Dispersion of Fibrin Using Cationic Detergent

Transcription

1 /. Biochem., 79, (1976) Dispersion of Fibrin Using Ctionic Detergent Susumu KURIOKA Deprtment of Biochemistry, Jikei University School of Medicine, Minto-ku, Tokyo 10 Received for publiction, My 1, 197 By treting fibrin with ctionic detergent in medium contining M ure, fibrinctionic detergent complex ws formed. The complex ws soluble in distilled wter, nd ws observed to be in dispersed stte by electron microscopy. The complex ws precipitted nd resolubilized in the presence of slt. This behvior of the complex ws due to dsorption of the slt nions onto the complex. When n queous solution of the complex ws incubted with fresh serum, the complex soon ggregted nd then ws converted into firm clot which ws not soluble in distilled wter. In the previous study (1), fibrinogen ws found to bind ctionic detergent effectively, nd the detergent molecules were shown to remin ttched to the protein even fter the ction of thrombin [EC ] nd to inhibit the series of rections which result in the formtion of fibrils. This suggested tht the fibrinogen-ctionic detergent complex ws converted into fibrin-ctionic detergent complex, nd tht fibrin-ctionic detergent complex might be formed if fibrin (ure-soluble fibrin) ws dispersed in suitble medium nd then ctionic detergent ws dded to it. In the present study, we ttempted to form fibrin-ctionic detergent complex in medium contining fibrin, ctionic detergent, nd ure. Abbrevitions: STA-CI, steryltrimethylmmonium chloride; F-STA(Cl), fibrin-steryltrimethylmmonium complex prepred from ure-soluble fibrin; F-STA(C1)T, fibrin-steryltrimethylmmonium complex prepred from FG-STA(Cl) by the ction of thrombin; FG-STA(Cl), fibrinogen-steryltrimethylmmonium complex. This pper describes the interction of fibrin with vrious detergents, nd some properties of the complex formed. MATERIALS AND METHODS Fibrinogen Humn fibrinogen ws purified by the method of Lki nd Steiner (2); its clottbility ws 98%. Thrombin Bovine thrombin [EC ] ws purchsed from Mochid Phrmceuticl Co. Detergent Synthetic detergents were obtined from Ko-Atls Co., nd used without further purifiction. Preprtion of Ure-soluble Fibrin A mixture of 2. pmoles of fibrinogen (m.w.=340,000) nd 2 units of bovine thrombin in 0 ml of solution contining 0.34 M NCl nd 1 mm EDTA, ph 9., ws incubted t 37 for 60 min. The resulting fibrin clot ws wshed with sline nd then squeezed. The clot obtined ws dispersed in 0 ml of solution contining M ure nd 0.34 M NCl. The solu- Vol. 79, No. 1,

2 118 S. KURIOKA tion ws centrifuged to remove insoluble mterils, nd the superntnt ws subjected to dilysis ginst 0.34 M NCl. Fibrin precipitted fter the dilysis ws collected by centrifugtion. Dispersion of Fibrin & suitble mount of purified fibrin ws dispersed in solution contining M ure nd 0.34 M NCl, nd djusted to 0.01 mm fibrin solution. A mixture of 4 ml of 0.01 mm fibrin nd 1 ml of 10 mm detergent in M ure-0.34 M NCl ws dilyzed ginst 1) distilled wter, 2) 2 mm detergent, 3) 0.34 M NCl t room temperture for 24 hr to remove ure. The mount of soluble protein in ech of the dilysis tubes ws mesured by the method of Lowry et l. (3). Precipittion of Fibrinipgen) with STA-Cl in Medium Contining Ure A mixture of //mole of fibrin(ogen) (m.w. =340,000) nd //moles of STA-Cl (m.w.