Detection of fragment D-fibrin complex by agarose chromatography

RESEARCH 14; 265-272 Printed Press Ltd.1979.

THROMBOSIS @Pergamon

in Great

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004g-3848/7g/ojo1-0265 #02.00/o

DETETIoIoOFFRA(IytEIppD-FIRRIKOOMEEX BYAGAROSECRROMATOGR4PRX

V.A.Beliteer, T.M.PoednJakova, V.M.TolstilPh Instituteof Biochmietry,Acadmy of Scienaee of the UkrainianS.S.R.,Xiev, U.S.S.R. (Received 12.5.1978; in revised form 5.10.1978. Accepted by Editor Z.S. Latallti)

ABSTRAOT

Botie fibrinmonmer_fvtD mlxhlma, afterprolongedincubation,rereamayedby qarooe gelahromatom. The elutionpeak of fragmentD wae found to besynmetricalwiththe oomstanteluticmvoltme,but fibrin eluticmprofile&an@ depa on frqqmt D ooncentration. Athi@fragamt D ooncmtratiapeapart of it elutedtogetherwith fibrin,apartfrom the free fr_entD, revealingfibrin-fr(LgPBllt D oomplexfoaation. Of the two fragmentD eubapeeieadiiferimgin POlecularweight the light one oouplerr with fibrin m&erably.In similareyperimente petiomtedwithfibL en substituting for fibrinno couplingoocured.

. IIVTRODDCTIOR Be well laxown, the protein-like degradation product6of fibrinogenand fibrin (fragment8 X, Y and D> are apeoificfib rin polymrieationinhibitors/I,2/.This propertyie due to thepresence inthesef~ents of orderedstruatuma oontaiaing a part of those binding(polymerization) sitesby uhieh fibrinmolecule8aocoqplishthe fibre formationproaesa.Poeaeeskrgbut a part of the binding-site eytiemchamwteri&ia of fibrin the fragmentsreaotto interierewith the ordered fibrinpolymerieation. led to the comluThe investigation of Lipimki et al./3/ sion that in fibrinogen-thrombin systemsinhibitow fragments 265

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incorporate partly into the insolubleclot and partly into solublefibrinmonomercomplexes.In the eqeriments of Lipinski et al an incubationtime 90 minuteswas chosen.I&all0 et al. /I/ while studyingthe fragment-effected retardationof clotting,noticedthat all of the fibrinformedby thrombinwas incorporated into the clot within 24 hours. In our reoemtwork on fib&+-fragmentD interaction/4/ observationtime aud fragxentconaentrationvaried in a wide rauge.It was found that after a latentperiodfibrin deposition took place. It continuedfor 48 hours or so, end came to an end leavingsome of the fibrin iu solution.The amouutof fibrin dissolvedincreasedwith increasingfragmentD concentration.Thus, the principleof fmgment-dependautdistribution of fibrinbetweensolid and fluid phasesproved correct. The problemconcerningsolublefibrinmonomercomplexesis not clear as yet. Originally,these complexeswere supposedto be stable.But this could be substantiated only for complexes of immobilizedfibrin /3,6/.In solutionscontainingfibrin and fragments Smith and Bang /7/ did not notice any complex formation.In their work agarosegel chromatography was used. By the same method,Latalloet al./8/failedto detectauy ente (exceptfor the clotinteractionbetweaufibrin and f r"$" table fragmentX), a resultaugges ing that no solubleco lexea of fibrin aud unolottablefragmentsexist at all. On "g t e other hand there are facts witnessingfor fragment-fi,bEcorn-plex formation;e.g.fibriusolubilization with fr In this comuunication we describeeqerimente% WhiCi fibrin-fragment D complex formation was directlyshownby agarosegel chromatography. Severalreasonsare possiblefor the previousnegativeresults.One of them may be the low anticosgulsntactivityof fragmentD aamplescomouly used. Sinco 1969 we use particularlyactivefTent D preparations izzed with trgpain/9/ or plasmin/I / in Ca l2 Containing .

