Interaction of fibrinogen and its derivatives with fibrin

The binding between complementary polymerization sites of fibrin monomers plays an essential role in the formation of the fibrin clot. One set of polymerization sites involved in the interaction of fibrin monomers is believed to pre-exist in fibrinogen, while the complementary set of binding sites is exposed after the cleavage of fibrinopeptides from fibrinogen. The polymerization sites present in fibrinogen and its derivatives mediate their binding to fibrin. Although the binding of fibrinogen and its derivatives to fibrin have been qualitatively studied, there has been no systematic, quantitative investigation of their interaction with forming or preformed clots. In the present study, the binding of fibrinogen and fragments DD, D1, and E1 was measured using a sonicated suspension of plasminogen- and thrombin-free human cross-linked fibrin as a model of a preformed clot. Dissociation constants of 0.056, 0.19, and 2.44 microM, and the number of binding sites corresponding to 0.10, 0.21, and 0.13/fibrin monomer unit of fibrin polymer were found for fibrinogen, fragment DD, and fragment D1, respectively. Fragment E1 did not bind to sonicated noncross-linked or cross-linked fibrin suspensions. However, it was bound to forming fibrin clots as well as to fibrin-Celite, suggesting that the binding sites on fibrin involved in the interaction with fragment E1 may have been altered upon sonication. Affinity chromatography of various fibrinogen derivatives on a fibrin-Celite column showed that only part of the bound fragment DD was displaced by arginine, whereas fragments D1 and E1 were completely eluted under the same conditions. The results indicate that interaction of fibrinogen with the preformed fibrin clots is characterized by affinity in the nanomolar range and that binding between fibrin monomers, in the process of clot formation, could be characterized by even a higher affinity.     

Expression of primary polymerization sites in the D domain of human fibrinogen depends on intact conformation

Fragments D1 and DD, plasmic degradation products of human fibrinogen and cross-linked fibrin, respectively, originate from the COOH-terminal domain of the parent molecule. Since a specific binding site for fibrin resides in the COOH-terminal region of the gamma chain, the primary structure of the two fragments was compared and their affinity for fibrin monomer measured. Fragments D1 and DD contained the same segments of the three fibrinogen chains, corresponding to the sequences alpha 105-206, beta 134-461, and gamma 63-411. Fragment DD had a double set of the same chain remnants. Fragments D1 and DD inhibited polymerization of fibrin monomer in a dose-dependent manner; 50% inhibition occurred at a molar ratio of fragment to monomer of 1:1 and 0.5:1, respectively. To prevent fibrin monomer polymerization and render it suitable for binding studies in the liquid phase, fibrinogen was decorated with Fab fragments isolated from rabbit antibodies to human fragment D1. Fibrinogen molecules decorated with 6 molecules of this Fab fragment did not clot after incubation with thrombin, and the decorated fibrin monomer could be used to measure binding of fragments D1 and DD in a homogeneous liquid phase. The data analyzed according to the Scatchard equation and a double-reciprocal plot gave a dissociation constant of 12 nM for fragment D1 and 38 nM for fragment DD. There were two binding sites/fibrin monomer molecule for each fragment. After denaturation in 5 M guanidine HCl, the inhibitory function on fibrin polymerization was irreversibly destroyed. Denatured fragments also lost binding affinity for immobilized fibrin monomer. The preservation of the native tertiary structure in both fragments was essential for the expression of polymerization sites in the structural D domain.     

The interference of plasmic degradation products of human crosslinked fibrin with clot formation.

The role of plasmic degradation products of human crosslinked fibrin on polymerization of fibrin monomer and clot formation was studied. Both reactions were inhibited by Fragment DD, which formed a complex with fibrin monomer in a molar ratio 1 : 1. The rate of polymerization was slightly increased by Fragment E but it was not affected by (DD)E complex and Fragment A. Approximately the same amount of fibrin was formed in the presence and absence of Fragments A, E and the complex. It was concluded that of the degradation products of crosslinked fibrin, only Fragment DD is a potent anticoagulant at physiologic pH. The (DD)E complex is inert and Fragments A and E have only marginal effects.     

The interference of plasmic degradation products of human crosslinked fibrin with clot formation

The role of plasmic degradation products of human crosslinked fibrin on polymerization of fibrin monomer and clot formation was studied. Both reactions were inhibited by Fragment DD, which formed a complex with fibrin monomer in a molar ratio 1 : 1. The rate of polymerization was slightly increased by Fragment E but it was not affected by (DD)E complex and Fragment A. Approximately the same amount of fibrin was formed in the presence and absence of Fragments A, E and the complex. It was concluded that of the degradation products of crosslinked fibrin, only Fragment DD is a potent anticoagulant at physiologic pH. The (DD)E complex is inert and Fragments A and E have only marginal effects.     

