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.