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Increased platelet-fibrinogen affinity in patients with myeloproliferative disorders

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Patients with myeloproliferative disorders (MPD) are known to have some abnormalities of platelet glycoproteins (Gp). Quantitative changes of the Gp Ib, IIb-IIIa, and/or their glucidic content have been reported. Since the Gp IIb-IIIa complex plays a
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  978  lood Vol71 ,No4 April ,1988:pp978-982 IncreasedPlatelet FibrinogenAffinityinPatients WithMyeloproliferativeDisorders ByRaffaeleLandolfi,RaimondoDeCristofaro,MassimoCastagnola,EricaDeCandia, GiuseppeD’Onofrio,GiuseppeLeone,andBrunoBizzi Patients withmyeloproliferativedisorders(MPD)areknowntohavesomeabnormalitiesofplateletglycopro-teins(Gp).QuantitativechangesoftheGpIb,lib-llla,and/ortheirglucidiccontenthavebeenreported.SincetheGpllb-Illacomplexplaysamajorroleinfibrinogenbindingbyactivatedplatelets,wemeasuredtheplateletfibrinogen affinityinninepatientswithpolycythemiavera (PV)andonesubjectwithchronicmyeloidleukemia(CML)bytheaggregometricmethodofMarguerie.InallpatientstheKdoftheplateletfibrinogenreactionwassignificantly decreased ascomparedtocontrols,withevidencein two P ATIENTS withmyeloproliferativedisorders(MPD)haveanincreasedincidenceofboththromboticandhemorrhagicepisodesthathavebeenattributedtoquantita- tive andqualitativeabnormalities.’Among qualitativechangesmajorabnormalitiesofMPDplateletmembraneandgranularglycoproteins(Gp)havebeenrecognized.2 In arecentstudyareducedsialylationandadefectinglucose- mannoseglycosylation ofGpIb,IIb,lIla,andIIIbhavebeen reported.5Because theGplIb-lIlacomplexplaysanimpor-tant roleinfibrinogen-specificbindingbyactivated plate- lets,7’8we investigatedtheplatelet-fibrinogeninteractionin patientswith polycythemiavera(PV)andchronicmyeboidleukemia(CML).Theaffinityforfibrinogenofplatelets fromnormal controlsandfromMPDsubjectswasmeasuredbytheaggregometrictechniqueofMarguerieeta19andbymeasuringtheplateletbindingof‘251-labeledfibrinogen.InMPD patients bothmethodsshowedaheterogeneityof plateletreceptorsforfibrinogenwithevidenceofbinding sites withincreasedaffinity. ClinicalMaterial MATERIALSANDMETHODS Thisstudywasconducted according to theprinciplesintheDeclarationofHelsinki.ThepatientswerefromtheDepartmentof HematologyoftheCatholicUniversityofRome.Elevenpatients with MPDwerestudied.NineofthesepatientshadPV,andtwohadCML.TheplateletcountofourpatientsvariedfromI.4to13.9x108/mL.Oneofthepatientshadbeensplenectomized.Twopatientshadhistories ofamajor thromboticepisode,andoneshowedclinical evidence ofhemorrhagictendency. Drugswerediscontinuedfor FromtheDepartmentofHematologyandtheDepartmentofChemistry,CatholicUniversity,Rome,Italy.SubmittedSeptember6.1987;acceptedNovember23.1987.AddressreprintrequeststoDrRaffaeleLandolfi,IstitutodiSemeioticaMedicaUniversit {224}Cattolica.LargoGemelli800168Roma,Italia.Thepublicationcostsofihisarticleweredefrayedinpartbypagechargepayment.Thisarticlemustthereforebeherebymarked “advertisement” inaccordancewith18U.S.C.§1734solelytoindicatethisfact. © I988byGrune&Stratton,Inc.0006-4971/88/7104-0013 3.00/0 caseswithPVofaheterogeneityofplatelet-fibrinogenreceptorsites.Themeasurementof125l-labeledfibrinogen-plateletbinding,performedinsevenpatients(fivePVandtwoCML).showedreceptorpopulationswithincreased(Kd1 = 0.58+0.3xi0 mol/L)andnormalaffinity(Kd25.12+3.1xi0 mol/L).Theseresultsdemon-strateaheterogeneityofplatelet-fibrinogenreceptorsinthesepatientsandmayexplainthethromboticdiathesisofMPDsubjects. a 1988 byGrune Stratton Inc patients receivingantiaggregatingtherapytendaysbeforethe