Structure of fragment E species from human cross-linked fibrin

Fragments E1, E2, and E3 are plasmic derivatives of fibrin encompassing the NH2-terminal region of the molecule. The first two species, but not the third, can bind to fragment DD, forming a (DD)E complex, and therefore probably contain binding sites involved in the polymerization of fibrin. For localization of these sites the structure of the fragments was determined by establishing the NH2- and COOH-terminal boundaries of the molecules and using the published amino acid sequence of fibrinogen. Fragment E1 encompasses Gly-alpha 17 to Lys-alpha 78, Gly-beta 15 to Lys-beta 122, and Tyr-gamma 1 to Lys-gamma 62, this representing the intact NH2-terminal region of fibrin. Fragment E2 is an asymmetric molecule which is lacking the sequence of Gly-beta 15 to Lys-beta 53 in one beta-chain remnant. This fragment E2 also lost Lys-beta 122 from the COOH terminal of the beta chain as compared with fragment E1. These cleavages did not affect the ability of fragment E2 to bind to fragment DD. Fragment E3 was heterogeneous, the main species encompassing Val-alpha 20 to Lys-alpha 78, Lys-beta 54 to Leu-beta 120, and Tyr-gamma 1 to Lys-gamma 53. Thus, the loss of the binding function involved in the formation of fibrin clot was associated with the removal of small fragments from all three polypeptide chains: alpha 17-19 (Gly-Pro-Arg), beta 15-53 from the remaining half of the molecule, beta 121 (Leu), and gamma 54-58 (Thr-Ser-Glu-Val-Lys).     

Conversion of fibrinogen to fibrin: mechanism of exposure of tPA- and plasminogen-binding sites

Conversion of fibrinogen into fibrin results in the exposure of cryptic interaction sites and modulation of various activities. To elucidate the mechanism of this exposure, we tested the accessibility of the Aalpha148-160 and gamma312-324 fibrin-specific epitopes that are involved in binding of plasminogen and its activator tPA, in several fragments derived from fibrinogen (fragment D and its subfragments) and fibrin (cross-linked D-D fragment and its noncovalent complex with the E(1) fragment, D-D. E(1)). Neither D nor D-D bound tPA, plasminogen, or anti-Aalpha148-160 and anti-gamma312-324 monoclonal antibodies, indicating that their fibrin-specific epitopes were inaccessible. The Aalpha148-160 epitope became exposed only upon proteolytic removal of the beta- and gamma-modules from D. At the same time, both epitopes were accessible in the D-D.E(1) complex, indicating that the DD.E interaction resulted in their exposure. This exposure was reversible since the dissociation of the D-D.E(1) complex made the sites unavailable, while reconstitution of the complex made them exposed. The results indicate that upon fibrin assembly, driven primarily by the interaction between complementary sites of the D and E regions, the D regions undergo conformational changes that cause the exposure of their plasminogen- and tPA-binding sites. These changes may be involved in the regulation of fibrin assembly and fibrinolysis.     

Primary soluble plasmic degradation product of human cross-linked fibrin. Isolation and stoichiometry of the (DD)E complex.

The formation of the (DD)E complex and fragments DD and E upon proteolysis of human cross-linked fibrin was studied by timed digestions using varying amounts of plasmin. The (DD)E complex was the primary soluble degradation product released form cross-linked fibrin. This complex contained fragments DD and E1. Upon further digestion (DD)E1 complex was cleaved to (DD)E2 complex whereby only the fragment E moiety was affected. However, when fragment E2 was digested to fragment E3, dissociation of the complex occurred. Thus, fragments DD and E3 are the terminal plasmic digestion products of cross-linked fibrin. This pattern was consistent regardless of the plasmin to fibrin ratio; however, the rate of production of the terminal degradation products was directly dependent upon enzyme concentration. Digestion conditions were modified so that either the (DD)E complex or fragment DD was the predominant degradation product, allowing for the purification of these species by one-step gel filtration. The molar ratio of fragment DD to fragment E in the (DD)E complex was investigated by dissociation of the complex and by reassociation of the purified components. The (DD)E complex contains one molecule of fragment DD and one molecule of fragment E.     

