Replacement of finger and growth factor domains of tissue plasminogen activator with plasminogen kringle 1. Biochemical and pharmacological characterization of a novel chimera containing a high affinity fibrin-binding domain linked to a heterologous protein.

A novel triple-kringle plasminogen activator protein, PK1 delta FE1X, has been produced which is a genetic chimera between the fibrin binding kringle 1 domain of plasminogen and the two kringles and serine protease domains of naturally occurring wild-type tissue plasminogen activator (wt t-PA). This chimera also contains a modification to prevent high mannose type N-linked glycosylation on kringle 1 of t-PA. PK1 delta FE1X is biochemically and fibrinolytically similar to wt t-PA in vitro but retains the decreased plasma clearance rate characteristic of other t-PA variants which lack fibronectin finger-like and epidermal growth factor domains. The serine protease domain of PK1 delta FE1X exhibits the amidolytic activity characteristic of wt t-PA. In an indirect coupled plasminogen activator assay, the specific activity of PK1 delta FE1X is approximately 1.4 times greater than that of wt t-PA. In a fibrin film-binding assay, greater binding to untreated fibrin is observed with wt t-PA than with PK1 delta FE1X. However, following limited plasmin digestion of the fibrin film, PK1 delta FE1X binding increases to the level observed with wt t-PA. The incremental binding to plasmin-digested fibrin observed with PK1 delta FE1X is eliminated if plasmin digestion of the fibrin film is followed by carboxypeptidase B treatment. This result suggests that plasminogen kringle 1 binds plasmin-digested fibrin even after recombination with a heterologous protein. The fibrinolytic activity of PK1 delta FE1X in human plasma clot lysis assays was similar to that of wt t-PA at activator concentrations of approximately 1 microgram/ml. At substantially lower concentrations, approximately 0.1 microgram/ml, PK1 delta FE1X was only slightly less active than wt t-PA. Pharmacokinetic analysis showed that wt t-PA activity is cleared approximately 15 times as rapidly as PK1 delta FE1X following intravenous bolus injection. In a rabbit jugular vein clot lysis model, intravenous bolus injection of 0.06 mg/kg of PK1 delta FE1X showed greater thrombolytic potency than a similar administration of 0.5 mg/kg of wt t-PA. Thus it appears that in vitro exon shuffling techniques can be used to generate novel fibrinolytic agents which biochemically and pharmacologically represent the combination of individual domains of naturally occurring proteins.     

Functional effects of asparagine-linked oligosaccharide on natural and variant human tissue-type plasminogen activator

The role of Asn-linked oligosaccharide in the functional properties of both human tissue-type plasminogen activator (t-PA) and a genetic variant of t-PA was studied. Nonglycosylated and glycosylated wild-type t-PA were produced in mammalian cells which express recombinant t-PA. These proteins were compared in fibrin binding and 125I-labeled fibrin clot lysis assays, using purified components. The nonglycosylated form showed higher fibrin binding, as well as higher fibrinolytic potency than the glycosylated form. Subsequently, prevention of glycosylation of a t-PA variant which lacked the finger and epidermal growth factor domains (delta FE), was carried out in an attempt to enhance its fibrinolytic activity. Glycosylation was prevented by changing Asn to Gln; at Asn-117 to produce delta FE1X t-PA, and at Asn-117, -184, and -448 to produce delta FE3X t-PA. All variants were similar to wild-type t-PA in their catalytic dependence on fibrinogen fragments, fibrinolytic activity in fibrin autography analysis, and plasminogen activator activity. In a clot lysis assay, using citrated human plasma, the fibrinolytic potency of the variants were comparable to that of wild-type t-PA at activator concentrations of 17-51 nM (approximately 1-3 micrograms/ml). At 0.5-5.1 nM (approximately 0.03-0.3 micrograms/ml), however, the variant proteins had lower fibrinolytic potency than wild-type t-PA. Fifty percent lysis in 1.5 h for wild-type, delta FE, delta FE1X, and delta FE3X t-PA, required 2.5, 10, 7.5, and 5.5 nM t-PA, respectively. The fibrinogenolytic activity in human plasma was measured for wild-type, delta FE, delta FE1X, and delta FE3X t-PA, and showed significant fibrinogen depletion after 3 h of incubation at 51 nM, decreasing to 11, 11, 50, and 72% of basal levels, respectively. These data indicate that partial or total nonglycosylated t-PA variants have a higher fibrinolytic versus fibrinogenolytic ratio than their fully glycosylated counterparts.     

