Description:
ChromogenixS-2765™isachromogenicsubstratefordeterminationofFactorXaactivity.Itisalsoverysensitivetotrypsin.
S-2765™ issuitable formeasuringFXainhibition inheparinanti-Xaassaysandantithrombinanti-Xaassays.
Composition:
EachvialcontainsthechromogenicsubstrateS-2765™,25mgandmannitol60mgasabulkingagent.
StABIlity:Lyophilizedsubstance:stableat25°Cuntilexpirydateprintedonthelabel.Thesubstanceishygroscopicandshouldbestoredinadryplace.
Solution:2mmol/LinH2Oisstableforsixmonthsat2to8°C.Suitablestocksolution:1-2mmol/LinH2O.
Chemicalname:N-a-Benzyloxycarbonyl-Darginyl-L-glycyl-L-arginine-pnitroaniline-dihydrochloride
Formula:N-a-Z-D-Arg-Gly-Arg-pNA·2HCl
Mol.wt.:714.6
FactorXaIUandEnzymeActivity:
FactorXa,whichhasamolecularweightof44KDa,istheactivatedformofFactorX(MW:59KDa).
TheInternationalUnitsofFactorXcorrespondtotheamountofFactorXcontainedin1mlofnormalplasma.Thisisabout8mg/lor0.13µmol/l.
SincethereisnoWHOstandardforFXa,onewouldassumethatifalltheFactorXinnormalplasmawasconvertedtotheactivatedform,theFactorXaconcentrationwouldbeapproximately5.7mg/l.
TheactivityofhumanFactorXaascalculatedfromthekinetictablesis1.5nkat/µgwiththechromogenicsubstrateS-2222™,and4.4µkat/µgwiththechromogenicsubstrateS-2765™.
Theactivityof1µgofFactorXaasdeterminedbyFrieberger(1)is1.9nkatchromogenicsubstrateS-2222™.
Thus,1plasmaequivalentunitofFactorXwouldcorrespondto15.2nkatchromogenicsubstrateS-2222™.
FribergerPetal.
Syntheticpeptidesubstrateassaysandfibrinolysisandtheirapplicationonautomates.In:SeminarsinThrombosisandHaemostasis,Vol.9,281-300(1983).
FactorXMethod:
DeterminationoffactorXinplasmawithChromogenicSubstrateS-2765™
MeasurementPrinciple
Themethodisbasedonatwo-stageprinciple.Instageone,FactorXisactivatedinthepresenceofcalciumtoFactorXa(FXa)usingtheactivatorRussell’sVipervenom(RVV).Instagetwo,thegeneratedFXahydrolysesthechromogenicsubstrateZ-D-Arg-Gly-Arg-pNA(S-2765™),thusliberatingthechromophoricgrouppNA(p-nitroaniline).Thecoloristhenreadphotometricallyat405nm.ThegeneratedFXa(andthustheintensityofcolor)isproportionaltotheFXactivityofthesample.
| FactorX | RVV | FactorXa |
| Z-D-Arg-Gly-Arg-pNA+H2O | FXa | Z-D-Arg-Gly-Arg-OH+pNA |
Reagents
- S-2765™,25mgArt.No.S821413
ReconstitutethesubstrateS-2765™(MW:714.6)with20mlsterilewater. - Russell’sViperVenom(RVV)
PrepareasolutionofRussell’sViperVenomataconcentrationof0.087mg/ml. - CaCl2
0.1mol/lcalciumchloridesolution. - TrisEDTABuffer
Dilutethebuffer1:10withdistilledwateraccordingtotheinsertsheetinstructions. - NormalPlasma
Calibrated,lyophilizedorfreshfrozenhumanplasmaisusedforthestandardizationoftheassay.Apoolednormalplasmacanbepreparedbytakingsamplesfrom20healthydonors.10-30mlcitrateblood(9volbloodand1vol0.1mol/lsodiumcitrate)fromeachdonoriscentrifugedat2000xgfor20minutesat15-25°C.Theplasmaispooledandsubsequentlydispensedinsmallvolumes,whicharefrozenrapidlyat-20°Corbelow.Toavoidlowtemperatureactivationofprekallikreintheplasmaiskeptat15-25°Cbeforeuseorfreezing.Thawingofplasmashouldbeperformedat37°Candthenkeptat15-25°Cuntilused. - RVV+CaCl2
Beforeuse,mix1volumeofRVVwith1volumeofCaCl2.Themixtureisstablefor48hoursat2-8°C. - Aceticacid20%
Aceticacidisusedasastoppersolutionintheend-pointmethod.
