Antibody-directed fibrinolysis. An antibody specific for both fibrin and tissue plasminogen activator

An investigation was made to determine whether it is possible to attract tissue plasminogen activator (tPA) to the site of a thrombus by means of an antibody with affinites for both tPA and fibrin. A bispecific antibody conjugate was constructed by cross-linking two monoclonal antibodies: one specific for tPA, the other specific for fibrin. The bispecific antibody enhanced fibrinolysis by capturing tPA at the site of a fibrin deposit. In an in vitro quantitative fibrinolysis assay, the relative fibrinolytic potency of tPA bound to the bispecific antibody was 13 times greater than that of tPA and 200 times greater than that of urokinase. When fibrin was treated with the bispecific antibody before being mixed with tPA, the relative fibrinolytic potency of tPA was enhanced 14-fold. This capture also occurred when the concentration of tPA present in the assay was less than the concentration of tPA present in normal human plasma. In a human plasma clot assay, samples containing both the bispecific antibody and tPA exhibited significantly more lysis than did samples containing tPA alone. In spite of the increased clot lysis effected by the presence of bispecific antibody, there was no significant increase in fibrinogen or alpha 2-antiplasmin degradation at equal tPA concentrations. The ability of the bispecific antibody to concentrate exogenous tPA in vivo was then examined in the rabbit jugular vein model. Systemic infusion of a small amount of tPA (10,000 units) produced no significant increment in thrombolysis over the level of spontaneous lysis (14 +/- 8%). However, the simultaneous infusion of 10,000 units of tPA and 2 mg of bispecific antibody resulted in 42 +/- 14% (p less than 0.01) lysis. These results suggest that a molecule capable of binding both fibrin and tPA with high affinity could enhance thrombolysis in the circulation by capturing endogenous tPA.     

Role of plasminogen activators in peritoneal adhesion formation

Intra-abdominal adhesion formation is a major complication of serosal repair following surgery, ischaemia or infection, leading to conditions such as intestinal obstruction and infertility. It has been proposed that the persistence of fibrin, due to impaired plasminogen activator activity, results in the formation of adhesions between damaged serosal surfaces. This study aimed to assess the role of fibrinolysis in adhesion formation using mice deficient in either of the plasminogen activator proteases, tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA). We hypothesize that, following serosal injury, mice with decreased peritoneal fibrinolytic activity will be more susceptible to adhesion formation. Adhesion formation was induced in tPA- and uPA-deficient and wild-type mice following either surgical trauma to the serosa with haemorrhage and acute or chronic intraperitoneal inflammation. Adhesion formation was assessed from 1 to 4 weeks post-injury. Mice deficient in tPA were more susceptible to adhesion formation following both a surgical insult and a chronic inflammatory episode compared with uPA-deficient and wild-type mice. In addition, the time of maximal adhesion formation varied depending on the nature of the initial insult. It is proposed that the persistence of fibrin due to decreased tPA activity following surgery or chronic inflammation plays a major role in peritoneal adhesion formation.     

Tissue plasminogen activator-mediated fibrinolysis protects against axonal degeneration and demyelination after sciatic nerve injury

Tissue plasminogen activator (tPA) is a serine protease that converts plasminogen to plasmin and can trigger the degradation of extracellular matrix proteins. In the nervous system, under noninflammatory conditions, tPA contributes to excitotoxic neuronal death, probably through degradation of laminin. To evaluate the contribution of extracellular proteolysis in inflammatory neuronal degeneration, we performed sciatic nerve injury in mice. Proteolytic activity was increased in the nerve after injury, and this activity was primarily because of Schwann cell-produced tPA. To identify whether tPA release after nerve damage played a beneficial or deleterious role, we crushed the sciatic nerve of mice deficient for tPA. Axonal demyelination was exacerbated in the absence of tPA or plasminogen, indicating that tPA has a protective role in nerve injury, and that this protective effect is due to its proteolytic action on plasminogen. Axonal damage was correlated with increased fibrin(ogen) deposition, suggesting that this protein might play a role in neuronal injury. Consistent with this idea, the increased axonal degeneration phenotype in tPA- or plasminogen-deficient mice was ameliorated by genetic or pharmacological depletion of fibrinogen, identifying fibrin as the plasmin substrate in the nervous system under inflammatory axonal damage. This study shows that fibrin deposition exacerbates axonal injury, and that induction of an extracellular proteolytic cascade is a beneficial response of the tissue to remove fibrin. tPA/plasmin-mediated fibrinolysis may be a widespread protective mechanism in neuroinflammatory pathologies.     

