Fibronectin decreases the stimulatory effect of fibrin and fibrinogen fragment FCB-2 on plasmin formation by tissue plasminogen activator.

Fibronectin is a dimeric glycoprotein (Mr 440,000) involved in many adhesive processes. During blood coagulation it is bound and cross-linked to fibrin. Fibrin binding is achieved by structures (type I repeats) which are homologous to the "finger" domain of tissue plasminogen activator. Tissue plasminogen activator also binds to fibrin via the finger domain and additionally via the "kringle 2" domain. Fibrin binding of tissue plasminogen activator results in stimulation of its activity and plays a crucial role in fibrinolysis. Since fibronectin might interfere with this binding, we studied the effect of fibronectin on plasmin formation by tissue plasminogen activator. In the absence of fibrin, fibronectin had no effect on plasminogen activation. In the presence of stimulating fibrinogen fragment FCB-2, fibronectin increased the duration of the initial lag phase (= time period until maximally stimulated plasmin formation occurs) and decreased the rate of maximal plasmin formation which occurs after that lag phase mainly by increasing the Michaelis constant (Km). These effects of fibronectin were dose-dependent and were similar with single- and two-chain tissue plasminogen activator. They were also observed with plasmin-pretreated FCB-2. An apparent Ki of 43 micrograms/ml was calculated for the inhibitory effect of fibronectin when plasminogen activation by recombinant single-chain tissue plasminogen activator was studied in the presence of 91 micrograms/ml FCB-2. When a recombinant tissue plasminogen activator mutant lacking the finger domain was used in a system containing FCB-2, no effect of fibronectin was seen, indicating that the inhibitory effect of fibronectin might in fact be due to competition of fibronectin and tissue plasminogen activator for binding to fibrin(ogen) via the finger domain.     

Bivalency of plasminogen monoclonal antibodies is required for plasminogen bridging to fibrin and enhanced plasmin formation

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+/-0.22 micromol/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.     

Specific binding of plasminogen to vitronectin. Evidence for a modulatory role of vitronectin on fibrin(ogen)-induced plasmin formation by tissue plasminogen activator.

Vitronectin immobilized onto polystyrene microtiter wells was demonstrated to specifically bind plasminogen in a concentration-dependent manner, yielding an estimated KD = 0.4 microM. Heparin only moderately interfered with the vitronectin-plasminogen interaction, whereas high concentrations of 6-amino-hexanoic acid inhibited binding. Utilizing a ligand-blotting procedure in which plasminogen was reacted with proteolytic fragments of vitronectin, transblotted onto nitrocellulose, the plasminogen-binding site of vitronectin was localized to the heparin-binding domain of the adhesive protein. Moreover, vitronectin was found to inhibit in a dose-dependent fashion the fibrin(ogen)-induced activation of plasminogen by tissue plasminogen activator. These results provide the first evidence for a novel vitronectin-mediated control of plasminogen activation potentially relevant for directional clot-lysis and plasmin-dependent proteolysis in extracellular matrices.     

Incorporation of fragment X into fibrin clots renders them more susceptible to lysis by plasmin

Bleeding, the most serious complication of thrombolytic therapy with tissue-type plasminogen activator (t-PA), is thought to result from lysis of fibrin in hemostatic plugs and from the systemic lytic state caused by unopposed plasmin. One mechanism by which systemic plasmin can impair hemostasis is by partially degrading fibrinogen to fragment X, a product that retains clottability but forms clots with reduced tensile strength that stimulate plasminogen activation by t-PA more than fibrin clots. The purpose of this study was to elucidate potential mechanisms by which fragment X accelerates t-PA-mediated fibrinolysis. In the presence of t-PA, clots containing fragment X were degraded faster than fibrin clots and exhibited higher rates of plasminogen activation. Although treatment with carboxypeptidase B, an enzyme that reduces plasminogen binding to fibrin, prolonged the lysis times of fragment X and fibrin clots, clots containing fragment X still were degraded more rapidly. Furthermore, plasmin or trypsin also degraded clots containing fragment X more rapidly than fibrin clots, suggesting that this effect is largely independent of plasminogen activation. Fragment X-derived degradation products were not preferentially released by plasmin from clots composed of equal concentrations of fibrinogen and fragment X, indicating that fragment X does not constitute a preferential site for proteolysis. These data suggest that structural changes resulting from incorporation of fragment X into clots promote their lysis. Thus, attenuation of thrombolytic therapy-induced fragment X formation may reduce the risk of bleeding.     

