凝血與血小板減少

BloodCoagulationOverviewandThrombophiliaCliffTakemotoM.D.PediatricHematologyTheJohnsHopkinsUniversityDisclosureInformationCliffTakemotoMDNONEObjectivesUnderstandthephysiologyandregulationofclotformationKnowwhattheaPTT/PTtellsyouKnowwhatthethrombophiliatestsmeanHowtotreataDVT(nextsessionprobably)Whatisinaclot?PlateletsFibrin(coagulationproteins)VENOUSSlowflowRED-(rbc’s)ARTERIALFastflowWHITE(plts)collagenTFHowweclotCutintheendotheliumExposesplateletbindingsitesExposestissuefactorforactivatingcoagulationcollagencollagenTFTFcollagenvWFvWFvWFvWFvWFvWFTFTFTFTFPlateletActivationPlateletgranulesecretionActivatefibrinogenreceptorProvidessiteforprothrombinasecomplexThrombinActivationAndfibrindepositionRED--plateletsGREEN—tissuefactorBLUE--fibrinBloodflowingthiswayThisishowaclotismadeFalatietal.,NatureMedicine2002(mpeg)withpermissionfromNaturePublishingGrouphowaclotismadeinvivoThrombinactivationINITIATEDbyTISSUEFACTOR(microparticles?)NEEDaPLATLETSURFACEforcoagulationfactorstoassembleCoagulationCascadesrealityinatesttube?…theoften-askedquestion:“Whyiscoagulationsocomplicated?”resolvesitselfintothequestion:“Whyaretheresomanystages?”R.G.Macfarlane,Nature1964CoagulationCascadesSomeconceptstoremember…Highlyregulatedcomplexesofserineproteasesandco-factorsAllpathsleadtothrombinactivationGoalistomakeafibrinclotTheyarenotjustanumber—theyhaveapersonalitytoo!!!thrombinfibrin…misunderstandingbreedsfearandhatred…SerineProteasesFVIIIFVTFCo-FactorsFIIPCTMPSFIXFXFVIIIIprothrombinIIathrombinFibrinpolymersFibrinogenXa

VaCa++,PL“Prothrombinase”IXaVIIIaCa++,PLintrinsictenaseprekallikreinHMWKXIIXIClotformation--physiologyTFVIIaextrinsictenaseIIIIaFibrinpolymersFibrinogenXa

VaCa++,PL“Prothrombinase”IXaVIIIaCa++,PLintrinsictenaseprekallikreinHMWKXIIXITFVIIaextrinsictenasePTPTTIIFibrinpolymersFibrinogenXa

VaCa++,PL“Prothrombinase”IXaVIIIaCa++,PLintrinsictenaseprekallikreinHMWKXIIXITFVIIaextrinsictenaseINITIATIONIIaAMPLIFICATIONPROPAGATIONenzymecomplexes:protease cofactorProthrombinase XaVaIntrinsictenase IXaVIIIaExtrinsictenase VIIaTF AlsoneedCalciumandaphosholipidsurfaceActivationofthrombin:3complexesIIprothrombinIIathrombinFibrinpolymersFibrinogenXa