Enhanced fibrinolysis by proteolysed coagulation factor Xa

We previously showed that coagulation factor Xa (FXa) enhances activation of the fibrinolysis zymogen plasminogen to plasmin by tissue plasminogen activator (tPA). Implying that proteolytic modulation occurs in situ, intact FXa (FXaα) must be sequentially cleaved by plasmin or autoproteolysis, producing FXaβ and Xa33/13, which acquire necessary plasminogen binding sites. The implicit function of Xa33/13 in plasmin generation has not been demonstrated, nor has FXaα/β or Xa33/13 been studied in clot lysis experiments. We now report that purified Xa33/13 increases tPA-dependent plasmin generation by at least 10-fold. Western blots confirmed that in situ conversion of FXaα/β to Xa33/13 correlated to enhanced plasmin generation. Chemical modification of the FXaα active site resulted in the proteolytic generation of a product distinct from Xa33/13 and inhibited the enhancement of plasminogen activation. Identical modification of Xa33/13 had no effect on tPA cofactor function. Due to its overwhelming concentration in the clot, fibrin is the accepted tPA cofactor. Nevertheless, at the functional level of tPA that circulates in plasma, FXaα/β or Xa33/13 greatly reduced purified fibrin lysis times by as much as 7-fold. This effect was attenuated at high levels of tPA, suggesting a role when intrinsic plasmin generation is relatively low. FXaα/β or Xa33/13 did not alter the apparent size of fibrin degradation products, but accelerated the initial cleavage of fibrin to fragment X, which is known to optimize the tPA cofactor activity of fibrin. Thus, coagulation FXaα undergoes proteolytic modulation to enhance fibrinolysis, possibly by priming the tPA cofactor function of fibrin.     

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.     

Characterization of monoclonal antibodies against human tissue plasminogen activator (tPA): quantitation of free tPA in human cell cultures by an ELISA.

Seven murine monoclonal antibodies produced against tissue plasminogen activator (tPA) were evaluated by means of enzyme-linked immunosorbent assays (ELISAs), and their effects on the enzymatic activities of tPA towards a synthetic substrate (S-2288) and plasminogen were investigated. One of the antibodies, TPA1-70, strongly inhibited the enzymatic activity of tPA in a fibrin agarose plate assay, while it did not affect the enzymatic activity towards the synthetic substrate or plasminogen. The antibody is directed to an epitope on the B-chain of tPA, which is necessary for the formation of a ternary complex of tPA, fibrin and plasminogen, but probably not to the active site. Another antibody, TPA2-14, partially inhibited the enzymatic activities of tPA towards the synthetic substrate and plasminogen, but it was not able to bind to the inactive tPA complexed with plasminogen activator inhibitor-1 (PAI-1). The antibody is directed to an epitope on the second kringle region, which is probably one of the PAI-1 binding sites. This property of the antibody enabled us to develop an ELISA for selective quantitation of free tPA in culture media conditioned with several human cell lines. The results indicate that tPA in these media exists either partially or almost entirely in a complex with PAI-1.     

Tissue-type plasminogen activator is a multiligand cross-beta structure receptor

Tissue-type plasminogen activator (tPA) regulates fibrin clot lysis by stimulating the conversion of plasminogen into the active protease plasmin. Fibrin is required for efficient tPA-mediated plasmin generation and thereby stimulates its own proteolysis. Several fibrin regions can bind to tPA, but the structural basis for this interaction is unknown. Amyloid beta (Abeta) is a peptide aggregate that is associated with neurotoxicity in brains afflicted with Alzheimer's disease. Like fibrin, it stimulates tPA-mediated plasmin formation. Intermolecular stacking of peptide backbones in beta sheet conformation underlies cross-beta structure in amyloid peptides. We show here that fibrin-derived peptides adopt cross-beta structure and form amyloid fibers. This correlates with tPA binding and stimulation of tPA-mediated plasminogen activation. Prototype amyloid peptides, including Abeta and islet amyloid polypeptide (IAPP) (associated with pancreatic beta cell toxicity in type II diabetes), have no sequence similarity to the fibrin peptides but also bind to tPA and can substitute for fibrin in plasminogen activation by tPA. Moreover, the induction of cross-beta structure in an otherwise globular protein (endostatin) endows it with tPA-activating potential. Our results classify tPA as a multiligand receptor and show that cross-beta structure is the common denominator in tPA binding ligands.     

