J Clin Pathol: first published as 10.1136/jcp.s3-14.1.10 on 1 January 1980. Downloaded from

J Clin Pathol, 33, Suppl (Roy Coll Path), 14, 10-17 Breakdown products of fibrin and fibrinogen: molecular mechanisms and clinical implications

PJ GAFFNEY From the National Institute for Biological Standards and Control, Holly Hill, Hampstead, London

The major terminal enzymatic expression of the Fibrin-plasmin breakdown products fibrinolytic cascade system in vivo is plasmin. This enzyme is piesumed to interact with fibrinogen and FRAGMENTATION MECHANISMS fibrin in vivo and is arguably man's major defence Investigations into molecular fragmentation began against the deposition of fibrin, an important com- in 1945 when Walter Seegers, present-day doyen of ponent in the general hazard of thrombosis. haemostasis research workers, showed that plasmin- Our understanding of the molecular details of digested fibrinogen consisted of two major electro- plasmin interactions with fibrinogen and fibrin has phoretic fragments which were called o-fibrinogen been established over the past 10 years. As a part of and /3-fibrinogen.5 In the first detailed examination the overall strategy to elaborate the primary sequence of the fragments obtained from the interaction of of human fibrinogen the precise locations of the fibrinogen with plasmin Nussenzweig et al.6 distin- peptide bonds ruptured by plasmin at various stages guished five major fractions by ion-exchange copyright. in the degradation process have been reported. Such chromatography. They called these A, B, C, D, and precise data are not available as yet for fibrin- E, and they described fragments D and E as plasmin- plasmin interactions. I shall discuss the major resistant or terminal core fragments with molecular aspects of fibrinogen-plasmin interactions only weights of about 83 000 and 37000 respectively. briefly since these have been recently reviewed by, Fragments D and E were 50% and 20% by weight among others, Mosesson and Finlayson1 and of the original fibrinogen molecule,7 represented Gaffney.2 I shall deal with the current knowledge of immunologically distinct regions of fibrinogen,8 and fibrin-plasmin interactions in more detail since new are synonymous with the o- and fl-fibrinogens of http://jcp.bmj.com/ and exciting data concerning fibrin fragmentation, Seegers.5 Nussenzweig et al.6 also observed inter- which may have significance in vivo, have recently mediate plasmin-labile degradation products of emerged. fibrinogen, subsequently named by other workers In this review I am supposing that the major X, Y fragments.9 breakdown products of fibrinogen and fibrin (FDPs) Examination of the polypeptide compositions of are mediated by plasmin in vivo. Thus the in-vitro the various fragments derived from plasmin- experiments conducted in many laboratories over fibrinogen interactions in conjunction with carbo- on September 27, 2021 by guest. Protected the years have concentrated on plasmin-mediated hydrate staining and thrombin susceptibility of their interactions. It is, however, well to point out that composite peptide chains has allowed a number of the interactions between fibrinogen and a number schemes of fibrinogen fragmentation'0-14 which have of other proteolytic enzymes have also been studied a number of common features. Fig. 1 shows the in vitro (for review see Latallo et al.3). Indeed, major features of a sequence of digestive reactions Moroz and Gilmore4 thought that a variety of and products formed withwhichmost oftheseworkers proteolytic enzymes of cellular origin may play a would now agree. The conservation of both the far more important part in the lysis of established NH2-terminal amino-acids1617 of the Ao-chains thrombin in vivo than does plasmin. Nevertheless, suggests that the initial lysis of the Aa-chain takes there is little doubt that plasmin interacts with place at their carboxy ends. Peptides of about forming fibrin, attempting to prevent its deposition 40 000 molecular weight are released rather rapidly, on the vessel wall. I shall expand this last opinion leaving the NH2-terminal remnant of the Ao-chain when discussing plasmin-fibrin interactions and their disulphide bonded to intact BP and y chains. The significance in vivo. next reaction involves the removal of peptides 10 J Clin Pathol: first published as 10.1136/jcp.s3-14.1.10 on 1 January 1980. Downloaded from

Breakdown products offibrin andfibrinogen: molecular mechanisms and clinical implications I1I

Fibrinogen Fragment X Fragment Y each subunit of the fibrinogen dimer is random. .1 344000) wt. 300000-260000) (nml wt.150000) N N N Further support has been provided for the asym- metrical mode of fibrinogen fragmentation and the release of D as a monomeric fragment by the finding that the NH2-terminal amino-acids of fibrinogen, X, Y, and E are essentially alanine and tyrosine while those of fragment D are aspartic, methionine, and valine. This locates the D fragment at the carboxy terminal half of the fibrinogen subunit. Subsequently, it has been shown24 that fragment Y

