Description:

ChromogenixS-2238™isachromogenicsubstrateforthrombin.Thesubstratehasbeenusedforthedeterminationof:

• Prothrombininplasma
• Antithrombininplasma
• Plateletfactor3inplasma
• Heparininplasma

Composition:

EachvialcontainschromogenicsubstrateS-2238™25mgandmannitol120mgasabulkingagent.StABIlity:Substance:Stableuntilexpirydateifstoredat2-8°C.Avoidexposuretolight.Thesubstanceishygroscopicandshouldbestoreddry.Solution:1mmol/LinH2Oisstableformorethan6monthsat2-8°C.

Chemicalname:H-D-Phenylalanyl-L-pipecolyl-Larginine-p-nitroanilinedihydrochloride.

Formula:H-D-Phe-Pip-Arg-pNA·2HCl

Mol.wt.:625.6

e316nm:1.27.104mol-1.L.cm-1

Solubility:>10mmol/LinH2O

Suitablestocksolution:1-2mmol/LinH2O.

ThrombinIUandEnzymeActivity:

ThecurrentInternationalStandardforthrombinistheHumana-thrombin89/588availablefromNIBSC.Thisisahighpuritypreparationofa-thrombinpreparedfromCohnfractionIIIandassayedbyaclottingtimemethodagainstthefirstInternationalStandardforthrombin,75/157.

TheNationalInstituteofHealthstandard(LotJ)isalsocommonlyusedforcalibrationandastudyconductedbyGaffneyPJetal.wasfocusedontherelationshipbetweenthetwostandards,andbetweentheInternationalUnitsandtheNIHUnits.Asaresultofthisstudy,basedbothonaclottingandachromogenicassay(withthechromogenicsubstrateS-2238™),1NIH-Ucorrespondsto1.15IU.

InanarticleitwasshownthatbovinethrombinhasahigheramidolyticactivitythanhumanthrombinwhenthesameNIH-Uarecompared.Itwasalsounderlinedthattheinfluenceofbandg-forms,thatwereprobablycontaminatingthebovineenzyme,mightbethereasonforthisdiscrepancy.

Inthesamearticleitwasconcludedthat1NIH-Ubovinethrombinwasequivalentto3.4nkatchromogenicsubstrateS-2238™,andthat1NIH-Uofhumanthrombinwasequivalentto2.7nkatchromogenicsubstrateS-2238™.

Fromanearlierpublication1NIH-Uofhumanthrombincorrespondedto 2.5nkatchromogenicsubstrateS-2238™.ThecorrespondencebetweenNIH-UorIUofthrombinandtheenzymeactivityexpressedinnkat,dependsonthesubstrate,theenzymepreparation(contentofa-,b-andg-thrombin)andtheassayconditions.

FromthearticleofFriberger,1µgthrombincorrespondsto2.2NIH-Uor5.5nkatchromogenicsubstrateS-2238™orto0.02plasmaequivalentunits.Inanotherstudy,1µgthrombincorrespondsto3.1NIH-U.

Intheexperimentsdoneby Chromogenix 1µgthrombinwasequivalentto3nkatchromogenicsubstrateS-2238™(human)or4.4nkatchromogenicsubstrateS-2238™(bovine).Itmightalsobeaddedthatifallprothrombinisactivatedin1mlofhumanplasma,about1.5nanomolesor17.5NIH-Uofthrombinareformed.

ThrombinMethod:

DeterminationofThrombininPlasmawithChromogenicSubstrateS-2238™

Reagents

1. ChromogenicSubstrateS-2238™: (0.56mM)25mgArt.No.S820324.Disolve25mglyophlizedsubstratein7.14mlsterilewater=5.6mMstocksolution.
2. Buffer:50nMTris,|0.15,pH8.3with0.2%BSA(10mlbuffer+100µl20%BSA)

Assay

Dilutetoasuitablesampleconcentration(0.5–1.0nkat/mlafterdilutionwithbuffer)

Usebufferasablankandsubtractfromsampleabsorbances

AntithrombinMethod:

