fibrin ratios

THROMBOSIS RESEARCH @Pergamon

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INCOMPLETE TRANSFORMATION OF PLASMA FIBRINOGEN TO FIB-RIN. FIBRINOGEN CONTENT AND STREPTOKINASE-INDUCED INTRA-CLOT LYSIS SUSCEPTIBILITY OF DES-AA OR DES-AABB FIBRIN CLOTS FORMED AT VARYING FIBRINOGEN/FIBRIN RATIOS. F. BROSSTAD and H.C. GODAL Hem3t010qical A%sewzhIabo~tc7~,

UllevQ

(Received 1.11.1978; in revised Accepted

by

Editor

E.

uwersityc1wc,~~~,

Norway

form 26.11.1978. Ikkala

MATERIALS AND METHODS Human fibrinogen (Kabi,Stockholm,Sweden) was prepared as previously described (2) The fibrinogen solutlon(17.2 g/l in 0.15 M TrisHCl buffer, pH 714) was kept at -20°C until used. 1125-labelled human fibrinoqen was prepared as previously described(2), accordinq to the method of McFarlane(3). Clottability was 507

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FIBRINOGEN/FIBRIN THANSFOBMATION

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more than 99%,and determined as follows:Labelled fibrinogen(75 ul, 17 g/l in 0.15M Tris-HCl buffer,pH 7.4) was added to 0.5 ml of inactivated serum(cfr.below).Subsequently,purified thrombin(4) (10 ul, 1000 NIH units /mh) was added,and the clot was removed after 20 min incubation at +20 C ,by winding it onto a wooden stick,sqeezing it maximally free of fluid.From radioactivity recorded on clot(using a Berthold-Friezeke Gammazint recorder) and on clot-deprived serum, the clottability was calculated. Human des-AA and des-AABB fibrin monomers ,I125-labelled or unlabel e ,were prepare fid,by exposing labelled and unlabelled fibrinogen in urea,to insolubilized reptilase or thrombin,respectively(2,5).Monomer concentration of the final preparations was 10 g of monomers per 1 of 0.15 M Tris-HCl buffer,pH 7.4,with 2.5 M urea,and they were kept on melting ice until used. Fibrinogen-boosted serum was prepared as follows:Whole blgod from a helathywed to clot in class tubes at +37 C for 2 h.The clots were removed,and serum centrifuged at 1500 x g for 20 min.The superna&ant was pipetted off,and icubated in a glass tube for 12 h at +37 C.To aliquots of this serum was added labelled or unlabelled fibrinogenlas above) to 2 g/l,and these sera further diluted with non-boosted serum to obtain sera containing fibrinogen of desired concentrations.Spontaneous clotting was not observed in these sera for 48 h. Preactivated human FSF.Factor XIII(Behringwerke,W.Germany~correspp unit= the amount of FSF in lml of plasma) was incubated @th insolubilized thrombin(4) (0.3 g),by mixing end over end at +20 C for 2 h in 0.15 M Tris-HCl buffer pH 7.4 with 2.5 mM CaCl .Subsequently,the insolubilized thrombin was filtered off,and the &F-containing filtratetl ml) was kept on melting ice until used.The activity of this preparation was measured as follows: To 0.5 ml samples of non-boosted serum was added 10 ul of preactivated FSF(corresponding to 1 unit/ml,cfr.above),and then 50 ul of des-AABB fibrin monomers(l0 g/l).The clots,which formed immediately upon the addition of monomers,were removedOat intervals (l-60 min) and their solubility in 5 M urea(1 h at +37 C) tested.Incubation for 1 min proved sufficient to develop,an urea resistant clot,whilst clots formed in serum to which no FSFa had been added,were soluble in urea irrespective of the incubation time,indicating a low or abscent spontaneous FSFa-activity in the serum used. Determination of the fibrinogen content of clots formed at varying fibrinogen/fibrin ratios. The following procedure was used:Des-AA or des-AABB fibrin monomers (10 g/l),either 10,20,40,60,80 or 90 ully3re applied to the plain -fibrinogen-boosted serum bottom center or 2 ml glass tubes,and I (with or without FSFa as above) to 0.5 ml(fina1 volume) added.The sum of fibrinogen + fibrin was always 2 g/l in all samples,and the radioactivity of the samples recorded as above.The clots,uniformly dispersed,were removed after 1 h,by winding them onto wooden sticks and squeezing them maximally free of fluid.The fibrin film was detached from the stick,and washed in 0.15 M NaCl,and the radioactivity recorded.Clot fibrinogen was calculated from the clot radioactivity relative to total radioactivity(as recorded above,prior to removal of clot). Determination of the fibrin content of clots formed at varying fibrinogen/fibrin ratios and of soluble fibrin remaining in plasma