Mechanism of fibrinolysis and thrombolytic therapy

The thrombolytic treatment with plasminogen activators, such as physiological tissue-type plasminogen activator (t-PA), suffers from a number of significant limitations. There is a resistance to reperfusion and acute coronary reocclusion. The peculiarity of t-PA and one-chain urokinase treatment is their using in very high doses. Thus the process of thrombolytic therapy is proceeding with a deviation from the fibrinolytic mechanism, which is needs of a little quantity of tissue-type plasminogen activator and provides the physiologic thrombolysis without systemic complication. The estimation of this disaccordance suggests, the possible reasons of these complications.     

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)     

New strategies in the development of thrombolytic agents

Recombinant DNA technology has allowed large-scale production of the physiological, fibrin-specific, plasminogen activators tissue-type plasminogen activator (t-PA) and single-chain urokinase-type plasminogen activator (scu-PA). The results of clinical trials with these agents, mainly for the treatment of acute myocardial infarction, have revealed a limited fibrin specificity at the large therapeutic doses required for efficient thrombolysis. Mutants and variants of t-PA and scu-PA have given important information on structure-function relationships in these proteins and have resulted in rt-PA variants with significantly prolonged half-lives in vivo. Construction of chimaeric plasminogen activators containing various portions of t-PA and scu-PA has produced functionally active enzymes, however with a lower fibrin-affinity than wild-type t-PA. The promise of antibody targeting and the use of synergistic combinations of thrombolytic agents remains to be further investigated. We anticipate that eventually these research lines will yield artificial plasminogen activators with improved efficacy, risk/benefit and cost/benefit ratios.     

Characterization of a tissue-type plasminogen activator from porcine urine

Porcine urine, unlike human urine, does not contain detectable amounts of urokinase-type plasminogen activator (u-PA). The plasminogen activator present in porcine urine is of tissue-type (t-PA) as identified by the following criteria. (1) Porcine urine PA exhibits an Mr of 65,000 similar to the Mr of human t-PA (64-70,000) but distinct from the Mr of human u-PA (55,000). (2) Antibodies against human t-PA bind and inhibit crude and purified porcine urine PA, while human u-PA-specific antibodies do not react with porcine urine PA. (3) Plasminogen activation by porcine urine PA is markedly stimulated in the presence of fibrinogen fragments. (4) Porcine urine PA activity is not affected by concentration of amiloride substantially suppressing human u-PA activity.     

Kinetic studies on the effect of heparin and fibrin on plasminogen activators.

1. Possible interactions between fibrin(ogen) and heparin in the control of plasminogen activation were studied in model systems using the thrombolytic agents tissue-type plasminogen activator (t-PA), urokinase and streptokinase.plasminogen activator complex and the substrates Glu- and Lys-plasminogen. 2. Both t-PA and urokinase activities were promoted by heparin and by pentosan polysulphate, but not by chondroitin sulphate or hyaluronic acid. The effect was on Km. 3. In the presence of soluble fibrin (and its mimic, CNBr-digested fibrinogen) the effect of heparin on t-PA was attenuated, although not abolished. In studies using a monoclonal antibody and 6-aminohexanoic acid, it was found that heparin and fibrin did not seem to share a binding site on t-PA. 4. The activity of t-PA B-chain was unaffected by heparin, so the binding site is located on the A-chain of t-PA (and urokinase). 5. Fibrin potentiated the activity of heparin on urokinase. The activity of streptokinase.plasminogen was unaffected by heparin whether or not fibrin was present. 6. If these influences of heparin and fibrin also occur in vivo, then, in the presence of heparin, the relative fibrin enhancement of t-PA will be diminished and the likelihood of systemic activation by t-PA is increased.     

