Antibody-directed fibrinolysis: a bispecific (Fab')2 that binds to fibrin and tissue plasminogen activator

A bispecific (Fab')2 molecule was constructed by linking the monovalent Fab' from an anti-fibrin monoclonal antibody to the Fab' from an anti tissue plasminogen activator (tPA, single chain) monoclonal antibody by means of inter-heavy-chain disulfide bonds. An immunochemical complex composed of the bispecific (Fab')2 molecule bound to tPA [tPA-bispecific (Fab')2 complex] was then generated and purified. Its molecular weight was 170 kDa [less than half the molecular weight of a previously described tPA-bispecific antibody complex containing the entire anti-fibrin and anti-tPA immunoglobulin molecules; Runge, M. S., et al. (1987) Trans. Assoc. Am. Phys. 100, 250-255]. The tPA-bispecific (Fab')2 complex was 8.6-fold more efficient in fibrinolysis than tPA alone and 94-fold more potent than urokinase. This enhancement in the fibrinolytic potency of tPA compares favorably with that observed for the bispecific whole-antibody complex. These results suggest that this smaller, less immunogenic molecule is capable of binding both fibrin and tPA with high affinity and of enhancing the thrombolytic efficiency of exogenous and, perhaps, endogenous tPA.     

PC-12 cells hydrolyze the fibrin clot by producing both plasminogen and its tissue activator

It has been shown that rat pheochromocytoma PC-12 cells degraded fibrin clots in vitro. SDS-PAGE performed in the gels of different density and Western blotting using monoclonal antibodies against DD- and D-fibrin fragments demonstrated that both high and low molecular weight degradation products similar to those of fibrin hydrolysis by plasmin had been formed. Enzyme electrophoresis, chromogenic assay and enzyme-linked immunosorbent assay using tPA-specific antibodies demonstrated that PC-12 cells constitutively secreted both plasminogen and tPA. The results obtained allow using PC-12 cells as a model to examine the interaction of nerve cells with the fibrin clot.     

Biochemical properties of conjugates of urokinase-type plasminogen activator with a monoclonal antibody specific for cross-linked fibrin

Equimolar mixtures of recombinant single chain urokinase-type plasminogen activator (rscu-PA) and a murine monoclonal antibody (MA-15C5) directed against fragment-D dimer of human cross-linked fibrin were conjugated, using the cross-linking agent N-succinimidyl 3-(2-pyridyldithio)propionate (PySSProSu). The conjugate (rscu-PA/MA-15C5), purified by immunoadsorption on a urokinase antibody and affinity chromatography on fibrin fragment-D dimer with a yield of 42 +/- 15% (mean +/- SD, n = 3), contained an average of 1.2 +/- 0.3 IgG molecules/rscu-PA molecule. On non-reduced SDS/PAGE it migrated as a main band with apparent Mr of 200,000. Specific amidolytic activities expressed/mass of u-PA were less than 250 IU/mg for rscu-PA/MA-15C5 and rscu-PA, 140,000 +/- 13,000 IU/mg and 100,000 +/- 17,000 IU/mg for their plasmin-generated two chain derivatives rtcu-PA/MA-15C5 and rtcu-PA respectively. Specific activities on fibrin plates were 100,000 +/- 24,000 IU/mg and 130,000 +/- 49,000 IU/mg for rscu-PA/MA-15C5 and rtcu-PA/MA-15C5 respectively, as compared to 180,000 +/- 15,000 IU/mg for both rscu-PA and rtcu-PA. Activation of plasminogen with rscu-PA/MA-15C5 (Km = 0.37 +/- 0.16 microM, k2 = 0.0063 +/- 0.0030 s-1 or rtcu-PA/MA-15C5 (Km = 19 +/- 3.0 microM, k2 = 2.0 +/- 0.10 s-1) in purified systems followed Michaelis-Menten kinetics with Km and k2 values comparable to those of rscu-PA and rtcu-PA. In an in vitro system composed of a 125I-fibrin-labeled whole human plasma clot immersed in citrated human plasma, dose- and time-dependent lysis was obtained; 50% lysis in 2 h required 1.4 microgram/ml of rscu-PA or 0.33 microgram/ml of rtcu-PA, but only 0.22 microgram u-PA/ml of rscu-PA/MA-15C5 or 0.15 microgram u-PA/ml of rtcu-PA/MA-15C5. Addition of purified fragment-D dimer reversed the increased fibrinolytic potency of rscu-PA/MA-15C5 in a concentration-dependent way (50% inhibition at 7.2 micrograms fragment-D dimer/ml). Thus, conjugation of u-PA moieties with the fibrin-specific antibody MA-15C5 targets the plasminogen activator to the clot, resulting in a significant increase of their fibrinolytic potencies as compared to their unconjugated counterparts: 6.4-fold for rscu-PA and 2.2-fold for rtcu-PA.     