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FIBRINOGEN/FIBRIN TRANSFORMATION

509

subsequent to clot removal. This was done by usins labelled des-AA or des-AABB fibrin monomers with unlabelled-fibrinogen as in the experiments above.The amounts of fibrin in the clots was calculated franthe clot radioactivity relative to total radioactivity(prior to removal of clot). The amount of fibrin remaining in the clot-deprived plasmas as soluble fibrin,was calculated from the radioactivity of the clot-deprived plasmas relative to total radioactivity(before removal of clots). Determination of the crosslinking extent of clot fibrinogen and clot fibrin,and of soluble plasma fibrinogen. To determine the crosslinkinc extent of clot fibrinocen,the clots formed,using labelled fibrinogen and unlabelled monomers,were harvested as above,and mercaptolyzed(clot dissolved in 0.2 ml of 10 M urea,cgntaining 2%(w/v) SDS and 5%(v/v) 2-mercaptoethanol,incubation at +37 C for 12 h),prior to electrophoresis in SDS-polyacrylamide, using 12%(w/v) gels,as previously described(2J.Radioactivity of sliced,Coomassie-stained,gamma-gsmma bands relative to that of the sum of gamma-gamma and gamma bands as well as the radioactivity of the alpha polymer bands relative to sum of alpha polymer and alpha bands were recorded,and the corresponding clot-fibrinogen crosslinking could be determined. To determine the crosslinking extent of the clot fibrin,the above procedures were used,now with labelled monomers and unlabelled fibrinogen. To determine the crosslinking extent of labelled fibrinogen in the abscence of fibrin,plasma samples as abovetbut without fibrin) were exposed to the same amounts of FSFa as above.After 1 h,EDTA to 20 mM (20 ~1,500 mM)was added,and after another 20 min,purified thrombin (4)(in 20 mM EDTA) to 10 NIH units were added.The clot was removed after 20 min,and treated as above.The mercaptolyzed clot material was electrophoresed,recording the radioactivity of the various bands after Coomassie-staining as above. Intra-clot lysis rate of clots formed at varying fibrinogen/fibrin ratios compared to that of clots formed in the abscence of fibrinogen.

Clots(formed at varying fibrinogen/fibrin ratios Jwere prepared as above,with and without FSFa present,but streptokinase(Kabi,Stockholm,Sweden) to 20 IU(10 ul,lOOO IV/ml) per ml was added just prior to fibrin monomers.The samples were incubated at +37 C,and the time of visual clot sysis recorded in seconds. Lysis of fibrin clots formed in the abscence of fibrinogen was tested by adding the same amounts of fibrin monomers as above to non-boosted serum,with or without FSFa present,containing streptokinase as above. RESULTS AND DISCUSSION As shown(figs.l&2).,substantiallymore fibrinogen was incorporated into clots formed at high fibrinogen/fibrin ratios than at low. Des-AA clots contained more fibrinogen than did des-AABB fibrin clots Furthermore‘the amount of fibrin remaining in solution after the removal of clots,was higher with des-AA than with des-AABB fibrin(2% versus 0.5% of the fibrinogen remaining in plasma subsequent to clot removal).The reason why larger amounts of fibrinogen were incorporated in des-AA than des-AABB clots,as well as why larger amounts of des-AA fibrin remained in plasma after clotting is obscure,but these differences might possibly reflect the different aggregability and

