Fibrinolysis is amplified by converting ? 2 -antiplasmin from a plasmin inhibitor to a substrate (original) (raw)
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Fibrinolysis is amplified by converting α2‐antiplasmin from a plasmin inhibitor to a substrate
Journal of Thrombosis and Haemostasis, 2007
a 2-antiplasmin (a 2-AP) is the fast serpin inhibitor of plasmin and appears to limit the success of treatment for thrombosis. We examined the mechanisms through which monoclonal antibodies (mAbs) against a 2-AP amplify fibrinolysis. The mAbs RWR, 49 and 77 interfered with the ability of a 2-AP to inhibit plasmin, microplasmin and trypsin. In solution, mAbs 49 and 77 bound to a 2-AP with 5-fold to 10-fold higher relative affinity than mAb-RWR, while mAb-RWR bound with greater avidity to immobilized or denatured a 2-AP. Binding studies with chimeric a 2-APs revealed that none of the mAbs bound to sites in a 2-AP that form putative contacts with plasmin, namely the carboxy terminal lysines of a 2-AP, or the reactive center loop in the serpin domain of a 2-AP. Rather, mAb-RWR recognized an epitope in the amino-terminus of a 2-AP (L 13 GNQEPGGQTALKSPPGVCS 32) near the site at which a 2-AP cross-links to fibrin. mAbs 49 and 77 bound to another conformational epitope in the serpin domain of a 2-AP. mAbs 49 and 77 markedly increased the stoichiometry of plasmin inhibition by a 2-AP (from 1.1 ± 0.1 to 51 ± 4 and 67 ± 7) indicating that they convert a 2-AP from an inhibitor to a substrate of plasmin. This was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis analysis showing cleavage of a 2-AP by plasmin in the presence of these mAbs. In summary, these mAbs appear to act at sites distinct from known a 2-AP-plasmin contacts to enhance fibrinolysis by converting a 2-AP from an inhibitor to a plasmin substrate.
Biochemistry, 2010
Covalent incorporation (cross-linking) of plasmin inhibitor R 2-antiplasmin (R 2-AP) into fibrin clots increases their resistance to fibrinolysis. We hypothesized that R 2-AP may also interact noncovalently with fibrin prior to its covalent cross-linking. To test this hypothesis, we studied binding of R 2-AP to fibrin(ogen) and its fragments by an enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance. The experiments revealed that R 2-AP binds to polymeric fibrin and surface-adsorbed fibrin(ogen), while no binding was observed with fibrinogen in solution. To localize the R 2-AP-binding sites, we studied the interaction of R 2-AP with the fibrin(ogen)-derived D 1 , D-D, and E 3 fragments, and the recombinant RC region and its constituents, RC connector and RC domain and its subdomains, which together encompass practically the whole fibrin(ogen) molecule. In the ELISA, R 2-AP bound to immobilized D 1 , D-D, RC region, RC domain, and its C-terminal subdomain. The binding was Lys-independent and was not inhibited by plasminogen or tPA. Furthermore, the affinity of R 2-AP for D-D was significantly increased in the presence of plasminogen, while that to the RC domain remained unaffected. Altogether, these results indicate that the fibrin(ogen) D region and the C-terminal subdomain of the RC domain contain high-affinity R 2-AP-binding sites that are cryptic in fibrinogen and exposed in fibrin or adsorbed fibrinogen, and the presence of plasminogen facilitates interaction of R 2-AP with the D regions. The discovered noncovalent interaction of R 2-AP with fibrin may contribute to regulation of the initial stage of fibrinolysis and provide proper orientation of the cross-linking sites to facilitate covalent cross-linking of R 2-AP to the fibrin clot.