VaCa++,PLIXaVIIIaCa++,PLClotformation--physiologyTFVIIaXinitiationpropagationAfewwordsaboutcontactactivation…RegulateInflammationHK,PK,FXIIdeficiency donotbleedFXIdeficiencyassociated withbleedingCofactorHighMolecular-WeightKininogen(HK)ProteasePrekallikrein(PK)FXIIFXIInibitorC1esteraseinhibitorC1inhThrombinIIaprocoagulantanticoagulantFibrinogenFV,FVIII,FXIFXIIITAFI(fibrinolysisinhibitor)PlateletActivationProteinC/S(boundtoThrombomodulin)intrinsicextrinsicThrombinhasbothpro-andanti-coagulantfunctionsFibrinogenStructureGorkunO.etal.,Blood1997;89:4407-44.WithpermissionfromtheAmericanSocietyofHematologyFibrinClotformationandfibrinolysisDDEDDEDDEDDEDDEDDEDDEDDEDDEDDEDDEfibrinogenFXIIIplasminThrombin(IIa)ABfibrinopeptidesD-dimerActivatorst-PA,u-PAInhibitorsPAI-1,PAI-2a2antiplasmina2macroglobulinTAFIPlasminogenregulationClinicalAspectsaboutFibrinolysisPAI-1--deficiencycausesbleeding --increasedinDIC,inflammation—prothrombotica2antiplasmin—deficiencycausesbleedingPlasminogen—newbornshavelowlevelscomparedtoadultsTestsforfibrinolyticpathway --measureindividualfactors --euglobulinclotlysis(shortwithincreasedfibrinolysis)FibrinogenDisordersDefect

Inheritance

ClinicalAfibrinogenemia recessive SevereBleeding umbilical,joint

Hypofibrinogenemia recessive MildBleeding or menorrhagia dominant miscarriagesDysfibrinogenemia dominant Mildbleeding poorwoundhealing thrombosis6polypeptidechains3genes:a,b,gEndogenousAnticoagulants:TurningofftheclotProteinC VIIIa,Va(thecofactors)ProteinS

Antithrombin IIa,Xa(serineproteases)TFPI VIIa/TFIIIIaVIIaXaVaIXaVIIIaProteinCProteinSATIIIthrombomodulinIIaTFPIHowtostoptheclotAntithrombinisaserineproteaseINHIBITOR

(SERPIN)PentasacchridesequenceofheparinbindsantithrombinATIIIIIaATIIIXaSerineproteasesSuicideinhibitorHeparinacceleratesthisreaction>1000foldProteinCandProteinSC4bbindingproteinThrombomodulinIIaVIIIaVaAPCSSCcofactorsVitaminKdependentProteinC-serineproteaseProteinS-cofactorActivatedbythrombin/TMHowtheendotheliuminhibitsclots…NitricOxidePGI2InhibitplateletaggregationthrombinthrombomodulinAPCantithrombinproteoglycans(heparan,dermatan)inactivateFVaandFVIIIaATinactivateserineproteasesplasminogenactivators(t-PA,u-PA)IIaTMTFPIinactivateTF/FVIIa/FXFactorLevelsandAgeVitKdependentFVIIIvWFAdaptedfromdatafromAndrewM.etal,AmJ.PedHemeonc1990ContactFactorssecondsaPTTPTChangesinaPTTandPTwithageAdaptedfromdatafromAndrewM.etal,AmJ.PedHemeonc1990AnticoagulantandProcoagulantProteinsarelowinneonatesAnti-coagulantproteinslowProcoagulantproteinslowProteinC/SAntithrombinplasminogenVitaminKdependentfactorsNormaltohigh:FVIII,FV,fibrinogenAreneonateshypercoagulable?ApproachtoabnormallaboratoryscreensElevatedaPTTElevatedPTElevatedaPTTandPTPointsaboutfactorsElevatedaPTTMixingstudy1:1ratiocontrol+patientplasmacorrectionBleedingFVIIIFIXFXINoBleedingContactfactors--FXII--PK--HMWKnocorrectionHeparinCirculatingInhibitorPhospholipiddependentLupusAnticoagulant--DRVVT--phospholipidneutralization--plateletneutralizationNon-specificElevatedaPTTandPTThrombinTimeProlongedHeparinFDP(DIC)FibrinogenLowAquiredLiverdiseaseDICNormalFactorDeficienciesCommonPathway--FV,FXCombinedPathway--Intrinsic/Extrinsic--vitaminKdeficiencyFibrinogenactivityInheritedFGNaglow/absent--hypofibrinogenemia--afibrinogenemiaFGNagnormal--dysfibrinogenemiaThrombinTime--prolongedReptilaseTime--normalheparinThrombinTime--prolongedReptilaseTime--prolongedLowfibrinogendysfibrinogenemiaFactor