Department of Protein Structure and Function--Volodimir Oleksandrovich Bielitser School of the Ukrainian Academy of Sciences. Studies on structure of fibrin fiber structure and mechanism (1975-1987

In this short historical review the records about foundation and research activity of the Department of Structure and Function of Protein--school of V. A. Belitser, Member of the National Academy of Sciences of Ukraine are presented. V. A. Belitser was the founder and indispensable chief of the department since the date of its creation (1944) till 1987. The main research interests (1975-1987) of the department were focused at the investigation of structure, biological function of the fibrinogen-fibrin system, mechanisms of the network assembly and of the fibrin fibers structure. Studying the molecular mechanisms of the fibrin fiber assembly, it was shown that the specificity of the building structure was shown is determined by the specific reactive sites with strong affinity of the molecules. The activity of the sites was investigated on protein molecules as well as the fragments. The physical nature of the bonds created by the active sites, that appearing during in the process of fibrinogen activation by thrombin, was revealed. Examination of the fibrin assembly in cooperation with electronmicroscopists and studies of the complex formation between active fragments and fibrin monomer were summarized. Both the fibrin monomer polymerization and protofibril lateral association are presented as two stages in the assembly of the fibrin network. In the research of the domain fibrinogen structure the specific sites of the fibrin assembly in each of the domains were found. COOH-terminal regions of the A alpha-chains play independent part in the fibrinogen and fibrin. That is why it is relevant to consider them as alpha C-domains. In the free fibrinogen molecules (in solution) these domains are responsible for globular shape, they are linked to domains D intramolecularly. When fibrin assembly takes place, alpha C-domains play significant carriage role in fibrin molecules interaction, linking to domains D intermolecularly. The model of the fibrinogen molecule structure and the general scheme of the fibrin fibers network formation were proposed. Physico-chemical basics of a biological structure assembly were elucidated using the process of the fibrin self-assembly as an example. Much attention was devoted to the problems of practical medicine. The quantitative methods of fibrinogen, soluble fibrin and active fibrin/fibrinogen fragments estimation in blood plasma were developed.     

A new direction for anticoagulants: Inhibiting fibrin assembly with PEGylated fibrin knob mimics

Current anticoagulants target coagulation factors upstream from fibrin assembly and polymerization (i.e., formation of fibrin clot). While effective, this approach requires constant patient monitoring since pharmacokinetics and pharmacodynamics vary from patient to patient. To address these limitations, we developed an alternative anticoagulant that effectively inhibits fibrin polymerization. Specifically, we investigated PEGylated fibrin knob “A” peptides, evaluating the effect of both polyethylene glycol (PEG) chain length (0, 2, 5, and 10–30 kDa) and knob peptide sequence (GPRPAAC, GPRPFPAC, and GPRPPERC) on inhibiting fibrin polymerization (i.e., clot formation). Thrombin‐initiated clotting assays with purified fibrinogen were performed to compare clot formation with each peptide–PEG conjugate. Results indicated a biphasic effect of PEG chain length, whereby, active‐PEG conjugates demonstrated increasingly enhanced inhibition of fibrin polymerization from 0 to 5 kDa PEG. However, the anticoagulant activity diminished to control levels for PEG chains above 5 kDa. Ultimately, we observed a 10‐fold enhancement of anticoagulant activity with active peptides PEGylated with 5 kDa PEG compared to non‐PEGylated knob peptides. The sequence of the active peptide significantly influenced the anticoagulant properties only at the highest 1:100 molar ratio where GPRPFPAC‐5 kDa PEG and GPRPPERC‐5 kDa PEG demonstrated significantly lower percent clottable protein than GPRPAAC‐5 kDa PEG. Moreover, human plasma treated with the active 5 kDa PEG conjugate exhibited delayed prothrombin time to within the therapeutic range specified for oral anticoagulants. Collectively, this study demonstrated the utility of PEGylated fibrin knob peptides as potential anticoagulant therapeutics. Biotechnol. Bioeng. 2011;108: 2424–2433. © 2011 Wiley Periodicals, Inc.     

