Modification of high molecular weight plasmic degradation products of human crosslinked fibrin

39

Biochimica et Biophysica Acta, 5 7 6 ( 1 9 7 9 ) 3 9 - - 5 0 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 3 8 0 7 3

MODIFICATION OF HIGH MOLECULAR WEIGHT PLASMIC DEGRADATION PRODUCTS OF HUMAN CROSSLINKED FIBRIN

S T E P H A N I E A. O L E X A , A N D R E I Z. B U D Z Y N S K I

and V I C T O R J. M A R D E R

"

Specialized Center of Research in Thrombosis and Department of Biochemistry. Temple University Health Sciences Center. Philadelphia. PA 19140 (U.S.A.) ( R e c e i v e d April 1 7 t h , 1 9 7 8 )

Key words, Fibrin degradation product; Plantain; (DD)E complex; Fragment DD

Summary The predominant high molecular weight products of plasmic digestion of human crosslinked fibrin Fragments DD, E and (DD)E complex were purified by column gel filtration in a non~iissociating buffer or by ion~xchange chromatography on DEAE~ellulose. The structure of the degradation products was studied by proteolytic degradation, polyacrylamide gel electrophoresis, immunodiffusion and sucrose density gradient centrifugation. Unaltered deriv~tires were very resistant to proteolytic degradation by plasmin. In the presence of 10 mM EDTA the (DD)E complex did not dissociate, but similar to Fragment DD, became susceptible to plasmic degradation forming Fragment D derivatives. The (DD)E complex dissociated in 3 M urea at pH 5.5, heal an altered conformation as evidenced by its aggregability and by its increased susceptibility to degradation by plasmin resulting in the formation of Fragment d. The 77 chain remnants of Fragment DD were attacked first, followed by cleavage of the ~ chain remnants. It is concluded that plasmin resistance is a function of the intact structure and it is not directly dependent on the presence of the crosslink bonds or calcium ions. Introduction P1asmic degradation of crosslinked fibrin leads to the formation of two distinctive products, Fragment DD and a polymer remnants [ I ~ 6 ] . A variable proportion of the Fragment DD molecules in plasmic digest of crosslinked fibrin is linked by non~ovalent bonds with Fragment E, forming a (DD)E com• Present a d d r e ~ : D ~ p a r t m e n t of Intern~ Medic/he and Dent~try, Un/ver~/ty o f R o c h e s t e r , R o c h e s t e r . NY 14642. U.S.A. Abbrev~t/ons: SDS, sod/urn dodecyl sulfate: CTA units, Committee on Thrombolytlc Aaents units: KIU. Kallik~eim inhibitor units.