Quantitative characterization of the binding of plasminogen to intact fibrin clots, lysine-sepharose, and fibrin cleaved by plasmin

The binding of human Glu- and Lys-plasminogens to intact fibrin clots, to lysine-Sepharose, and to fibrin cleaved by plasmin was quantitatively characterized. On intact fibrin clots, there was one strong binding site for Glu-plasminogen with a dissociation constant, Kd, of 25 microM and one strong binding site for Lys-plasminogen with a Kd of 7.9 microM. In both cases, the number of plasminogen binding sites per fibrin monomer was 1. Also, a much weaker binding site for Glu-plasminogen was observed with a Kd of about 350 microM. Limited digestion of fibrin by plasmin created additional binding sites for plasminogen with Kd values similar to the binding of plasminogen to lysine-Sepharose. This was predictable given the observations that plasminogen binds to lysine-Sepharose and can be eluted with epsilon-aminocaproic acid [Deutsch, D.G., & Mertz, E.T. (1970) Science (Washington, D.C.) 170, 1095-1096] and that plasmin preferentially cleaves fibrin at the carboxy side of lysyl residues [Weinstein, M.J., & Doolittle, R.F. (1972) Biochim. Biophys. Acta 258, 577-590], because the structures of the lysyl moiety in lysine-Sepharose and of epsilon-aminocaproic acid are identical with the structure of a COOH-terminal lysyl residue created by plasmin cleavage of fibrin. The Kd for the binding of Glu-plasminogen to lysine-Sepharose was 43 microM and for fibrin partially cleaved by plasmin 48 microM. The Kd for the binding of Lys-plasminogen to lysine-Sepharose was 30 microM. With fibrin partially cleaved by plasmin, there were two types of binding sites for Lys-plasminogen, one with a Kd of 7.6 microM and the other with a Kd of 44 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibrin and plasminogen structures essential to stimulation of plasmin formation by tissue-type plasminogen activator

Plasminogen activation catalysed by tissue-type plasminogen activator (t-PA) has been examined in the course of concomitant fibrin formation and degradation. Plasmin generation has been measured by the spectrophotometric method of Petersen et al. (Biochem. J. 225 (1985) 149-158), modified so as to allow for light scattering caused by polymerized fibrin. Glu1-, Lys77- and Val442-plasminogen are activated in the presence of fibrinogen, des A- and des AB-fibrin and the rate of plasmin formation is found to be greatly enhanced by both des A- and des AB-fibrin polymer. Plasmin formation from Glu1- and Lys77-plasminogen yields a sigmoidal curve, whereas a linear increase is obtained with Val442-plasminogen. The rate of plasmin formation from Glu1- and Lys77-plasminogen declines in parallel with decreasing turbidity of the fibrin polymer effector. In order to study the effect of polymerization, this has been inhibited by the synthetic polymerization site analogue Gly-Pro-Arg-Pro, by fibrinogen fragment D1 or by prior methylene blue-dependent photooxidation of the fibrinogen used. Inhibition of polymerization by Gly-Pro-Arg-Pro reduces plasmin generation to the low rate observed in the presence of fibrinogen. Antipolymerization with fragment D1 or photooxidation has the same effect on Glu1-plasminogen activation, but only partially reduces and delays the stimulatory effect on Lys77- and Val442-plasminogen activation. The results suggest that protofibril formation (and probably also gelation) of fibrin following fibrinopeptide release is essential to its stimulatory effect. The gradual increase and subsequent decline in the rate of plasmin formation from Glu1- or Lys77-plasminogen during fibrinolysis may be explained by sequential exposure, modification and destruction of different t-PA and plasminogen binding sites in fibrin polymer.     