DeterminationofAntithrombinActivityinPlasmawithChromogenicSubstrateS-2238™

MeasurementPrinciple

Theantithrombinactivityinplasmaismeasuredafteradditionofanexcessofheparin,toformanAT•Heparincomplex.Anexcessofthrombinisthenaddedandallowedtoreactquantitativelyina1:1stoichiometricrelationshipwiththeATHeparincomplexpresent.Theresidualthrombinsplitsoffp-nitroaniline(pNA)fromthechromogenicsubstrateH-D-Phe-Pip-Arg-pNA(S-2238™).TherateatwhichpNAisreleasedismeasuredphotometricallyat405nm.Thiscanbefollowedonarecorder(initialratemethod)orreadafterstoppingthehydrolysiswithacid(acidstoppedmethod).Thecorrelationbetweenthechangeinabsorbanceperminute(ΔA/min)orabsorbance(A)andtheATactivityislinearandinverselyproportionalinthe5-125%rangeofnormalplasma.

AT+Heparin(excess)		[AT·Heparin]
[AT·Heparin]+Thrombin(excess)		[AT·Heparin·Thrombin] +Thrombin(residual)
H-D-Phe-Pip-Arg-pNA+H2O	Thrombin(residual)	H-D-Phe-Pip-Arg-OH+pNA

Reagents

1. ChromogenicSubstrateS-2238™,25mgArt.No.S820324
   ReconstitutethesubstrateS-2238™(MW:625.6)in53mlofdistilledwater.Note:Polybrene®canbeaddedtothechromogenicsubstratesolutionatafinalconcentrationof0.33mg/ml.
2. Thrombin,53nkat,Art.No.DPGBT-10
   Reconstitutewith1.5mlsterilewater.Thesolutionisstablefor4weeksat2-8°C.
3. Buffer–Tris/Heparin,pH8.4(25°C)
4. Tris6.1g(50mmol/l)
   NaCl10.2g(175mmol/l)
   Na2EDTA-2H2O2.8g(7.5mmol/l)
   Distilledwater800ml
   AdjustthepHto8.4at25°Cbyaddinganappropriateamount(approx.22ml)of1mol/lHCl.Add3000IUofheparin.Fillupto1000mlwithdistilledwater.Thebuffer,ifnotcontaminated,willremainstablefortwomonthsat2-8°C.
5. Aceticacid20%
   Aceticacidisusedintheacid-stoppedmethod.

Specimencollection

Ninepartsoffreshlydrawnvenousbloodarecollectedintooneparttrisodiumcitrate.
Centrifugation:2000xgfor10-20minat20-25°C.

Standardcurve

Normalplasmahasanantithrombinactivityof100%.Twostandards(e.g.25%and100%)madeupfreshshouldbeincludedineachtestrun.CheckwhetherΔA/minorAforthetwostandardscorrespondwiththestoredstandardcurve.Thetolerancelimitis±0.1absorbanceunits.Preparethestandardsaccordingtothetablebelow:

Method

Dilutesamplesandstandardsasfollows:
Tris/HeparinBuffer:3000µl
Testplasmaorstandard:50µl

Transferimmediatelytoa1cmsemi-microcuvette(preheatedto37°C)formeasurementoftheabsorbancechangeinaphotometerat405nmandat37°C,calculateΔA/min.

Readtheabsorbance(A)ofthesampleagainstdistilledwaterat405nmwithin4hours.

Limitationsoftheprocedure

Insomepathologicalstates(DIC,sepsis)plasmaalonemayhydrolysethechromogenicsubstrateS-2238™.Thisinterferingreactionmaybedeterminedbyassayofatestsampleintheabsenceofaddedthrombin.Thisactivityrarelycorrespondstomorethan1%ofthatoftheaddedthrombin.Toimprovethevalidityoftheassaythevalueobtainedintheabsenceofaddedthrombincanbesubtractedfromthesamplevalue.Bilirubin,haemoglobinandplasmafromhyperlipaemicpatientsinterfereinabsorbancereADIng.Patientsplasmablanksarenecessaryintheseinstancesfortheacidstoppedmethodonly.Atconcentrationsbelow25%ATitisrecommendedtodoubletheplasmaconcentration(100µlplasma+3mlbuffer).Theresultisthendividedbytwo.