Characterization of an antibody-urokinase conjugate. A plasminogen activator targeted to fibrin

The plasminogen activator urokinase was linked covalently to a monoclonal antibody specific for the amino terminus of the beta chain of human fibrin by means of the unidirectional cross-linking reagent N-succinimidyl-3-(2-pyridyldithio)propionate. N-Succinimidyl-3-(2-pyridyldithio)propionate allowed the amino groups on urokinase to be coupled to the sulfhydryl groups on iminothiolane (which had been introduced into the antibody before the coupling reaction). The inter-heavy chain sulfhydryl of the Fab' of this antibody was also linked to N-succinimidyl-3-(2-pyridyldithio)propionate-substituted urokinase. The antibody- or Fab'-urokinase complexes were purified by two affinity chromatography steps. In the first, benzamidine was used as ligand for urokinase, in the second, a heptapeptide consisting of the 7 amino-terminal residues of the beta chain of fibrin (beta peptide) was used as ligand for the antibody. The activity of the purified conjugates was compared with that of urokinase alone in an assay measuring lysis of 125I-fibrin monomer covalently linked to Sepharose CL-4B. For any concentration of either urokinase alone or urokinase-antifibrin antibody conjugate, an equivalent amount of lysis (release of labeled peptide from fibrin monomer-Sepharose) was obtained with 1/250 the concentration (with respect to urokinase content) of antifibrin antibody-urokinase conjugate. The antifibrin Fab'-urokinase conjugate exhibited a similar enhancement of activity in comparison with urokinase. Enhanced fibrinolysis was fully inhibited by beta peptide. These results suggest that antibody targeting enhances the concentration of urokinase in the vicinity of immobilized fibrin monomer, thereby also increasing the local conversion of plasminogen to plasmin, which in turn degrades its substrate, fibrin. Univalent antigen-antibody binding is sufficient for optimal efficiency.     

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.     

Plasminogen activator-anti-human fibrinogen conjugate

A covalent conjugate between the plasminogen activator urokinase and polyclonal rabbit anti-human fibrinogen has been formed using the heterobifunctional coupling reagent N-succinimidyl 3-(2-pyridyldithio) propionate. The resultant urokinase-anti-human fibrinogen conjugate was separated from unreacted material by gel filtration. The conjugate exhibited amidase activity against the small chromogenic substrate pyroglutamyl-glycyl-arginine-p-nitroanilide as well as plasminogen activator activity in an assay employing plasminogen and the plasmin substrate D-valyl-leucyl-lysine-p-nitroanilide. Retention of antibody specificity for fibrinogen was demonstrated using an enzyme linked immunoassay procedure. The conjugate was found to have greater stability in human plasma than unconjugated urokinase.     

Expression of a Novel Chimeric-Truncated tPA in Pichia pastoris with Improved Biochemical Properties

Thrombolytic therapy by plasminogen activators (PAs) has been a main goal in the treatment of acute myocardial infarction. Despite improved outcomes of currently available thrombolytic therapies, all these agents have different drawbacks that may result in less than optimal outcomes. In order to make tissue plasminogen activator (tPA) more potent, while being more resistant to plasminogen activator inhibitor-1 (PAI-1) and having a higher affinity to fibrin, a new chimeric-truncated form of tPA (CT tPA) was designed and expressed in Pichia pastoris. This novel variant consists of a finger domain of Desmoteplase, an epidermal growth factor (EGF) domain, a kringle 1 (K1) domain, a kringle 2 (K2) domain, in which the lysine binding site (LBS) was deleted, and a protease domain, where the four amino acids lysine 296, arginine 298, arginine 299, and arginine 304 were substituted by aspartic acid. The chimera CT tPA showed 14-fold increase in its activity in the presence of fibrin compared to the absence of fibrin. Furthermore, CT tPA showed about 10-fold more potency than commercially available full-length tPA (Actylase^®) and provided 1.2-fold greater affinity to fibrin. A residual activity of only 68 % was observed after incubation of Actylase^® with PAI-1, however, 91 % activity remained for CT tPA. These promising findings suggest that the novel CT tPA variant might be an acceptable PA with superior characteristics and properties.     