Functional role of Bbeta-chain N-terminal fragment in the fibrin polymerization process

Four mAbs of the IgG(1) class to the thrombin-treated N-terminal disulfide knot of fibrin, secreted by various hybridomas, have been selected. Epitopes for two mAbs, I-3C and III-10d, were situated in human fibrin fragment Bbeta15-26, and those for two other mAbs, I-5G and I-3B, were in fragment Bbeta26-36. Three of these mAbs, I-5G, I-3B and III-10D, as well as their Fab-fragments, decreased the maximum rate of fibrin desAA and desAABB polymerization up to 90-95% at a molar ratio of mAb (or Fab-fragment) to fibrin of 1 or 2. The fourth mAb, I-3C, did not influence the fibrin desAABB polymerization and inhibited by 50% the maximum rate of fibrin desAA polymerization. These results suggest that these mAb inhibitors block a longitudinal fibrin polymerization site. As the mAbs retard both fibrin desAABB and fibrin desAA polymerization, one can conclude that the polymerization site does not coincide with polymerization site 'B' (Bbeta15-17). To verify this suggestion, the polymerization inhibitory activity of synthetic peptides BbetaSARGHRPLDKKREEA(12-26), BbetaLDKKREEA(19-26), BbetaAPSLRPAPPPI(26-36), BbetaAPSLRPAPPPISGGGYRARPA(26-46) and BbetaGYRARPA(40-46), which imitate the various sequences in the N-terminal region of the fibrin Bbeta-chain, have been investigated. Peptides Bbeta12-26 and Bbeta26-46, but not Bbeta40-46, Bbeta19-26, and Bbeta26-36, proved to be specific inhibitors of fibrin polymerization. The IC(50) values for Bbeta12-26 and Bbeta26-46 were 2.03 x 10(-4) and 2.19 x 10(-4) m, respectively. Turbidity and electron microscopy data showed that peptides Bbeta12-26 and Bbeta26-46 inhibited the fibrin protofibril formation stage of fibrin polymerization. The conclusion was drawn that fibrin fragment Bbeta12-46 took part in fibrin protofibril formation simultaneously with site 'A' (Aalpha17-19) prior to removal of fibrinopeptide B. A model of the intermolecular connection between fragment Bbeta12-46 of one fibrin desAA molecule and the D-domain of another has been constructed.     

A critical role of lysine residues in the stimulation of tissue plasminogen activator by denatured proteins and fibrin clots

Two-chain 70 000-dalton plasminogen activator of tissue origin displays only weak activity toward plasminogen in a two-component system. The rate of activation is enhanced a minimum of 50-fold by the presence of fibrin clots or denatured proteins. The stimulation must depend on both chemical determinants and spatial configuration, since native proteins, including fibrinogen, lack significant stimulatory activity. These studies employed chemical modifications of four stimulatory proteins (fibrin, denatured fibrinogen, denatured IgG and denatured ovalbumin) to identify a critical role for lysine residues. Arginine, aspartic acid, cysteine, cystine, glutamic acid, histidine, methionine, tyrosine and tryptophan were found not to be essential. The critical spatial determinant(s) remain(s) unknown.     

Identification of the domains of tissue-type plasminogen activator involved in the augmented binding to fibrin after limited digestion with plasmin

The binding of recombinant tissue-type plasminogen activator (rt-PA) to fibrin increases upon digestion of fibrin with plasmin. Optimal binding is observed following a limited plasmin digestion of fibrin, coinciding with the generation of fibrin fragment X polymers. We studied the involvement of the separate domains of the amino-terminal "heavy" (H) chain of rt-PA in this augmentation of fibrin binding. The fibrin-binding characteristics of a set of rt-PA deletion mutants, lacking either one or more of the structural domains of the H chain, were determined on intact fibrin matrices and on fibrin matrices that were subjected to limited digestion with plasmin. The augmented fibrin binding of rt-PA is partially abolished when the plasmin-degraded fibrin matrices are subsequently treated with carboxypeptidase B, demonstrating that this increased binding is dependent on the generation of carboxyl-terminal lysine residues in the fibrin matrix. Evidence is provided that this increase of fibrin binding is mediated by the kringle 2 (K2) domain that contains a lysine-binding site. Further increase of the fibrin binding of rt-PA is independent of the presence of carboxyl-terminal lysines. It is shown that the latter increase is not mediated by the K2 domain. Based on our data, we propose that the increase in fibrin binding, unrelated to the presence of carboxyl-terminal lysine residues, is mediated by the finger (F) domain, provided that this domain is correctly exposed in the remainder of the protein.     

