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At least 19 recordsLinked to original sources

PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗

PubMed1995

Urokinase-type plasminogen activator/type-2 plasminogen-activator inhibitor complexes are not internalized upon binding to the urokinase-type-plasminogen-activator receptor in THP-1 cells. Interaction of urokinase-type plasminogen activator/type-2 plasminogen-activator inhibitor complexes with the cell surface.

The urokinase-type plasminogen activator (uPA) and its inhibitor PAI-2 form a covalent complex that, upon binding to the uPA receptor (uPA-R), is cleaved into two fragments of molecular masses 70 kDa and 22 kDa. The 70-kDa fragment results from the interaction of the B chain of uPA and PAI-2 whereas the 22-kDa fragment is the A chain of the enzyme [13]. We prove that, at 37 degrees C, the 70-kDa fragment is released into the medium, whereas the 22-kDa fragment remains bound to the cell surface. uPA complexed with its other specific inhibitor, PAI-1, is cleaved into fragments of identical sizes, but the 70-kDa component is internalized via the alpha 2-macroglobulin receptor. At 4 degrees C, both uPA/PAI-2 complex degradation products remain bound to the uPA-R. We propose that the 70-kDa molecule, which lacks the uPA binding region for uPA-R, is bound to uPA-R via a new binding site, unmasked only when uPA-R is occupied by uPA/PAI-2 complexes.

Cell Line↗

PubMed2002

Gonadotropin surge-induced up-regulation of the plasminogen activators (tissue plasminogen activator and urokinase plasminogen activator) and the urokinase plasminogen activator receptor within bovine periovulatory follicular and luteal tissue.

This study examined the effect of the preovulatory gonadotropin surge on the temporal and spatial regulation of tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), and uPA receptor (uPAR) mRNA expression and tPA, uPA, and plasmin activity in bovine preovulatory follicles and new corpora lutea collected at approximately 0, 6, 12, 18, 24, and 48 h after a GnRH-induced gonadotropin surge. Messenger RNAs for tPA, uPA, and uPAR were increased in a temporally specific fashion within 24 h of the gonadotropin surge. Localization of tPA mRNA was primarily to the granulosal layer, whereas both uPA and uPAR mRNAs were detected in both the granulosal and thecal layers and adjacent ovarian stroma. Activity for tPA was increased in follicular fluid and the preovulatory follicle apex and base within 12 h after the gonadotropin surge. The increase in tPA activity in the follicle base was transient, whereas the increased activity in the apex was maintained through the 24 h time point. Activity for uPA increased in the follicle apex and base within 12 h of the gonadotropin surge and remained elevated. Plasmin activity in follicular fluid also increased within 12 h after the preovulatory gonadotropin surge and was greatest at 24 h. Our results indicate that mRNA expression and enzyme activity for both tPA and uPA are increased in a temporally and spatially specific manner in bovine preovulatory follicles after exposure to a gonadotropin surge. Increased plasminogen activator and plasmin activity may be a contributing factor in the mechanisms of follicular rupture in cattle.

Animals↗

PubMed1988

Plasminogen activation in diabetes mellitus. Kinetics of plasmin formation with tissue plasminogen activator and plasminogen from individual diabetic donors and with in vitro glucosylated plasminogen.

Functional abnormalities of tissue plasminogen activator (t-PA) (high Km in the presence of fibrin) and plasminogen (Pg) (substrate inhibition in the fibrin-stimulated system) from uncontrolled type I diabetics are reversible upon normalization of metabolic parameters. Therefore the effect of in vitro glucosylation of Pg was studied and similar but less pronounced substrate inhibition as with diabetic Pg was observed. However, the activation of the Pg fraction most likely containing bound cis-OH groups (e.g. glucose) was normal. These data suggest that either glucosylation in vitro is less stable than in vivo or that in diabetics protein modifications other than glucosylation occur. In this respect we can also show that acetone, a representative of ketone bodies and also elevated in poorly controlled diabetics, does not affect Pg activation in vitro.