Journal of Biological Chemistry, 2004
Activated thrombin-activable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase B-like plasma enzyme that can slow clot lysis by removing lysine residues exposed on fibrin as it is cleaved by plasmin. Previously, it was shown that fibrin treated with TAFIa is less able to promote plasminogen activation by tissue-type plasminogen activator. In this study, the effect of TAFIa modification of a fibrin surface on the rate of plasmin inhibition by antiplasmin was studied using high molecular weight fibrin degradation products (HMw-FDPs) as a soluble model for intact plasmin-modified fibrin. To quantify the inhibition, a novel end point assay was employed where plasmin, antiplasmin, and cofactors were mixed in the presence of a chromogenic substrate and the end point in the substrate hydrolysis reaction was used to measure the second order rate constant of inhibition. When HMw-FDPs were titrated in the presence of plasmin and antiplasmin, the rate constant for inhibition decreased by 16-fold at saturation (9.6 ؋ 10 6 M ؊1 s ؊1 to 0.59 ؋ 10 6 M ؊1 s ؊1). When HMw-FDPs were pretreated with TAFIa, nearly two-thirds of the protective effect was lost. When 730 nM HMw-FDPs were treated for 20 min with TAFIa, the rate constant for plasmin inhibition was increased 3-fold from 1.9 ؋ 10 6 M ؊1 s ؊1 to 6.2 ؋ 10 6 M ؊1 s ؊1. Therefore, a novel mechanism was identified whereby TAFIa can modulate plasmin levels by increasing the susceptibility of plasmin to inhibition by antiplasmin.
Biochemistry, 2010
Covalent incorporation (cross-linking) of plasmin inhibitor α 2-antiplasmin (α 2-AP) into fibrin clots increases their resistance to fibrinolysis. We hypothesized that α 2-AP may also interact noncovalently with fibrin prior to its covalent cross-linking. To test this hypothesis, we studied binding of α 2-AP to fibrin(ogen) and its fragments by ELISA and Surface Plasmon Resonance. The experiments revealed that α 2-AP binds to polymeric fibrin and surface-adsorbed fibrin(ogen) while no binding was observed with fibrinogen in solution. To localize the α 2-AP-binding sites, we studied the interaction of α 2-AP with the fibrin(ogen)-derived D 1 , D-D and E 3 fragments, and the recombinant αC region and its constituents, αC-connector and αC-domain and its sub-domains, which together encompass practically the whole fibrin(ogen) molecule. In ELISA, α 2-AP bound to immobilized D 1 , D-D, αC region, αC-domain and its C-terminal sub-domain. The binding was Lys-independent and was not inhibited by plasminogen or tPA. Furthermore, the affinity of α 2-AP to D-D was significantly increased in the presence of plasminogen while that to the αC-domain remained unaffected. Altogether, these results indicate that the fibrin(ogen) D region and the Cterminal sub-domain of the αC-domain contain high affinity α 2-AP-binding sites that are cryptic in fibrinogen and exposed in fibrin or adsorbed fibrinogen, and the presence of plasminogen facilitates interaction of α 2-AP with the D regions. The discovered non-covalent interaction of α 2-AP with fibrin may contribute to regulation of the initial stage of fibrinolysis and provide proper orientation of the cross-linking sites to facilitate covalent cross-linking of α 2-AP to the fibrin clot.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2002
Binding of plasminogen to fibrin and cell surfaces is essential for fibrinolysis and pericellular proteolysis. We used surface plasmon resonance and enzyme kinetic analyses to study the effect of two mAbs (A10.2, CPL15) on plasminogen binding and activation at fibrin surfaces. A10.2 is directed against the lysine-binding site (LBS) of kringle 4, whereas CPL15 recognises a region in kringle 1 outside the LBS. In the presence of CPL15 and A10.2 mAbs, binding of plasminogen (K d = 1.16 F 0.22 Amol/l) to fibrin was characterised by a mAb concentration-dependent bell-shaped isotherm. A progressive increase in the concentration of mAbs at the surface was also detected, and reached a plateau corresponding to the maximum of plasminogen bound. These data indicated that at low mAb concentration, bivalent plasminogen-mAb-plasminogen ternary complexes are formed, whereas at high mAb concentration, a progressive shift to monovalent plasminogen-mAb binary complexes is observed. Plasmin formation in the presence of mAbs followed a similar bell-shaped profile. Monovalent Fab fragments of mAb A10.2 showed no effect on the binding of plasminogen, confirming the notion that a bivalent mAb interaction is essential to increase plasminogen binding and activation at the surface of fibrin.