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.     

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.     

Kinetic analysis of the interactions between plasminogen activator inhibitor 1 and both urokinase and tissue plasminogen activator

Plasminogen activator inhibitor 1 (PAI-1) was purified from medium conditioned by cultured bovine aortic endothelial cells by successive chromatography on concanavalin A Sepharose, Sephacryl S-200, Blue B agarose, and Bio-Gel P-60. As shown previously for conditioned media (C. M. Hekman and D. J. Loskutoff (1985) J. Biol. Chem. 260, 11581-11587) the purified PAI-1 preparation contained latent inhibitory activity which could be stimulated 9.4-fold by sodium dodecyl sulfate and 45-fold by guanidine-HCl. The specific activity of the preparation following treatment with 0.1% sodium dodecyl sulfate was 2.5 X 10(3) IU/mg. The reaction between purified, guanidine-activated PAI-1 and both urokinase and tissue plasminogen activator (tPA) was studied. The second-order rate constants (pH 7.2, 35 degrees C) for the interaction between guanidine-activated PAI-1 and urokinase (UK), and one- and two-chain tPA are 1.6 X 10(8), 4.0 X 10(7), and 1.5 X 10(8) M-1 S-1, respectively. The presence of CNBr fibrinogen fragments had no affect on the rate constants of either one- or two-chain tPA. Steady-state kinetic analysis of the effect of PAI-1 on the rate of plasminogen activation revealed that the initial UK/PAI-1 interaction can be competed with plasminogen suggesting that the UK/PAI-1 interaction may involve a competitive type of inhibition. In contrast, the initial tPA/PAI-1 interaction can be competed only partially with plasminogen, suggesting that the tPA/PAI-1 interaction may involve a mixed type of inhibition. The results indicate that PAI-1 interacts more rapidly with UK and tPA than any PAI reported to date and suggest that PAI-1 is the primary physiological inhibitor of single-chain tPA. Moreover, the interaction of PAI-1 with tPA differs from its interaction with UK, and may involve two sites on the tPA molecule.     

Nonclottable fibrin obtained from partially reduced fibrinogen: characterization and tissue plasminogen activator stimulation.

Out of 29 disulfide bonds in human fibrinogen, 7 were cleaved during limited reduction under nondenaturing conditions in calcium-free buffer: 2 A alpha 442Cys-A alpha 472Cys and 2 gamma 326Cys-gamma 339Cys intrachain disulfide bonds in the carboxy-terminal ends of the A alpha- and gamma-chains and the symmetrical disulfide bonds at gamma 8Cys, gamma 9Cys, and A alpha 28Cys. We studied the loss of thrombin clottability that followed limited reduction and the increase in the susceptibility of the fibrinogen A alpha 19-A alpha 20 bond to hydrolysis by thrombin. Using differential scanning calorimetry, we show that the extent of unfolding and denaturation of specific domains following limited reduction is small. Heat absorption peaks corresponding to the melting of the major regions of compact structure give high calorimetric enthalpies, as in untreated nonreduced fibrinogen, indicating that substantial regions of native structure are still present in partially reduced fibrinogen. Thrombin releases fibrinopeptide A at an identical rate as in nonreduced fibrinogen while fibrinopeptide B release is slower. Sedimentation velocity studies show that thrombin treatment leads to complex formation; however, gelation does not occur. Amino-terminal analysis indicates that the second thrombin cleavage in the A alpha-chain at A alpha 19-A alpha 20 takes place only after fibrinopeptide A release. Thus, the loss of clottability appears to result from perturbation of carboxy-terminal polymerization sites, probably a consequence of gamma 326Cys-gamma 339Cys intrachain disulfide bond cleavage. The thrombin-treated partially reduced fibrinogen remains soluble in buffered saline and fully expresses at least one epitope, B beta 15-21, unique to fibrin. Furthermore, this nonclottable form accelerates the tissue plasminogen activator dependent conversion of plasminogen to plasmin.     