40 plex [5,7]. The recognition and characterization of structural features of plasmic degradation products of crosslinked fibrin may provide a basis for the detection and direct monitoring of the rate and extent of fibrinolysis occurring in vivo. In this work attention was focused on alterations of Fragment DD and the (DD)E complex induced by EDTA, urea and low pH. The modifications were assessed by polyacrylamide gel electrophoresis, sucrose density gradient centrifugation and susceptibility to the enzymatic degradation by plasmin. Analysis of sequence of events, studied by polypeptide chain fragmentation, provided clues to the location of enzyme-susceptible regions in Fragment DD. Materials and Methods Crosslinked fibrin was obtained from human fibrinogen (Grade L, A.B. Kabi, Stockholm, Sweden) as previously described [4]. Digestion of crosslinked fibrin. 1 g freeze~lried fibrin was suspended in 20 ml pre-warmed (37°C) 0.15 M Tris-HC1 buffer, pH 7.8. An aliquot of 0.6 ml human plasmin (10.2 CTA units/ml, 12.7 CTA units/mg protein, kindly provided by Dr. David L. Aronson, Bureau of Biologics, Food and Drug Administration, Rockville, Md.) was added and the reaction mixture was incubated with gentle magnetic stirrin~ at 37°C for 24 h. Fractionation of [ibrin digests. (1) Gel filtration through Sepharose CL-6B (Pharmacia, Piscataway, N.J.). Columns (2.5 × 170 cm) were equilibrated and developed with 0.05 M Tris-HC1 buffer containing 0.028 M sodium citrate, 0.2 M e-aminocaproic acid, and 0.02% sodium azide, pH 7.4, at a flow rate of 60 ml/h.. Approx. 300 mg of the digest were applied to the column. The fractions within protein peaks were combined and concentrated. (2) Ion-exchange chromatography was performed according to the methods of Nussenzweig et al. [8] and Pizzo et al. [6], on a Whatman DE-52 microgranular cellulose column (1.5 × 50 cm) (Whatman, Clifton, N.J.), which had been equilibrated with 0.01 M sodium carbonate buffer, pH 8.9, and eluted with a linear gradient of 0--0.2 M NaC1 in the same buffer at 60 ml/h flow rate. After collecting approx. 950 ml, the column was washed with 0.5 M NaC1 in the same buffer. Fractions under, protein peaks were pooled, dialyzed against distilled water and concentrated. (3) Gel filtration through Sephadex G-200 (Pharmacia, Piscataway, N.J.). Columns (2.5 × 170 cm) were equilibrated and developed with 3 M urea/ 0.025 M sodium citrate solution, pH 5.5, at 30 ml/h flow rate. Digestion of purified degradation products. An aliquot containing 2 mg protein in 0.3 ml was dialyzed in 0.15 M Tris-HC1 buffer, pH 7.8, then mixed with 0.016 CTA units human plasmin and incubated at 37°C. The digestion was terminated by addition of 0.02 ml trasylol (aprotinin, 10 000 KIU/ml, Mobay Chemical Corp., N.Y.) after different time intervals. Po!yacrylamide gel electrophoresis. (1) Non-reduced proteins were analyzed in a non
41 were applied; electrophoresis was conducted at a constant current of 2 mA per gel for 18 h. All gels were stained and destained according to the procedure of Fairbanks et al. [1-1]. (3) Electrophoresis in polyacrylamide gradient slab gels at pH 8.6 used a di~'continuous sulfate-borate buffer system according to Neville [12]. Electrophoresis was performed at a constant current of 12 mA for approx. 4 h, or until a bromophenol blue marker reached the bottom of the slab gel. The gel was stained and destained according to Falrbanks et al. [11], soaked in methanol/acetic acid/glycerol ( 5 0 : 1 0 : 2 0 , v/v) for 2 h and dehydrated under vacuum on a filter paper sheet for storage. Two
Results

Fractionation of a crosslinked fibrin digest under non-dissociating conditions The protein eluted from a Sepharose CL45B column was divided into five fractions, the compositions of which were tested by double immunodiffusion and by polyacrylamide gel electrophoresis in a non-dissociating (Tris-glycine) system at pH 8.6 and in the presence of SDS (Fig. 1). Fraction I eluted in the void volume and had both D and E antigenic determinants. In Fraction II two bands of approx, equal staining intensity were seen in the non
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tion III consisted mainly of Fragment E, contaminated by a small a m o u n t of a 100 000 molecular weight moiety, perhaps explaining the trace immunoprecipitation line seen with anti-D antiserum (Fig. 1B). Fraction IV contained polypeptide material with a mean molecular weight of approx. 21 000, which did not precipitate with anti-Fragment E or D antiserum but did react with anti-An chain antiserum. Peak V contained peptides with molecular weights less than 15 000.

Crosslint~ed fibrin digest fractionated on DEAE-cellulose This was resolved into two main peaks {Fig. 2). Fractions I and II contained predominantly Fragment DD. Two components with molecular weights of 81 000 and 102 000 were demonstrable in the presence of SDS. Fragment E was not detected in these fractions. Fractions III and IV contained the (DD)E complex and Fragment DD {Fig. 2C, top) as well as those two components present in Fractions I and II (Fig. 2C, b o t t o m ) . Fraction V contained all these fragments. The incomplete separation of Fragments DD and E on DEAEcellulose may reflect a strong association of the these fragments in the {DD)E complex. Dissociation of the (DD)E complex by acid urea In order to isolate Fragment DD, a crosslinked fibrin digest was incubated in

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3 M urea/0.025 M sodium citrate, pH 5.5, then chromatographed on Sephadex G-200 using the same solution (Fig. 3). The relative amounts of Fractions I and II varied in different experiments, but usually Fraction I was larger; both reacted only with antiserum against Fragment D (Fig. 3B). Since the electrophoretic mobility of the major band was the same in Fractions I and II as tested in the presence of SDS (Fig. 3C, bottom), and since the material present in these fractions did not enter the Trist~lycine gels (Fig. 3C, top), it was concluded that they contain mostly an aggregate of modified Fragment DD. Fraction II contained aggregates of lower molecular weight as judged from its elution pattern (Fig. 3A). Fractions III, IV and V resembled those eluted from the Sepharose CL-6B column (Fig. 1).