=347) in 6 ml of solution contining M ure, 0.17 M NCl, nd mm sodium citrte, ph 6., ws left to stnd t room temperture for 30 min, then centrifuged. The mount of protein precipitted ws mesured. Precipittion of fibrinogen with STA-Cl in the bsence of ure ws lso nlyzed in the sme mnner. Preprtion of Fibrin-steryltrimethylmmonium Complex (F-STA(Cl)) Approximtely 0.3 mmole of STA-Cl ws dded to 0 ml of 0.03 mm fibrin solution contining M ure nd 0.17 M NCl. The mixture ws left to stnd t room temperture for 30 min. The resulting precipitte ws collected, nd suspended in 40 ml of distilled wter, followed by dilysis ginst distilled wter. The precipitte grdully dispersed, forming colloidl solution of F-STA(Cl). Preprtion off-sta(cl) T (Fibrin-steryltrimethylmmonium Complex Obtined by the Hydrolysis of FG-STA(CT) with Thrombin) FG-STA(Cl) ws prepred from mixture of intct fibrinogen nd STA-Cl in 0.34 M NCl s described previously ( / ). A mixture of 0 ml of 0.03 mm FG-STA(Cl) nd 0 units of bovine thrombin in distilled wter ws incubted t 37 for 6 hr. The ph of the mixture ws 6.0. After incubtion, NCl ws dded to the mixture to finl concentrtion of 0.2 M. The resulting precipitte ws collected by centrifugtion, nd then suspended in 40 ml of distilled wter, followed by dilysis ginst distilled wter. The precipitte ws grdully dispersed, forming colloidl solution of F- STA(C1)T. Precipittion nd Solubiliztion of F- STA(Cl) nd F-STA(Cl) T Depending on the Slt Concentrtion The effects of slt concentrtion on the precipittion of F-STA(Cl) nd F-STA(Cl) r were estimted by 2-fold dilution in 2 ml of distilled wter; 0. ml of 0.01 mm F-STA(Cl) (determined on the bsis of protein concentrtion) ws dded to ech tube. The mixtures were left to stnd t room temperture for 30 min, nd then centrifuged. The minimum concentrtion of slt giving the mximum precipittion ws determined by mesurement of the protein in the superntnt. The minimum concentrtion of slt giving complete solubiliztion of the precipitted F- STA(Cl) ws lso determined in the sme mnner. The effects of slt concentrtion on the precipittion nd solubiliztion of F-STA(C1)T were lso estimted by the sme method. Anlysis of Slt Anions Adsorbed on F- STA(Cl) nd F-STA(Cl) T A 4 ml liquot of queous solution contining 0.01 ^mole of F- STA(Cl) or F-STA(C1)T ws mixed with 1 ml of K s Fe(CN)e solution (-0.10 /*mole per ml). The resulting precipitte ws collected nd the mount of precipitte ws mesured. The nion content dsorbed on the precipitte ws clculted by mesurement of Fe(CN)e~ 8 concentrtion in the superntnt in terms of the bsorbncy t 420 nm. Precipittion by sodium deoxycholte ws nlyzed in the sme mnner using 0.01 pmole of F-STA(Cl) or F- STA(C1)T nd ftmole of deoxycholte. Deoxycholte in the superntnt ws mesured by the method of Eriksson (4). Anlysis of the slt nion contents in the following mixtures ws crried out using Diflo Ultrfilters (Amicon) equipped with MX-0 membrnes, the retentivity of which ws X 10* (moleculr weight): 1) mixture of mm F-STA(Cl) nd 60 mm K,Fe(CN),, 2) mixture of mm F-STA(Cl>r nd 60 mm K,Fe(CN)6, 3) mixture of mm F-STA(Cl) nd 6 mm sodium deoxycholte, 4) mixture f. Bioch$m.