MAT-

AID

MXTHODS

Fibrinogen.Bovinefibrinogenwas preparedaccordingto Varetskaja /11,12/. Fibrinmonomer.An acid solutionof fibrinwas obtainedas describedby Belitaerand Varetakaja/I3/. Frran;+T_ was a roduct of t sin bovine fibrinsen hydra1.3% f$btiogeP tiTO63 M phosphate b f er pH 7.0 wit: ionic strength0.2 (adjustedwith XaCl),CaC12 0.63~1O'~Mwas digestedat en-e to aubst~~~?&w?r% I:2300during100 hours.Temperature22023'C. Trypsiuwas tha inactivatedby soy-besninhibitor.Fragment D waa isolated using CM-cellulose ion-exchange chromatography and lyophilised /g/. Molecular weight of the fragment was found by sedimaPtation-di.ffusion methodto be 89 000 daltona /Is/. The ultraviolet fluorescence spectra demonstrated that

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FRAGMENTD-FIBRIN

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the fragmentpreservesmuch of the conformation order characteristicof fibrinogenin its native state.Like fibrinogen fragmentD is highly susceptible to denaturating agents/16/. BRaroeegel &ronatoRl?aahs One ml of the solutionto be tested (1.0-3.0mg of proteinjwas appliedto 0.6x45cm Sepharose 6B oolumn.Equilibration and elutionwere carriedout with 0.1 M phoephatebufferpH 7.6, ionic strength0.2 or 0.4 (rJaCl>. 'Pentperature 24'Cg 0.5 ml fractionswere collected. Polsacmlamide- sodiumdodecylmUhate (SDS)Rel electmmhoresis. The method of Weber and Osbom /17/ was used in the -described earlier/18/.Gel concentration was 5 per cent. lea. Mixtureswere prepared containing1.0 mg fibrinmonomer an 0.4-2.0mg fragmentD per ml in 0.1 Y phosph$zebuffer =% H 7.6, ionicetrength0.2 or 0.4 (WaCl),CaC12 '3x10IL Soxitamaside IO' Y servedas antiseptic.These mixtureswere k t at rool~temperaturefor one day. IX a fibrinclot appear3 it wa8 removedby winding on a glass rod! fibrinauspensionswere precipitated by centrlfugation. was determinedepectrophotometrically PIP,extinotioncoefficients 15.67for fibrin (fibrinogen) and 20.0 for fr ent D being ueed 1151. Fibrinclot6were careful washedZYth 0.15 M l!MZland d&ssolvedin 0.1 IdWaOH at I3 QCpriorto photometry. RESUIIPS AHD

DISCUSSIOR

Our experimental resultsare reportedhere as elutionprofiles and electrophoregzwna of representative fractions.Fig.1 relatesto an experimentin which the originalmirturecontained 1.0 n&ml fibrinmonomer and 0.4 m&ml fragmentD. During

1

a/

FIG.1 Sepharoee=6Bgelchromatographyofa le oontaiaing 0.4 mg/ml of "pf brin and 0.4 /ml of f_entD. Ionic Y! 8 reagthO.2. Dottedlinefibrinogauelutlonprofile. On the left polyacrylamide gel8 remaltingfrom electrophoreticanal sis of the originaltma@e 9S) andofpeak z?tiom (I.21are reprodu.

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FIG.2 Electrophoregrsms of the dissolvedolot (C snd remainingsolutioutS). See tart.

24 hours standinga clot appeared.After its removalonly 0.4 n&ml fibrin remainedin the solutian.Thus, the final fragment D, fibrinweightratiowas I:1 which oorrespondsto a molar ratio of 4tI. On agarosegel chromatography of this solutionfib rin snd frsgmentD separatedfrom each other.The band op the top of el V1* representsfibrin,the following atitg E E D, the lowestline - steelwire indicat fragman L3 of the buffer.From the area of fibrin elution,beginuingat the void volume of the column,frsgmentD was practicallyabsent.Consequently, there was no detectablefibrin-frsgment complexformation. The fibrin clot, withdrawnfrom the reactionmixture,was washedwith 0.13 II. IVaClsolutionsnd dissolvedin 2% SDS - 4 M urea. It was found virtuallyfree from fragmentD /Fig.2/.This could be expectedas frsgmentsdo not incorporateinto soluble fibrinpolymers/7/. Then, we tried to achievefragment-fibrin complexaccumulationby fncreasingioniestreugthandfragmentD concentration. A very marked,e.g. fivefold,concentartion increase was desirable.We had first to ascertainthat our Sepharosecolumn would not be overloadedwith frsgmentD at such concentrations. Controlassayswith fragmentD at concentration 2.0 &ml or over 4.0 &ml did not show overloading. Thus, in our next experimentfibrinconcentration was 1.0 molar ratio n&ml, fragment D- 2.0 mg/ml.The fragment-fibrin was 8. Ionic strengthwas increasedto 0.4. This increasefavours the inhibitoryeffectof the fragmentD /4,15/.Fibrin resultsare depositiondid not occur in this e;lcperiment.Its presentedin Fig.3.Considerablechsngeis seen in the fibrin area of the elutionprofile.The peak is flattenedand shifted a littlefartherfrom the void volume.Obviously,the mean size of polymerizedfibrinparticlesdiminished.(CompareFig.1). As evidentfrom the electrophoregrams fragmentD was presentin all of the effluentfractionscontainingfibrin.The appearsnce of fragmentD in the whole fibrin elutionarea withessesfor complexformatian. Our fragmmt D preparationconsistsof two discretefractions differingin molecularweight.(See electrophoregrams). Yet, in the regionof fibrin-f ent complexelutionthe 7 light fragmentD was mainlyfoun (Fig.3).This form of frag-