Functional characterization of monoclonal antibodies directed against fibrin binding domains of tissue-type plasminogen activator

Fibrin interacts with tissue-type plasminogen activator (tPA) via the finger and the kringle 2 domains. Three monoclonal antibodies against tPA, designated MPW3VPA, MPW6VPA, and MPW7VPA, which react with epitopes in the tPA molecule involved in fibrin binding, were characterized. The IgM monoclonal antibody MPW6VPA, directed against an epitope close to the finger and epidermal growth factor domains, stimulated plasminogen activation only in the absence of CNBr-fibrinogen fragments by increasing kcat in a dose-dependent fashion, an effect which was not restricted to the intact molecule. These results suggest that MPW6VPA mimics the initial effect of fibrin bound to the tPA molecule, which results in a change of kcat values. The MPW6VPA effect was reversed by another antibody, MPW3VPA, also directed against epidermal growth factor and finger domains. The latter antibody also inhibited plasminogen activation by tPA in the presence of CNBr-fibrinogen fragments in a dose-dependent, apparently noncompetitive way. No effect of MPW3VPA was seen in the absence of CNBr-fibrinogen fragments. MPW7VPA directed against kringle 2 of tPA inhibited plasminogen activation by tPA only when CNBr-fibrinogen fragments were present. This inhibition was apparently competitive and dose-dependent. These data suggest that MPW3VPA interferes with the first phase of fibrin binding to tPA, whereas MPW7VPA interferes with the second phase of fibrin binding to the tPA molecule via kringle 2, resulting in Km changes.     

Vampire bat salivary plasminogen activator exhibits a strict and fastidious requirement for polymeric fibrin as its cofactor, unlike human tissue-type plasminogen activator. A kinetic analysis.

The vampire bat salivary plasminogen activator (BatPA) is virtually inactive toward Glu-plasminogen in the absence of a fibrin-like cofactor, unlike human tissue-type plasminogen activator (tPA) (the kcat/Km values were 4 and 470 M-1 s-1, respectively). In the presence of fibrin II, tPA and BatPA activated Glu-plasminogen with comparable catalytic efficiencies (158,000 and 174,000 M-1 s-1, respectively). BatPA's cofactor requirement was partially satisfied by polymeric fibrin I (54,000 M-1 s-1), but monomeric fibrin I was virtually ineffective (970 M-1 s-1). By comparison, a variety of monomeric and polymeric fibrin-like species markedly enhanced tPA-mediated activation of Glu-plasminogen. Fragment X polymer was 2-fold better but 9-fold worse as cofactor for tPA and BatPA, respectively, relative to fibrin II. Fibrinogen, devoid of plasminogen, was a 10-fold better cofactor for tPA than fibrinogen rigorously depleted of plasminogen, Factor XIII, and fibronectin; the enhanced stimulatory effect of the less-purified fibrinogen was apparently due to the presence of Factor XIII. By contrast, the two fibrinogen preparations were equally poor cofactors of BatPA-mediated activation of Glu-plasminogen. BatPA possessed only 23 and 4% of the catalytic efficiencies of tPA and two-chain tPA, respectively, in hydrolyzing the chromogenic substrate Spectrozyme tPA. However in the presence of fibrin II, BatPA and tPA exhibited similar kcat/Km values for the hydrolysis of Spectrozyme tPA. Our data revealed that BatPA, unlike tPA, displayed a strict and fastidious requirement for polymeric fibrin I or II. Consequently, BatPA may preferentially promote plasmin generation during a narrow temporal window of fibrin formation and dissolution.     

The interaction of streptokinase.plasminogen activator complex, tissue-type plasminogen activator, urokinase and their acylated derivatives with fibrin and cyanogen bromide digest of fibrinogen. Relationship to fibrinolytic potency in vitro.

The effects of purified soluble fibrin and of fibrinogen fragments (fibrin mimic) on the activation of Lys-plasminogen (i.e. plasminogen residues 77-790) to plasmin by streptokinase.plasminogen activator complex and by tissue-type plasminogen activator were studied. Dissociation constants of both activators were estimated to lie in the range 90-160 nM (fibrin) and 16-60 nM (CNBr-cleavage fragments of fibrinogen). The kinetic mechanism for both types of activator comprised non-essential enzyme activation via a Rapid Equilibrium Ordered Bireactant sequence. In order to relate the fibrin affinity of plasminogen activators to their fibrinolytic potency, the rate of lysis of supported human plasma clots formed in the presence of unmodified or active-centre-acylated precursors of plasminogen activators was studied as a function of the concentration of enzyme derivative. The concentrations of unmodified enzyme giving 50% lysis/h in this assay were 0.9, 2.0 and 11.0 nM for tissue-type plasminogen activator, streptokinase.plasmin(ogen) and urokinase respectively. However, the potencies of active-centre-acylated derivatives of these enzymes suggested that acylated-tissue plasminogen activator and streptokinase.plasminogen complexes of comparable hydrolytic stability were of comparable potency. Both types of acyl-enzyme were significantly more potent than acyl-urokinases.     