3 DISPERSION OF FIBRIN 119 of mm F-STA(C1)T nd 6 mm sodium deoxycholte. The mount of slt nions in the nitrte ws mesured by the method described bove. Electron Microscopy An queous solution of F-STA(Cl) contining pproximtely 0.01 mg of protein per ml, ph.8, ws prepred for electron microscopy. The mteril ws spryed onto freshly cleved mic surfce, nd ws shdowcst with pltinum. A JEOL type JEM- U electron microscope ws employed in the present study. Incubtion of F-STA(Cl) with Serum A mixture of 10 ml of fresh humn serum nd 10 ml of 0.03 mm F-STA(Cl) ws incubted t 37 for 60 min in the presence of 2 mm CCli or 1 mm EDTA. A piece of ure-soluble fibrin clot prepred from 10 ml of 0.03 mm purified fibrinogen in 0.34 M NCl ws lso incubted with the serum in the sme mnner. RESULTS AND DISCUSSION Interction of Fibrin with Detergent in Medium Contining Ure A medium contining fibrin, ure, detergent, nd NCl ws used to test the interction of fibrin with detergent. The results re summrized in Tble I. Fibrin remined soluble in medium contining ny type of detergent. This is consistent with the fct tht fibrinogen becomes incogulble s result of the ction of thrombin in the presence of detergent. A remrkble effect ws observed when ctionic detergent ws employed, nd the fibrin ws soluble even in distilled wter. Precipittion of Fibrin by STA-Cl Figure 1 shows the precipittion of fibrin by STA-Cl in M ure-0.17 M NCl (ph 6.). Fibrin did not precipitte until the molr rtio (STA-Cl/ fibrin) ws more thn, but most of the fibrin precipitted t molr rtio of 160. The resulting precipitte ws solubilized in distilled wter. Figure 1 lso shows the pttern of fibrinogen precipittion by STA-Cl in the presence or bsence of ure. It is interesting tht the mximum precipittion occurred t the sme molr rtio of STA-Cl to fibrin(ogen) in spite of the observed difference in the pproch to the mximum, nd the fct tht no STA-Cl ws observed in the filtrte on ultrfiltrtion using n MX-0 membrne until the molr rtio ws more thn 160 in every cse. This suggests tht ll the STA-Cl dded ws used for the formtion of fibrin(ogen)-steryltrimethylmmonium complex, nd tht STA-Cl modified fibrin by forming F-STA(Cl) in M ure in the sme wy tht STA-Cl modified fibrinogen by forming FG-STA(Cl) in 0.34 M NCl (7, ). TABLE I. Effect of detergent on fibrin in M ure. The bsl mixture consisted of 0.0 //mole of fibrin, 2 mmole of ure, 10 /imole of the detergent indicted in this tble, nd 1.7 mmole of NCl in totl volume of ml. The mixture ws dilyzed ginst 1) distilled wter, 2) 2 mm detergent, 3) 0.34 M NCl, respectively, to remove ure t room temperture for 24 hr. Ctionic Anionic Detergent Dodecylpicolinium Steryltrimethylmmonium Deoxycholte Nonionic Dodecylsulfte Sorbitn monosterte Polyoxyethylene dodecylether Dist-H.O (%) Yield of soluble fibrin fter dilysis ginst 2mM detergent (%) 0.34 M NCl (%) Vol. 79, No. 1, 1976

4 120 S. KURIOKA looi Molr rtio, STA/ fibrinfcxjen) Fig. 1. Precipittion of fibrin(ogen) by STA-C1. The bsl mixture contined //mole of fibrin- (ogen) (m.w. =340,000), /zmoles of STA(Cl) (m.w.=347), 1.02 mmoles of NCl, nd 30 /^moles of sodium citrte in totl volume of 6 ml t ph 6. nd 20. O, fibrin precipittion in the presence of ure , fibrinogen precipittion in the presence of ure., fibrinogen precipittion in the bsence of ure. Arrows mrk the mximum precipittion of fibrin(ogen) by STA-C1. In the previous study () we found tht exposed crboxyl groups of fibrinogen were cpble of intercting with ctionic detergent, leding to the binding of STA-C1 nd to precipittion of the protein fter sturted binding of STA-C1. It is well-known tht ure cn unfold protein structures. Therefore, it is possible, tht the number of exposed crboxyl groups on fibrin(ogen) increses when fibrin- (ogen) is present in medium contining ure. The observed difference in the precipittion pttern of fibrin(ogen) my be interpreted, s result of difference in the number of exposed crboxyls of the protein. A remrkble difference in the precipittion ptterns of fibrin nd fibrinogen in M ure suggests tht ure cn unfold fibrin more esily thn fibrinogen. The reson for this remins to be studied. Approximtely 160 crboxyl groups seem to be exposed on the surfce of fibrin dispersed in M ure, nd thus precipittion occurs when most of the crboxyls re sturted with STA- Cl t molr rtio (STA-Cl/fibrin) of On the other hnd, mny of the crboxyl groups of fibrinogen seem to be still embedded in the interior of the protein in M ure, resulting in grdully incresing binding of STA-C1 followed by fibrinogen precipittion. Precipittion of F-STA(Cl) nd F- STA(Cl) T by Vrious Slts Tble II demonstrtes the effects of vrious slts on the precipittion of F-STA(Cl) nd F-STA(C1) T dispersed in distilled wter. Anions cuse mrked precipittion, nd both nions with lrge hydrocrbon moiety nd those such s Fe(CN)r 3 nd Fe(CN)~* re very effective for precipit- TABLE II. Effects of vrious slts on the precipittion nd solubiliztion of F-STA(Cl) nd F-STA(C1)T. The bsl mixture contined mm F-STA(Cl) or F-STA(C1)T nd the slts in totl volume of 2. ml t ph 6. nd room temperture. The concentrtions shown re the minimum concentrtions of slt required for precipittion nd solubiliztion. NCl CCl, Slt NjSO, K s Fe(CN), K 4 Fe(CN), Cprylte (N) Deoxycholte (N) Dodecylsulfte (N) mm cone. for precipittion of F-STA(Cl) F-STA(C1)T FG-STA(Cl) mm cone, for solubiliztion of precipitted F-STA(Cl) F-STA(C1)T FG-STA(Cl) * Not completely soluble. /. Biochtm.