510

FIBRINOGEN/FIBRINTRANSFORMATION

Vol.l$No.2(3

solubility of the two types of fibrin(6,7). Incomplete transformationof plasma fibrinogen to fibrin is most likely to occur in connection with heparinized systems( 8). It should be stressed that fibrin clots harvested at incipient gelation thus may contain substantial amounts of fibrinogen,whichmay lead to misinterpretationas to the amount of fibrin actually formed,if the above findings are not considered. FIG.1 zJM3mtsof fibrincgen(***~-*~ anl fibrin(-) inngillclotsfonnsdupcnadditicnof (un)b i3 label.led,prefoxmsddes-z4Afibrinxsxmers(in c zaIKnlntsasindicatedonfig)to 0.5ml sanples offUxirqeWbcostedsensn(called"plamna"on : fig)withvaryingmrcxlnts of (WI)-labelled fii brincgelLSunfUxinogW +fibrin=lnyinall 1.0 samples.AmnmtsoffiLnirogen andfibrinill:

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labelle3fiixinogenwithunlabelled fitafn orunlabelledfibrincgenwithlabelled fibrin,alternatively.Pardetails,cfr.Waterial al-d Wthods". clCts,usbq

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DOS-AA tikln monomorr rddod to plauna (mg)

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of fibrinogen(..~.~ and fibrin ) inmginclotsformsdlqonadditionof (un)-l.abelled,~onc&desAhBBfiixinxmrxxlWs(inanuuntsasabave, and as indicated on fig)to 0.5nilsamplesof fibrinogen-boosted senrn(called "plasma" on fig)withvaryingarrpunts of (un)-labelledfib&Mgen .Sunfibrinogen [email protected]&nique forassessing clot-fibrirqen ard clotfibrinis as indicated in legendto fig.1 andasoutlinedin?MaterialsandMeUxxW Altn.mt

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0.2

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Der-AAIB fibrin monomers addad to plasma (mg)

Vol.l'+,No.2/3

FIBRINOGEN/FIBRIN TRANSFORMATION

511

Activated Fibrin Stabilizing Factor(FSFa) did not affect the fibrinogen/fibrin ratio of the clots formed at various fibrinogen/ fibrin ratios,but in accordance with previous findings(g)hig percentages of des-AABB than of des-AA fibrin molecules were crosslinked(75% versus 65% of the gamma-chains and 55% versus 40% of the alpha chains).Furthermore,it was noted that the fibrinogen present in the clots was crosslinked to gamma dimers at physiological levels of FSFa,whilst the fibrinogen remaining in solution was not.However, the fibrinogen in the clots did not participate in formation of alpha polymers.Moreover,at a fibrinogen/fibrin ratio of 9:l,about 25% of the fibrinogen incorporated into des-AA clots were gamma dimerized increasing to 40% in clots formed at a fibrinogen/fibrin ratio= 1:9. In contrast,only 10% of the fibrinogen in the des-AABB fibrin clots were gamma dimerized,irrespective of the fibrinogen/fibrin ratio at which they were formed.The present data do not,however,permit any conclusion as to wether the,gamma-gamma dimers were formed between fibrinogen and fibrin molecules,or wether dimerization between gamma chains of fibrinogen only,predominated, but according to previous documentatfon,the former possibility is most likely(lO,ll) TABLE I. Intra-clotlysis~~ofclatsfonaedby~~~ofvarying~~of des-AAordes-AABBfibrirl xIonaWrstoi.nactivatedsenlmorinactiWuzedserum conWningvaryinganKnmtsoffibrinogen,intbepIwence of 20 Iu of streptot&inase(forde+zI.ls,cfr.slaterials and Methods") Typeofclot 0.2m30ffibrinpo1ymsrised in the 7 of 0.8 ragof fibrinogeniJlsennn 0.2 lugof fibrinpolymerized inserun 0.5Iq0ffibrinpo1ymWised in the m of 0.5 a?gof fibrincgeninserum 0.5 Iq of fibrinlzolymerized insenml 0.81rgoffibrinpolym&ed in the presmce of 0.2 mg of fibrilvgeninsensn9 0.8 nrgof fibrinpolymerized insens&