High resolution analysis of functional determinants on human tissue-type plasminogen activator

Sixty-four variants of human tissue-type plasminogen activator (tPA) were produced using recombinant DNA techniques. Charged residues were converted to alanine in clusters of from one to four changes per variant; these clusters spanned all the domains of the molecule. The variants were expressed by mammalian cells and were analyzed for a variety of properties. Variants of tPA were found that had reduced activity with respect to each tested property; in a few cases increased activity was observed. Analysis of these effects prompted the following conclusions: 1) charged residues in the nonprotease domains are less involved in fibrin stimulation of tPA activity than those in the protease domain, and it is possible to increase the fibrin specificity (i.e. the stimulation of tPA activity by fibrin compared to fibrinogen) by mutations at several sites in the protease domain; 2) the difference in enzymatic activity between the one- and two-chain forms of tPA can be increased by mutations at several sites on the protease domain; 3) binding of tPA to lysine-Sepharose was affected only by mutations to kringle-2, whereas binding to fibrin was affected most by mutations in the other domains; 4) clot lysis was influenced by mutations in all domains except kringle-2; 5) sensitivity to plasminogen activator inhibitor-1 seems to reside exclusively in the region surrounding residue 300. A model of the tPA protease domain has been used to map some of the critical residues and regions.     

Kinetics of the inhibition of plasminogen activators by the plasminogen-activator inhibitor. Evidence for ''second-site'' interactions.

The reactions between plasminogen-activator inhibitor (PAI) and different plasminogen activators were studied in the presence of chromogenic peptide substrates for the enzymes. Our findings suggest that the rate constants for the reactions of PAI with single-chain tissue plasminogen activator (tPA), two-chain tPA, high-Mr urokinase and low-Mr urokinase are high and quite similar (1.6 X 10(7)-3.9 X 10(7) M-1.s-1). A free active site in the enzymes seems to be necessary for their reaction with PAI. Amino acids with antifibrinolytic properties did not interfere with the reactions. However, di-isopropyl phosphorofluoridate-inactivated tPA inhibited the reaction between PAI and all plasminogen activators in a similar way. These findings clearly demonstrated that a 'second-site' interaction, in addition to that between the enzyme active site and the inhibitor 'bait' peptide bond, is of importance for the high reaction rate. The reaction rate between PAI and single-chain tPA in the presence of an activator substrate (D-Ile-Pro-Arg p-nitroanilide) was decreased in the presence of fibrin. Fibrin caused a decrease in the Km for the single-chain tPA-substrate reaction. As a consequence, the 'free' concentration of single-chain tPA in the system decreased in the presence of fibrin, affecting the reaction rate between PAI and single-chain tPA. The phenomenon might be of physiological relevance, in the sense that single-chain tPA bound to fibrin in the presence of plasminogen would be protected against inactivation by PAI.     

Tissue plasminogen activator binds to human vascular smooth muscle cells by a novel mechanism. Evidence for a reciprocal linkage between inhibition of catalytic activity and cellular binding.

Human vascular smooth muscle cells (VSMC) bind tissue plasminogen activator (tPA) specifically, saturably, and with relatively high affinity (K(d) 25 nM), and this binding potentiates the activation of cell-associated plasminogen (Ellis, V., and Whawell, S. A. (1997) Blood 90, 2312-2322). We have observed that this binding can be efficiently competed by DFP-inactivated tPA and S478A-tPA but not by tPA inactivated with H-D-Phe-Pro-Arg-chloromethyl ketone (PPACK). VSMC-bound tPA also exhibited a markedly reduced inhibition by PPACK, displaying biphasic kinetics with second-order rate constants of 7. 5 x 10(3) M(-1) s(-1) and 0.48 x 10(3) M(-1) s(-1), compared with 7. 2 x 10(3) M(-1) s(-1) in the solution phase. By contrast, tPA binding to fibrin was competed equally well by all forms of tPA, and its inhibition was unaltered. These effects were shown to extend to the physiological tPA inhibitor, plasminogen activator inhibitor 1. tPA.plasminogen activator inhibitor 1 complex did not compete tPA binding to VSMC, and the inhibition of bound tPA was reduced by 30-fold. The behavior of the various forms of tPA bound to VSMC correlated with conformational changes in tPA detected by CD spectroscopy. These data suggest that tPA binds to its specific high affinity site on VSMC by a novel mechanism involving the serine protease domain of tPA and distinct from its binding to fibrin. Furthermore, reciprocally linked conformational changes in tPA appear to have functionally significant effects on both the interaction of tPA with its VSMC binding site and the susceptibility of bound tPA to inhibition.     

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.