Involvement of residues 296-299 in the enzymatic activity of tissue-type plasminogen activator.

The tetra-alanine substitution variant KHRR 296-299 AAAA of tissue-type plasminogen activator (t-PA) was previously shown to have enhanced fibrin specificity and enhanced activity in the presence of fibrin compared with the wild-type form of the molecule. The structural requirements for these alterations in enzymatic activity were investigated by constructing several amino acid substitution variants at each of the positions from 296 to 299 and evaluating their activities under a variety of conditions. Effects on plasminogen activator activity were common among the point mutants at positions 296-299; nearly all had a phenotype similar to the KHRR 296-299 AAAA variant. The greatest effects on enzymatic function were found with multiple substitution variants, but some single charge reversals and proline substitutions had substantial effects. The enhanced fibrin specificity of KHRR 296-299 AAAA t-PA results in less fibrinogenolysis than seen with wild-type t-PA. Approximately four times greater concentration of KHRR 296-299 AAAA compared with wild-type t-PA was required to consume 50% of the fibrinogen in human plasma.     

Characterization of interaction of active-site serine mutants of tissue-type plasminogen activator with plasminogen activator inhibitor-1

To define determinants of interactions of tissue-type plasminogen activator (t-PA) with plasminogen activator inhibitor type-1 (PAI-1), we utilized site-directed mutagenesis to substitute either threonine or glycine for the active-site serine of tissue-type plasminogen activator. Assays of conditioned media of transfected cells demonstrated that the threonine substitution markedly decreased but did not entirely abolish plasminogen activating activity. In contrast, the glycine substitution yielded a mutant with absolutely no detectable plasminogen activating activity. Wild-type t-PA formed stable complexes with PAI-1. However, even when exogenous inhibitor was present in the medium or purified mutant was added to plasma that had been rendered PAI-1-rich in vivo, the mutants were present in the free form exclusively judging from results of fibrin autography and Western blot analysis. Thus, despite maintenance of some residual plasminogen-activating activity associated with preservation of the hydroxyl group at the active site, the threonine mutant did not form stable complexes with inhibitor. The glycine mutant, developed so that steric hindrance or other unfavorable interactions at the modified active site would be minimal, was similarly incapable of forming complexes with PAI-1. These results show that the presence of an active site serine residue is necessary for formation of stable complexes between t-PA and PAI-1.     

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.     

Involvement of finger domain and kringle 2 domain of tissue-type plasminogen activator in fibrin binding and stimulation of activity by fibrin.

Human tissue-type plasminogen activator (t-PA) catalyses the conversion of inactive plasminogen into active plasmin, the main fibrinolytic enzyme. This process is confined to the fibrin surface by specific binding of t-PA to fibrin and stimulation of its activity by fibrin. Tissue-type plasminogen activator contains five domains designated finger, growth factor, kringle 1, kringle 2 and protease. The involvement of the domains in fibrin specificity was investigated with a set of variant proteins lacking one or more domains. Variant proteins were produced by expression in Chinese hamster ovary cells of plasmids containing part of the coding sequence for the activator. It was found that kringle 2 domain only is involved in stimulation of activity by fibrin. In the absence of plasminogen and at low concentration of fibrin, binding of t-PA is mainly due to the finger domain, while at high fibrin concentrations also kringle 2 is involved in fibrin binding. In the presence of plasminogen, fibrin binding of the kringle 2 region of t-PA also becomes important at low fibrin concentrations.     

Kinetic analysis of the interaction between plasminogen activator inhibitor-1 and tissue-type plasminogen activator.

The kinetics of inhibition of tissue-type plasminogen activator (t-PA) by the fast-acting plasminogen activator inhibitor-1 (PAI-1) was investigated in homogeneous (plasma) and heterogeneous (solid-phase fibrin) systems by using radioisotopic and spectrophotometric analysis. It is demonstrated that fibrin-bound t-PA is protected from inhibition by PAI-1, whereas t-PA in soluble phase is rapidly inhibited (K1 = 10(7) M-1.s-1) even in the presence of 2 microM-plasminogen. The inhibitor interferes with the binding of t-PA to fibrin in a competitive manner. As a consequence the Kd of t-PA for fibrin (1.2 +/- 0.4 nM) increases and the maximal velocity of plasminogen activation by fibrin-bound t-PA is not modified. From the plot of the apparent Kd versus the concentration of PAI-1 a Ki value of 1.3 +/- 0.3 nM was calculated. The quasi-similar values for the dissociation constants between fibrin and t-PA (Kd) and between PAI-1 and t-PA (Ki), as well as the competitive type of inhibition observed, indicate that the fibrinolytic activity of human plasma may be the result of an equilibrium distribution of t-PA between both the amount of fibrin generated and the concentration of circulating inhibitor.     