InvivoT1/2

Synthesis

Function

vitK

Fibrinogen(I)2-4daysliver -Prothrombin(II)3daysliver SP+V 36hrsliver/mega CoF-VII 3-6hrsliver SP+VIII 8-12hrsliver/EC CoF-IX 22hrsliver SP+X 40hrsliver SP+XI 80hrsliver SP-XII 50-70hrsliver SP-XIII 10daysliver TG-VWF 12hrsmega/ECCharacteristicsofClottingFactorsmega—megakaryocyte;EC—endothelialcell;SP—serineprotease;CoF—Cofactor;TG--transglutaminaseSummaryofthispartEnzymecomplexesthatturnontheclotandturnofftheclot(therearenotthatmany—itsnothard!)Prothrombinase,intrinsicandextrinsictenaseProteinC/S,antithrombin,TFPITheaPTTisnotanexactsimulationofclotformationinvivocontactfactorsAssemblyoffactorsonplateletmembraneInitiationofextrinsicpathway,amplificationofintrinsicWhydoyouclot?(toomuch)BloodFlow(VenousStasis)VascularWall(Endothelialdamage)CirculatingBloodFactors(Hypercoagulability)Dr.Virchow1866ThrombophiliaWhototest?Whattotest?HowtoTreat?WhotoTest?Doesitchangeyoumanagment? AcuteManagment—No FutureRisk—maybeNon-catheterrelatedVTE—IthinksoCatheterrelatedVTE—debatableCatheterrelatedasymptomatic—probablynotFamilyHistory—individualizePriortocatheterplacement—probablynot

menuWhatisyour“l(fā)ist”forthrombophilicworkup? TEST venousarterialAnti-thrombin + +/-ProteinC/S + +/-ActivatedProteinCresistance + +/-FactorVLeiden + +/-Prothrombin20210 + +/-HighFVIII + +/-Antiphospholipidantibodies + +MTHFR/Homocysteine +/- +Lp(a) +/- +etcWhatdothetestsmeaninChildren?

(meta-analysisof35studies,Youngetal.2008) TEST

1stVTERecurrence(OR)Anti-thrombin 9

3ProteinC 8 2.5 ProteinS

6 3Prothrombin20210 3 2FactorVLeiden 4 1Lp(a) 5 1>2traits 10 5TieredApproachtoworkupFirstTierTestingAntiphospholipidAntibodies (DRVVT,ACA,B2GPI)AntithrombinProteinC/SActivatedProteinCResistance(FVL)Prothrombin20210Homocysteine/MTHFRLp(a)ElevatedFVIIISecondTierTestingDysfibrinogenemiaFXI,FIXelevationFibrinolyticpathwayFindacquiredrisksandremovethem!CVL,malignancy,obesity,immobility,inflammation,OCPs,smoking,obstructiononvein,etc.LaboratoryTesting?summaryCase15yearoldboywithhemoptysisfor3weeksSeenatEasternShoreERandspiralCTshowslargePETransportTeamcallsyou—asksformanagementadviceandrecommendationforbloodworkbeforetreatment?CaseAntithrombin,proteinC/S,APC,prothrombin20210,Homocysteine,etc—allnormal.Onetestyouwerenotabletogetwastheantiphospholipidantibodies.Heisalreadyonheparin.Whencanyoudothetest?Doesitmatter?Case5.DRVVTwastestedoncoumadinandprolonged.The“confirmratio”washigh,suggestinganantiphospholipidantibodyRVVT: 74 (27-45)1:1mix 56.2 (27-45)4:1mix 65.3 (27-45)aPTT 30 (24-34)PT 10.9 (10.4-12.0)RVVT:confirm 1.8 (1-1.4)ratioAntiphospholipidantibodies(APA)LAACAB2GPILupusAnticoagulant prolongPTTtestANDisphospholipiddependent (mixingstudywithpltneutralization,DRVVT) --usuallybenignandtransient --canbeseeninassociationwiththrombosis --bleedingwhendirectedagainstspecificfactor (FII,FV,FVIII)