Sucrose density gradient centrifugation It was used to assess the extent of aggregation of unmodified and modified Fragment DD. The protein distribution profile of a crosslinked fibrin digest showed the presence of two peaks and the absence of material at the bottom of the gradient (Fig. 4A). Fraction II (Fig. 1) and Fraction II (Fig. 2) showed only single symmetrical peaks (Fig. 4B and C). The protein distribution profile of freshly isolated, modified Fragment DD (Fractions I and II, Fig. 3) showed

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heterogeneous material of vet3' high molecular weight (Fig. 4 I ) a n d E). Both fractions also contained non-aggregated species similar to material in gradients shown in Fig. 4B and C. After storage at -20~C for 3 months, modified Fragment I)D showed that the entire protein material was aggregated, more so in Fraction I than in Fraction II (Eig. 4F and G). After aggregation, Fragment DD did not di~ociate into the 190 000 molecular species after dialysis in 0.15 M NaCI or 0.15 M Tris-HCl buffer at pH 7.4. The (DD)E complex and unmodified Fragment DD were unaffected by repetitiw, freezing and thawing and by storage.

Plasmin susceptibility The possibility was tested that the increased a~gregability of Fragment DD is associated with such structural alterations that ren(ter is susceptible to proteolysis. 'Fhe (I)D)E complex and modified Fragment DD were incubated with plasmin for various time intervals, after which the reaction was stopped with trasylol and the digest analysed by electrophoresis in SDS-polyacrylaznide slab gels. The (DD)E complex (Fraction II, Fig. 1) was resistant to plasmic digestion. There was no noticeable change in the electrophoretic pattern of the Fragment DD moiety in either reduced or non-reduced SDS gels. Under identical incubation conditions, modified Fragment DD was very susceptible to plasmic degradation, as was evident in both non-reduced (Fig. 5, top) and

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reduced systems (Fig. 5, bottom). Compared with the starting material, which contained minor amounts of degradation products, further proteolysis was apparent after only 1 min. Fragment DD disappeared after 15 min and after 4 h only fragments of 45 000 molecular weight and low molecular weight peptides were present. Reduced plasmic digests of modified Fragment DD showed a complex pattern of polypeptide chains (Fig. 5, bottom). To clarify these results the 5 min plasmic digest of modified Fragment DD (see Fig. 5) was electrophoresed in the first (horizontal) direction in non-reduced form, then in the second (vertical) direction under reducting conditions (Fig. 6).. The twodimensional pattern clearly showed that the 77 chain remnant (81 000) was the most susceptible to plasmic degradation and that its gradual breakdown determined the formation of the multiple forms of Fragment DD derivatives. Abrupt cleavage of the ~3 chain remnant (43 000) was associated with formation of the 45 000 molecular weight species. The a chain remnants were not susceptible to proteolysis. The 45 000 molecular weight species produced by plasmic degradation of modified Fragment DD have been identified as Fragment d [14,15] by gel

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REDUCED Fig. 5. E l e c t r o p h o r e t i c p a t t e r n s of t i m e d p l a s m i c digests o f acid u r e a m o d i f i e d F r a g m e n t D D in a slab gel w i t h a 6 - - 1 0 % linear p o l y a c r y l a m i d e g r a d i e n t in n o n - r e d u c i n g c o n d i t i o n s ( t o p ) a n d in a slab gel w i t h a 9 - 15% l i n e a r p o l y a c r y l a m i d e g r a d i e n t u n d e r r e d u c i n g c o n d i t i o n s ( b o t t o m ) . D i g e s t i o n t i m e i n d i c a t e d b e t w e e n t h e t w o sets of gels.

electrophoresis in non-reduced and reduced systems, and by direct comparison with Fragment d kindly provided by Dr. M. Furlan (Inselspital, University of Berne, Switzerland). An increased susceptibility of crosslinked fibrin and Fragment DD to plasmic degradation has also been found in the presence of EDTA. Samples of crosslinked fibrin, (DD)E complex and Fragment DD were incubated in 10 mM disodium EDTA at room temperature for 24 h. Half of each sample was then dialyzed in 0.15 M Tris-HC1 buffer, pH 7.8, and all samples were digested by plasmin for 24 h. Digestions performed in the presence of EDTA degraded the

