-stimulatedphosphoprotein(VASP)[25,96,107,108].
Ideallyresponsesaremon-itoredpre-andpost-drug.
LTAusing5or20μMADPcanbeusedtoarbitrarilyclassifypatientsbaseduponmeasuringthechangein(delta)aggregationatbaselineandpost-drug[109].
Non-responderscanbedefinedwithadeltaaggregationofb10%.
Studieshaveshownthatthereisconsiderablevariationinpatientresponsetoclopidogrelandupto30%ofpatientsmaybenon-responders.
Thelargestanalysissofarhasfound4%of544patientstobehypo-responsivetoclopidogrel[101].
Morerecentdatasuggestthataproportionofpatientsareprobablyunder-dosedandthata600mgloading330P.
Harrisonetal.
dosesignificantlyreducesthenumberofnon-responderswhencomparedto300mg[109–111].
Thereisstillthecriticalunresolvedquestionastowhetherinvitrolackofresponsivenesstoclopido-grelcorrelateswiththeanincreasedincidenceofadverseevents.
ADP-inducedLTAisprobablynotverypracticaltotestonlargenumbersofclinicalsamples.
Also,asresidualP2Y1functioncanpotentiallywidelyvarydespiteP2Y12inhibition,thiscouldnotonlyexplainsomeoftheheterogeneityobservedwithLTAbutsuggeststhatADPalonemaynotbespecificenoughtomeasuretheeffectofclopidogrelandotherP2Y12antagonists[112].
Despitetheseproblems,Matetzkyetal.
foundevidencethatADP-inducedLTApredictedadverseevents[113].
TheVerifyNowinstrumentwasoriginallydesignedtoovercomethemajorlimitationsofLTAandcanbeusedasaPOCtest.
TheVerifyNowP2Y12cartridgehasbecomeavailableformonitoringclopidogrelandotherP2Y12antagonists.
Theassayusesprostaglandin(PG)E1inadditiontoADPtoincreaseintracellularcyclicadenosinemonophosphate(cAMP),theoreticallyenhancingthesensitivityandspecificityofthetestforADP-inducedactivationofplateletsviaP2Y12[114,115].
ThePGE1shouldsuppresstheactivationofplateletsbyP2Y1.
TheVERITAS(TheVerifyThrombosisRiskAssessment)trialwilldetermineiftheVerifyNowP2Y12testisareliableandsensitivemeasureformonitoringclopidogreltherapy.
ThecombinationofADPandPGE1isalsousedintheflowcytometric-basedVASPassay(BioCytex,Marseilles,France)[112,116].
TheprincipleofthisassayistomeasurethephosphorylationofVASP,whichistheoreticallyproportionaltothelevelofinhibitionoftheP2Y12receptor.
ComparisonoftheVASPassaywithLTAshowsthatthelevelofinhibitionishigherintheflowcytometryassay,becausenon-specificaggregationcanoccurviaADPstimulationofP2Y1[112].
Recentdataindeedshowthatthephosphor-ylationofVASPcorrelateswithinhibitionofLTA[107].
ThePFA-100isconsideredunsuitableformonitoringclopidogrel[32,117–119].
TheoreticallythePFA-100CADPcartridgemaybesuitableformonitoringP2Y12butbothcollagenactivationandADPactingthroughtheP2Y1receptor,alongwiththehighshearconditions,maybenormallysufficienttolargelyovercomeP2Y12blockade[96].
ArecentsmallstudyprovidesevidencethattheP2Y1recep-tormaybeanimportantvariableindeterminingtheCTinresponsetoP2Y12blockade[120].
Theremaybealsobeadegreeoftime-anddose-dependence.
Ithasalsobeenobservedthatthereissynergywithclopidogrel/aspirincombinationtherapy,reflectedasprolongationofbothCADPandCEPICTs[121,122].
AssessmentofplateletfunctionbyavarietyoftestsincorrelationwithclinicaloutcomeswillbenecessarytodefineresponsivenesstoclopidogrelandotherP2Y12antagonists.