5 DISPERSION OF FIBRIN It 3 3- I Fe(CN)f dded, Detycholle dded, prnote urttole Fig. 2. Anion-induced precipittion of F-STA(Cl), F-STA(C1)T, nd FG-STA(Cl) in medium contining K 3 Fe(CN), or sodium deoxycholte. The bsl mixture contined 0.01 //mole of F-STA(Cl) or F-STA(C1)T nd the slt indicted in totl volume of ml t ph 6.0 nd 20. ) KjFe(CN) 6 medium, b) sodium deoxycholte medium. tion. Tble II shows the results using FG- STA(Cl) ( /); these were similr to the present results. Figure 2 shows the results of nion-induced precipittion of F-STA(Cl), F-STA(C1) T in the presence of K 3 Fe(CN) 6 nd sodium deoxycholte. Adsorption of nions incresed until ll the F-STA(Cl), F-STA(C1) T, or FG-STA(Cl) ws precipitted. This suggests reltionship between the dsorption of nions onto the complex nd precipittion. Solubiliztion of the Precipitted F-STA{Ct) nd F-STA(Cl) T As shown in Tble II, F- STA(Cl) nd F-STA(C1)T were precipitted t definite slt concentrtion, nd the resulting precipittes becme soluble t higher slt concentrtion. Tble III summrizes the results of ultrfiltrtion of complexes solubilized in medium contining K 3 Fe{CN) e or sodium deoxycholte. Most of the nions in the medium did not pper in the filtrte, indicting tht the nions were bound to the complexes which were retined on the membrne. Thus, the solubiliztion is due to further dsorption of nions onto the precipitted complexes. It is worthy of note tht solubiliztion of FG- STA(Cl) by K,Fe(CN), or sodium deoxycholte hs lso been performed in the sme mnner FG-STA(Cl) hs been found to relese TABLE III. Recovery of nions in the filtrte using n MX-0 membrne (retentivity: X10 4 moleculr weight). The mount of nions in the nitrte ws mesured by the methods described in the experimentl section. 1) 2) 3) 4) Mixture 60 mm KjFe(CN), 60 mm K,Fe(CN), plus mm F-STA(Cl) 60 mm KjFe(CN), plus mm F-STA(C1) T 6mM deoxycholte 6mM deoxycholte plus mm F-STA(Cl) 6mM deoxycholte plus mm F-STA(C1)T Recovery of nions in the nitrte (%) 7 9 (%) ' fibrinopeptides when cted upon by thrombin nd it remined soluble in distilled wter (1). This my be explined by supposing tht F- STA(C1)T is lmost the sme complex s F- STA(Cl). This is supported by the observtions tht we could not distinguish the behvior of F-STA(Cl) from tht of F-STA(C1) T in slt solution (Tbles II & III nd Fig. 2) nd tht both fibrinogen nd fibrin precipitted mximlly t the sme molr rtio of Vol. 79, No. 1, 1976

6 122 S. KURIOKA iooo A Fig. 3. Electron microgrph of F-STA(Cl). STA-Cl/fibrin(ogen). However, we cnnot rule out the possibility tht F-STA(Cl) my differ from F-STA(C1) T in the higher structure of fibrin, becuse the preprtion of the former ws crried out in M ure. As cn be seen in Tble II nd Fig. 2, we could not distinguish FG-STA(Cl) from F-STA(Cl) or F-STA(C1) T s regrds behvior in slt solution. This is probble due to similrity of the surfce chrcteristics of these complexes, which re modified with STA-C1. Electron Microscopic Observtion of F- STA(Cl) The ttempt to visulize the shpe of F-STA(Cl) by electron microscopy ws not very successful, s shown in Fig. 3. However, this electron microgrph certinly demonstrtes tht there re no fibrils or intermedite fibrils. It shows mny globulr prticles which seem to join nd form liner rry of nodulr elements, lthough they re irregulr in size nd shpe. Different types of fibrin(ogen) model hve been proposed on the bsis of