The potential improvement of thrombolytic therapy by targeting with bispecific monoclonal antibodies: Why they are used and how they are made

The generation of the proteolytic enzyme plasmin from its inactive precursor plasminogen, mediated by so called plasminogen activators, is the essential step in thrombolytic therapy. Plasmin is responsible for the degradation of the insoluble fibrin, the major component of a thrombus, to soluble fibrin degradation products. So far, the use of the more recently developed thrombolytic agents single-chain urokinase-type plasminogen activator (scu-PA) and tissue-type plasminogen activator (t-PA) were disappointing, mainly due to some of their negative propertiesin vivo, i.e., rapid inhibition and/or hepatic clearance. Besides some background information on the haemostatic balance; t-PA and scu-PA structure; and mechanisms of action, we here review some reported attempts to improve on these agents for thrombolytic therapy following various strategies. One of the more potential strategies, antibody-targeted thrombolytic therapy using bispecific monoclonal antibodies, is discussed somewhat more extensively, as are the several procedures that can befollowed for bispecific antibody preparation.     

Human tissue-type plasminogen activator gene located near chromosomal breakpoint in myeloproliferative disorder.

Plasminogen activators (PA) convert the inactive proenzyme plasminogen into plasmin, which is involved in the process of fibrinolysis, tissue remodeling, and cell migration. There are two distinct forms of PA: urokinase (u-PA) and tissue-type plasminogen activator (t-PA). t-PA has higher affinity for fibrin and is the main form involved in thrombolysis. By in situ chromosomal hybridization and Southern blot analysis of somatic cell hybrid DNA, we have assigned the human t-PA gene to chromosome 8, bands 8p12----q11.2. We have detected a common EcoRI restriction fragment length polymorphism within the t-PA gene that thus provides a precisely localized highly informative marker for genetic linkage studies. The t-PA gene localization coincides with a translocation breakpoint observed in myeloproliferative disorders. Whereas leukemic cells usually secrete both types of PA, a correlation exists between acute myeloid leukemic cells that release only t-PA and failure to respond to chemotherapy.     

The Effect of Tranexamic Acid on the Fibrinolytic System During Anaphylaxis in Rabbits; The Importance of the Fibrinolytic System During Anaphylaxis

Abstract

We previously reported (Shimaya et al. (1992) Enzyme, 46, 204) that a rapid and strong increase of plasminogen activator (PA) was induced during anaphylaxis, and that the main plasma fibrinolytic enzyme which increased in the anaphylaxis group was shown to be tissue-type plasminogen activator (t-PA). Anaphylaxis was induced in rabbits by giving BSA after t-AMCHA injection. 44% of those rabbits died within 3 h after BSA injection. In the dead group, the euglobulin fibrinolytic activity (EFA) could not be detected by the plasminogen-rich fibrin plate method and the t-PA activity, using the natural substrate plasminogen, did not rise significantly reaching a peak at 10–15 min. However, the EFA and t-PA activity increased significantly in the surviving group. A significant prolongation of the activated partial thromboplastin time (AFTT) and the prothrombin time (PT) was observed during anaphylaxis in both groups. These findings suggest that increased PA activity during anaphylaxis is an important defense mechanism against the rapid increase in the blood coagulation system.     

A rapid and strong increase of plasminogen activator induced by experimental anaphylaxis in rabbits.

Anaphylactic shock was induced in rabbits by injecting bovine serum albumin (BSA) as an antigen. Measurements of the enzyme activities in the fibrinolytic system confirmed that a rapid and strong increase of plasminogen activator (PA) was induced during anaphylaxis. The euglobulin fibrinolytic activity (EFA) as estimated by the plasminogen-rich fibrin plate method rose significantly, peaking at 15 min after the BSA injection (when the arterial pressure was minimum). However, EFA was not detected by the plasminogen-poor fibrin plate method. The tissue-type PA (t-PA) activity using the natural substrate plasminogen increased significantly with a peak at 15 min. The amidolytic activity also simultaneously increased significantly using the t-PA substrate, H-D-Ile-Pro-Arg-pNA. The plasminogen activator inhibitor (PAI) activity remained at baseline levels until 30 min, but rose fourfold at 90 min. The main plasma fibrinolytic enzyme which increased in anaphylaxis was proved by zymography to be t-PA with a molecular weight (MW) of 69,000.     