FragmenltE contains 2 mol fibrinopeptide A and a set each of (m.1 .t 50000) heavy and light polypeptide chains. These latter C C data seem to have confirmed beyond doubt the Fragment D Fragment D M,t9400) (moL.r.94000) asymmetric scheme of fibrinogen degradation by plasmin shown in Fig. 1. Fig. 1 Diagram ofplasmin-mediated conversion of fibrinogen (Fg) to its core fragments D and E, showing intermediate fragmentts X and Y. Fibrin-plasmin interactions The scheme is constructed so that the polypeptide chain origin of the constituent subunits of each fragment The thrombin-mediated reactions which accompany can be related to those of the preceding fragment and the conversion of fibrinogen to various types of in turn to chains of the originating fibrinogen molecule fibrin must first be outlined. (Aoa, B,, and y). The amino (N)- and carboxy (C)- Fibrin formation is initiated by the cleavage of the terminal ends of the fragments are shown to aid in fibrinopeptides A and B from fibrinogen (ACX2Bfl2y2) visualising the site of structural breakdown more easily. by thrombin.25 The resultant fibrin monomer MWs ( x JO-S) are shown in parentheses after each (U2P2y2) can polymerise to non-stabilised polymers fragment designation. The range of MWs offragment X (a2fl2y2)p, which can subsequently be cross-linked depends on the amount of peptide material removed copyright. from the carboxy ends of the A ce-chains and whether in the presence of calcium and factor XIII (activated the peptides have been hydrolysed from the amino- by thrombin) to form stabilised fibrin.26 The terminal ends of the Bf3 chains. Other details of this glutamyl-lysyl27 28 cross-links form rapidly between asymmetrical scheme offibrinogen fragmentation are the y chains to form y dimers (y-y) while the oa chains in the text. (Taken from Gaffney15; reproduced by kind cross-link more slowly to form x chain polymers permission of the Editor and Publishers of Haemostasis.) (aP) of a molecular weight in excess of 400 000.29 It seems that the y chain cross-links can form in fibrin

(MW 6000) from the NH2-terminal ends of the when it is still in the soluble state in plasma2l and http://jcp.bmj.com/ Bf-chainls'2 17-21 followed by the asymmetric splitting the incorporation of of chain cross-links seems to of the three chains at one side of the partly degraded depend on the y chains being already in situ. dimer. These latter cleavages release fragment D, The importance of these cross-links in fibrin and and thus all lysis points must be adjacent to the whether they have the opportunity of forming in vivo NH2-terminal ends of all three chains, allowing that will be discussed later. For the moment it is sufficient fragment D is located near the carboxy end of each to indicate that an understanding is required of the fibrinogen subunit.22 The remaining major fragment interactions of plasmin with at least three

major on September 27, 2021 by guest. Protected is called y,9 23 and the subsequent Iysis of its Act, forms of fibrin: (1) non-cross-linked fibrin (NXL- B,B, and y chain remnants converts fragment Y to FN); (2) partly (y-y) cross-linked fibrin (PXL-FN); fragments D and B. Evidently the terminal or 'core' and (3) totally (cx chains and y chains cross-linked) fragments D are made up of three polypeptide chain cross-linked fibrin (TXL-FN). Part of the diagram remnants of the Act, B,B, and y chains of fibrinogen in Fig. 2 shows three fibrin molecules with the while fragment E is a dimer containing disulphide approximate locations of the factor XIII cross-links. bonded subunits, each subunit being made up of The (x chain cross-links have been placed near the three polypeptide chains. carboxy termini for ease of presentation, whereas This scheme of degradation owes much to the we now know that they are located at least 20 000 original scheme of Marder et al.23 in which they MW from the carboxy ends of the cx chains.3' suggested an asymmetrical cleavage of the fibrinogen dimer by plasmin. This type of digestive splitting NON-CROSS-LINKED FIBRIN (NXL-FN) may suggest that the two subunits of this fibrinogen From a chemical viewpoint it is reasonable to dimer differ discretely in primary structure or else regard NXL-FN as being quite similar to fibrinogen, that the sequential release of the D fragments from since its chemical structure lacks only fibrinopeptides J Clin Pathol: first published as 10.1136/jcp.s3-14.1.10 on 1 January 1980. Downloaded from