electron microscopy (6 9). It hs been suggested tht fibrinogen might be "coiled spring," which unrvels to different degrees under different conditions (10). Electron microscopic studies on F-STA(Cl) will be continued under better conditions. Incubtion of F- STA(Cl) with Fresh TABLE IV. Effect of humn serum on F-STA(Cl). A mixture of 10 ml of 0.03 mm F-STA(Cl) nd 10 ml of fresh humn serum ws incubted t 37 for 60 min. After incubtion, the F-STA(Cl) ws dispersed in definite volume of distilled wter or M ure, nd then the soluble prt ws determined from the bsorbncy t 280 nm. The dt were expressed s percentges of the mount of F-STA(Cl) employed in the incubtion mixture. A piece of ure-soluble fibrin clot prepred from purified fibrinogen nd thrombin ws put into 10 ml of fresh humn serum nd incubted t 37 for 60 min, nd then nlyzed in the sme mnner. 1) A mixture of 0.03 mm F-STA(Cl) nd 10 ml of humn serum. 2) A mixture of fibrin clot prepred from 10 ml of 0.03 mm purified fibrinogen nd 10 ml of humn serum. 1 Percent of soluble protein in Incubtion mixture - Dist-H,0 M ure 1) F-STA(Cl) with 2mM CCl, with lmm EDTA 2) Fibrin* with 2mM CCl, with lmm EDTA Ure-soluble fibrin. 89 (%) 91 7 (96) 7 92 /. Biochem.

7 DISPERSION OF FIBRIN 123 Humn Serum F-STA(Cl) becme insoluble nd formed semi-trnsprent gel soon fter the ddition of humn serum due to the slt nions contined in the serum. When the mixture ws incubted in the presence of clcium ions, the semi-trnsprent gel becme elstic nd stretchble, nd ws insoluble in distilled wter. On the other hnd, when the mixture ws incubted in the presence of EDTA, the gel remined soluble in distilled wter. Ure-soluble fibrin ws found to become ure-insoluble fter incubtion with the serum nd clcium ions. These results seem to indicte tht ctive fctor XIII (cross-linking enzyme) introduces covlent bond (e-(rglutmyl)lysine bridge) between side chins of neighboring monomers of F-STA(Cl) s well s ure-soluble fibrin (11). Mny types of fibrin product re vilble in different forms (fibrin fom, powder, nd film) to id hemostsis nd heling in diffuse nd prenchymtous bleeding(12). F-STA(Cl) my be lso pplied to such bleeding surfce or into the cvity of wound. Concentrted F-STA(Cl), which looks like jelly, my be esily fshioned to the size of the wound, nd cn form firm clot. Ctionic detergent is known to hve sterilizing power, nd therefore bcteril infection my be prevented. The uthors wish to thnk Dr. S. Tkski (Division of Morphology, Bio-Medic Reserch Lbortories, Jikei University School of Medicine) for the electron microscopic nlysis of F-STA(Cl). We lso thnk Miss Y. Kwrbt for her help in prt of the study. REFERENCES 1. Kuriok, S. & Inoue, F. (197) /. Biochem. 77, Lki, K. & Steiner, R.F. (192) /. Poly. Set. 8, Lowry, O.H., Rosebrough, N.J., Frr, A.L., & Rndll, R.J. (191) /. Biol. Chem. 193, Eriksson, S. & Sjovll, J. (19) Arkiv Kemi 8, Kuriok, S., Inoue, F., & Mtsud, M. (197) /. Biochem. 78, Hll, C.E. & Slyter, H.S. (199) /. Biophys. Biochem. Cytol., Ky, D. & Cuddign, B.J. (1967) Brit.]. Hemtol. 13, Bng, N.U. (1966) Thromb. Dith. Hemorrh. Supple. 13, Koppel, G. (1967) Z. ZeUforxh. 77, Doolittle, R.F. (1973) Advnces in Protlin Chemistry Vol. 23, pp , Acdemic Press, New York & London 11. Tkgi, T. & Iwng, S. (1970) Biochem. Biophys. Res. Commun. 38, Gerends M. (1968) Fibrinogen (Lki, K., ed.) pp , Mrcel Dekker, Inc., New York Vol. 79, No. 1, 1976