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48 Fragment DD moiety to Fragment D species of molecular weights 81 000 and 87 000. The reduced polyacrylamide gels showed two 3" chain remnants of 26 000 and 32 000 molecular weight, in addition to the characteristic a and ~ chain remnants [16]. The EDTA-induced susceptibility of the Fragment DD moiety for plasmic degradation was reversible in that dialysis in 0.15 M Tris-HC1 buffer at pH 7.8, rendered the (DD}E complex and Fragment DD again resistant to proteolysis b y plasmin (Fig. 7). Discussion The (DD)E complex and Fragment DD native forms are resistant to degradation b y plasmin and do not dissociate during gel filtration (Fig. 1). The present work indicates that the resistance of the (DD)E complex and Fragment DD to degradation is not related to the e(3'-glutamyl)lysine bonds, occupying the 3' chain crosslink donor and acceptor sites, as has been suggested [3], but rather that it is a function of the intact structure. An alteration of this structure renders it susceptible to proteolysis. In fact, the 3' chain remnants of modified Fragment DD are the first polypeptide chains to be cleaved (Figs. 5 and 6). The resistance of the (DD)E complex to plasmin is not related to the presence of calcium or trace metal ions [17,18], since plasmin susceptibility after incubation with EDTA is reversed following removal of EDTA b y dialysis in buffer lacking calcium. It appears that EDTA affects the fibrinogen molecule by a time-dependent and reversible alteration of conformation and not by the removal of metal ions [18--20]. Fragment D retains much of the original conformation of the fibrinogen molecule [21], and the same is probably true for Fragment DD and the (DD)E complex, so the EDTA effect on all these proteins may be qualitatively similar. The structure of Fragment DD purified b y gel filtration in the presence of 3 M urea at pH 5.5 (Fig. 3) has been significantly altered, as inferred b y its irreversible aggregability (Figs. 3C and 4D--G). Aggregated Fragment DD was easily and extensively digested by plasmin (Fig. 5), in contradistinction to the (DD)E complex, which was not affected by the same experimental conditions (Fig. 7). The structural alteration in Fragment DD induced in EDTA is reversible and less extensive than that caused b y urea at pH 5.5. Upon incubation with plasmin only the 3'3' chain, b u t n o t the fi chain remnant, was cleaved, resulting in the formation of Fragment D species (Figs. 5 and 7). The Fragment D species which accumulated in EDTA-treated, plasmin-degraded Fragment DD explained the observation of Mosesson and Finlayson [22], which shows Fragment D rather than Fragment DD predominant in plasmic digests of human crosslinked fibrin obtained in the presence of 0.01 M EDTA. It appears that there also exists an alternative pathway of Fragment DD degradation. All of the fractions eluted from DEAE-cellulose contained two derivatives with molecular weights of 81 000 and 102 000, the sum of which is compatible with the molecular weight of Fragment DD (Scheme I). Their presence in the digest suggests that some Fragment DD molecules are cleaved into unequal parts forming t w o products of slightly different molecular weight having the same a and fi chain remnants b u t differing in the 3' chain remnants.

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S c h e m e I. D e ~ d a b o n p a ~ w a y s of F ~ e n t DD afar m~GcaUo~. U~lfled F~ent DD ~ p~In ~ t . An ~Uon of i ~ I ~ c ~ ~ ~ q u ~ e d to ~ow i~ de~dabon. ~e mol~u~r we~ of ~ e c ~ afar ~ucbon of ~l~de ~n~ ~ no~ in ~ o n t o f e ~ h c ~ Acid u ~ a m ~ i f l ~ F~ent DD ~ c l e a v e d i n l U ~ y by p ~ at ~ e N H ~ - ~ end of ~ e ~ c ~ ~m~L ~Ibn~ in ~ e f o ~ a U o n o f m ~ y d e ~ b o n pr~ue~ ~ mol~l~ wel~ ~ t w e e n 102 0 ~ ~ d I ~ 0 ~ ~ ] o f w h i c h ~ v e t w o c r a n k ~n~ per m o l ~ e . FoUowins ~e de~daUon o f ~ e ~ chMn ~ m ~ ~e ~ c~n ~mn~ unde~o fr~en~bon; ~e o chin ~mn~ ~ not ~Jflc~Uy a l l , t e d by p ~ i n . ~e pro~oly~ a t ~ c k of p ~ i n on E D T A - m ~ f l ~ F~ent DD ~eun • t ~e COOH-~In~ end of ~e ~ chin remit ~d r~I~ in ~ e e x c ~ o n o f t w o ( ( ? - ~ u ~ y l ) l y ~ n e bon~ ~d ~e fo~abon of F ~ e n ~ D of m ~ wel~ 81 ~ ~ d 87 0 ~ . F r a ~ e n t D s~i~ of m o I ~ u ~ w e i ~ t 94 ~ ( w i ~ 39 O~ ~ c h i n ~ m ~ t ) ~ n o t f o x e d in ~ pa~way ~e ~e COOH-~ m ~ of ~ e 7 c ~ ~ cut out to~e~er wi~ ~e c ~ bo~. ~ e ~ d d p a ~ w a y foUows s m ~ i f l e a U o n ~ u c ~ by ~ ~ o w n ~ e n t (e.~. • o p e ~ d ~ ion-excb~e chroma~mphy on D E A E ceUuJo~). ~ ] b n ~ In ~ ~ y m m e ~ c c l e a v ~ e and ~ e f o m a b o n of two d e ~ d a b o n pr~uc~ wi~ mol~u~ wei~ 81 ~ ~ d 102 ~ . ~e ~t~r c o n ~ ~e two e(7-~u~yl)iy~ne bon~.