12 Gaffney CROSS-LINKED FIBRIN (XL-FN) The interaction of plasmin with XL-FN will deal with both PXL-FN and TXL-FN together since the spectrum of fragments obtained from both forms of fibrin is quite similar.38 It would seem that the major property endowed on fibrin by o chain cross-linkage is resistance to lysis by plasmin.39 40 Information about the fragments yielded on lysis of XL-FN by plasmin has slowly unfolded. In 1973 XL-FN

llll l l ~~~r-S-5-1 digests were shown to contain a major distinct I ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~fragment not found in fibrinogen digests.33 4142 This E was called D dimer32 since it was composed of two D D dimer domains from adjacent fibrin molecules, joined Fig. 2 Schematic representation ofpolypeptide chains by their cross-linked y chain remmants.41 of cross-linked human fibrin and its plasmin-mediated A scheme showing the origin of the D dimer and degradation products D dimer and E. E fragments from XL-FN is shown in Fig. 2. Later The cross-linked y-chain dimers, the cross-linked it was found that some D dimer and E molecules a-chain polymers, and the uncross-linked fl-chains, are could be present in a digestion mixture as an electro- here related to each other schematically. All the phoretically stable but non-covalent complex known cross-links are shown for schematic convenience close as the D dimer-E complex.384344 Subsequent to the carboxy (C) ends of the respective chains. experiments, in which the lysis of XL-FN was While this localisation is true for the y-chain cross-links performed in serum or plasma, indicated that all the the a-chain cross-links are at least 20 000 MW distance from the C-terminal end. Disulphide (S-S) bonds D dimer and E molecules were present as a stoicho- (not shown here except for one S-S bondjoining the metrically satisfied complex, having the general two amino (N)-terminal ends of each fibrin molecule) formula (D-D)E.45 46 The schematic link the individual polypeptide chains (x, fi, y) of each representation of the D dimer-E fibrin molecule. Non-covalent bonds also hold the units complex (Fig. 3) indicates that each domain origin- copyright. of the fibrin polymer together but are omittedfor the ates in a distinct fibrin molecule. More important sake ofsimplicity. The location of the antigenically is that the D dimer-E complex must contain the distinct sections offibrin D and E are shown in each of major polymerisation sites of fibrin located in the three fibrin molecules of the fibrin polymer. Plasmic D and E domains.47 Olexa and Budzynski48 showed digestion a yields fragment D which is cross-linked to that one mole of D dimer and one mole of E form another D fragment from an adjacent fibrin molecule. one This type is called D dimer. Fragment E and a variety mole of the complex and that conversion of the ofcross-linked and uncross-linked peptides (not shown) complex to free D dimer and E fragments is mediated http://jcp.bmj.com/ are also released during the lysis of cross-linked fibrin. by digestion of the E domain. The D dimer-E com- The lengths of the lines representing the polypeptide plex can dissociate in 8 M urea and reforms after chains are not proportional to their MWs. (Taken from removal of the detergent.45 Specific ant-genic markers Gaffney et al.30; reproduced by kind permission of the have been recognised in D dimer and the D dimer-E Editor, Cardiovascular Research.) complex,49 but since these crosslink-related markers represent only a small proportion of the total antigenicity of the D dimer fragment it is difficult A and B while conformational differences are due to to devise a useful test for these fragments in vivo. on September 27, 2021 by guest. Protected aggregation during the clotting process. Thus, not In view of the evidence that the D dimer-E surprisingly, the digestion of NXL-FN by plasmin complex is a major product of cross-linked fibrin yields an array of fragments similar in molecular lysis in vivo,5051 a variety of lytic regimens was weight to those observed for fibrinogen.32-34 The investigated to determine whether this complex was susceptibility to plasmin of the individual chains in the earliest soluble expression of XL-fibrin lysis. the intact NXL-FN is the same as that observed for It transpired that besides the D dimer-E complex fibrinogen.35 Obviously, of course, X, Y, and E from higher molecular weight complexes are released fibrinogen will at least differ from those fragments from XL-FN which have been classified as cross- obtained from NXL-FN in that the former contain linked fragment X-oligomers, Y-D, and D dimer fibrinopeptide A while the latter do not. Other complexes. Schematic structures of these fibrin discrete structural differences between the respective degradation products (Fig. 4) were elucidated from fragments from fibrinogen and NXL-FN exist which sodium dodecyl sulphate polyacrylamide gel electro- can be detected by radioimmunoassay using phoresis, carbohydrate staining, and 125I-label appropriate antisera.3637 distribution.52 Thus it would seem that cross-linked J Clin Pathol: first published as 10.1136/jcp.s3-14.1.10 on 1 January 1980. Downloaded from

Breakdown produlcts offibrin and fibrinogeni: molecular mechanisms and clinical implications 13

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Fig. 3 Schematic view ofprobable fibrin subunit origins of the three component parts of the D dimer-E complex. The intermolecular y chain cross-links are shown as solid lines while the shaded area shows that two D fragments from adjacent fibrin subunits and an Efragment from another fibrin subunit are associated in the D dimer-E conmplex copyright. (D1-D2)E4. The suggestion is inherent that the COOH-terminal ends of the y chains of fibrin which contain the cross-linking residues are also involved in the binding of one E fragment between the two D fragments.