Interleukin 1 preferentially stimulates the production of tissue-type plasminogen activator by human articular chondrocytes

Interleukin 1, derived from human placenta, stimulates plasminogen activator activity in human articular chondrocytes. The stimulation of plasminogen activator activity can be abolished by preincubation of placental interleukin 1 with an antiserum to homogeneous 22K factor, a species of interleukin 1 beta, indicating that the stimulation of plasminogen activator activity is due to interleukin 1 and not contaminating factors. Chondrocytes produce three species of plasminogen activator, with apparent Mr approximately 50,000, 65,000 and 100,000 as determined after sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis with gels containing casein and plasminogen. Both placental interleukin 1 and 22K factor enhance the production of the species of Mr approximately 65,000 and 100,000. Comparison of the mobility of the plasminogen activator species on SDS-polyacrylamide gel electrophoresis with human urokinase (u-PA) and human melanoma tissue-type plasminogen activator (t-PA) and studies with antibodies to these enzymes indicate that the Mr approximately 50,000 species is a u-PA and the Mr approximately 65,000 a t-PA. The Mr approximately 100,000 species is possibly an enzyme-inhibitor complex. Interleukin 1 therefore appears to enhance the production of t-PA and a putative enzyme-inhibitor complex. Abolition of plasminogen activator activity in the fibrin plate assay with antibodies to t-PA and u-PA also confirms enhanced t-PA production on interleukin 1 stimulation, though there is also evidence for increased cell-associated production of u-PA.     

Characterization of a modified human tissue plasminogen activator comprising a kringle-2 and a protease domain

To study structure/function relationships of tissue plasminogen activator (t-PA) activity, one of the simplest modified t-PA structures to activate plasminogen in a fibrin-dependent manner was obtained by constructing an expression vector that deleted amino acid residues 4-175 from the full-length sequence of t-PA. The expression plasmid was introduced into a Syrian hamster cell line, and stable recombinant transformants, producing high levels of the modified plasminogen activator, were isolated. The resulting molecule, mt-PA-6, comprising the second kringle and serine protease domains of t-PA, produced a doublet of plasminogen activator activity having molecular masses of 40 and 42 kDa. The one-chain mt-PA-6 produced by cultured Syrian hamster cells was purified in high yield by affinity and size exclusion chromatography. The purified mt-PA-6 displayed the same two types of microheterogeneity observed for t-PA. NH2-terminal amino acid sequencing demonstrated that one-chain mt-PA-6 existed in both a GAR and a des-GAR form. Purified mt-PA-6 also existed in two glycosylation forms that accounted for the 40- and 42-kDa doublet of activity produced by the cultured Syrian hamster cells. Separation of these two forms by hydrophobic interaction chromatography and subsequent tryptic peptide mapping demonstrated that both forms contained N-linked glycosylation at Asn448; in addition, some mt-PA-6 molecules were also glycosylated at Asn184. Plasmin treatment of one-chain mt-PA-6 converted it to a two-chain molecule by cleavage of the Arg275-Ile276 bond. This two-chain mt-PA-6, like t-PA, had increased amidolytic activity. The fibrinolytic specific activities of the one- and two-chain forms of mt-PA-6 were similar and twice that of t-PA. The plasminogen activator activity of one-chain mt-PA-6 was enhanced greater than 80-fold by CNBr fragments of fibrinogen, and the one-chain enzyme lysed human clots in vitro in a dose-dependent manner. The ability to produce and purify a structurally simple plasminogen activator with desirable fibrinolytic properties may aid in the development of a superior thrombolytic agent for the treatment of acute myocardial infarction.     

Tissue plasminogen activator is released into cultured medium by cultured human uveal melanocytes

Melanoma cells produce tissue plasminogen activator (t-PA) that plays an important role in tumor invasion and metastasis. The production of t-PA by normal human uveal melanocytes has not been reported previously. In order to explore this possibility, we studied the production of t-PA by cultured human uveal melanocytes and compared that with the production by cultured human uveal melanoma cells and epidermal melanocytes. Human adult uveal melanocytes were isolated and cultured from donor eyes. The cells were cultured in serum-free medium for 48 h and the conditioned medium then collected for the plasminogen activator (PA) activity assay. Free PA activity was tested in an amidolytic assay using a t-PA standard curve. PA type was identified by fibrinography and antihuman t-PA and urokinase plasminogen activator (u-PA) blocking antibodies. Free PA activity was found in the conditioned medium of normal melanocytes and melanoma cells. The predominant PA activity was t-PA. Normal uveal melanocytes produced more t-PA (3.23 +/- 0.73 IU/105 cells/24 h) than that of epidermal melanocytes (1.25 IU/105 cells/24 h) but much less than uveal melanoma cells (11.0 +/- 3.39 IU/105 cells/24 h). Western blot analysis revealed that most t-PA in conditioned media were one-chain t-PA with molecular weight of 69 kDa. Our study indicates that uveal melanocytes may contribute to the free t-PA activity previously found in aqueous humor and choroidal eye cup superfusions. Therefore, this function of uveal melanocytes may play a role in intraocular matrix remodeling, fibrinolysis and aqueous humor outflow.     