Diabetes Mellitus, Type 1↗

PubMed1995

Recombinant lys-plasminogen, but not glu-plasminogen, improves recombinant tissue-type plasminogen activator-induced coronary thrombolysis in dogs.

OBJECTIVES: This study examined the modification of recombinant tissue-type plasminogen activator (rt-PA)-induced thrombolysis by recombinant lys-plasminogen. BACKGROUND: Recombinant tissue-type plasminogen activator restores flow in the thrombosed coronary artery, but the artery often reoccludes. The rt-PA-induced thrombolysis is a result of activation of plasminogen bound to fibrin in the thrombus and results in generation of the fibrinolytic enzyme plasmin. Small amounts of lys-plasminogen are formed when rt-PA is used. Lys-plasminogen binds to fibrin with a 10-fold greater affinity than the predominant native glu-plasminogen, leading to a loose fibrin structure. METHODS: Dogs with electrically induced occlusive intracoronary thrombus were treated with saline solution (n = 9), glu-plasminogen (2 mg/kg body weight, n = 5) or lys-plasminogen (2 mg/kg, n = 5), followed by infusion of rt-PA (1 mg/kg over 20 min) 10 min later. RESULTS: Reperfusion rates were similar in all groups of dogs, but the time to reflow was lowest in dogs given lys-plasminogen compared with those given saline solution or glu-plasminogen before rt-PA (mean [+/- SE] 14 +/- 2 vs. 22 +/- 2 and 23 +/- 3 min, respectively, p < 0.05). None of the reperfused coronary arteries reoccluded in the lys-plasminogen plus rt-PA group, whereas 75% reoccluded in dogs given saline solution plus rt-PA, and 50% reoccluded in those given glu-plasminogen plus rt-PA. Accordingly, duration of reflow was greater in the lys-plasminogen plus rt-PA group (> 120 vs. 39 +/- 7 and 82 +/- 21 min, respectively, p < 0.05). Plasminogen activator inhibitor-1 activity decreased during rt-PA infusion and thereafter increased in all dogs, but less so in dogs given lys-plasminogen (p < 0.05 vs. those given saline solution before rt-PA). CONCLUSIONS: Treatment with recombinant lys-plasminogen before rt-PA reduces time to reflow and sustains reflow after thrombolysis, whereas glu-plasminogen has no such effect.

Animals↗

PubMed1996

Cell-surface cytokeratin 8 is the major plasminogen receptor on breast cancer cells and is required for the accelerated activation of cell-associated plasminogen by tissue-type plasminogen activator.

Cytokeratin 8 (CK 8) has been identified on the external surfaces of viable, unpermeabilized epithelial cells (Hembrough, T. A., Vasudevan, J., Allietta, M. M., Glass, W. F., and Gonias, S. L. (1995) J. Cell Sci. 108, 1071-1082). In this study, we demonstrated that CK 8 is the major plasminogen-binding protein in plasma membrane fractions isolated from three breast cancer cell lines, BT20, MCF-7, and MDA-MB-157. To assess the function of CK 8 as a plasminogen receptor, monoclonal antibody 1E8 was raised against the carboxyl-terminal 12 amino acids of CK 8. The 1E8 epitope was present on the external surfaces of breast cancer cells, as determined by immunofluorescence microscopy. 125I-1E8 bound to MCF-7 cells; the maximum binding capacity (1.5 x 10(6) sites per cell) was comparable with that determined for plasminogen. When MCF-7 cells were incubated with Fab fragments of 1E8, specific 125I-plasminogen binding was decreased up to 82%. Specific plasminogen binding was decreased up to 67%, even when the unbound 1E8 Fab was removed by washing the cells prior to adding 125I-plasminogen. Preincubation with 1E8 Fab decreased plasminogen binding to BT20 and MDA-MB-157 cells, although to a lesser extent than with MCF-7 cells. Plasminogen activation by tissue-type plasminogen activator was greatly accelerated, due to a large decrease in Km, when the plasminogen was bound to MCF-7 cells. Pretreatment with 1E8 Fab decreased the rate of plasminogen activation by up to 83%, implicating CK 8 in the MCF-7 cell-accelerated reaction. These studies identify cell-surface CK 8 as a major plasminogen receptor in breast cancer cells and as a required component for the rapid activation of cell-associated plasminogen by tissue-type plasminogen activator.