PreliminarydatafromtheCRESTstudy(ClopidogrelResistanceandStentThrombosisstudy)byGurbeletal.
showdifferenceswithVASP,LTAandactivatedGPIIb–IIIaresponsive-nesstoADPbetweenpatientswithandwithoutsubacutestentthrombosis(SAT)[123].
Comparingdatafrompatientswith(n=20)andwithoutSAT(n=100)suggeststhatclopidogrelresponsevariabil-itytoADPissignificantlyassociatedwithanincreasedriskofSAT[123].
This,coupledwithotherstudiesonpost-dischargeandpost-PCIevents,suggeststhathighpost-treatmentexvivoreactivitytoADPmayindeedbeanimportantriskfactorforadverseclinicalevents[113,116,124].
Carefullycontrolled,largerandomizedtrialswillberequiredtodefineaninadequateresponsetoP2Y12inhibitionforanindividualtestandtoshowthatthiscorrelateswithadverseclinicalevents.
Withoutsuchdata,therapyshouldnotbealteredbasedupontheresultsofanyoftheteststhatpurporttodetermineresponsivenesstoaP2Y12antagonist.
TheRESISTOR(ResearchEvaluationtoStudyIndivi-dualswhoShowThromboxaneorP2Y12Resistance)trialthatiscurrentlyunderwayin600PCIpatientsmaydetermineifthelevelofP2Y12inhibitioncorrelateswithclinicaloutcomeandifchangingtherapyinresistantpatientsimprovesoutcome.
ThedevelopmentandclinicalapplicationofthienopyridinessuchasclopidogrelhasproventhattheP2Y12receptorisanattractivetargetfornewdrugdevelopment.
Becausethienopyridinesaremetabolizedtotheiractivederivativesbytheliver,anumberofdirectantagonistsareindevelopment(e.
g.
,cangrelorandAZD6140)[96].
Somenewthienopyridines(e.
g.
,prasugrel)havealsobeendevelopedwhichexhibitsuperiorproper-ties(e.
g.
,higherefficacy,fasteronsetandlongerdurationofaction)comparedwithclopidogrel[96].
Assomeoftheobservedinter-individualheteroge-neityofclopidogrelresponsivenessmaybecausedbydifferencesinlivermetabolism,itwillbeinterestingtodeterminewhethertheincidenceofnon-responsivenessisloweroreveneradicatedwiththesenewdrugsandwhetherhighpost-treatmentreactivitytoADPremainsapotentialsignificantproblem.
MonitoringofGPIIb–IIIaantagonistsTheidentificationoftheimportanceoftheGPIIb–IIIacomplexinmediatingplateletaggregation(i.
e.
,thefinalcommonpathwayofplateletactivation)suggestedthatthisreceptorwouldbeanattractive331Measuringantiplateletdrugeffectsinthelaboratorytargetforantithrombotictherapy.
TheFDA-ap-provedGPIIb–IIIaantagonists(abciximab,tirofibanandeptifibatide)havenowbecomeanimportantclassofantiplateletagentsthatarewidelyusedforthepreventionofthromboticcomplicationsinpatientsundergoingPCIorpresentingwithacutecoronarysyndromes.
EarlyobservationsontheinhibitionofthrombusformationwithinanimalmodelsnotonlyestablishedastrongcorrelationbetweenthelevelofGPIIb–IIIablockadeandthepreventionofthrombusformationbutdemonstrat-edsteepdose-responsecurves[125,126].
ItbecamerapidlyapparentthatacertainlevelofGPIIb–IIIainhibitionwasrequiredfortheoptimalefficacyofGPIIb–IIIaantagonists.
Thisstronglysuggestedthatmonitoringofplateletinhibitioncouldbeimportantinpatientstreatedwiththeseagents.
MonitoringGPIIb–IIIaantagonistscanbeperformedbyavarietyoftestsincludingLTA,WBA,flowcytometry,andradiolabeledantibodybindingassays[127].
Howev-ersomeofthesetestsaretime-consuming,expen-siveandareusuallyperformedwithinspecializedlaboratories.