Specimencollection
Blood(9vol)ismixedwith0.1mol/lsodiumcitrate(1vol)andcentrifugedat2000xgfor20minutesat20-25°C.Storage:1weekat2-8°Cor3-4monthsat-20°C.
Standardcurve
Predilution | FinalDilution | |||
| FX% | NormalPlasma ml | Buffer ml | Predilplasma ml | Buffer ml |
| 0 | – | – | – | 1000 |
| 25 | 25 | 75 | 50 | 1000 |
| 50 | 50 | 50 | 50 | 1000 |
| 75 | 75 | 25 | 50 | 1000 |
| 100 | – | – | 50 | 1000 |
| 124 | – | – | 50 | 800 |
Method
| SampleDilution | |
| Buffer | 1000 |
| Testplasmaorstandard | 50 |
| Mix | |
| AcidStoppedMethod | A | B |
| DilutedSample | 200 ml | 50 ml |
| Incubateat37°C | 3-4min | 3-4min |
| Substrate(37°C) | 200 ml | 50 ml |
| Mixandaddwithin30sec | ||
| RVV+ CaL2 | 200 ml | 50 ml |
| Mixandincubateat37°C | 3min | 3min |
| Aceticacid20% | 200 ml | 50 ml |
A=testtubemethod
B=microplatemethod
Sampleblankactivitiesshouldbedeterminedandsubtractedwhenanalyzingstronglycoloredplasma,e.g.lipemicandhemolytic.Thesampleblanksarepreparedbymixingthedilutedsample,aceticacid20%andwaterinsteadofthereagents(400µlfortesttubesand100µlformicroplates).Readtheabsorbanceofthesamplesandblanksat405nm.Thecolorisstableforatleastfourhours.WhenpossIBLe,useadualwavelengthmodewith490nmasthereferencewavelength.
Initialratemethod
Whenperformingtheinitialratemethod,transferthemicroplatetoamicroplatereaderimmediatelyaftertheadditionofRVV+CaCl2andreadthechangeinA/min.Themicroplatereadermustbepre-incubatedat37°C.
Calculation
PlotAorΔA/minforthestandardsagainsttheirconcentrationofFactorX.ReadtheFactorXvalueforthecorrespondingAorΔA/minoftheunknowntestsamplefromthestandardcurve.
Bibliography
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- LindhoutMJetal.ActivationofdecarboxyfactorXbyaproteinfromRussell’sViperVenom.PurificationandpartialcharacterisationofactivateddecarboxyfactorX.BiochemBiophysActa533,327-341,(1978).
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- ChabbatJetal.AprotininisacompetitiveinhibitorofthefactorVIIa-tissuefactorcomplex.ThrombRes71,205-215(1993).
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- KoppakaVetal.SolublephospholipidsenhancefactorXa-catalyzedprothrombinactivationinsolution.Biochemistry35,7482-7491(1996).
- RiesbeckKetal.HumantissuefactorpathwayinhibitorfusedtoCD4bindsbothFXaandTF/FVIIaatthecellsurface.ThrombHaemost78,1488-1494(1997).
- RomischJetal.Comparativeinvitroinvestigationofprothrombincomplexconcentrates.SeminThrombHemost24,175-181(1998)
- FariaF,etal.AnewfactorXainhibitor(lefaxin)fromtheHaementeriadepressaleec.ThrombHaemost82,1469-73(1999).