Calculation

PlotAorΔA/minforthestandardsagainsttheirknownantithrombinactivity.
PercentofnormalATactivityisdeterminedbyplottingtheAorΔA/minforthetestsampleonthestandardcurveandreadingthecorrespondingATvalue.

BIBLiography

1. OdegardORetal.Heparincofactoractivitymeasuredwithanamydolyticmethod.ThrombRes6,287-294(1975).
2. OdegardORetal.Evaluationofanamidolyticheparincofactormethod.ThrombRes7,351-360(1975).
3. AbildgaardUetal.Antithrombin(heparincofactor)assaywithnewchromogenicsubstrates.ThrombRes11,549-553(1977).
4. KahléLHetal.AntithrombinIII,EvaluationofanautomatedantithrombinIIImethod.ThrombRes12,1003-1014(1975).

HeparinMethod:

DeterminationofHeparininPlasmawithChromogenicSubstrateS-2238™

MeasurementPrinciple

Heparinisanalysedasacomplexwithantithrombin(AT)presentinthesample.TheconcentrationofthiscomplexisdependentontheavailabilityofAT.InordertoobtainamoreconstantconcentrationofAT,purifiedATisaddedtothetestplasma.Thrombininexcessisneutralizedinproportiontotheamountofheparin,whichdeterminestheamountofheparin-ATcomplex.TheremainingamountofthrombinhydrolysesthechromogenicsubstrateH-D-Phe-Pip-Arg-pNA(ChromogenicSubstrateS-2238™)thusliberatingthechromophoricgroup,pNA.Thecoloristhenreadphotometricallyat405nm.

Heparin+AT		[Heparin·AT]
[Heparin·AT]+Thrombin(excess)		[Heparin·AT·Thrombin]+Thrombin(residual)
H-D-Phe-Pip-Arg-pNA+H2O	Thrombin(residual)	H-D-Phe-Pip-Arg-OH+pNA

Reagents

1. ChromogenicSubstrateS-2238™,25mgArt.No.S820324
   ReconstitutethesubstrateS-2238™(MW:625.6)with40mlofdistilledwater.
2. Thrombin
   Humanthrombinorbovinethrombincanbeusedin0.15mol/lNaClsolution.
   Theactivityofthesolutionshouldbe14nkat/ml(about6NIH-U/ml).
   Ifbovinethrombin53nkatfromDiapharma (Art.No.DPGBT-10)isused,dissolvethecontentofonevialwith3.8mlsaline.
3. Antithrombin10IUArtNo.B820720
   Reconstitutewith5mlwatertoobtainaconcentrationof2IU/ml.
4. TrisBuffer,pH8.4(25°C)
   Tris6.1g(50mmol/l)
   NaCl10.2g(175mmol/l)
   Na2EDTA2.8g(7.5mmol/l)
   Distilledwater800ml
   AdjustthepHto8.4at25°Cbyaddinganappropriateamount(approx.22ml)of1mol/lHCl.Fillupto1liter.
5. Normalplasma
   Bloodshouldbetakenfromnormaldonors.10-30mlofcitratedblood(9volbloodand1vol0.1mol/lsodiumcitrate)aretakenfromeachdonor.Thefirstmlofbloodisdiscardedandthetubeiskeptinanicebath.Plasmaispreparedbycentrifugationat2000xgfor20minutesat4°C.Equalamountsofplasmafromthedonorsaremixedanddispensedinsmallvolumes.Thenormalplasmaisstablefor3monthsat-20°Corbelow.Thawat37°Candthenkeeponice.
6. Aceticacid20%
   Aceticacidisusedintheacid-stoppedmethod.