GiventhewidespreadclinicaluseoftheseGPIIb–IIIaantagonistsincardiology,thereexistedthepotentialdemandforasimple,inex-pensiveandrapidmethodthatcouldbeutilizedasaPOCtesteitheratthebedsideorintheclinic,sothatthedegreeofGPIIb–IIIablockadecouldbealsobepotentiallydeterminedbynon-specialists.
TheVerifyNowsystemwasoriginallydevelopedtomeetthisdemand.
Theassayprinciplewasdevel-opedbaseduponexperimentsusingfibrinogen-coatedbeadsandTRAPwhichfacilitatedtherapidvisualanalysisofthedegreeofGPIIb–IIIablockade[20].
ThebasisoftheVerifyNowIIb/IIIaassayisthatfibrinogen-coatedbeadswillagglutinateinwholebloodindirectproportiontothedegreeofplateletactivationreflectedbyexposureofthefibrinogenbindingsiteonGPIIb–IIIa[18].
ThepresenceofaGPIIb–IIIaantagonistwillthereforedecreasetheamountofagglutinationinproportiontothelevelofinhibitionachieved.
InitialinvitroevaluationsoftheVerifyNowIIb/IIIaassaydemonstratedgoodcorrelationswithbothLTAinPRPandradiolabeledreceptorbindingassays[18].
StudiesinpatientsreceivingabciximaborotherGPIIb–IIIaantagonistsalsodemonstratedgoodcorrelationswithLTA[128,129].
GOLD(AU—Asses-singUltegra),alargeprospectivemulticenterstudy,showedasignificantassociationbetweenthelevelofplateletinhibitionbytheVerifyNowIIb/IIIaassayandclinicaloutcomes[130].
Thissuggeststhatthedevicehasclinicalutility,althoughnostudyhasyetbeenperformedtodeterminewhethertitrationofGPIIb–IIIatherapybasedupontheVerifyNowIIb/IIIatestresultdecreasesadverseevents.
AslightlymodifiedPlateletworksPOCassaywasrecentlyreportedtocorrelatemorestronglythanVerifyNowIIb/IIIawithLTAinmeasuringplateletinhibitionbyGPIIb–IIIaantagonists[26].
ThePFA-100hasalsobeenutilizedtomonitorGPIIb–IIIablockadeandcorrelateswellwithLTAandreceptoroccupancymeasurements[131–133].
Althoughmanypatientsgivenon-closureorN300sCTinthePFA-100followingGPIIb–IIIaantagonisttreatment,onestudysuggeststhatfailuretoobservenon-closuremaybeassociatedwithanincreasedriskofcardiacevents[133].
ConclusionsAssummarizedinthisreviewarticlemanytestsofplateletfunctionarenowavailableforclinicaluse,andsomeofthesetestshavebeenshowntopredictclinicaloutcomesafterantiplatelettherapy.
How-ever,inmostofthesestudies,thenumberofmajoradverseclinicaleventswaslow,andadditionalstudiesarethereforeneeded.
Mostimportantly,nopublishedstudiesaddresstheclinicaleffectivenessofalteringtherapybasedontheresultsofmonitor-ingantiplatelettherapy.
Therefore:1)thecorrecttreatment,ifany,of"resistance"toantiplatelettherapyisunknown;2)otherthaninresearchtrials,itisnotcurrentlyappropriatetomonitorantiplate-lettherapyinpatientsortochangetherapybasedonsuchtests[55–57].
Aclinicallymeaningfuldefinitionof"resistance"toantiplateletdrugsneedstobedeveloped,basedondatalinkingdrug-dependentlaboratoryteststoclinicaloutcomesinpatients.
Nevertheless,the2006AmericanCollegeofCardi-ology/AmericanHeartAssociationguidelinesforPCIprovidedaClassIIBrecommendation(basedonlevelCevidence)that,inpatientsinwhomsubacutestentthrombosismaybecatastrophicorlethal,plateletaggregationstudiesmaybeconsideredandthemaintenancedoseofclopidogrelincreasedfrom75mgto150mgperdayiflessthan50%inhibitionofplateletaggregationisdemonstrated[134].
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