Interaction of plasminogen and fibrin in plasminogen activation

Glu1-, Lys77-, miniplasminogens, kringle 1-3, kringle 1-5A, and kringle 1-5R were able to bind with fibrin, while microplasminogen and kringle 4 did not bind significantly. Kringle 1-5A, but not kringle 1-3, effectively inhibited the binding of Glu1-, Lys77-, and miniplasminogens with fibrin. Miniplasminogen also inhibited the binding of Glu1-plasminogen with fibrin. The binding of kringle 1-3 with fibrin was blocked by mini- or Glu1-plasminogen. It is therefore evident that there are two fibrin-binding domains in plasminogen and that the one in kringle 5 is of higher affinity than that in kringle 1-3. CNBr cleavage products of fibrinogen effectively enhanced the activation of Glu1-, Lys77-, or miniplasminogens, but not microplasminogen, by tissue-type plasminogen activator. Kringle 1-5, but not kringle 1-3, dose-dependently inhibited the enhancement by fibrinogen degradation products of Glu1-plasminogen activation by the activator. Lysine and epsilon-aminocaproic acid could inhibit the binding of plasminogens and plasminogen derivatives with fibrin and block the enhancement effect of fibrinogen degradation products on plasminogen activation. The data clearly illustrate that the binding of plasminogen with fibrin, mainly determined by kringle 5, is essential for effective activation by tissue-type plasminogen activator. However, the presence of kringle 1-4 in the plasminogen molecule is required for the full enhancing effect since the kcat/Km of miniplasminogen activation in the presence of fibrinogen degradation products was 8.2 microM-1 min-1 which is significantly less than 52.0 microM-1 min-1 of Glu1-plasminogen.     

Amino-acid sequence of the cyanogen-bromide fragment from human plasminogen that forms the linkage between the plasmin chains.

The complete amino acid sequence of a cyanogen bromide fragment (122 residues) obtained from plasminogen is described. This fragment forms the overlap between heavy (A) and light (B) chains of human plasmin. The particular arginyl-valyl bond cleaved in the second step of the activation process is shown to be Arg98-Val99 in this fragment. This site is not very similar to the one in the NH2-terminal part of the molecule (Arg68-Met69). Remarkable homologies with the 'triple loops' ('kringle structures') found in the non-thrombin part of prothrombin are demonstrated. Homologies occurred during evolution of this chain.     

The role of DL and DH degradation products in the reactions of the plasmin hydrolysis of fibrinogen and fibrin]

The mechanism of effect of plasmin hydrolysis degradation products, fragments DL and DH, on fibrinolysis and fibrinogenolysis processes was investigated on the basis of their various structural and functional characteristics. Using electrophoresis of unreduced samples and degradation products concentrations changing kinetics, DH was shown to be the only fragment which possessed an antifibrinolytic effect. Rauleigh's light scattering analysis indicated the ability of fragments DL and DH to co-form with plasminogen reversible equimolar complexes.     

Binding of synthetic B knobs to fibrinogen changes the character of fibrin and inhibits its ability to activate tissue plasminogen activator and its destruction by plasmin