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.     

Identification of determinants involved in binding of tissue-type plasminogen activator-plasminogen activator inhibitor type 1 complexes to HepG2 cells

Complexes between tissue-type plasminogen activator (t-PA) and its rapidly acting inhibitor plasminogen activator inhibitor type 1 (PAI-1) are bound, internalized, and degraded by HepG2 cells. The mechanism involves endocytosis mediated by a specific high-affinity receptor. However, the particular domains of the complex that are recognized by the receptor have not been elucidated. To identify the determinants involved in ligand binding to the receptor, several variants of t-PA were assessed for their ability to form complexes with PAI-1 and thereby to inhibit specific cellular binding of complexes between structurally unmodified 125I-t-PA and PAI-1. Catalytically active variants lacking selected structural domains form complexes with PAI-1 and inhibit 125I-t-PA.PAI-1 binding to HepG2 cells. In addition, several forms of the plasminogen activator urokinase (u-PA), which shares partial structural homology with t-PA, were evaluated as competitors of cellular binding. The catalytically active two-chain forms of u-PA, but not the inactive proenzyme single-chain form, complex with PAI-1 and inhibit specific binding of 125I-t-PA.PAI-1, suggesting that the serine protease domain, rather than other domains, may confer the determinants required for cellular binding. However, a mutant t-PA with markedly reduced catalytic activity, resulting from replacement of the active site serine with threonine, not only forms complexes with PAI-1 but also inhibits specific cellular binding of unmodified 125I-t-PA.PAI-1. These data indicate that specific binding of t-PA.PAI-1 to HepG2 cells does not require a serine-containing catalytic site in the protease domain. To determine whether binding of the complex is mediated through other components of t-PA or through structural elements of PAI-1, both t-PA and PAI-1 were examined separately for capacity to bind directly to HepG2 cells. To exclude potential interactions with components of the extracellular matrix which contains binding sites for PAI-1, ligand binding to HepG2 cells in suspension was assessed. Although neither t-PA nor PAI-1 alone binds specifically to HepG2 cells, the preformed t-PA.PAI-1 complexes do. These findings suggest that specific binding of t-PA.PAI-1 requires elements of the PAI-1 moiety and/or parts of the protease domain of t-PA.     

Artificial exon shuffling between tissue-type plasminogen activator (t-PA) and urokinase (u-PA): a comparative study on the fibrinolytic properties of t-PA/u-PA hybrid proteins

We constructed two human tissue-type plasminogen activator/urokinase (t-PA/u-PA) hybrid cDNAs which were expressed by transfection of mouse Ltk- cells. The properties of the secreted proteins were compared with those of recombinant t-PA (rt-PA) and high molecular weight (HMW) u-PA. The hybrid proteins each contain the amino-terminal fibrin-binding chain of t-PA fused to the carboxy-terminal serine protease moiety of u-PA but differ by a stretch of 13 amino acid residues between kringle 2 of t-PA and the plasmin cleavage site of u-PA. Hybrid protein rt-PA/u-PA I contains amino acids 1-262 of t-PA connected with amino acids 147-411 of u-PA, whereas hybrid protein rt-PA/u-PA II consists of the same t-PA segment and residues 134-411 of u-PA. We demonstrated fibrin binding for rt-PA, whereas the hybrid proteins bind to a lesser extent and HMW u-PA has no affinity for fibrin. Plasminogen activation by either one of the hybrid proteins in the absence of a fibrin substitute was similar to that by HMW u-PA, while rt-PA was much less active. The catalytic efficiency, in the presence of a fibrin substitute, increases more than 2000-fold for rt-PA, about 250-fold for hybrid proteins I and II, and 12-fold for HMW u-PA, respectively. Under these conditions the hybrid proteins are more efficient plasminogen activators than the parental ones. The hybrid molecules form a 1:1 molar complex with the human endothelial plasminogen activator inhibitor (PAI-1), analogous to that formed by rt-PA and HMW u-PA. The relative affinity of rt-PA for PAI-1 is 4.6-fold higher than that of HMW u-PA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Type 1 plasminogen activator inhibitor binds to fibrin via vitronectin