study. PreparationofPlatelets Venousbloodfrompatientsandcontrolswascollectedin plastic tubes containing theanticoagulantsodiumcitrate(finalconcentra- tion 0.38%).Plateletrichplasma(PRP)wasobtainedbycentrifuga-tionat120gfor20minutes.Plateletswerethengelfilteredtwiceona25x1.5-cmSepharose 2B columnequilibratedwithHEPES-bufferedmodifiedTyrode’ssolution(I-IBMT),pH7.2,containing 0.2 bovineserum albumin(BSA),0.13mol/LNaCI,2.6mmol/LKC1,0.39mmol/LNaH2PO4,12mmol/LNaHco3,10mmol/L HEPES, and 5.5 mmol/Lglucose.Plateletconcentrationwasthenadjustedto1.5to2.0x108/mL. PreparationofFibrinogen Humanadultfibrinogen(gradeL),purchasedfromKabi(Stock-holm),waspurifiedbygelfiltrationona2x100-cmSephadexG 200 columnequilibratedwithHBMTwithoutalbuminandglucose. Thepurityofthispreparationwascontrolledbysodiumdodecylsul- fatepolyacrylamide gelelectrophoresis(PAGE)performedunderbothreducingandnonreducingconditions.’ {176}Fibrinogenconcentra-tionwasmeasuredspectrophotometricallyusinganextinctioncoeffi- cient F ofI.51  “ Clottingabilityofthisfibrinogenpreparation,as assessedbypurifiedthrombin,wasconsistently>95%.Fibrinogen solutionaliquotswerestoredat - 70 {176}C. AggregometricStudies Evaluationofplatelet-fibrinogeninteractionwasperformedby testing theaggregatoryresponseofwashedplateletstoexcess adenosinediphosphate (ADP)inthepresenceofvariousconcentra-tionsoffibrinogen.Inatypicalexperiment0.25mLofplateletsuspensioninaplasticcuvettewasstirredat1,000rpmat37 {176}CnanElvi840dual-channelaggregometerconnectedtoadouble-pen recorder. At 30-secintervals10MLof0.28mol/LCaCl2,10zLof fibrinogensolution (finalconcentrations0.010to0.21mg/mL)and10 zLof280zmol/LADPwereconsecutivelyadded.Theinitialslopeofeachaggregometriccurvewasusedtoevaluatetheeffectofthevariousfibrinogenconcentrationsonplateletaggregatory response.9Inallcasesaggregometricstudieswereperformedwithin twohoursofbloodcollection. ‘251-LabeledFibrinogenBindingStudiesLabelingprocedures. Purified humanfibrinogenwaslabeled with carrier-freeNa 251 using thelodogentechnique.’2Thespecific activity of‘“I-labeledfibrinogenwas200 Ci/mgofprotein.  PLATELET-FIBRINOGENINTERACTIONINMPD 979 Log Ml Table 1   KdValuesofthePlateletsFibrinogenReactioninNormal Subjects andinTenMPDPatientsCalculatedbytheAggregometricMethod Normal subjects n   10Kd mo1/L)   MPDpatientst0.26 ± 0.060.98 ± 0.01 CF PV 0.050.96DP PV 0.050.96 SA PV 0.100.89 GF PV)0.050.97 NG PV 0.060.98 MM PV)0.080.90 LG PV 0.050.95 CM PV)0.090.98BE PV)0.060.96 MS CML 0.060.96 r = correlationcoefficientoflinearregressionplot. C Meanvalues ± SDarereported. t .001. Clottingability offibrinogenwasnotsignificantlyaffectedbythelabelingprocedure. Measurementofplatelet‘25I fibrinogenbinding Twohundredandfiftymicrolitersofgel-filteredplateletsuspensionswasincu- bated for20minutesat37 {176}CithImmol/LCaCl2,10 mol/LADP,and‘251-labeledfibrinogenatfinalconcentrationsranging between 15 and1,200nmol/L.Tomeasurethenonspecificbindingat the variousfibrinogenconcentrations,ADPandCaC12were omitted. At theendofincubation100 Loftheplateletsuspensionswerestratifiedon400;.tLof20%sucroseinplasticcuvettes.After centrifugation fortwominutesatI2,000rpm,thesupernatantswere aspirated, andthetipsofthetubescontainingtheplateletpellets werecutand countedinaHewlettPackardgammacounter.TheequilibriumdissociationconstantandthenumberofreceptorsiteswerecalculatedbyplottingthedataaccordingtoKlotz’3and ‘4 StatisticalMethods The beststraight-linefittotheexperimentalpointswascalculated by theleastsquare-fitlinearregression.Comparisonofresults Fig1   Hillplotsofdataobtainedbymeasuringforeachfibrinogenconcentrationtheinitialslopeofaggregationcurveat10 mol/LADP. A)Con-trol; B)PatientwithPV; CandD)PatientswithPV in