Polymerization site in the beta chain of fibrin: mapping of the B beta 1-55 sequence

The formation of a fibrin clot occurs through binding of putative complementary sites, called fibrin polymerization sites, located in the NH2- and COOH-terminal domains of fibrin monomer molecules. In this study, we have investigated the structure of the NH2-terminal fibrin polymerization site by using fibrinogen-derived peptides and fragments. Fibrinogen was digested with Crotalus atrox protease III, to two major molecular species: a Mr 325,000 derivative (Fg325) and a peptide of Mr 5000. The peptide and its thrombin-cleavage product were purified by ion-exchange and reverse-phase HPLC; the authenticity of the B beta 1-42 and beta 15-42 peptides, respectively, was confirmed by amino acid sequencing. Since Fg325 had decreased thrombin coagulability, we addressed the question of whether the peptide B beta 1-42 contained a fibrin polymerization site. In order to identify and map the site, the peptides B beta 1-42 and beta 15-42 were tested for their ability to inhibit fibrin monomer polymerization. In addition the following peptides prepared by chemical synthesis were also tested: beta 15-18, beta 15-26, beta 24-42, beta 40-54, beta 50-55, and alpha 17-19-Pro. While B beta 1-42 had no inhibitory activity, the peptide devoid of fibrinopeptide B, beta 15-42, was a strong inhibitor. The peptides beta 15-18, beta 15-26, and beta 15-42 decreased the rate of fibrin polymerization by 50% at a molar excess of the peptide to fibrin monomer of 500, 430, and 50, respectively. The peptides beta 24-42, beta 40-54, and beta 50-55 were inactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of a fibrin polymerization site

The formation of a fibrin clot is initiated after the proteolytic cleavage of fibrinogen by thrombin. The enzyme removes fibrinopeptides A and B and generates fibrin monomer which spontaneously polymerizes. Polymerization appears to occur though the interaction of complementary binding sites on the NH2-terminal and COOH-terminal (Fragment D) regions of the molecule. A peptide has been isolated from the gamma chain remnant of fibrinogen Fragment D1 which has the ability to bind to the NH2-terminal region of fibrinogen as well as to inhibit fibrin monomer polymerization. The peptide reduces the maximum rate and extent of the polymerization of thrombin or batroxobin fibrin monomer and increases the lag time. The D1 peptide does not interact with disulfide knot, fibrinogen, or Fragment D1, but it binds to thrombin-treated disulfide knot with a Kd of 1.45 X 10(-6) M at approximately two binding sites per molecule of disulfide knot. Fibrin monomer formed either by thrombin or batroxobin binds approximately two molecules of D1 peptide per molecule of fibrin monomer, indicating that the complementary site is revealed by the loss of fibrinopeptide A. The NH2-terminal sequence (Thr-Arg-Trp) and COOH-terminal sequence (Ala-Gly-Asp-Val) of the D1 peptide were determined. Therefore the gamma 373-410 region of fibrinogen contains a polymerization site which is complementary to the thrombin-activated site on the NH2-terminal region of fibrinogen.     

Binding phenomena of isolated unique plasmic degradation products of human cross-linked fibrin.

Proteolysis of human cross-linked fibrin by plasmin results in the formation of a DD . E complex, and Fragments DD and E as the major degradation products. Three species of Fragment E, which differ both in molecular weights (E1, Mr = 60,000; E2, Mr = 55,000; E3, Mr = 50,000) and in charge, have been isolated from a digest of cross-linked fibrin. Each Fragment E species reacts with monospecific anti-E antiserum. Fragments E1 and E2 bind with Fragment DD to form a DD . E complex but Fragment E3 is inactive. This binding is specific since these Fragments E do not bind to fibrinogen or to degradation products of fibrinogen or of noncross-linked fibrin. Fragments E1 and E2 incubated with plasmin are degraded to Fragment E3, suggesting that the three species represent sequential degradation products. Plasmin-treated Fragments E1 and E2 no longer bind with Fragment DD; therefore, it appears that the peptides cleaved from Fragment E2 by plasmin contain or modify the sites responsible for complex formation. On the other hand, Fragment DD binds not only to Fragments E1 and E2, but also to fibrinogen, Fragments X (Stage 1), X (Stage 2), Y, and NH2-terminal disulfide knot, but only after thrombin treatment, suggesting that Fragment DD binds to complementary sites on the NH2-terminal region of fibrinogen which are exposed after thrombin treatment.     

Three-dimensional structural studies on fragments of fibrinogen and fibrin

Fibrinogen is a 340 kDa glycoprotein found in the blood plasma of all vertebrates. It is transformed into a fibrin clot by the action of thrombin. Recent X-ray structures of core fragments of both fibrinogen and fibrin have revealed many details about this polymerization event. These include structures of a 30 kDa recombinant γC domain, an 86 kDa fragment D from human fibrinogen and a cross-linked double-D fragment from fibrin.     