Interaction of one-chain and two-chain tissue plasminogen activator with intact and plasmin-degraded fibrin

Tissue-type plasminogen activator (t-PA) plays a central role in fibrinolysis in vivo. Although it is known to bind to fibrin, the dissociation constant (Kd) and number of moles bound per mole of fibrin monomer (n) have never been measured directly. In this study, the binding of both the one-chain form and the two-chain form of recombinant, human t-PA to fibrin was measured. Although more one-chain t-PA than two-chain t-PA is bound to fibrin, the Kd's and n's were within experimental error of each other. Significantly more t-PA is bound to clots made from fibrinogen which has been digested with plasmin than to clots made from intact fibrinogen. The additional binding was shown to be due to the formation of new set(s) of binding site(s) with dissociation constants that are 2-4 orders of magnitude tighter than the binding site present on clots made from intact fibrinogen. epsilon-Aminocaproic acid was capable of competing for the loose binding site present on both intact and degraded fibrin but had little effect on the binding of t-PA to the new site(s) formed by plasmin digestion. This increase in binding caused by plasmin-mediated proteolysis of fibrin suggests a possible mechanism for a positive regulation capable of accelerating fibrinolysis.     

Staphylokinase--a specific plasminogen activator

Staphylokinase is a 135 amino acid protein produced by certain strains of Staphylococcus aureus. It belongs to fibrin-specific plasminogen activator. Staphylokinase converts plasminogen--the inactive proenzyme--to the plasmin, which dissolves the fibrin of a blood clots. This review will focus on the biochemical and thrombolytic properties of staphylokinase and its derivatives, which would make use of treatment in acute myocardial infarction and other cardiovascular diseases.     

Plasminogen activation by tissue plasminogen activator on fibrin clots with different surface structures

Transformation of fibrinogen into fibrin with consequent formation of the fibrin clot trimeric structure is one of the final steps in the blood coagulation system. The plasminogen activation by the tissue plasminogen activator (t-PA) is one of the fibrinolysis system key reactions. The effect of different factors on transformation of plasminogen into plasmin is capable to change essentially the equilibrium between coagulation and fibrinolytic sections of haemostasis system. We have studied the plasminogen activation by tissue plasminogen activator on fibrin clots surface formed on the interface between two phases and in presence of one phase. The t-PA plasminogen activation rate on fibrin clots both with film and without it the latter has been analyzed. These data allow to assume that the changes of fibrin clot structure depend on its formations, as well as are capable to influence essentially on plasminogen activation process by means of its tissue activating agent.     

A region of tissue plasminogen activator that affects plasminogen activation differentially with various fibrin(ogen)-related stimulators.