whomtwodifferentKdvaluescouldbecalculated.S.slopeofaggregationcurve;F.fibrin- ogen concentration molar);n,Hillcoefficient;Kd, dissociation constantoftheplatelet-fibrinogen reaction. ForexperimentalconditionsandHillplot analysis, seetext. ‘I’ S   obtainedincontrolsandMPDpatientswasperformedbyWilcox-on’s test,’5 AggregometricStudiesRESULTSWashedplateletsfromeachsubject werefirsttestedforADP-inducedaggregationinabsenceoffibrinogen.Undertheseconditionsnoaggregationcouldbeinduced.Platelet- fibrinogenaffinity wasevaluatedaccordingtotheclassicalreceptoroccupancy Accordinglydose-responsecurvesmeeteachofthefollowingequations:and 1/S = 1/FxKd/Smax + 1/SmaxLogS/(Smax - 5)=nLogF - LogKd whereF,Kd,5, andnarefibrinogenconcentration,dissocia-tionconstantofthereaction,theinitialslopeofaggregation, andHillcoefficient,respectively.Smax,obtainedfromthe aggregationexperiments,wasalsocontrolledbyplottingthedataaccordingtoKlotz.’3Theplatelet-fibrinogenreactionwasanalyzedbybothdouble-reciprocalplot(equation1)and  ill plot  equation2 .The Kdvaluesobtainedfromthetwotypesofanalyseswerenotsignificantlydifferent.OnlyKdvaluescalculatedbydouble-reciprocalplothavebeen reported Table 1).IntennormalsubjectstheKdofthe platelet-fibrinogen reactionrangedfrom1.6to3.5,withanaveragevalueof2.6 ± 0.6xiO mol/L.InMPDpatientstheKdvaluesrangedfrom0.2to0.9xl0 mol/L(mean 0.65 ± 0.18).Therewasnocorrelationbetweenblood plateletcounts andKdvaluesinthesepatients.Exceptintwopatients withPV,correlationcoefficients ofstraightlinesofdouble-reciprocalplotswerealways>0.94.Hillcoefficients in the samegroupwerenotsignificantlydifferentfrom1   In two PV patientsin whomlowercorrelationcoefficientsindouble-reciprocal plot werefound,Hill plot analysissug-gestedthe presenceoftwodifferentgroupsofreceptors. As  a ‘ z I(5 M.RO.I mm. t S FIBRINOGEN cad2 ADP0.03MG/MI.1mM   MM 2520 BF s (M x io3) 10S . MPD S N N....._   35404550 Fig 3.Scatchardplotof‘2 -labeIed-fibrino-gen-bindingexperimentsperformedinanormal subjectand inapatientwithCML.Thebestfittotheexperimentalpointsshows two classesofsiteswithdifferentaffinities.980 LANDOLFI ETAL S1015202530 B ( M x io {176}) Fig2.Comparisonofaggregatoryresponseofgel-filtered plateletsfromanormalsubjectandfromapatientwithPVat similarfibrinogen.CaCl andADPconcentrations.(Forexperi- mentalconditionsseeMaterialsandMethods.) shown in Fig 1   twodifferentKdinplateletsfromthesamedonorcould becalculated.OfthesetwovaluesoneisclosetothemeanKdofnormalsubjects,whiletheotherissimilartothevaluegenerallyobservedinMPDpatients.Inbothcases the Hillcoefficientsinthehalf-saturationregionwere0.36and0.38,respectively.InMPDpatientsSmaxandslopevaluesatthevariousfibrinogenconcentrationswereconsis-tentlyhigherthaninnormalsubjects(Fig2). BindingStudies ‘25I-labeledfibrinogenbindingstudieswereperformedinsevenpatients,sixofthem(fivePVandoneCML)previ- ouslystudiedwith theaggregometricmethod.ResultsobtainedinnormalcontrolsandMPDpatientsaresumma-rizedinTable2.InsixnormalcontrolsthemeanKdvaluewas3.5 ± 1  5x l0 mol/L,with 18 000 ± 2,500 receptorsites/platelet.InallcasesKlotz’s’3andScatchard’s’4analysisofbindingdata providedevidence ofasingleclassofreceptors(Figs3and4).BindingstudiesofpatientswithMPDshowedtwodif- ferentplatelet-fibrinogenbinding siteswithmeanKdvaluesof0.58 ± 0.3and5.12 ± 3.1xiO mol/Landwith1,830 ± 75 and8,100 ± I,800receptorsites/platelet,respectively(Figs3and5).TheresultsobtainedinpatientswithPVand Table 2.BindingStudiesWith‘al-LabeledFibrinogeninNormal Subjects andinMPDPatients Kd   Normal SUbJeCtS n-6 MPDpatients ( 1 0.35±0.15 8,000 ± 2.500) Kd, Kd2 CF PV 0.10.65 (2,500)(10,000)DP(PV)0.0340.2  1,500 6,200 SA(PV)0.0350.44 (1,000) (6.500) NG(PV)0.091.14 (1.000) (6.500)BE(PV)0.090.65  