Antibodies, Monoclonal↗

PubMedDate not supplied

Differential effects of acute and chronic wound fluids on urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor, and tissue-type plasminogen activator in cultured human keratinocytes and fibroblasts.

The effect of wound fluids collected from acute well-healing wounds and chronic nonhealing venous leg ulcers on the plasminogen activation system of keratinocyte and fibroblast cell cultures was studied in a simplified wound-healing model. Acute wound fluid was collected from donor sites of split skin grafts at different time points representing the progressive healing of the wound. Urokinase-type plasminogen activator, tissue-type plasminogen activator, urokinase-type plasminogen activator receptor, and plasminogen activator inhibitor 1 expression were studied. The methods used were immunocapture assay and immunocytochemistry. The results indicated that the later the acute wound fluid was collected, the greater the urokinase-type plasminogen activator and the lower the plasminogen inhibitor-1 level in treated cells. In contrast, the level of urokinase-type plasminogen activator receptor remained stable irrespective of wound fluid treatment. Immunostaining for urokinase-type plasminogen activator of acute wound fluid-treated cells showed a disseminated punctate pattern over the cell surface, but with chronic wound fluid, urokinase-type plasminogen activator was localized to focal contacts. Our findings support the view that in the acute wound environment the plasminogen activator system is proteolytically active and that in chronic leg ulcers urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor may also be organized for cell adhesion and migration.

Acute Disease↗

PubMed1984

Importance of the interaction between plasminogen and fibrin for plasminogen activation by tissue-type plasminogen activator.

The potentiating effect of fibrin monomer on plasminogen activation by tissue-type plasminogen activator is much more important with lys-plasminogen than with mini-plasminogen (which lacks the high affinity lysine-binding site important for binding to fibrin). Furthermore, this potentiating effect is totally abolished when lys-plasminogen is eluted from fibrin by the addition of 1 mM epsilon-amino caproic acid. Binding does however not seem to be the only condition required since it was found that fragment D is a much stronger potentiator of the activation of plasminogen by tissue-type plasminogen activator than fragment E although plasminogen binds to both fragment D and fragment E. Furthermore, fragment E has the same effect on the activation of lys-and mini-plasminogen by tissue-type plasminogen activator. Therefore, it is suggested that binding of plasminogen to fibrin involves a conformational change in the plasminogen molecule, facilitating its activation by tissue-type plasminogen activator.

Aminocaproic Acid↗

PubMed1983

Differences in effects of fibrin(ogen) fragments on the activation of 1-glu-plasminogen and 442-val-plasminogen by tissue-type plasminogen activator.

The activation rate of plasminogen by tissue-type plasminogen activator can be increased by fibrin(ogen) fragments. There is a remarkable difference in the effect of these fragments on the stimulation of 1-glu-plasminogen activation and 442-val-plasminogen (mini-plasminogen) activation. Fibrin monomer as well as plasmic fragments Y, D EGTA and D-dimer have a stimulating effect on both 1-glu-plasminogen and 442-val-plasminogen activation, whereas cyanogen bromide fragment FCB2 stimulates only the activation of 1-glu-plasminogen. Results indicate that two types of sites may be operational in fibrin and fibrin(ogen) fragments Y, D EGTA and D-dimer. One type of site (FCB2-related) interacts probably with plasminogen and may be dependent on the kringle 1-4 region; the other type of site probably interacts either with plasminogen in a non-kringle 1-4 region-dependent manner or with tissue-type plasminogen activator.