AnticardiolipinAntibodies ELISAbaseddetectionAnti-Beta2GlycoproteinIAntibodies ELISAbaseddetectionDiluteRussellvipervenomtime

(DRVVT—likeaPTT)XXaVaIIIIadRVVT(sec)dRVVT+PL(sec)ConfirmRatio60sec.30sec.=2(1-1.4)NormalActivateXSensitivetoinhibitionbyAntiphospholipidAbIftheconfirmratioishighAnaphospholipidabispresentKhamashitaetal,NEJM,1995HighIntensitycoumadinforLupusAnticoagulants?Warfarin>3Warfarin<3AretrospectivestudySuggestingthathighIntensitywarfarinshouldBeusedforpatientswithLupusanticoagulantCrowtheretal,NEJM,2004HighIntensitycoumadinforLupusAnticoagulants?ProspectiveTrialshowingHigherincidenceofrecurrenceInhighintensitygroupBleeding2.2inmoderate3.3inhighBacktotestsCase10yearoldboyisreferredtoyoubecausemotherhadaclotandwasdiagnosedwith“factor5deficiencyorsomethingorother.”Therearenorecordsforyourreview.YoufindoutmomisheterozygousforFVLeiden.ShedevelopedafemoralDVT1monthafterdeliveringbabysister.Shealsohadrecurrentmiscarriages.CaseParentsareveryanxiousabouttheirchildren.Whatdoyourecommend?2yearslater,theboyisadmittedforspinalsurgeryforscoliosis.Theorthopedicsteamwantstoknowaboutprophylaxis.Whatdoyourecommend?FactorVLeidenPrevalence5%(0-15%)AutosomaldominantPreventsinactivationofFVaThrombosisrisk:FVL+/-=5X FVL+/+=50XFVL+/-andOCPs=30X306506679APCFactorVaFactorViArg506Arg306Arg679FVa39sec30secR506QistheFVleidenmutationWhatisAPCresistance?FVL:PTT(APC)PTT75sec30sec=1.3=2.5normal:BacktotestsAntithrombinDeficiencyPrevalence1/5000Riskofthrombosis15-20XAcquiredforms-nephroticsyndrome,liverdiseaseLab-AntithrombinactivityIIaIXaXaXIaATIIIheparinIIiXiIXiXIiBacktotestsProteinCandSdeficiencyProteinC-1/250-500ProteinS-1/1000Increaserisk5-10XAcquiredcauses: Both:Vit.KdeficiencyProteinS:estrogens,pregnancyHomozygousproteinCpresentswithneonatalpurpurafulminansLab-ProteinCandSactivityTTMCAPCSSVIIIaVaVIIIiViC4bBPSThromboemboliceventswithotherprothromboticriskfactorsBacktotestsFactorII20210genemutationPrevalence:1-2%AutosomaldominantIncreasedmRNAprocessing=increasedprothrombinlevelsVenousthrombosisrisk2.8XProthrombingeneProthrombinRNAProthrombinProthrombin20210isaGtoAchangeinthe3’untranslatedportionofthegeneProthrombin20210isaGtoAchangeinthe3’untranslatedportionofthegeneGehringetal,NatureGenetics2001Thisisa“gainoffunction”PolymorphismthatresultsinElevatedprothrombinactivityNorthernblotBacktotestsThenyouhavetoknowabouthomocysteinemetabolismhomocysteinemethionineMethylationpathwayTransulfurationpathwaycysteineRegenerationoffolatePooldependentonMTHFRB12folateMSB6CBSDoyouwanttoknowwhatMTHFRis?DecreasedMTHFRactivityresultsinhighhomocysteineMTHFRgeneratesthefolatepoolrequiredforhomocysteinemethylationMutationsofMTHFRresultsinhighhomocysteinelevelsWhatistheC677TMTHFRmutation???C677TpolymorphismintheMTFHRgeneresultsinathermolabileenzymewithdecreasedactivity.HomozygocityforC677T(TT)isprevalent(1.4%to15%ofthepopulation)Homozygotes(TT)havehighfastinghomocysteinelevelsFrostetal.,NatureGenetics1995normalhomozygoteBacktotestsThromboembolismisamultigenicandenvironmentaldiseaseThresholdForclotnoclotclotSurgery,planeride,ocp’s,etc.ThromboembolismisamultigenicandenvironmentaldiseaseThresholdForclotnoclotclotSurgery,planeride,ocp’s,etc.FVLeidenmenuHowtotreataclotYougetcallfromtheNICUabouta32weekpremie,bornwithanIVCandrenalveinthrombus.Whatdoyourecommendforworkupandtreatment?TreatmentofDVT’s—Why?