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.     

Biochemical and functional characterization of human tissue-type plasminogen activator variants obtained by deletion and/or duplication of structural/functional domains

Five cDNA encoding human tissue-type plasminogen activator (t-PA) variants with deletion and/or duplication of structural/functional domains were cloned and expressed in Chinese hamster ovary cells. The mutants included: rt-PA-delta FE (where r represents recombinant), with deletion of the finger (F) and growth factor (E) domains; rt-PA-delta K1 delta K2, with replacement of kringle 1 (K1) by a second copy of kringle 2 (K2); and rt-PA-delta FK1 delta K2, rt-PA-delta EK1 delta K2, and rt-PA-delta FEK1 delta K2, with deletions in rt-PA-delta K1 delta K2 of the finger or growth factor domain or both, respectively. The variant rt-PAs, purified to homogeneity, were obtained essentially as single-chain molecules. CNBr-digested fibrinogen enhanced plasminogen activation between 110-fold with rt-PA-delta EK1 delta K2 and 150-fold with rt-PA-delta FEK1 delta K2 as compared to 140-fold with rt-PA. All rt-PA moieties showed a comparable concentration-dependent binding to fibrin, except rt-PA-delta FE, which had significantly reduced binding that was, however, partially restored by additional replacement of K1 with K2. All the rt-PA variants with two copies of K2 showed increased binding to lysine-Sepharose as compared to rt-PA, whereas rt-PA-delta FE had reduced binding. All rt-PA moieties induced a similar time- and concentration-dependent lysis of a 125I-fibrin-labeled plasma clot immersed in human plasma. Equally effective concentrations (causing 50% clot lysis in 2 h) ranged between 1.0 microgram/ml for rt-PA-delta K1 delta K2 and 1.6 micrograms/ml for rt-PA-delta FE as compared to 0.5 microgram/ml for rt-PA. Thus, replacement in rt-PA of K1 by a second copy of K2, which is known to contain a lysine-binding site, significantly enhances its affinity for lysine, with maintenance of its affinity for intact fibrin. Deletion of the finger and growth factor domains results in decreased fibrin affinity and fibrinolytic potency in a plasma milieu, which are partially restored by replacement of K1 by K2.     

Extracellular matrix produced by cultured corneal and aortic endothelial cells contains active tissue-type and urokinase-type plasminogen activators

Incubation of plasminogen with the subendothelial extracellular matrix (ECM) synthesized by cultured bovine corneal and aortic endothelial cells resulted in generation of fibrinolytic activity, indicated by proteolysis of ¹²⁵I-fibrin in a time-and dose-dependent manner. Both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) were identified in the ECM by fibrin zymography, immunoblotting, and inhibition of plasminogen activation by anti-u-and anti-t-antibodies. Most of the ECM-resident plasminogen activator (PA) activity did not originate from intracellular PA release occurring when the endothelial cells were lyzed and the ECM exposed, since a comparable amount of PA was associated with the ECM when the cells were lyzed with Triton X-100 or removed intact by treatment with 2 M urea. Active u-PA and t-PA were released from ECM by treatment with heparanase (endo-β-D-), indicating that some of the ECM-resident PA activity is sequestered by heparan sulfate side chains. These results indicate that both u-PA and t-PA produced by endothelial cells are firmly sequestered in an active form by the subendothelial ECM. It is suggested that ECM-resident plasminogen activators participate in sequential matrix degradation during cell invasion and tumor metastasis. PA activity may also function in release of ECM-bound growth factors (i.e., basic fibroblast growth factor) and activation of proenzymes (i.e., prothrombin), resulting in modulation of the ECM growth-promoting and thrombogenic properties. © 1993 Wiley-Liss, Inc.     