Specimencollection

Blood(9vol)ismixedwithsodiumcitrate(1vol)cooledto0°Cwithiceandcentrifugedat2000xgfor20minat4°C.

Diluteplasma1:5withTrisBufferpH8.4.

Standardcurve

Thesameheparinasisusedforthepatientisdilutedto1IU/mlwithsaline0.9%.Then100µldilutionisfurtherdilutedwith1.9mlbuffertoobtainaconcentrationof0.05IU/ml.

Method

Transfersampleimmediatelytoa1cmmicro-cuvette(preheatedat37°C)formeasurementoftheabsorbancechangeat405nm.CalculateΔA/min.Readtheabsorbanceagainstanormalplasmablankinaphotometerat405nm.

Note:Asaruleanormalplasmablankorevenwaterisusedasablank.Ifbilirubinexceeds100mmol/lorthetestplasmaisopaque,readthetestplasmasampleagainstitsownblank.

Calculation

PlotAorΔA/minforthestandardsagainsttheirknownheparinconcentration.
HeparinconcentrationisdeterminedbyplottingtheAorΔA/minforthetestsampleonthestandardcurveandreadthecorrespondingheparinvalue.

Bibliography

1. BhargavaASetal.Characterizationofanewpotentheparin.2ndcommunication:chemicalanalysisofthecarbohydratecontentanddeterminationoftheBIOLOGicalactivityofanewpotentheparinpreparationinvitro,usingprotaminenutralizationandamidolyticmethodsforfactorXaandthrombin.Arzneimittelforschung30,1071-1074(1980).
2. SacheEetal.Studiesonahighlyactiveanticoagulantfractionofhighmolecularweightisolatedfromporcinesodiumheparin.ThrombRes25,443-458(1982).
3. VanPuttenJetal.Determinationoflowmolecularweightheparininclinicallaboratory.Haemostasis14,205-210(1984).
4. VanPuttenJetal.Automatedspectrophotometricheparinassays.Comparisonofmethods.Haemostasis14,195-204(1984).
5. BerryCNetal.Effectsofthesyntheticthrombininhibitorargatrobanonfibrin-orclot-incorporatedthrombin:comparisonwithheparinandrecombinanthirudin.ThrombHaemost72,381-386(1994).
6. ByunYetal.EffectoffibronectinonthebindingofantithrombinIIItoimmobilizedheparin.JBiomedMaterRes30,95-100(1996).

ProthrombinActivityMethod:

DeterminationofprothombinactivityinplasmawithChromogenixS-2238™

Background

Anumberofstudiesduringthelastfewyearssupportthenotionthatvenousthromboembolism(VTE)isamultifactorialdiseasemostoftentriggeredbycircumstantialriskfactors(trauma,surgery,pregnancy,oralcontraceptives,immobilizationandage)incombinationwithoneormoregeneticoracquiredcoagulationdisorders(seeref.1ofareview).
Elevatedactivityofprothrombinintheabsenceofaknownunderlyinggeneticdisorderisalsoassociatedwithanincreasedthromboticrisk².

AmutationG→Aintheuntranslated3’-regionoftheprothrombingeneatnucleotideposition20210constitutesariskfactorforVTEwithanoddsratioof3-5^2-10.About90%ofthecarriersofthismutationhaveelevatedlevels(>115%)ofprothrombinactivity^2,7,8.Levelsabovetheupperlimitofthenormalrange(75–130%)arecommonlyhetero-andhomozygotes^2,7-9.

Sofar,thereisnoexplanationwhyacomparativelymildincreaseofprothrombinactivityconstitutesariskfactorforthrombosisandthisisthereforeanareaofactiveclinicalandbiochemicalresearch.Chromogenicmethodsforaccuratedeterminationofelevatedactivitiesofprothrombinandothercoagulationfactors,suchasfactorVIII^11,12areimportanttoolsforassessingtheriskforVTEinpatientsandfamilymembers.

MeasurementPrinciple

ProthrombinisactivatedtomeizothrombinbythesnakevenomenzymeEcarinfromEchisCarinatus.