Chemical Phenomena↗

PubMed2003

Critical role for conversion of glu-plasminogen to Lys-plasminogen for optimal stimulation of plasminogen activation on cell surfaces.

When Glu-plasminogen, the native circulating form of the zymogen, is bound to cell surfaces, its activation is markedly enhanced compared with the reaction in solution. This results in localization of the broad-spectrum proteolytic activity of plasmin on cell surfaces. The cell-associated plasmin plays a key role in fibrinolysis, cell migration, and prohormone processing. It is well established that the localization of plasminogen and plasminogen activators on cell surfaces promotes the enhanced plasminogen activation on the cell surface. The focus of this article is to review recent studies demonstrating that the conversion of Glu-plasminogen to the more readily activated Lys-plasminogen derivative is necessary for optimal stimulation of plasminogen activation on the cell surface, and that the interaction of Glu-plasminogen with cells serves to increase processing of Glu-plasminogen to Lys-plasminogen, thereby enhancing plasminogen activation on the cell surface.

Fibrinolytic Agents↗

PubMed1996

Cytokeratin 8 released by breast carcinoma cells in vitro binds plasminogen and tissue-type plasminogen activator and promotes plasminogen activation.

Cell-surface activation of plasminogen may be important in diseases that involve cellular migration, including atherosclerosis and tumour invasion/metastasis. Cytokeratin 8 (CK 8) has been identified as a plasminogen-binding protein expressed on the external surfaces of hepatocytes and breast carcinoma cells [Hembrough, Vasudevan, Allietta, Glass and Gonias (1995) J. Cell Sci. 108, 1071-1082]. In this investigation, we demonstrate that a soluble form of CK 8 is released into the culture medium of breast cancer cell lines. The released CK 8 is in the form of variably sized polymers that bind plasminogen and promote the activation of [Glu1]plasminogen and [Lys78]plasminogen by single-chain tissue-type plasminogen activator (sct-PA). To assess the mechanism by which CK 8 promotes plasminogen activation, CK 8 was purified from rat hepatocytes and immobilized in microtitre plates. Immobilized CK 8 bound 125I-plasminogen and 125I-sct-PA in a specific and saturable manner. The KDs were 160 +/- 40 nM and 250 +/- 48 nM, respectively. Activation of plasminogen bound to immobilized CK 8 was accelerated compared with plasminogen in solution, as determined using a coupled-substrate fluorescence assay and SDS/PAGE. The ability of CK 8 to promote plasminogen activation may be important in the pericellular spaces surrounding breast cancer cells and at the cell surface.

Animals↗

PubMed1991

Interactions of plasminogen and tissue plasminogen activator (t-PA) with amphoterin. Enhancement of t-PA-catalyzed plasminogen activation by amphoterin.

The heparin-binding p30 protein amphoterin is proposed to mediate adhesive interactions of the advancing plasma membrane in migrating and differentiating cells. Since the NH2-terminal part of amphoterin is exceptionally rich in lysine residues, we have studied its interactions with plasminogen and tissue plasminogen activator (t-PA). On immunostaining of N18 neuroblastoma cells, amphoterin and t-PA showed a close co-localization in the filopodia of the leading membrane and in the substrate-attached material. In purified systems, both t-PA and plasminogen bound to immobilized amphoterin, and their binding was inhibited by the lysine analogue epsilon-aminocaproic acid. Plasminogen bound to immobilized amphoterin was activated by t-PA, and this resulted in effective degradation of the immobilized amphoterin. Correspondingly, amphoterin-bound t-PA activated plasminogen. In solution amphoterin accelerated t-PA-catalyzed plasminogen activation maximally 46-fold. The results indicate that t-PA and plasminogen form through their lysine-binding sites a complex with amphoterin, which results in acceleration of plasminogen activation and effective degradation of amphoterin. We suggest that local acceleration of t-PA-catalyzed plasminogen activation by amphoterin at the leading membrane enhances the penetration of growing cytoplasmic processes through extracellular materials during cell migration, differentiation and regeneration. The amphoterin-mediated adhesion at the leading membrane may be transient in nature, because the protein also enhances its own breakdown by accelerating t-PA-catalyzed plasminogen activation.