(ie.Hownottoclot)Whatarethegoalsoftherapy?PreventmorbidityandmortalityfrompulmonaryemboliReducemorbidityfromacutethrombusMinimizepostphlebiticsymptomsPreventthromboembolicpulmonaryhypertensionHowtotreataclotAnticoagulanttherapy—heparin,warfarinThrombolytictherapySurgicalthrombectomyVenacavalinterruptionWhatarethegoalsoftherapy?InitialClotmanagementCandidateforthrombolysis?yesunfractionatedheparinCVDLconsultationLong-termanticoagulationLMWHorcoumadinnounfractionatedheparinorlowmolecularweightheparinthrombolysisInitialClotmanagementStartHeparinizationfor5to7daysStandardHeparinIV:Load75units/kg<1year—28units/kg/hr>1year—20units/kg/hrKeepAPTT60to85secOrcontrolratio1.5to2.5XLMWHeparinsq:<2months—1.5mg/kgq12hr>2months—1.0mg/kdq12hrMonitorantiXalevel4to6hoursafter1stor2nddose.Striefetal2003,ArchDisChildFetalNeonatalEd.CanyouuseLMWHinpremies?Heparin--physicalcharacteristicsPentasacchrideATIIIbindingsequenceHeparin—physicalcharacteristics

ItbindstoAntithrombinheparinpentasaccharidebindingtoantithrombinActivationloopbindstoreactiveSiteinserineproteaseHeparin—physicalcharacteristicsUnfractionatedheparin—longerpolysaccaridechainsstabilizeIIaATIIIinteractionsandenhanceIIainactivationPentasacchrideSequence—bindsATIIILowmolecularweightheparin—preferentialXainactivationATIIIIIaXaATIIIHeparin—physicalcharacteristics

SizemattersIIaXafibrinOligo

mw

effectpentasaccharide 1500 anti-Xa>18 5400 anti-IIa anti-Xa>24 7200 anti-IIa anti-Xa bindsfibrin anti-HCII ATIIIATIIIATIIIIIaOutpatientClotmanagementChoosebetweencoumadinorLMWheparinforcontinuedanticoagulationCoumadin:Initiation0.2mg/kgmax7.5mgnomogramtoadjustfor5daysTargetINR2.0to3.0INR2.5to3.5forheartvalvesKeepheparinonuntiltherapeutic(4to5days)LMWHeparin:Osteoporosiswithlongtermuse?Targetanti-Xalevel0.5to1.0TheStoryofWarfarin…

or,whydidmycowdie???

Wisconsin

Alumni

Research

FoundationHepA

R

I

NSweetgrassvanillaThecanofmilkwithbloodthatdidnotclotThedeadheiferandthespoiledcloverhaymelilotVitaminKmetabolismAct