Afteracertainincubationtime,theamountofmeizothrombinformedismeasuredwiththethrombinselectivesubstrateChromogenixS-2238™,whichalsoiscleavedbymeizothrombin.

Theabsorbancerecordedat405nmisproportionaltotheprothrombinactivityinthesample.

Prothrombin	Ecarin	Meizothrombin
S-2238™	Meizothrombin	pNA+Peptide

Reagents

1. TrisBSABuffer(catalog#TB031-20)Bufferforsampledilution,containing0.5mol/lTrisHClpH7.3,I=2.0withNaCland2%bovineserumalbumin.Beforeuse,dilutethestocksolution1+9withsterilewatertoobtainabufferworkingsolution.Thebufferworkingsolutionispreparedandusedwithinthesameday.
2. EcarinDiluent(catalog#ED0413-20)BufferfordilutionofEcarin,containing0.05mol/lTrisHClpH7.6,I=0.15withNaCl,bovineserumalbumin,polyethyleneglycolandafibrinpolymerizationinhibitor.
3. Ecarin(catalog#ECARIN50B)ReconstitutewithsterilewateraccordingtotheEcarinpackageinsert.Freezeinsuitablealiquotsat-20°Corat-70°C.Stablefor3monthsatbothstoragetemperatures.Beforeuse,dilutewithProthrombinActivatorDiluenttoobtainaconcentrationof2.4U/ml.Stablefor8hoursat20-25°Candfor1weekat2-8°C.Note:EchisCarinatuscrudevenomcanalsobeused.Asuitablefinalconcentrationofthisreagentisapproximately5µg/ml;however,thismayvarybetweendifferentsources.10-20%lossofactivitymayoccuruponfreezingat-20°C.
4. S-2238™(catalog#S820324)Reconstitutewith13mlofsterilewatertoobtaina3mmol/lsolution.

SpecimenCollection

Blood(9volumes)ismixedwith0.1mol/lsodiumcitrate(1volume)andcentrifugedat2000xgfor20minat20-25°C.Separateplasmacarefullyfrombloodcells.Performtheanalysiswithin24hourswhenplasmaisstoredat2-25°C.Alternatively,freezealiquots≤1mlat-20°Corbelow.Performtheanalysisoffrozensampleswithintwomonthswhenstoredat-20°Corwithinoneyearwhenstoredat-70°Corbelow.Nosignificantlossofprothrombinactivityoccursuponfreezingonce,providedfreezingismadeinsmallaliquots(<1ml)andthawingisperformedinawaterbathorinanelectricheaterat25-37°C.

SampleandStandardDilutions

Standards
Calibratednormalplasmaisdiluted1:23–1:160toprovidestandardconcentrationsof25-175%.Thefollowingtableprovidesasuggestionofstandarddilutions.

Samples
Plasmasamplesarediluted1:40inTrisBSABufferworkingsolutionforapplicationonmicroplateanddiluted1:80forapplicationonACL(seebelow).

MicroplateAssayProcedure

DeterminetheabsorbancedifferenceA405nm-490nmforthestandarddilutionandthesamples.Drawastandardcurvefromtheabsorbancesobtainedforthestandarddilutions.Readtheprothrombinactivityforthesamplesfromthestandardcurve.

Fig.1.Standardcurvewiththemicroplatemethod.

ApplicationonACL

Usetheplasminogenchannelprogram.Prepareastandarddilution1:40,whichcorrespondstoanominalprothrombinactivityof100%(seeaboveregardingcalibration).Standarddilutionscorrespondingto25%and50%arethenautomaticallypreparedbytheinstrument.Inordertoallowdeterminationofprothrombinactivityupto200%,sampleplasmashouldbediluted1:80andtheobtainedresultshouldbemultipliedwithtwo.

Fig.2.StandardcurvewiththeACLmethod

Expectedvalues¹³

Thenormalrangeis75–130%(mean102%2SD)asdeterminedfromanalysisinmicroplateandontheACL300of101healthyindividuals(49menand52women;agerange20–68years).Analysisofplasmafrom42carriersoftheG20210Amutation,whowerenotonoralanticoagulanttreatmentatthetimeofbloodsampling,resultedinanactivityrangeof94–164%(mean128%2SD).