Binding Sites↗

PubMed1989

Potentiation by Lys-plasminogen of clot lysis by single or two chain urokinase-type plasminogen activator or tissue-type plasminogen activator.

Study has been made of the influence of addition of human NH2 terminal glutamic acid plasminogen (Glu-Plg) or human NH2 terminal lysine plasminogen (Lys-Plg) to normal citrated plasma upon the rate of lysis of fully crosslinked plasma clots in the presence of single or two chain urokinase type plasminogen activator (scu-PA/tcu-PA) or tissue plasminogen activator (t-PA). The specificity of any thrombolytic property was evaluated by measurement of plasma fibrinogen levels. Lys-Plg added to a concentration of 20% of normal plasma plasminogen caused 5 to 6 fold increase in the extent of lysis observed at 6 hours by 100 units/ml of scu-PA and with a small increase in fibrinogenolysis. Glu-Plg added at 20% of normal level had no influence on thrombolysis but at 50% of normal caused increased thrombolysis with rapid depletion of plasma fibrinogen. An apparently synergistic effect of addition of tcu-PA on scu-PA activity was increased by addition of plasminogen (e.g. addition of 20% Lys-Plg increased the lysis rate 4 to 5 fold over the first hour equivalent to an increase of potency of approximately three to four fold). Addition of plasminogen up to double the normal plasma concentration was observed to have no influence on clot lysis in the presence of t-PA. Plasminogen potentiated the rate of lysis by scu-PA/t-PA synergic mixtures with an approximately 1.5 to 1.9 fold increase in potency. Potentiation occurred without increase in the depletion of plasma fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Coagulation Tests↗

PubMed1990

Plasminogen activation with single-chain urokinase-type plasminogen activator (scu-PA). Studies with active site mutagenized plasminogen (Ser740----Ala) and plasmin-resistant scu-PA (Lys158----Glu).

The mechanism of the activation of plasminogen by single-chain urokinase-type plasminogen activator (single-chain u-PA, scu-PA) was studied using rscu-PA-Glu158, a recombinant plasmin-resistant mutant of human scu-PA obtained by site-specific mutagenesis of Lys158 to Glu, and rPlg-Ala740, a recombinant human plasminogen in which the catalytic site is destroyed by mutagenesis of the active-site Ser740 to Ala. Conversion of 125I-labeled single-chain plasminogen to two-chain plasmin was quantitated on reduced sodium dodecyl sulfate-gel electrophoresis combined with autoradiography and radioisotope counting of gels bands. The efficiencies of both rscu-PA-Glu158 and rscu-PA for the activation of rPlg-Ala740 and of natural plasminogen were comparable and were 250-500-fold lower than that of recombinant two-chain u-PA (rtcu-PA) for rscu-PA-Glu158 and 100-200-fold lower for rscu-PA. Pretreatment of rscu-PA-Glu158 or rscu-PA with excess alpha 2-antiplasmin, which efficiently neutralizes all contaminating rtcu-PA, did not significantly reduce the catalytic efficiency of these single-chain moieties, indicating that they have a low but significant intrinsic plasminogen activating potential. The low intrinsic catalytic efficiency of rscu-PA for the conversion of plasminogen to plasmin may be sufficient to generate trace amounts of plasmin, which may regulate plasminogen activation by converting poorly active rscu-PA to very active rtcu-PA.

Alanine↗

PubMed1996

Plasminogen activation in bullous pemphigoid immunohistology reveals urokinase type plasminogen activator, its receptor and plasminogen activator inhibitor type-2 in lesional epidermis.