Synthetic peptides corresponding to the amino-terminal sequence of the beta chain of fibrin increase the turbidity of fibrin clots, whether they are generated by the direct interaction of thrombin and fibrinogen or by the reassociation of fibrin monomers. The turbidity of batroxobin-induced clots, which are characteristically "fine," is increased even more dramatically. Pentapeptides are more effective than tetrapeptides. Surprisingly, the same peptides also delay fibrinolysis, whether activated by exogenously added plasmin or from the fibrin-enhanced stimulation of tissue plasminogen activator (tPA) activation of plasminogen. The peptides have only a very slight effect on the plasmic hydrolysis of a chromogenic peptide, either by the direct addition of plasmin or by plasmin generated from plasminogen by tPA. The synthetic peptides mimicking the B knobs appear to exert their action in two ways. First, they render fibrin less vulnerable to attack by plasmin. Second, they delay the fibrin activation of tPA. The latter is attributed to their ability to prevent the binding of the authentic B knob, which itself is located at the end of a flexible 50-residue tether and which needs time to find its elusive "hole". We propose that, when after a while the tethered knob does become inserted, it locks the betaC domain in a conformation that allows access to tPA-plasminogen-binding sites, whereas the untethered synthetic knobs restrict the fibrin to a conformation in which those sites remain inaccessible. Thus, although the interaction involving the A knob and gammaC hole is the basis for the polymerization of fibrin, the comparable but delayed interaction involving the B knob and the betaC hole is ultimately directed at preparing the clot for its eventual destruction.     

The role of tissue plasminogen activator in methamphetamine-related reward and sensitization

In the central nervous system, tissue plasminogen activator (tPA) plays a role in synaptic plasticity and remodeling. Our recent study has suggested that tPA participates in the rewarding effects of morphine by regulating dopamine release. In this study, we investigated the role of tPA in methamphetamine (METH)-related reward and sensitization. Repeated METH treatment dose-dependently induced tPA mRNA expression in the frontal cortex, nucleus accumbens, striatum and hippocampus, whereas single METH treatment did not affect tPA mRNA expression in these brain areas. The METH-induced increase in tPA mRNA expression in the nucleus accumbens was completely inhibited by pre-treatment with R(+)-SCH23390 and raclopride, dopamine D1 and D2 receptor antagonists, respectively. In addition, repeated METH treatment increased tPA activity in the nucleus accumbens. There was no difference in METH-induced hyperlocomotion between wild-type and tPA-deficient (tPA-/-) mice. On the other hand, METH-induced conditioned place preference and behavioral sensitization after repeated METH treatment were significantly reduced in tPA-/- mice compared with wild-type mice. The defect of behavioral sensitization in tPA-/- mice was reversed by microinjections of exogenous tPA into the nucleus accumbens. Our findings suggest that tPA is involved in the rewarding effects as well as the sensitization of the locomotor-stimulating effect of METH.     

Identification of the mechanism responsible for the increased fibrin specificity of TNK-tissue plasminogen activator relative to tissue plasminogen activator

TNK-tissue plasminogen activator (TNK-t-PA), a bioengineered variant of tissue-type plasminogen activator (t-PA), has a longer half-life than t-PA because the glycosylation site at amino acid 117 (N117Q, abbreviated N) has been shifted to amino acid 103 (T103N, abbreviated T) and is resistant to inactivation by plasminogen activator inhibitor 1 because of a tetra-alanine substitution in the protease domain (K296A/H297A/R298A/R299A, abbreviated K). TNK-t-PA is more fibrin-specific than t-PA for reasons that are poorly understood. Previously, we demonstrated that the fibrin specificity of t-PA is compromised because t-PA binds to (DD)E, the major degradation product of cross-linked fibrin, with an affinity similar to that for fibrin. To investigate the enhanced fibrin specificity of TNK-t-PA, we compared the kinetics of plasminogen activation for t-PA, TNK-, T-, K-, TK-, and NK-t-PA in the presence of fibrin, (DD)E or fibrinogen. Although the activators have similar catalytic efficiencies in the presence of fibrin, the catalytic efficiency of TNK-t-PA is 15-fold lower than that for t-PA in the presence of (DD)E or fibrinogen. The T and K mutations combine to produce this reduction via distinct mechanisms because T-containing variants have a higher K(M), whereas K-containing variants have a lower k(cat) than t-PA. These results are supported by data indicating that T-containing variants bind (DD)E and fibrinogen with lower affinities than t-PA, whereas the K and N mutations have no effect on binding. Reduced efficiency of plasminogen activation in the presence of (DD)E and fibrinogen but equivalent efficiency in the presence of fibrin explain why TNK-t-PA is more fibrin-specific than t-PA.