InterferenceandLimitations

Noinfluenceintheassayisobtainedfromvariationofantithrombinactivityintherange50–150%ofnormal.Sincemeizothrombinisformedandmeasured,noinfluenceintheassayisobtainedfromheparinlevels≤1IU/mlplasma.SinceEcarinalsoactivatesdecarboxyprothrombin,whichisproducedduringoralanticoagulanttherapywithanti-vitaminKdrugs,plasmafrompatientsundergoingsuchtreatmentshouldnotbeanalysedwiththismethod.

Repeatability

Theimprecision,expressedasCV,withinandbetweenseries(7series,5replicatesineachseries)is≤4%at50%and100%prothrombinactivity.

Bibliography

1. LaneDA,MannucciPM,BauerKA,BertinaRM,BochkovNP,BoulyjenkovV,ChandyM,DahlbäckB,GinterEK,MiletichJp,RoosendaalFR,SelingsohnU.
   InheritedThrombophilia:part1.
   ThrombHaemost76,651-662(1996)
2. PoortSR,RoosendaalFR,ReitsmaPH,BertinaRM.Acommongeneticvariationinthe3’-untranslatedregionoftheprothrombingeneisassociatedwithelevatedplasmaprothrombinlevelsandanincreaseinvenousthrombosis.
   Blood88,3698-3703(1996)
3. HillarpA,ZöllerB,SvenssonPJ,DahlbäckB.
   The20210alleleoftheprothrombingeneisacommonriskfactoramongSwedishout-patientswithverifieddeepvenousthrombosis.
   ThrombHaemost78,990-992(1987)
4. CummingAM,KeeneyS,SaldenA,BhavnaniM,ShweRH,HayCRM.TheprothrombingeneG20210Avariant:prevalenceinaUKanticoagulantclinicpopulation.
   BrJHaematol98,353-355(1997)
5. BrownK,LuddingtonR,WilliamsonD,BakerP,BaglinT.
   RiskofvenousthromboembolismassociatedwithaGtoAtransitionatposition20210inthe3’-untranslatedregionoftheprothrombingene.
   BrJHaematol98,907-909(1997)
6. MakrisM,PrestonFE,BeuchampNJ,HamptonKK,DalyME,CooperP,BaylissP,PeakeIR.Co-inheritanceofthe20210Aalleleoftheprothrombingeneincreasesthethromboticriskinsubjectswithfamilialthrombophilia.
   ThrombHaemost78,Suppl.,165(1997)
7. FerraresiP,MarchettiG,LegnaniC,CavallariE,CastoldiE,MascoliF,ArdissinoD,PalaretiG,BernardiF.Theheterozygous20210G/Aprothrombingenotypeisassociatedwithearlyvenousthrombosisininheritedthrombophiliasandisnotincreasedinfrequencyinarterydisease.
   ArteriosclThrombVascBiol17,2418-2422(1997)
8. KapurRK,MillsLA,SpitzerSG,HultinMB.Aprothrombingenemutationissignificantlyassociatedwithvenousthrombosis.
   ArteriosclThrombVascBiol17,2875-2879(1997)
9. HowardTE,MarusaM,ChannelC,DuncanA.Apatienthomozygousforamutationintheprothrombingene3’-untranslatedregionassociatedwithmassivethrombosis.
   BloodCoagFibrinol8,316-319(1997)
10. MartinelliI,SacchiE,LandiG,TaioliE,DucaF,MannucciPM.Highriskofcerebral-veinthrombosisincarriersofaprothrombin-genemutationandinusersoforalcontraceptives.
    NewEnglJMed338,1793-1797(1998)
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13. RosénS,AnderssonM,GhoshR.Anewchromogenicprothombinmethodprovidingaccuratedeterminationofelevatedprothombinactivityinplasmasamples.
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