Mechanism of functioning of specific sites of interdomain D-E binding of fibrin molecules: interaction of fibrinogen with the N-terminal disulfide node of fibrin

The intermolecular noncovalent binding of complementary fibrin polymerization sites localized in fibrin domains D and E was investigated in the model system. In this system fibrinogen molecules represent the active D domains and the N-terminal disulphide knot of fibrin (N-DSK) represents the active E domain. Quantitative definition of insoluble fibrinogen and N-DSK copolymer and light scattering data of their mixtures before the appearance of visible precipitate show that complexing of these structures decreases with an increase of the temperature and ionic strength. The character of this dependence permits certain conclusions to be made on the functioning mechanism for two types of the D-E binding sites. These conclusions are based on an idea of their different affinity. The interdomain binding is primarily realized by D1-E1 sites which are characterized by a high affinity and work mainly on the basis of electrostatic forces. This binding directs the D2-E2 binding which is characterized by lower affinity and which determines the final degree of fibrinogen and N-DSK complexing. These sites function mainly by the H-binding.     

Modification of fibrin network ultrastructure by Fab fragments specific for different domain of fibrinogen

Kinetics of inhibition of fibrin monomer polymerization produced by Fab fragments prepared from immunochemically purified monospecific antibodies to the surface epitopes of different domains of fibrinogen molecule has been correlated with electron microscopic observations of resulting specimens. Fab fragments prepared from anti FgD antisera were the most efficient inhibitors of thrombin-catalysed conversion of fibrinogen to fibrin; polymerization of fibrin monomers as detected spectrophotometrically was abolished at 2:1 molar ratio of anti FgD Fab fragments to fibra monomer. These Fab fragments acting as a steric hindrance of polymerization sites inhibited the first stage of fibrin monomer aggregation. Interaction of Fab fragments derived from antibodies specific for alpha 239-476 with corresponding segment of fibrinogen molecule resulted in a weak inhibition of fibrin monomer polymerization. However, fibrin obtained in the presence of these Fab fragments was significantly modified and showed no periodicity. This observation may suggest that anti alpha 239-476 Fab impaired the course of the second stage of fibrin monomer polymerization, i.e. lateral association of fibrin fibrils.     

Localization of the binding site on fibrin for the secondary binding site of thrombin

Affinity chromatography of active site inhibited thrombin on immobilized fragments derived from the central (desAB-NDSK) and terminal (D1) globular domains of fibrinogen revealed that the site responsible for the binding of thrombin at its secondary fibrin binding site is located in the central domain. Chromatography of various domains of the central nodule (desAB-NDSK, fibrinogen E, and fibrin E) having nonidentical amino acid sequences showed that all of these fragments are capable of binding to PMSF-thrombin-Sepharose, suggesting that the thrombin binding site resides within the peptide regions common to all of these fragments: alpha(Gly17-Met51), beta(Val55-Met118), and gamma(Tyr1-Lys53). Competitive affinity chromatography of the same binding domains revealed that there is no detectable difference in their binding constants to PMSF-thrombin-Sepharose, indicating that the alpha(Lys52-Lys78), beta(Gly15-Lys54)/(Tyr119-Lys122), and gamma(Thr54-Met78) peptide segments do not contribute significantly to the binding of thrombin. Chromatography of the isolated chains of fibrinogen E showed that the alpha(Gly17-Lys78) peptide region itself contains a strong binding site for PMSF-thrombin-Sepharose. The location of the binding site suggests that the secondary site interaction may play an important role in determining the cleavage specificity of thrombin on fibrinogen and can affect the rate of release of the fibrinopeptides. Affinity chromatography of fragments prepared from polymerized fibrin showed that cross-linked DD (D x D) itself does not bind to thrombin, whereas the D x DE complex remained attached to the column, suggesting that the binding site on fragment E for thrombin is distinct from its binding site for D x D.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thrombin-induced fibrinopeptide B release from normal and variant fibrinogens: influence of inhibitors of fibrin polymerization