Type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA), circulates as a complex with the abundant plasma glycoprotein, vitronectin. This interaction stabilizes the inhibitor in its active conformation In this report, the effects of vitronectin on the interactions of PAI-1 with fibrin clots were studied. Confocal microscopic imaging of platelet-poor plasma clots reveals that essentially all fibrin-associated PAI-1 colocalizes with fibrin-bound vitronectin. Moreover, formation of platelet-poor plasma clots in the presence of polyclonal antibodies specific for vitronectin attenuated the inhibitory effects of PAI-1 on t-PA-mediated fibrinolysis. Addition of vitronectin during clot formation markedly potentiates PAI-1-mediated inhibition of lysis of (125)I-labeled fibrin clots by t-PA. This effect is dependent on direct binding interactions of vitronectin with fibrin. There is no significant effect of fibrin-associated vitronectin on fibrinolysis in the absence of PAI-1. The binding of PAI-1 to fibrin clots formed in the absence of vitronectin was characterized by a low affinity (K(d) approximately 3.5 micrometer) and rapid loss of PAI-1 inhibitory activity over time. In contrast, a high affinity and stabilization of PAI-1 activity characterized the cooperative binding of PAI-1 to fibrin formed in the presence of vitronectin. These findings indicate that plasma PAI-1.vitronectin complexes can be localized to the surface of fibrin clots; by this localization, they may modulate fibrinolysis and clot reorganization.     

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.     

Concentration of plasminogen activators and inhibitor in the human endometrium at different phases of the menstrual cycle.

The concentrations of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA) and plasminogen activator inhibitor (PAI-1) have been determined in endometrial curettings obtained from 46 subfertile women during proliferative, early or late secretory phases of the menstrual cycle. t-PA activity and antigen concentrations was significantly higher (P < 0.001) in late secretory endometrium than in proliferative or early secretory endometrium. Higher concentrations of PAI-1 antigen (P < 0.05) were also noted in late secretory phase than in proliferative and early secretory endometrium. However, u-PA concentration was not significantly different and no PAI activity could be demonstrated in the menstrual phases studied. Zymography studies confirmed the presence of both t-PA and u-PA in the endometrium. Ovarian hormonal patterns may therefore influence the activity of plasminogen activators especially of t-PA in the endometrium during various phases of the menstrual cycle.     

Fibrinolytic components in nasal mucosa and nasal secretion

 We evaluated a possible role for fibrinolytic components in nasal secretion by tissue localization with immunohistochemical techniques and by measuring their antigen concentrations in nasal discharge by means of ELISA and fibrin autography. Nasal mucosa was obtained surgically from the inferior turbinate. Urokinase-type plasminogen activator (u-PA) specific staining was observed in pseudostratified ciliated epithelium and was predominant in mucous cells of the seromucinous gland, while serous cells were almost devoid of stain. The pattern of staining of plasminogen activator inhibitor-2 was similar to that of u-PA. In contrast, plasminogen activator inhibitor-1(PAI-1) immunoreactive material was localized exclusively in serous cells of seromucinous glands. Positive staining for tissue-type plasminogen activator (t-PA) was observed in endothelial cells and basal cells, which differentiate into either ciliated or goblet cells. Nasal secretions were partially fractionated by immunospecific antibody-immobilized Sepharose. Subsequent fibrin autography patterns indicated the presence of u-PA, PAI-1, and t-PA. After methacholine provocation, the level of t-PA increased transiently but decreased rapidly with subsequent challenges. These differential stainings of fibrinolytic components and the existence of PAs and PAI-1 in the nasal discharge suggest that the fibrinolytic system may play a role in the movement and fluidity of nasal secretion. Accepted: 25 May 1998     

Functional role of proteolytic cleavage at arginine-275 of human tissue plasminogen activator as assessed by site-directed mutagenesis