X'Polymer Y-D D-D http://jcp.bmj.com/

;~~~~~tfDSp0~~~ ( 1= i'YB42

_ _ p _~~~~~~~~~~~~~~~I a. 25 on September 27, 2021 by guest. Protected 594 Y-Y -6Y-T' Bs p3 56 txD5ep 56 ^ DcP) 42 D(p4422 i - 25 a' D( 12 D (p 12

Fig. 4 Diagrammatic presentations of the various cross-linked fragments found in the plasmin-mediated lysates of cross-linked fibrin. The polypeptide chain components of the various cross-linked classes offragments are shown below the relevant diagram. The polyacrylamide gel separation of the various polypeptide chains which comprise the combined high molecular weight cross-linked fragments is shown to the right of the diagram. The nomenclature usedfor the chains has been described elsewhere.22 There is evidence to suggest that all these cross-linked fragments can remain associated with each other, possibly through the D dimer-E polymerisation site, and separate only when subjected to detergent treatment before electrophoresis. J Clin Pathol: first published as 10.1136/jcp.s3-14.1.10 on 1 January 1980. Downloaded from

14 Gaffney X, Y, and D fragments can be released from XL-FN have the same molecular composition as those as soluble fragments, as suggested elsewhere53 and formed from the simultaneous enzymatic competition in contradiction to our earlier proposals.54 of plasmin and thrombin for fibrinogen. Our unpublished data also indicate that the cross-linked X-oligomeis are the first fragments Practical implications of fibrin degradation derived from XL-fibrin by plasmin, are the pre- products (FDPs) cursors of the D dimer-E complex, and are present during lysis of fibrin performed under near-physio- Whether fibrinogen in circulation is normally logical conditions in vitro. These oligomers have also susceptible to plasmin-mediated digestion is a been isolated from the plasma of patients during subject of controversy. Most concede, however, that episodes of fulminant disseminated intravascular the major source of FDPs in plasma is from second- coagulation (DIG)54 and other forms of intravascular ary fibrinolysis56-namely, the digestion of fibrin coagulation.55 The molecular interactions which after its generation by thrombin from fibrinogen. It allow the XL X-oligomers to be formed during DIC can be argued that the rapidly acting 012-anti- are rather difficult to interpret in the light of our plasmin57 neutralises any plasmin formed in circula- understanding of the formation of such high tion, while such a reaction cannot effectively take molecular weight derivatives from a XL-FN clot. place when the generated plasmin occurs in the The scheme outlined in Fig. 5 incorporates some of protective environment of soluble or insoluble fibrin. Nevertheless, if exercise3' and conventional DIC streptokinase therapy54 58 can be regarded as examples of primary fibrinogenolysis in their FG-X Tb FN-X ultimate plasmic effect on fibrinogen then the P Ca Y-YXL X-oligomners significance and speed of ac2-antiplasmin activity in FS *FN Pi FN blood may need reappraisal. These notions are in \Ca+ part supported by the fact that streptokinase \Fxiul induces a greater reduction in circulating oC2-anti- copyright. P plasmin than that observed during ancrod infusion, Y-yXL-FN Y-Y XL X-oligomers which induces secondary and far more effective fibrinolysis (Gaffney, unpublished data). Th rombolysis Thus our consideration of the practical value of the above molecular data seems best considered Th Pi FG XL-fN Y-YXL X-oligomers against a background of thrombin-plasmin mediated Ca'+Fxnii DIC and direct plasmin attack on preformed