The dissolution of blood clots by plasmin is normally initiated in vivo by the activation of plasminogen to plasmin through the activity of tissue plasminogen activator (t-PA). The rate of plasminogen activation can be stimulated several orders of magnitude by the presence of fibrin-related proteins. Here we describe the kinetic analysis of both recombinant human t-PA (wild-type) and a t-PA variant produced by site-directed mutagenesis in which the original sequence from amino acids 296 to 299, KHRR, has been altered to AAAA. This tetra-alanine variant form of t-PA, K296A/H297A/R298A/R299A t-PA, we refer to as "KHRR" t-PA here. The plasminogen activating kinetics of wild-type t-PA (Activase alteplase) showed a catalytic efficiency which changed over 100-fold dependent on the stimulator in the assay. The lowest rate was in the absence of a stimulator. The following stimulators showed increasing ability to accelerate the catalytic efficiency of the reaction: fibrinogen, fragments of fibrinogen obtained by digestion with plasmin, fibrin, and slightly degraded fibrin. This increase in efficiency was driven primarily by decreases in the Michaelis constant (KM) of the reaction, whereas the catalytic rate constant (kcat) of the reaction did not change significantly. The "KHRR" variant of t-PA displayed novel kinetics with all stimulators tested. In the absence of a stimulator or with the poorer stimulators (fibrinogen and fibrinogen fragments), the KM values of the reaction with Activase alteplase and "KHRR" t-PA were similar. The kcat however, was lower with "KHRR" t-PA than with wild-type t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasminogen activator inhibitor-1 binds to fibrin and inhibits tissue-type plasminogen activator-mediated fibrin dissolution.

Plasminogen activator inhibitor-1 (PAI-1) accumulates within thrombi and forming whole blood clots. To explore this phenomenon at the molecular level, PAI-1 binding to fibrin was examined. The experiments were performed by adding 125I-PAI-1, which retains its complete tissue-type plasminogen (t-PA) inhibitory activity, to fibrin matrices formed in 2-cm2 tissue culture wells. Guanidine HCl-activated PAI-1 binding was reversible and was inhibited in the presence of excess, unlabeled PAI-1. Activated 125I-PAI-1 recognized 2 sites on fibrin: a very small number of high affinity sites (Kd less than 1 nM) and principally a large number of low affinity sites with an approximate Kd of 3.8 microM. Latent PAI-1 bound to fibrin at a site indistinguishable from the lower affinity site recognized by activated PAI-1. Fibrin, pretreated with activated PAI-1, was protected from t-PA-mediated plasmin degradation in a PAI-1 dose-responsive manner (IC50 = 12.3 nM). Clot protection correlated with partial occupancy of the low affinity PAI-1 binding site on fibrin and was due to the formation of sodium dodecyl sulfate-stable, PAI-1.t-PA complexes. Latent PAI-1 (27 nM) did not protect the fibrin from dissolution. The localization of PAI-1 to a thrombus by virtue of its fibrin binding potential could result in significant protection of the thrombus from the degradative effects of the fibrinolytic system.     

Synergistic fibrinolysis: The combined effects of tissue plasminogen activator and recombinant staphylokinase in vitro

BackgroundMechanisms of fibrin-specificity of tissue plasminogen activator (tPA) and recombinant staphylokinase (STA) are different, therefore we studied in vitro the possibility of the synergy of their combined thrombolytic action.MethodsThrombolytic effects of tPA, STA and their combinations were measured by lysis rate of human plasma clot and side effects were evaluated by decreasing in fibrinogen, plasminogen and α₂-antiplasmin levels in the surrounding plasma at 37 °C in vitro.ResultsSTA and tPA induced dose- and time-dependent clot lysis: 50% lysis in 2 h was obtained with 30 nM tPA and 75 nM STA, respectively. At these concentrations, tPA produced greater degradation of plasma fibrinogen than STA. According to a mathematical analysis of dose–response curves by the isobole method, combinations of tPA and STA caused a considerable synergistic thrombolytic effect. The simultaneous and sequential combinations of tPA (< 4 nM) and STA (< 35 nM) induced a significant fibrin-specific synergistic thrombolysis, which was more pronounced in 2 h at simultaneous combinations than at sequential addition of STA after 30 min of tPA action. Simultaneous combination of 2.5 nM tPA and 15 nM STA showed a maximal 3-fold increase in thrombolytic effect compared to the expected total effect of the individual agents. Sequential combinations caused a lower depletion of plasma proteins compared to simultaneous combinations.ConclusionsThe simultaneous and sequential combinations of tPA and STA possessed synergistic fibrin-specific thrombolytic action on clot lysis in vitro.General significanceThe results show that combined thrombolysis may be more effective and safer than thrombolysis with each activator alone.     