Production, purification and characterization of the plasminogen activator in teratocarcinoma stem cells induced with sodium butyrate

Suspension cultures of pluripotent teratocarcinoma cells were induced with sodium butyrate to produce plasminogen activator (PA), generally regarded as a marker enzyme of differentiation of the teratocarcinoma. The induction of plasminogen activator was very efficient, resulting in production of sufficient enzyme to allow its purification. The activator was inactivated by rabbit anti-human melanoma plasminogen activator antiserum, indicating that it was a tissue-type activator (t-PA). The enzyme was purified by column chromatograph on phosphocellulose, zinc-chelate agarose, Con-A Sepharose and Sephadex G-150. The preparation at the final step of purification gave a single peak of enzyme activity at pH 7.3 +/- 0.1 on isoelectric focusing, and showed a molecular weight of approximately 77,000 on SDS PAGE.     

Isolation, characterization, and cDNA cloning of a vampire bat salivary plasminogen activator

Vampire bat saliva contains a plasminogen activator that presumably assists these hematophagous animals during feeding. Here, we report that the vampire bat salivary plasminogen activator, Bat-PA, is homologous to tissue-type plasminogen activator (t-PA) but contains neither a kringle 2 domain nor a plasmin-sensitive processing site. Three Bat-PA species corresponding to full-length, finger-, and finger- epidermal growth factor homology domain- forms of t-PA have been isolated. Bat-PA(H), the full-length form, was purified and its activity has been characterized. Bat-PA(H) and t-PA are of similar efficacy when monitored for their abilities to catalyze plasminogen activation in the presence of a fibrin cofactor. Interestingly, Bat-PA activity toward plasminogen is stimulated 45,000-fold in the presence of fibrin I; the corresponding value for t-PA is only 205-fold. Bat-PA(H) is the only Bat-PA species which binds tightly to fibrin, although each of the three species exhibit remarkable stimulation by a fibrin cofactor.     

Molecular mechanisms of fibrinolysis and their application to fibrin-specific thrombolytic therapy

The fibrinolytic system comprises a proenzyme, plasminogen, which can be converted to the active enzyme, plasmin, which degrades fibrin. Plasminogen activation is mediated by plasminogen activators, which are classified as either tissue-type plasminogen activators (t-PA) or urokinase-type plasminogen activators (u-PA). Inhibition of the fibrinolytic system may occur at the level of the activators or at the level of generated plasmin. Plasmin has a low substrate specificity, and when circulating freely in the blood it degrades several proteins including fibrinogen, factor V, and factor VIII. Plasma does, however, contain a fast-acting plasmin inhibitor, alpha 2-antiplasmin, which inhibits free plasmin extremely rapidly but which reacts much slower with plasmin bound to fibrin. A "systemic fibrinolytic state" may, however, occur by extensive activation of plasminogen and depletion of alpha 2-antiplasmin. Clot-specific thrombolysis therefore requires plasminogen activation restricted to the vicinity of the fibrin. Two physiological plasminogen activators, t-PA and single-chain u-PA (scu-PA) induce clot-specific thrombolysis, via entirely different mechanisms, however. t-PA is relatively inactive in the absence of fibrin, but fibrin strikingly enhances the activation rate of plasminogen by t-PA. This is explained by an increased affinity of fibrin-bound t-PA for plasminogen and not by alteration of the catalytic rate constant of the enzyme. The high affinity of t-PA for plasminogen in the presence of fibrin thus allows efficient activation on the fibrin clot, while no significant plasminogen activation by t-PA occurs in plasma. scu-PA has a high affinity for plasminogen (Km = 0.3 microM) but a low catalytic rate constant (kcat = 0.02 sec-1). However, scu-PA does not activate plasminogen in plasma in the absence of a fibrin clot, owing to the presence of (a) competitive inhibitor(s). Fibrin-specific thrombolysis appears to be due to the fact that fibrin reverses the competitive inhibition. The thrombolytic efficacy and fibrin specificity of natural and recombinant t-PA has been demonstrated in animal models of pulmonary embolism, venous thrombosis, and coronary artery thrombosis. In all these studies intravenous infusion of t-PA at sufficiently high rates caused efficient thrombolysis in the absence of systemic fibrinolytic activation. The efficacy and relative fibrinogen-sparing effect of t-PA was recently confirmed in three multicenter clinical trials in patients with acute myocardial infarction.(ABSTRACT TRUNCATED AT 400 WORDS)