Fig. 5 Two schemes proposed to explain the presence XL-fibrin as present in established thrombi.30 In http://jcp.bmj.com/ of the cross-linked (XL) high molecular weight discussing fibrin-plasmin interactions I have com- X-oligomers during DIC and thrombolysis. A number mented on the presence of cross-linked X-oligomers ofpathways are proposed interrelating thrombin (Th), in the of plasmin (Pl), and Ca++-m ediated polymerisation and plasma patients during DIC and in XL- cross-linking (factor XII) activities during DIC. All fibrin digests. Fig. 5 outlines pathways by which could allow the formation of cross-linked X-oligomers. 'giant' fibrin fragments (X-oligomers) could be It is also proposed that such fragments should be formed during the competitive interactions ofplasmin formed during the lysis ofpreformed thrombi in vivo and thrombin for circulating fibrinogen, while on September 27, 2021 by guest. Protected since they are present during the lysis of XL-fibrin similar fragments should be found in the plasma of in vivo under near-physiological conditions. FG = patients during the lysis of preformed thrombi. fibrinogen; FN = fibrin; the X derivatives of both FG During the DIC-like condition induced during and FN are formed by the PL-mediated removal of defibrination by ancrod infusion fragments similar peptide material from the carboxy terminal ends of the to X, Y, and D are formed in vivo, while D dimer is Aca and cx chains (see Fig. 1). It is surmised that during DIC these reactions take place in solution absent.'559 In contrast, plasma from patients who without deposition offibrin. have been bitten by the snake Echis carinatus contains significant amounts of D dimer.60 The conversion of prothrombin to thrombin by the snake venom our thoughts with those of Graeff and his col- allows the formation of factor XIII-mediated leagues55 and suggests that the XL X-oligomers cross-lnked fibrin and its subsequent digestion to could occur by a number of pathways which involve cross-linked D fragments. The latter clinical con- the competition of plasmin and thrombin for dition bears some of the hallmarks of advanced fibrinogen and fibrin during DIC. It seems that the DIC in contradistinction to the benign condition of earliest fragments formed during fibrin clot lysis the patient during defibrination with ancrod, a J Clin Pathol: first published as 10.1136/jcp.s3-14.1.10 on 1 January 1980. Downloaded from

Breakdown products offibrin andfibrinogen: molecular mechanisms and clinical implications 15 procoagulant from the venom of Agkistrodon The hypothesis presented in Fig. 6 suggests that rhodostoma. Coagulation disorders (mostly DIC) in the composition of the fragments formed during the obstetric patients were accompanied by the presence competition between thrombin and plasmin should in the plasma of large fibrin fragments similar to the shed light on the severity of the so-called hyper- X-oligomers described above in the XL-fibrin coagulable state in the patient's blood. The presence digests,55 while cases of fulminant DIC had cross- of non-cross-linked fragments would suggest that linked X-oligomers and the D dimer-E complex in the fibrinolytic system was rapidly digesting the plasma.51 forming fibrin, whereas large cross-linked fragments Emerging from case reports such as those referred (X-oligomers) together with cross-linked D dimer-E, to above is a hypothesis (Fig. 6) which echoes and Y-Y, and Y-D complexes would suggest that the hypercoagulability was quite severe in that the N o r m a I Pathological fibrinolytic system was being overpowered and barely coping with the fibrin polymerisation process. The latter instance could be detected by sensitive Fg Fn [pilsol Fn [PASO assays for these high molecular weight cross-linked AA + complexes, and such assays might be useful in high- Th Ca+ FN lighting the clinically-dangerous hypercoagulable or 000 F xiii CLOT thrombotic state. Pi Pi co Similar concepts about the haemostatic balance during DIC have been outlined by others62 using assay procedures for fibrinopeptide A and the X,Y,D,E D-1, E NH2-terminal B,B peptide (1-42) to determine the XL Y-Y,Y-D X oligomers influence of thrombin and plasmin respectively. We Fig. 6 Diagram outlining possible sequences of events believe, however, that the presence of the cross- in vivo during the hypercoagulable or prethrombotic linked fragments described above (and shown in state. Figs. 4 and 6) in a patient's plasma would indicate a copyright. After the removal offibrinopeptide A from fibrinogen more advanced state of hypercoagulability than (Fg) by thrombin (Th) soluble fibrin polymers (Fnp) would be detected by the tests suggested by Nossel form which can be converted by plasmin (Pl) to fibrin and colleagues.63 Whereas these cross-linked frag- degradation products (X, Y, D, E). Such reactions are ments have been found in the plasma of patients thought to occur at a local level regularly in healthy during DIC, assays have yet to be developed of subjects. Should either the available Th production be to monitor their in excessive or the level ofplasmin depressed large fibrin sufficient sensitivity presence polymers may form and cross-link under the plasma during thrombolytic therapy for thrombosis. (Fnp2) http://jcp.bmj.com/ influence of Ca++ andfactor XIII. These cross-linked To this end antisera have been raised in rabbits fibrin polymers can act as a focus for clot formation which contain conformational neo-antigens for the or can be digested by plasmin to generate cross-linked cross-linked X-oligomers. These antisera do not fragments-for example, (D-D)E, Y- Y, Y-D, and react with fibrinogen and their use is contemplated X-oligomers. The diagram proposes that the detection in radioimmunoassay to detect the advanced in plasma of XL fibrin polymers or their plasmin- prethrombotic state and to clarify the role of mediatedfragments defines a 'pathological' fibrinolysis as man's major defence against throm- prethrombotic state. bosis. on September 27, 2021 by guest. Protected extends the thoughts of Astrup6' about coagulation and fibrinolysis being systemically balanced to References maintain blood fluidity and competent haemostasis. It is now more probable that this balance operates Mosesson MW, Finlayson JS. The search for the at a local rather than a systemic level. The amount of structure of fibrinogen. In: Spaet TH, ed. Progress in available thrombin depends on the amount of haemostasis and thrombosis, vol 3. New York: circulating inhibitors (notably At III) which operate Grune and Stratton, 1976:61-107. at various in the coagulation cascade. The 2Gaffney PJ. The biochemistry of fibrinogen and fibrin points degradation products. In: Ogston D, Bennett B, amount of plasmin available for fibrin digestion eds. Haemostasis, biochemistry, physiology, and seems to be independent of the concentrations of pathology. New York: John Wiley, 1977:105-68. fibrinolytic inhibitors (notably the fast-acting 3 Latallo ZS, Teisseyre E, Ardelt W, Wegrzynowitz Z, a,2-antiplasmin) since plasminogen seems to be Kopec M. Fibrino(geno)lysis by enzymes other than protected from inhibition by the forming fibrin plasmin. In: Gaffney PJ, Balkov-Ulutin S, eds. polymers. Fibrinolysis: current fundamental and clinical J Clin Pathol: first published as 10.1136/jcp.s3-14.1.10 on 1 January 1980. Downloaded from