Activation of the zymogen form of a serine protease is associated with a conformational change that follows proteolysis at a specific site. Tissue-type plasminogen activator (t-PA) is homologous to mammalian serine proteases and contains an apparent activation cleavage site at arginine-275. To clarify the functional consequences of cleavage at arginine-275 of t-PA, site-specific mutagenesis was performed to convert arginine-275 to a glutamic acid. The mutant enzyme (designated Arg-275----Glu t-PA) could be converted to the two-chain form by Staphylococcus aureus V8 protease but not by plasmin. The one-chain form was 8 times less active against the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine-p-nitroanilide (S-2288), and the ability of the enzyme to activate plasminogen in the absence of fibrinogen was reduced 20-50 times compared to the two-chain form. In contrast, one-chain Arg-275----Glu t-PA has equal activity to the two-chain form when assayed in the presence of physiological levels of fibrinogen and plasminogen. Fibrin bound significantly more of the one-chain form of t-PA than the two-chain form for both the wild-type and mutated enzymes. One- and two-chain forms of the wild-type and mutated plasminogen activators slowly formed complexes with plasma protease inhibitors, although the one-chain forms showed decreased complex formation with alpha 2-macroglobulin. The one-chain form of t-PA therefore is fully functional under physiologic conditions and has an increased fibrin binding compared to the two-chain form.     

Longistatin, a novel plasminogen activator from vector ticks, is resistant to plasminogen activator inhibitor-1

Thrombo-occlusive diseases are major causes of morbidity and mortality, and tissue-type plasminogen activator (t-PA) is recommended for the treatment of the maladies. However, both t-PA and u-PA are rapidly inactivated by plasminogen activator inhibitor-1 (PAI-1). Here, we show that longistatin, a novel plasminogen activator isolated from the ixodid tick, Haemaphysalis longicornis is resistant to PAI-1. Longistatin was relatively less susceptible to the inhibitory effect of SDS-treated platelet lysate than physiologic PAs. Platelet lysate inhibited t-PA and tcu-PA with the IC₅₀ of 7.7 and 9.1 μg/ml, respectively, whereas for longistatin inhibition IC₅₀ was 20.1 μg/ml (p < 0.01). Similarly, activated PAI-1 (20 nM) inhibited only 21.47% activity of longistatin but almost completely inhibited t-PA (99.17%) and tcu-PA (96.84%). Interestingly, longistatin retained 76.73% initial activity even after 3 h of incubation with 20 nM of PAI-1. IC₅₀ of PAI-1 during longistatin inhibition was 88.3 nM while it was 3.9 and 3.2 nM in t-PA and tcu-PA inhibition, respectively. Longistatin completely hydrolyzed fibrin clot by activating plasminogen efficiently in the presence of 20 nM of PAI-1. Importantly, unlike t-PA, longistatin did not form complex with PAI-1. Collectively, our results suggest that longistatin is resistant to PAI-1 and maybe an interesting tool for the development of a PAI-1 resistant effective thrombolytic agent.     

Interaction of tissue-type plasminogen activator and plasminogen activator inhibitor 1 on the surface of endothelial cells

The site of the reaction between plasminogen activators and plasminogen activator inhibitor 1 (PAI-1) was investigated in cultures of human umbilical vein endothelial cells. In conditioned medium from endothelial cells, two forms of a plasminogen activator-specific inhibitor can be demonstrated: an active form that readily binds to and inhibits plasminogen activators and an immunologically related quiescent form which has no anti-activator activity but which can be activated by denaturation. In conditioned medium, only a few percent of PAI-1 is the active form. However, the addition of increasing concentrations of tissue-type plasminogen activator (t-PA) or urokinase to confluent endothelial cells produced a saturable (3.0 pmol/5 x 10(5) cells), dose-dependent increase of the activator-PAI-1 complex in the conditioned medium even in the presence of actinomycin D or cycloheximide. This resulted also in a dose-dependent decrease of the residual PAI activity measured by reverse fibrin autography both in the conditioned medium and cell extracts. Short-time exposure of endothelial cells to a large amount of t-PA caused almost complete depletion of all cell-associated PAI activity. Although there was no detectable PAI activity even after activation of PAI by denaturants or antigen in the culture medium at 4 degrees C without the addition of t-PA, the addition of t-PA at 4 degrees C not only resulted in the formation of 70% of the amount of the t-PA.PAI complex in conditioned medium at 37 degrees C, but also induced PAI-1 antigen in a time and dose-dependent manner in the conditioned medium. Moreover, 125I-labeled t-PA immobilized on Sepharose added directly to endothelial cells formed a complex with PAI-1 in a dose-dependent manner. On the other hand, no detectable complex was formed with PAI-1 when Sepharose-immobilized 125I-labeled t-PA was added to endothelial cells under conditions in which the added t-PA could not contact the cells directly but other proteins could pass freely by the use of a Transwell. All these results suggest that a "storage pool" on the surface of endothelial cells or the extracellular matrix produced by endothelial cells contains almost all the active PAI-1, and reaction between PA and PAI-1 mainly occurs on the endothelial cell membranes, resulting in a decrease of the conversion of active PAI-1 to the quiescent form.     