Fibrinolysis, 1993
Fibrin enhances the rate of plasminogen activation by t-PA. We have described a site in fibrin(ogen), i.e. Aa-(14%160), which can mimic part of the rate enhancement induced by fibrin. During the fibrinogen-to-fibrin conversion, Ao-(14fM60) appears to become accessible to proteins, since monoclonal antibodies against synthetic Ao-(148-160) react with fibrin, but not with fibrinogen. In previous publications we have reported on the role of position Aa-157. In this study we investigated the role of "%a1 on the rate-enhancing effect of Aa-(M-160). ls2Val was replaced by charged residues (Arg, Lys, Glu). Also incorporated were some uncharged polar residues (Ser, Tyr), uncharged nonpolar residues (Ala, Nle), and Gly and Pro. The results clearly indicate that, to maintain stimulatory activity, Val (which possesses an uncharged nonpolar side chain) at position Aa-may be exchanged by another uncharged nonpolar residue, e.g. Ala or Nle or polar residues e.g. Tyr (with its aromatic, though polar side chain) or Ser. With the amino acids Gly, Glu, Arg, Lys or Pro at position Ad-152 the peptide becomes virtually inactive. Our results indicate that the amino acid residue at position Aais important for (inducibility of) a three-dimensional structure in Aa-(148-160) which is required for stimulatory activity of the peptide. An activated coagulation system leads to the formation of thrombin from its precursor prothrombin. Thrombin converts the soluble blood protein fibrinogen to insoluble fibrin. Up to a limited concentration fibrin is, however, kept in solution by complexing with fibrinogen. At higher concentrations, fibrin starts to aggregate and forms the insoluble protein matrix of a blood clot. Fibrin has only a temporary function; after it has fulfilled its role, e.g. in tissue repair, it will be converted to soluble degradation products, with concomitant lysis of the blood clot. The degradation of fibrin is catalysed by plasmin, the product of an activated fibrinolytic system. Plasminogen, the precursor of plasmin, is activated by plasminogen activators such as tissuetype plasminogen activator (t-PA).'** Fibrin is not merely the substrate of fibrinolysis, but is also a cofactor in the fibrinolytic system i.e. it enhances the rate of plasmin formation. Both t-PA and plasminogen interact with fibrin, and the accelerating effect of fibrin on the plasminogen activation by t-PA may be explained by these interactions.3 Fibrinogen has been
Biochemical Journal, 1990
The mechanism of activation of human Glu-plasminogen by fibrin-bound tissue-type plasminogen activator (t-PA) in a plasma environment or in a reconstituted system was characterized. A heterogeneous system was used, allowing the setting of experimental conditions as close as possible to the physiological fibrin/plasma interphase, and permitting the separate analysis of the products present in each of the phases as a function of time. The generation of plasmin was monitored both by spectrophotometric analysis and by radioisotopic analysis with a plasmin-selective chromogenic substrate and radiolabelled Glu-plasminogen respectively. Plasmin(ogen)-derived products were identified by SDS/PAGE followed by autoradiography and/or immunoblotting. When the activation was performed in a plasma environment, the products identified on the fibrin surface were Glu-plasmin (90 %) and Glu-plasminogen (10 %), whereas in the soluble phase only complexes between Glu-plasmin and its fast-acting inhibitor were detected. Identical results were obtained with a reconstituted system comprising solid-phase fibrin, t-PA, Glu-plasminogen and a2-antiplasmin. In contrast, when a2-antiplasmin was omitted from the solution, Lys-plasmin was progressively generated on to the fibrin surface (30 %) and