16 Gaffney concepts. London: Academic Press, 1978:129-36. 313. 4 Moroz LA, Gilmore NJ. Fibrinolysis in normal plasma 21 Kierulf P. N-terminal analysis of fibrins from plasmin and blood; evidence for significant mechanisms hydrolysed fibrinogen-evidence for lack of fibrino- independent of the plasminogen-plasmin system. peptide B. Thromb Res 1972;1:527-32. Blood 1976;48:531-45. 22 Gaffney PJ. Localisation of carbohydrate in the 5 Seegers WH, Nieft ML, Vandenbelt JM. Decomposi- subunits of human fibrinogen and its plasmin- tion products of fibrinogen and fibrin. Arch Biochem induced fragments. Biochim Biophy Acta 1972;263: 1945 ;7:15-9. 453-8. 6 Nussenzweig V, Seligmann M, Pelmont J, Grabar P. 23 Marder VJ, Shulman NR, Carroll WR. High mole- Les produits de degradation du fibrinogene humain cular weight derivatives of human fibrinogen par la plasmine. I. Separation et proprietes physico- produced by plasmin. 1. Physicochemical and chimiques. Ann Inst Pasteur (Paris) 1961 ;100:377-89. immunological characterization. J Biol Chem 1969; Jamieson GA, Gaffney PJ. Nature of the high mole- 244:2111-9. cular weight fraction of fibrinolytic digests of human 24 Budzynski AZ, Marder VJ, Shainoff JR. Structure of fibrinogen. Biochim Biophys Acta 1968;154:96-109. plasmin degradation products of human fibrinogen. 8 Marder VJ. Identification and purification of fibrin- Fibrinopeptide and polypeptide chain analysis. ogen degradation products produced by plasmin: JBiol Chem 1974;249:2294-302. considerations on the structure of fibrinogen. 25 Bettelheim FR, Bailey K. The products of the action Scand J Haematol (Suppl) 1971 ;13:21-36. of thrombin on fibrinogen. Biochim Biophys Acta 9 Larrieu MJ, Marder VJ, Inceman S. Effects of fibrin- 1952;9 :578-9. ogen degradation products on platelets and co- 26 Lorand L, Konishi K. Activation of the fibrin stabi- agulation. Thromb Diath Haemorrh 1966;20 (Suppl): lizing factor of plasma by thrombin. Arch Biochem 215-26. Biophys 1964;105 :58-67. 10 Mills DA. A molecular model for the proteolysis of 27 Matacic S, Loewy AG. The identification of isopeptide human fibrinogen by plasmin. Biochim Biophys Acta cross-links in insoluble fibrin. Biochem Biophys 1972 ;263 :619-30. Res Commun 1968;30:356-62. Mills DA, Karpatkin S. The initial macromolecular 28 Pisano JJ, Finlayson JS, Peyton MP. Cross-link in derivatives of human fibrinogen produced by fibrin polymerized by factor XIII; E-(y-glutamyl)