2.800 10,000 MS(CML)0.0280.25 (1,500)(10,000) QP CML 0.0350.29 (2,500)(7.500) Thenumberofthefibrinogenreceptorsites/platelet for eachreceptorclassisreportedinbrackets. CMLwerecomparable.ThedatareportedinTable2derivedfromScatchard’sanalysis.’4Klotz’splot’3wasusedtoevalu-atethesaturationdegreeofreceptorsandgavevaluesofKdandnumberofsitescomparabletoScatchard’splot.’4 DISCUSSION Platelet-fibrinogeninteractioniscentraltothephysiologicprocessofplateletaggregation.’8Plateletaffinityforfibrino-genhasbeenstudiedbybothradioisotopicandaggregomet- nc techniques.InnormalsubjectsKdvaluesrangingfrom 0.03 to5.6 ±mol/Lhavebeenreported.’9ThedifferentestimationofthenormalKdvaluesbythevariousauthors can probablybeattributedtodifferentmethodsofplateletpreparation.Whentheaggregometricandradioisotopic methods werecompared inthesame study,similarKdvalueswereobtained.9’ {176}AlsointhisstudythetwotechniquesgavesimilarKdvaluesinnormalsubjects.Inaddition,ahomoge- neousreceptor-sitepopulation wasfound.BoththeKdvaluesandthehomogeneityofreceptorpopulationareinagreement withthefindingsofMarguerieetal. {176} InpatientswithMPD,aggregometricstudiesand25I fibrinogen-bindingexperimentsgaveapparentlydiscordant  . KdO.3O M MOLRCULRSBOUND ,‘ PLATELET 1.8s1O I,-.- 200 4, 4) aa. ‘0 0 K -a C 0 .0 1: 76.56 0 a  {149}a i  0 S., 0 S   .04   e3E ii  {149} {149}  Ed1 0.03  M   Ed2 O.3 MMOLECULESBOUNDIPLATELET : 2.io MOLECULESBOUNDiPLATELET:6.55 1O .. I I-j.I Fig5.Titrationplotofa‘ -labeled-fibrino- gen-bindingexperimentinapatientwithCML.The curve shows two fibrinogen-receptorclasseswith differentsaturationpoints. PLATELET-FIBRINOGENINTERACTIONINMPD 981 -‘LegFibrinog.n(M)Fig4.Titrationplotof‘251-labeledfibrinogen-bindingexperimentperformedinanormalcontrol.Thiscurveshowsaclearsaturationpointaccord-ingtoKlotz’3andasingleclassofplatelet-fibrino- gen receptorsites. results.Infact,whiletheradioisotopicmethodshowedinall casesadoubleclassofplatelet-fibrinogen receptors,theaggregometrictechniqueresultedinthesamefindinginonlytwoMPDsubjects.Sincethehigh-affinityreceptorsites foundby theradioisotopicmethodhad aKdvaluecompara-ble to theKdvaluederivedfromtheaggregometricmethod, itis likelythat thebindingoffibrinogentothesesitescould induce thefullbiologicaleffect ie,aggregation),which masksthenormal-affinity-receptor-populationoccupancy. 0 7 57 6.56 -LogFibrinogen M Theexistence ofa doubleclassofplatelet-fibrinogenreceptorsinMPDcouldprobablybeattributedtoahetero-geneityof plateletpopulationratherthantothepresenceof differentreceptorclassesinthesameplatelet.Thishypothe- sisrequires furtherstudy.Thereducednumberoffibrinogen receptor sitesonMPDplateletsdidnotcauseareductionofmaximalMPDplateletaggregatoryresponse;onthecon-trary,maximalMPDplateletaggregatoryresponseincreased Fig2).  982 LANDOLFIETAL Althoughamodifiedsensitivityto ADP cannot beexcluded,thisfindingsuggeststhatwithinareasonablerangeofreceptorsites theaggregatoryresponsedependslargely upon thethermodynamicfactorsthatregulatethe fibrinogen-receptorinteraction.Themolecularmodifications that causetheincreaseofMPDplateletaffinityforfibrino-genmustbedemonstratedbyfurther studies.Thereduced glycosylationofGpIIbandlilainMPDplateletsreported recently byClezardineta15mayaccountforthemodifiedaffinityforfibrinogen.In factthefunctionofGpsugarsin modulatingthereceptor ligandaffinity is wellknown.2’The enhancedplatelet-fibrinogenaffinitymayplayarole in thethromboticdiathesisofMPDpatients.Indeed modificationsofotherplatelet membraneGpcouldcauseanabnormal MPD plateletinteractionwithotheradhesiveproteinsandwithsubendothelium 22thuscontributingtothethrombo hemorrhagictendencyofthesepatients. 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