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D-FIBRIN

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FIG.3 Sepharosegelchromatograof aeaqplecontaining $3 irin I.OIpg/ml andfragmentD t2.0 &ml. 1 Ionic strength0.4. On the left polyacrylamide gele resulting from electrophoretic analysieof the aaqple (S) and of eff'luentfraotiona denotedby corresponding arabicnumbereare shown.

FIG.4 !Chesame eyatema8 inthe first experiment(F&.1>, but at ionic atmngth 0.4 *-

.

amountof me&Dmustbepreferably bound by fibrin. we ggehfyerfwemtD didnot appeartillthe beginningof errtelution. A se eotivebindin&of the frqguentD fractiodby immobi"f$ limd fibrinmonmer was observedearlier/6/. But it was heavier forr that was bound. !Twofactors,iwe& D concentration and ionic et h, 7.3 out were alteredin the precediq experiment. In order to f which of them was re onsiblefor the effectobservedan me% whiehionic atremgthwaohi& (0.4) rilnelltwa8perfowed other oonditionsbeing the 8ame as ia the firet experiment (Flg.1).Becauseof ionic strengthincreaseno fibrindepoBition took place.But,as Fig.4 ~&OWS,ooqplexfOWatiOn was stillnegligible.Evidently the high concentration of iragmat& D was the decisivefaocor.

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FIG.5

gel chromatogra hy of a samplecontainingfiri% no en (Img/ml) andfra@llent D f2.0 &ml). Otherwiseas in the precedingexperiment Sepharose

(Fig.31

I 2

4

vtx%nr fw ‘u

‘a

specificrole of fibrin in complexformationwas further evidencedby experimentsin whichfibrinogenwas substituted for fibrinmonomer,all other conditionsbeing the same as ia the experimentshown in Fig.3. See Fig.5. As could be expected the two ~eaka were nearer to each other.In spite of this, chromatogrf&c separationwas good and there was no fragment D in the main fibrinogen-containing effluentfractions.So, in contrastto fibrin,fibrinogendoes not bind fragmentD. !l!he

DISCUSSION Accordingto Kudrik et al /6/ sepharose-bound fibrinmonomer combineswith fragmentD to form complexeswhich do not dissociatespontsneously at neutralpH even in absenceof free fragmentD. On the contrary,in solutionscomplexesare usually not detectable/?,S/.They seem to be extremelylabile.ITevetheless,as reportedhere, in the presenceof excessive amountsof fragmentD solublefibrin-frsgment D complexesarise, and they are stableenoughto be chromatographically separated from the free fragment. The relativelystrongcouplingof fragmentD with fibrin monomer fixed on Sepharosemay be due to some ohaqes of conformationproducedby the interaction with the solidmatrix. But otherpossibilities exist.For example,fragmentD may be set free througha reactionin which mobilefibrin (e.g.monomeric)withdrawsfibrinfrom the complexto combinewith it, i.e. polymerise.Immobilization of fibrinprecludessuch a reactionmakingfixed complexesratherstable.This problemis being studiedin our laboratory.

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8.

~L$!ATAk;.S.)MATTIZR,LE., BANG, B.U., HMSEN, M.S., Analysisof solublefibrin c lexee by agarose &el'&omatography end protamineeulfa "$ e gelation. Biochim.B~OD~IYS. Acta. 420, 69-80, 1976.

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