plasmin. Biochim Biophys Acta 1972;271 :163-73. lysine. Science 1968;169:892-3. copyright. 12 Mosesson MW, Finlayson JS, Galanakis DK. The 29 McKee PA, Mattock P, Hill RL. Subunit structure of essential covalent structure of human fibrinogen human fibrinogen, soluble fibrin and cross-linked evinced by analysis of derivatives formed during insoluble fibrin. Proc Nat! Acad Sci USA 1970;66: plasmic hydrolysis. J Biol Chenm 1973;248:7913-29. 738-44. 13 Furlan M, Beck EA. Plasmin degradation of human 30 Gaffney PJ, Brasher M, Lord K, et al. Fibrin subunits fibrinogen. 1. Structural characterisation of degrada- in venous and arterial thromboembolism. Cardiovasc tion products. Biochim Biophys Acta 1972;263: Res 1976;10:421-6. 631-44. 31 Finlayson JS, Mosesson MW. Crosslinking of a chain 14 Pizzo SV, Schwartz ML, Hill RL, McKee PA. The remnants in human fibrin. Thromb Res 1973;2:467- http://jcp.bmj.com/ effect of plasmin on the subunit structure of human 78. fibrinogen. J Biol Chem 1972;247:636-45. 32 Gaffney PJ. Subunit relationships between fibrinogen 15 Gaffney PJ. Fibrin(ogen)interactions with plasmin. and fibrin degradation products. Thromb Res 1973; Haemostasis 1977 ;6:2-25. 2 :201-17. 16 Sherman LA, Mosesson MW, Sherry S. Isolation and 33 Pizzo SV, Schwartz ML, Hill RL, McKee PA. The characterization of the clottable low molecular effect of plasmin on the subunit structure of human weight fibrinogen derived by limited plasmin fibrin. J Biol Chem 1973;248:4574-83. hydrolysis of human fraction 1-4. Biochemistry 1969; 34 Slade CL, Pizzo SV, Taylor LM Jr, Steinman HM, on September 27, 2021 by guest. Protected 8:1515-23. McKee PA. Characterisation of fragment E from 17 Mosesson MW, Finlayson JS, Umfleet RA, Galanakis fibrinogen and cross-linked fibrin. J Biol Chem DK. Human fibrinogen heterogenetics. I. Structural 1976 ;251 :1591-6. and related studies of plasma fibrinogens which are 35 Gaffney PJ, Dobos P. A structural aspect of human high solubility catabolic intermediates. J Biol Chem fibrinogen suggested by its plasmin degradation. 1972;247:5210-9. FEBS Lett 1971 ;15:13-6. 18 Shainoff JR, Lahiri B, Bumpus FM. Ultracentrifuge 36 Edgington TS, Plow EF. Conformational and structural studies on the reaction between thrombin and plas- modulation of the NH2-terminal regions of fibrin- minized fibrinogen. Thromb Diath Haemorrh ogen and fibrin associated with plasmin cleavage. (Suppl) 1970;39:203-17. J Biol Chem 1975;250:3393-8. 19 Lahiri B, Bumpus FM, Shainoff JR. Fate of the 37 Edgington TW, Plow E. Differentiation between fibrinopeptides in the reaction between human fibrinogen and fibrin cleavage fragments based on plasmin and fibrinogen. Fed Proc 1970;29 :648. immunochemical correlates of structure and clinical 20 Budzynski AZ, Marder VJ, Shainoff JR. The content of implications. In: Neri Serneri GG, Prentice CRM, fibrinopeptides A and B in purified degradation eds. Haemostasis and thrombosis (Proceedings of the products of human fibrinogen. Fed Proc 1973;32: Serono Foundation Symposia, vol 15). London: J Clin Pathol: first published as 10.1136/jcp.s3-14.1.10 on 1 January 1980. Downloaded from