released to the soluble phase. In the presence of a2-antiplasmin or in plasma, the amount of active plasmin generated on the fibrin surface was lower than in the absence of the inhibitor: in a representative experiment the initial velocity of plasmin generation was 2.8 x 10-3, 2.0 x 10-3 and 1.8 x 10-3 (AA405/min) for 200 nM-plasminogen, 200 nM-plasminogen plus 100 nM-a2-antiplasmin and native plasma respectively. Our results indicate that in plasma or in a reconstituted purified system containing plasminogen and cx2-antiplasmin at a ratio similar to that found in plasma (1) the activation pathway of native Glu-plasminogen proceeds directly to the formation of Glu-plasmin, (2) Lys-plasminogen is not an intermediate of the reaction and therefore (3) Lys-plasmin is not the final active product. However, in the absence of the inhibitor, Lys-plasmin and probably Lys-plasminogen, which is more readily activated to plasmin than is Gluplasminogen, are generated as well. Abbreviations used: Glu-plasminogen, native human plasminogen with N-terminal glutamic acid; Lys-plasminogen, plasmin-modified forms of Glu-plasminogen with N-terminal lysine, valine or methionine (mostly lysine) obtained by hydrolysis of the Arg-68-Met-69, Lys-76-Lys-77 or Lys-77-Val-78 peptide bonds; Glu-plasmin and Lys-plasmin, activator-modified forms of Glu-plasminogen and Lys-plasminogen respectively; t-PA, tissuetype plasminogen activator; CBS 3308, chromogenic substrate D-norleucylcyclohexylalanylarginine p-nitroanilide; GGACK, dansyl-L-glutamylglycylarginylchloromethane. * To whom correspondence should be addressed.
Overview on fibrinolysis: Plasminogen activation pathways on fibrin and cell surfaces
Chemistry and Physics of Lipids, 1994
Plasminogen activation at the surface of fibrin or of cell membranes is a sophisticated specialized system for localized extracellular proteolysis implicated in a large variety of biological functions (fibrinolysis, cell migration and extracellular matrix degradation). Assembly of plasminogen and/or activators at specific binding sites induces conformational changes that make accessible the scissile peptide bond of plasminogen and exposes the active centre of the tissue-type plasminogen activator. The mechanism of activation by pro-urokinase, a second type of activator that binds to cell membrane but not to fibrin, is far from being understood. It may be able, however, in contrast to urokinase, to specifically activate plasminogen bound to partially degraded fibrin. An extremely low K m and high catalytic rate are characteristic of the process of activation at surfaces. In contrast, activation in liquid phase by tissue-type plasminogen activator proceeds at an extremely low catalytic rate. The initiation and amplification of plasminogen activation depend on specific interactions between the modular constitutive units of these proteins and binding sites present on cell or fibrin surfaces. Thus, the most important mechanism for the acceleration of fibrinolysis and pericellular proteolysis is the unveiling of carboxy-terminal lysine residues on these surfaces, to which plasminogen may bind. Since plasminogen bound to carboxy-terminal lysines of progressively degraded firbrin or membranes is readily transformed into plasmin by fibrin-bound t-PA, this mechanism represents the most important pathway for the acceleration and amplification of fibrinolysis. Alpha-2-antiplasmin, by inhibiting plasmin release from surfaces, regulates the extent and rate of this process but has no effect on fibrin-bound or membrane-bound plasmin. Lipoprotein(a), a particle possessing a plasminogen-like apolipoprotein, apo(a), may interfere with this mechanism by inhibiting the specific binding of plasminogen to lysine residues in membrane or fibrin surfaces.