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.     

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.     

Catabolism of tissue-type plasminogen activator by the human hepatoma cell line Hep G2. Modulation by plasminogen activator inhibitor type 1

Catalytic activity of tissue-type plasminogen activator (t-PA) in plasma is regulated in part by formation of complexes with specific inhibitors as well as by hepatic clearance. Potential interaction of these two regulatory mechanisms was examined in the human hepatoma cell line Hep G2. These cells secrete plasminogen activator inhibitor type-1 (PAI-1) and initiate catabolism of exogenous t-PA by receptor-mediated endocytosis. Specific binding of 125I-t-PA to cells at 4 degrees C results in dose-dependent formation of a 95-kDa species recognized by monospecific anti-PAI-1 and anti-t-PA antibodies and stable in the presence of low (0.2%) concentrations of sodium dodecyl sulfate (SDS). Specific binding of 125I-t-PA and formation of the 95-kDa SDS-stable species are inhibited in a concentration-dependent manner following preincubation of cells with anti-PAI-1 antibodies. High and low molecular weight forms of urokinase plasminogen activator (u-PA) capable of forming specific complexes with PAI-1 complete for 125I-t-PA binding sites. However, the proenzyme form of u-PA (scu-PA), incapable of forming complexes with PAI-1, does not compete for 125I-t-PA binding sites. The role of the serine protease active site of t-PA in mediating both interaction with PAI-1 and specific binding was examined using 125I-t-PA that had been functionally inactivated with D-phenylalanyl-L-propyl-L-arginyl-chloromethyl ketone (PPACK). 125I-t-PA-PPACK, despite a 6-fold lower affinity than active 125I-t-PA, exhibited specific binding to cells without detectable formation of SDS-stable complexes with PAI-1. Both surface-bound 125I-t-PA and 125I-t-PA-PPACK are internalized and degraded by cells at 37 degrees C. 125I-t-PA is internalized as a stable complex with PAI-1, whereas 125I-t-PA-PPACK is internalized with similar kinetics but without the presence of an SDS-stable complex. Thus, PAI-1 appears capable of modulating t-PA catabolism in the human hepatocyte.     

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.     

Vitronectin governs the interaction between plasminogen activator inhibitor 1 and tissue-type plasminogen activator.

The "serpin" plasminogen activator inhibitor 1 (PAI-1) is the fast acting inhibitor of plasminogen activators (tissue-type (t-PA) and urokinase type-PA) and is an essential regulatory protein of the fibrinolytic system. Its P1-P1' reactive center (R346 M347) acts as a "bait" for tight binding to t-PA/urokinase-type PA. In vivo, PAI-1 is encountered in complex with vitronectin, an interaction known to stabilize its activity but not to affect the second-order association rate constant (k1) between PAI-1 and t-PA. Nevertheless, by using PAI-1 reactive site variants (R346M, M347S, and R346M M347S), we show that the binding of vitronectin to the PAI-1 mutant proteins improves plasminogen activator inhibition. In the absence of vitronectin the PAI-1 R346M mutants are virtually inactive toward t-PA (k1 less than 1 x 10(3) M-1 s-1). In contrast, in the presence of vitronectin the rate of association increases about 1,000-fold (k1 of 6-8 x 10(5) M-1 s-1). This inhibition coincides with the formation of serpin-typical, sodium dodecyl sulfide-stable t-PA.PAI-1 R346M (R346M M347S) complexes. As evidenced by amino acid sequence analysis, the newly created M346-M/S347 peptide bond is susceptible to attack by t-PA, similar to the wild-type R346-M347 peptide bond, indicating that in the presence of vitronectin M346 functions as an efficient P1 residue. In addition, we show that the inhibition of t-PA and urokinase-type PA by PAI-1 mutant proteins is accelerated by the presence of the nonprotease A chains of the plasminogen activators.