Breakdown products offibrin andfibrinogen: molecular mechanisms and clinical implications 17 Academic Press, 1979:27-43. crosslinked fibrin. Blood 1979;54:794-804. 38 Gaffney PJ, Lane DA, Brasher M. Soluble high 50 Gaffney PJ. Distinction between fibrinogen and fibrin molecular weight E fragments in the plasmin- degradation products in plasma. Clin Chim Acta induced degradation products of crosslinked human 1975;65:109-15. fibrin. Clin Sci Mol Med 1975;49:149-56. 51 Whitaker AN, Rowe EA, Masci PP, Gaffney PJ. 39 Schwartz ML, Pizzo SV, Hill RL, McKee PA. The Identification of D dimer-E complex in disseminated effect of fibrin-stabilizing factor on the subunit intravascular coagulation (DIC). Thromb Haemostas structure of human fibrin. J Clin Invest 1971;50: 1979 ;42 :274. 1506-13. 52 Gaffney PJ, Joe F. The D dimer-E complex and its 40 Gaffney PJ, Whitaker AN. Fibrin crosslinks and lysis aggregates in crosslinked fibrin digests. Thromb rates. Thromb Res 1979 ;14 :85-94. Haemostas 1979;42:427. 41 Gaffney PJ, Brasher M. Subunit structure of the 53 Regafion E, Vila V, Aznar J. Identification of high plasmin-induced degradation products of cross- molecular weight derivatives of plasmic digests of linked fibrin. Biochim Biophys Acta 1973 ;295:308-13. cross-linked human fibrin. Thromb Haemostas 42 Koped M, Teisseyre E, Dudek-Wojciechowska G, 1978 ;40:368-76. Kocewiak M, Pankiewicz A, Latallo ZS. Studies on 54 Gaffney PJ. The molecular and functional condition the 'double D' fragment from stabilized bovine of plasma fibrinogen during thrombolytic therapy fibrin. Thromb Res 1973;2:283-91. with streptokinase. Thromb Res 1973;2:105-14. 43 Hudry-Clergeon G, Paturel L, Suscillon M. Identi- 55 Graeff H, Hafter R, Bachmann L. Subunit and macro- fication d'un complexe (D-D) . . . E dans les produits molecular structure of circulating fibrin from obstet- de degradation de la fibrine bovine stabilisee par le ric patients with intravascular coagulation. Thromb factor XIII. Pathol Biol (Suppl) 1974;22:47-52. Res 1979;16:313-28. 44 Gaffney PJ, Lane DA, Kakkar VV, Brasher M. 56 Nilsson IM, ed. Haemorrhagic and thrombotic disease. Characterisation of a soluble D dimer-E complex London: John Wiley, 1971 :111-62. in crosslinked fibrin digests. Thromb Res 1975;7: 57 Collen D. Identification and some properties of a new 89-99. fast-acting plasmin inhibitor in human plasma. Eur 45 Gaffney PJ. The relevance of the D dimer-E complex J Biochem 1976;69:209-16. to the lysis of cross-linked fibrin. In: Davidson JF, 58 Gaffney PJ, Chesterman CN, Allington MJ. Plasma

Cepelak V, Samama MM, Desnoyers PC, eds. fibrinogen and its fragments during streptokinase copyright. Progress in chemical fibrinolysis and thrombolysis, treatment. Br J Haematol 1974;26:285-93. vol 4. Edinburgh: Churchill Livingstone, 1979: 59 Prentice CRM, Edgar W, McNicol GP. Character- 424-32. isation of fibrin degradation products in patients 46 Gaffney PJ, Joe F. The lysis of crosslinked human on ancrod therapy: comparison with fibrinogen fibrin by plasmin yields initially a single molecular derivatives produced by plasmin. Br J Haematol complex, D dimer-E. Thromb Res 1979;15:673-97. 1974;27:77-87. 47 Kudiyk BJ, Collen D, Woods KR, Blomback B. 60 Edgar W, Warrell MJ, Warrell DA, Prentice CRM. Evidence for localization of polymerization sites in The structure of soluble fibrin complexes and fibrin fibrinogen. J Biol Chem 1974;249 :3322-5. degradation products after Echis carinatus bite. http://jcp.bmj.com/ 48 Olexa SA, Budzynski AZ. Primary soluble plasmic Br J Haematol 1980 (in press). degradation product of human cross-linked fibrin. 61Astrup T. The biological significance of fibrinolysis. Isolation and stoichiometry of the (DD)E complex. Lancet 1956 ;2:565-8. Biochemistry 1979 ;18 :991-5. 62 Nossel HL, Canfield R, Kaplan K, et al. Fibrinogen 43 Budzynski AZ, Marder VJ, Parker ME, Shames P, BP chain proteolysis by thrombin and plasmin-a Brizuela BS, Olexa SA. Antigenic markers on regulatory mechanism in hemostasis and throm- fragment DD, a unique plasmic derivative of human bosis. Thromb Haemostas 1979 ;42 :427. on September 27, 2021 by guest. Protected