An experimental and theoretical study on the dissolution of mural fibrin clots by tissue-type plasminogen activator.

During thrombolytic therapy and after recanalization is achieved, reduction in the volume of mural thrombi is desirable. Mural thrombi are known to contribute to rethrombosis and reocclusion. The lysis rate of mural thrombi has been demonstrated to increase with fluid flow in different experimental models, but the mechanisms responsible are unknown. An experimental and a theoretical study were developed to determine the contribution of outer convective transport to the lysis of mural fibrin clots. Normal human plasma containing recombinant tissue-type plasminogen activator (tPA; 0.5 microg/mL) was (re)perfused over mural fibrin clots with fluorescently labeled fibrin at low arterial, arterial, or higher wall shear stresses (4, 18, or 41 dyn/cm(2), respectively) and lysis was monitored in real time. Flow accelerated lysis, but significantly only at the highest shear stress: The average lysis front velocity was 3 x 10(-5) cm/s at 41 dyn/cm(2) vs. almost half of that at the lower shear stresses. Confocal microscopy showed fibrin fibers dissolving only in a narrow region close to the surface when permeation velocity was predicted to be low. A heterogeneous transport-reaction finite element model was used to describe mural fibrinolysis. After scaling the effects of outer and inner convection, inner diffusion, and chemical reactions, a simplified inner diffusion/reaction model was used. Correlation to fibrin lysis data in purified systems dictated higher rates of plasmin(ogen) and tPA adsorption onto fibrin and a decreased catalytic rate of plasmin-mediated fibrin degradation, compared with published parameters. At each shear stress, the model predicted a temporal pattern of lysis of mural fibrin (similar to that observed experimentally), and protease accumulation in a narrow fibrin region and significant lysis inhibition by plasma alpha(2)-antiplasmin (according to the literature). Increasing outer convection accelerated mural fibrinolysis, but the model did not predict the big increase in lysis rate at the highest shear stress. At higher than arterial flows, additional mechanisms not accounted for in the current model, such as fibrin collapse at the fibrin front, may regulate the lysis of mural clots and determine the outcome of thrombolytic therapy.     

Influence of various structural domains of fibrinogen and fibrin on the potentiation of plasminogen activation by recombinant tissue plasminogen activator

Fibrinogen, fibrin, and related fragments have varying stimulatory effects on the initial rate of the activation of human plasminogen ([Glu¹]Pg) by recombinant tissue plasminogen activator (rt-PA). A detailed analysis of this enhancement was undertaken using various purified and complexed forms of the known domains of fibrin(ogen) with a view to gaining additional knowledge regarding the substructures of fibrinogen and fibrin that are important for their stimulatory capacities. Both arvin-mediated fibrin, as well as fibrinogen fragments generated as a result of its cleavage with CNBr, stimulate the activation in a biphasic manner, most likely as a result of changes in the promoter molecule accompanying the denaturation processes that are normally employed to either solubilize or generate these particular promoters. Using purified fibrinogen and fibrin fragments, it was found that fragment E, which binds to [Glu¹]Pg, does not enhance the activation reaction, while fragment D₁ has a potentiating effect. This suggests that the binding of [Glu¹]Pg to fibrin(ogen) alone is not, in itself, sufficient for stimulation of activation to occur, but that the rt-PA-fibrin(ogen) interaction is fundamental to this same process. All purified and mixtures of fragments containing the fragment D domain (e.g., D₂E, X-oligomer, fragment X) stimulate the reaction to a greater degree than fibrinogen and fragment D₁. It is concluded that the fibrinogen D domain is asine qua non for the enhancement reaction, while structures containing the E domain had a symbiotic effect on enhancement.On study leave from the National Institute for Biological Standards and Control, South Mimms, HERTS EN6 3QG, England.