Clotlysistiirs!insec. des-AAfibrin des-AABBfibrin I.20

120

210

180

150

150

300

220 I

240

200

370

300

The streptokinase-induced intra-clot lysis time of clots formed in the presence or abscence of fibrinogen is listed in Table I.As is evident,clots formed in the presence of fibrinogen were lysed at higher rates than the corresponding "complete clots".Moreover,a tendency for des-AA clots formed in the abscence of fibrinogen to be lysed at slower rates than the corresponding des-AABB clots,was noted.This finding is contradictory to the findings of Kwaan and Barlow(lZl,and no explanation for this discrepancy can be offered. Furthermore, no difference in intra-clot lysis rate between FSFa crosslinked clots and non-crosslinked clots was observed,thus confirming the findings of Haverkate(l3) and Rampling(l4).

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FIBRINOGEN/FIBRINTRANSFORMATION

Vol.lk,No.2/3

The present observation,thatdes-AA fibrin clots formed at high fibrinogen/fibrinratios,thus containing substantial amounts of fibrinogen,lysemore rapidly than the corresponding fibrin clots(fonned in the abscence of fibrinogen)mightpossibly contribute to explain the low rate of thrombo-emboliccomplicationsduring treatment with reptilaae or ancrod.Thus,it is reasonable to believe that the clots formed under such treatment are of the fibrinogen-containing des-AA fibrin type.

.REFERENCES.. l.ABIIDGAARD,U. ~analysisduringcoagulationofpllrified~fibrinogen,frm I,andplasma.Scand.J.Clin.Iab.Invest.~,529,1965 ~.BX~SIAD,F.,~IYXQH.C. and KIERUIF,P. Preparation of fibrinB fraQhl.mmn fikinogenin urea,using soluble or insolubilized thra&in.Haemostasis&,225, 1977 of 1131-fibr~en.J.Clin.Ivest.~,346,1963 3.w,A.S. In vivabehaviaur 4.BKXHTAD,F. Rrrification, characterizatia3ntiinso1ubilizati~0fbovinethranbin.Thranb.ks. l&119,1977 5.BWGSIIAD,F.,NIERE.F ,P.,GRAVR$X. and CXXXL,H.C. Purification and insolubilizatianof~~forthe~~oftnrmandes_AAfitain~Fnutea. ~.Faes.Inpress. ,P.and aXN&,H.C.Ccnparison betwzenm des-AAand'des6.BMXslBD,F.,KIERULF AAHBfibrinlrpaarrersasto solubility,aff~tytowardsagarose-fixedfibr_ genandsusceptibilitytoww&FSFtiplas&l.Abstra&no:896p.114.XVII Cnqress ofltheInternational Societyof Henatology.Paris 1978 ,P.atd GODAL,H.C. The fibrin-solubilizing effectof fibri7.I3RXDBD,F.,KIERULF nogen.Thranb.Res.Inpress. B.~,H.c. The influence 0f heparinon the fibrind.0t.Scand.ir.ciin.r.db.Imrest. l-3,306,1%1 susceptibilityofhumandes-AAvecsusdes~fibri.ntowards S.BEIDSSIIAD,F. = FibrinStabilizingFactor. Thmnb.Pes.Inpress. 10.LY,B.,~,P.,JAEOB!iEN,E. and GPAVl&K.Characterization of soluble,FSF-stahilizedf~~~l~eswithspecialreferencetothel~leoff~~. Thranb.*s.1,21,1974 ll.KANAIDE,H. and SHAINXF,J.R.CrosslWdngoffibrinogenandfibrinbyfikinstabilizing-factartfactor XIII)J.Lab.Clin.M&85,574,1975 of actionof arvinand reptilase. lZXWAAN,H.C and =,G.H. Themechanism l!hranb.Diathes.Hamuorrh. Suppl.47,361,1971 13,HAVEHKiQE,F. Lysisof cross-linked and non-crosslinkd purified fikin.T'h?xmb. Diathes.HaemDrrh. %,584,1975 14.PX@LING,M.W. FactorXIIIcross-linking ard the rateof fibrtilysis ticed by sv andurokinase.Thran.Res. l&287,1978