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50

The heavier of these products should contain both e(7~glutamyl)lysine crosslink bonds. The data presented here indicated that the change in the susceptibility to proteolytic degradation by plasmin represents a subtle approach for testing the integrity of Fragment DD structure (Scheme I). The presence of Fragment D in plasmic digests of Fragment DD would reflect changes in the structure of 7 chain remnants; the formation of Fragment d [14,15] would be associated with additional structural changes, these involving the ~ and ~/chain remnants. The latter conclusion is valid also for the degradation pathway of Fragment D from fibrinogen. Acknowledgements

This work was supported by grant No. 14217 from the National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, and by Grant No. 76826 from the American Heart Association. References 1 Gaffney, P~. (1973) Thromb. Res. 2,201--218 2 Kopek, M., Teisseyre, E., Dudek-Wojciechowska, G., Kloczewiak, M., Pankiewicz, A. and Latallo, Z.S. (1973) Thromb. Res. 2,283--291 3 Ferguson, E.W., Fretto, L. and McKee, P.A. (1975) J. Biol. Chem. 250, 7210--7218 4 Marder, V~I., Budzynski, A.Z. and Barlow, G.H. (1.976) Biochim. Biophys. Acta 427, 1--14 5 Gaffney, P.J., Lane, D.A., Kakkar, V.V. and Brasher, M. (1975) Thromb. Res. 7, 89~99 6 Pizzo, S.V., Taylor, L.M., Jr., Schwartz, M.L., Hill, R.L. and McKee, P.A. (1973) J. Biol. Chem. 248, 4584--4590 7 Hudry-Clergeon, G., Paturel, L. and Suscillon, M. (1974) Pathol. Biol. Suppl. 22, 47--52 8 Nussenzweig, V., Seligmann, M., Pelmont, J. and Grabar, P. (1961) Ann. Inst. Pasteur 100,377--389 9 Davis, B J . (1964) Ann. N.Y. Acad. Sci. 1 2 1 , 4 0 4 ~ 4 2 7 10 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406--4412 11 Fairbanks, G., Steck, T.L. and Wallach, D.F.H. (1971) Biochemistry 10, 2606--2617 12 Neville, Jr., D.M. ( 1 9 7 1 ) J . Biol. Chem. 246, 6328--6334 13 Ouchterlony, ~. (1958) Progr. Allergy 5, 1--78 14 Furlan, M. and Beck, E.A. (1973) Biochim. Biophys. Acta 310, 205--216 15 Kemp, G., Furlan, M. and Beck, E.A. (1973) Thromb. Res. 3, 553--564 16 Marder, V.J. and Budzynski, A.Z. (1975) Thromb. Diath. Haemorrh. 33, 199--207 17 Haverkate, F. and Timan, G. (1977) Thromb. Res. 10,803--812 18 Bithell, T.C. (1964) Biochem. J. 93,431--439 19 Capet-Antonini, F.C. (1970) Biochirn. Biophys. Acta 200, 497--507 20 Endres~ G.F. and Scheraga, H.A. (1971) Arch. Biochem. Biophys. 144, 519--528 21 Budzynski, A.Z. (1971) Biochim. Biophys. Acta 229,663--671 22 Mosesson, M.W. and Finlayson, J.S. (1976) Progr. Haemost. Thromb. 3, 61--107