Thrombin preferentially cleaves fibrinopeptides A (FPA) from fibrinogen resulting in the formation of desAA-fibrin from which most of the fibrinopeptides B (FPB) are then released with an enhanced rate. Kinetics of fibrinopeptide release from normal and dysfunctional fibrinogens were investigated in order to further characterize the mechanism of accelerated FPB release during desAA-fibrin polymerization. Dysfunctional fibrinogens London I and Ashford, exhibiting primary polymerization abnormalities (i.e., an abnormality present when all fibrinopeptides have been cleaved), which in the case of fibrinogen London I is believed to be caused by a defect in the D-domain, were shown to exhibit a decreased rate of FPB release compared with normal fibrinogen. While Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, was shown to decrease the rate of FPB release from normal fibrinogen by a factor of 5, normal fragment D1, although inhibiting clot formation of normal fibrinogen, did not influence the acceleration of FPB release. On the other hand, the presence of fragment D1 did not enhance FPB release from fibrinogen London I, suggesting that interaction of D-domains in functional isolation with desAA-fibrin E-domains is not sufficient to enhance FPB release. Although clot formation was inhibited by the concentrations of fragment D1 used, the formation of small desAA-fibrin oligomers was hardly affected. Thus, small fibrin polymers, but not desAA-fibrin monomers, act as optimal substrates for the release of FPB by thrombin.     

Lysine- and arginyl-binding sites of plasminogen domains

Hydrolysis of plasminogen permits obtaining its nine fragments. The method of differential scanning microcalorimetry reveals seven domains in plasminogen, and the affinity chromatography--three lysin- and three arginyl-binding sites. The lysin-binding sites of domains (Kringles) K1 and K4 differ in ligand specificity. Benzamidine-binding sites of domain K5 and of plasmin light chain are simultaneously arginine-binding ones. The third arginyl-binding site differing from the benzamidine-binding one is found in fragment K1-3. In the plasminogen-fibrin interaction only lysin-binding sites of plasminogen take part; in the plasminogen fragments-fibrinogen fragments interaction both types of plasminogen sites participate. The heavy chain of plasmin interacts with the E-fragment of fibrinogen by the lysin-binding sites, and the light chain of plasmin interacts with D-fragment of fibrinogen by arginyl-binding sites. Sites complementary to arginyl binding sites of plasminogen are located on the DH-fragment and sites of interaction with lysin- and arginyl-binding sites--on the DL-fragment. The plasmin-fibrin interaction mediated by sites of the first four cringles is not associated with changes in the catalytic function of the active centre. Interaction of Lys-plasminogen with fibrin accelerates polymerization of the latter. The effect of Lys-plasminogen is conditioned by the lysin-binding sites. Glu-plasminogen has no effect on the polymerization process.     

Localization of a fibrin polymerization site complementary to Gly-His-Arg sequence

Dansyl-labeled tetrapeptide Gly-His-Arg-Pro which mimics the central fibrin polymerization site was used to investigate its binding to a number of fibrinogen fragments containing different numbers of domains. The tetrapeptide was found to bind to fragments D_H(95 kDa), D_L(82 kDa) and D_Y(63 kDa) but not to the TSD(28 kDa) fragment. The D_Y fragment differs from the TSD by the presence of β and βC domains. Therefore these domains, which are formed by the C-terminal part of the β chain, possess a polymerization site complementary to the Gly-His-Arg containing counterpart.     

Thrombin-activable fibrinolysis inhibitor attenuates (DD)E-mediated stimulation of plasminogen activation by reducing the affinity of (DD)E for tissue plasminogen activator. A potential mechanism for enhancing the fibrin specificity of tissue plasminogen activator

A complex of d-dimer noncovalently associated with fragment E ((DD)E), a degradation product of cross-linked fibrin that binds tissue plasminogen activator (t-PA) and plasminogen (Pg) with affinities similar to those of fibrin, compromises the fibrin specificity of t-PA by stimulating systemic Pg activation. In this study, we examined the effect of thrombin-activable fibrinolysis inhibitor (TAFI), a latent carboxypeptidase B (CPB)-like enzyme, on the stimulatory activity of (DD)E. Incubation of (DD)E with activated TAFI (TAFIa) or CPB (a) produces a 96% reduction in the capacity of (DD)E to stimulate t-PA-mediated activation of Glu- or Lys-Pg by reducing k(cat) and increasing K(m) for the reaction; (b) induces the release of 8 mol of lysine/mol of (DD)E, although most of the stimulatory activity is lost after release of only 4 mol of lysine/mol (DD)E; and (c) reduces the affinity of (DD)E for Glu-Pg, Lys-Pg, and t-PA by 2-, 4-, and 160-fold, respectively. Because TAFIa- or CPB-exposed (DD)E produces little stimulation of Glu-Pg activation by t-PA, (DD)E is not degraded into fragment E and d-dimer, the latter of which has been reported to impair fibrin polymerization. These data suggest a novel role for TAFIa. By attenuating systemic Pg activation by (DD)E, TAFIa renders t-PA more fibrin-specific.