European Journal of Biochemistry, 1994
It is well established that tissue-type plasminogen activator (t-PA) binds to the D region of fibrin(ogen) and that two distinct CNBr fragments of fibrinogen (FCB), FCB-2 and FCB-5, comprising parts of this region, stimulate plasminogen activation by t-PA. In the present work, ligandbinding studies were performed to characterize the interactions between t-PA and the corresponding fibrin regions using a well defined model of a fibrin surface and both FCB-2 and FCB-5 in liquid and solid phase. Binding isotherms showed a characteristic Langmuir adsorption saturation profile. The dissociation constants determined for the binding of t-PA to immobilized FCB-2 (& = 0.70 2 0.10 nM) and FCB-5 (& = 0.47 * 0.08 nM) were of the same order of magnitude as the Kd for fibrin binding (Kd = 1 ? 0.2 nM). The specificity of the binding was demonstrated by the ability of soluble FCB-2 and FCB-5 to inhibit t-PA binding to solid-phase fibrin (K, = 3.3 pM and 6.4 pM, respectively). The binding of t-PA to fibrin and to immobilized FCB-2 was partially inhibited by the lysine analogue 6-aminohexanoic acid (K, = 123 * 47 pM and 364 pM, respectively) but was not modified by carboxypeptidase B, thus indicating involvement of internal lysine residues. Removal of lysine residues by treatment with, successively, plasmin and carboxypeptidase B, produced only a partial inhibition of t-PA binding, thus confirming the existence of both a lysine-dependent and a lysine-independent mechanism of binding of t-PA to both fibrin and FCB-2. In contrast, the binding of t-PA to F C B J was not significantly affected by 6-aminohexanoic acid. Altogether, these data indicate that the mechanism of binding of t-PA to fibrin involves mainly a lysine-independent interaction with the D region which is contributed by sequences present in FCB-5 and FCB-2; contribution to binding by a lysine-dependent interaction was detected only in FCB-2 and is probably of minor relevance as suggested by the limited effect of 6-aminohexanoic acid.
European Journal …, 1993
A well characterized model of an intact and a degraded surface of fibrin that represents the states of fibrin during the initiation and the progression of fibrinolysis was used to quantitatively characterize the molecular interplay between tissue-type plasminogen activator (t-PA), plasminogen and fibrin. The molecular assembly of t-PA and plasminogen on these surfaces was investigated using combinations of proteins that preclude complications due to side reactions caused by generated plasmin : native plasminogen with di-isopropylphosphofluoridate-inactivated t-PA, and a recombinant human plasminogen with the active-site Ser741 mutagenized to Ala which renders the catalytic site inactive. Under these conditions, neither the affinity nor the maximal number of binding sites for plasminogen were modified by the presence of t-PA, indicating that binding sites for plasminogen pre-exist in intact fibrin and are not dependent on the presence of t-PA. In contrast, when plasminogen activation is allowed, increasing binding of plasminogen to the progressively degraded fibrin surface is directly correlated (1. = 0.98) to the appearance of the fibrin E-fragment as shown using a monoclonal antibody (FDP-14) that has its epitope in the E domain of fibrin. t-PA was shown to bind with a high affinity to both the intact (K, = 3.3 % 0.6 nM) and the degraded surface of fibrin (Kd = 1.2 i 0.4 nM). Binding of t-PA to carboxy-terminal lysine residues of degraded fibrin was shown to be efficiently competed by physiological concentrations of plasminogen (2 pM), indicating that the affinity of t-PA for these residues was lower than that of plasminogen (K,, = 0.66 i 0.22 pM) and unrelated to the high affinity of t-PA for specific binding sites on intact fibrin. These data confirm and establish that the generation of carboxy-terminal lysine residues on fibrin during ongoing fibrinolysis, and the binding of plasminogen to these sites, is an important pathway in the acceleration of clot dissolution. Intravascular fibrinolysis is a heterogeneously catalyzed process triggered by tissue-type plasminogen activator (t-PA) [l], and evolving from the plasmdfibrin interface to the interior of the clot [ 2 ]. In this process, polymerized fibrin acts both as a stimulator of t-PA activity and as a substrate for in situ generated plasmin.