Keratinocytes synthesize urokinase-type plasminogen activator (uPA) and a specific cell surface receptor for uPA (uPA-R, CD 87). Plasminogen is present in plasma and interstitial fluids from where it is bound to cell surfaces via plasmin(ogen) binding sites. uPA binds to the uPA-R in an autocrine manner and activates cell-bound plasminogen: a mechanism, which provides plasmin for pericellular proteolysis. Cell-bound uPA is regulated by plasminogen activator inhibitor type-1 (PAI-1) or type-2 (PAI-2). Bullous pemphigoid is an autoimmune inflammatory skin disease characterized by subepidermal blisters. Although circumstantial evidence suggested plasminogen activation in lesional epidermis of bullous pemphigoid, immunohistological data on the type of plasminogen activators, on the uPA-receptor or the type of plasminogen activator inhibitors in the lesions of bullous pemphigoid are lacking so far. To obtain this information we have performed the present immunohistological study. The presence of uPA and its receptor as well as PAI-2 was disclosed in epidermal keratinocytes in the roof of the subepidermal blisters. Moreover, keratinocytes at the bottom of the blister, which most likely represent keratinocytes during reepithelialization were stained. Co-localization was found for uPA and its receptor, uPA and plasmin(ogen) as well as for uPA and PAI-2. In non-lesional epidermis of bullous pemphigoid only PAI-2 was found. We propose that the expression of uPA and uPA-R, as well as the upregulation of PAI-2 in keratinocytes of lesional epidermis is part of the repair and reepithelialization process following lesion formation, i.e. epidermo-dermal dyshesion, in bullous pemphigoid.

Aged↗

PubMed1990

Comparison of the effects of fibrinogen and fibrin products and isolated peptide chains on the fibrin-mediated stimulation of plasminogen activation by tissue-type plasminogen activator, and on the fibrin-dependent enhancement of the amidolytic activity of one-chain tissue-type plasminogen activator.

Intact fibrin monomer, the early fibrin degradation product (X-fragment), late fibrinogen degradation products (fragments D and E), fibrinogen cyanogen bromide fragment FCB-2, and isolated peptide chains of fibrinogen and fibrin were investigated for their ability to replace fibrin in the stimulation of one-chain tissue-type plasminogen activator. They were also investigated for their ability to stimulate plasminogen activation by one-chain tissue-type plasminogen activator, which occurs via ternary complex formation. The stimulatory effect of the different fibrin/ogen products decreased in the order: fibrin X-fragment greater than fibrin monomer greater than CNBr-fragment FCB-2 greater than fibrin alpha-chain. Fibrin beta/gamma-chains and fibrinogen peptide chains were found to be weak stimulators. Fibrinogen fragments D and E have almost no effect. The amidolytic activity of one-chain tissue-type plasminogen activator was stimulated by intact fibrin monomer and somewhat more strongly by fibrin X-fragment. This stimulation by fibrin monomer, which occurred via an increase in the kcat value, was competitively inhibited by isolated fibrin alpha-chain (Ki = 0.12 mumol/l). The results show that the fibrin-mediated stimulation of plasminogen activation occurs when both one-chain tissue-type plasminogen activator and plasminogen are bound to fibrin, and that this process is essentially independent of the conformation of the fibrin molecule. In comparison, the fibrin-dependent stimulation of the amidolytic activity of one-chain tissue-type plasminogen activator is a more complex process, which depends on the correct conformation of the fibrin molecule.

Amino Acid Sequence↗

PubMed1986

Interaction of heparin with plasminogen activators and plasminogen: effects on the activation of plasminogen.

The amidolytic plasmin activity of a mixture of tissue plasminogen activator (tPA) and plasminogen is enhanced by heparin at therapeutic concentrations. Heparin also increases the activity in mixtures of urokinase-type plasminogen activator (uPA) and plasminogen but has no effect on streptokinase or plasmin. Direct analyses of plasminogen activation by polyacrylamide gel electrophoresis demonstrate that heparin increases the activation of plasminogen by both tPA and uPA. Binding studies show that heparin binds to various components of the fibrinolytic system, with tight binding demonstrable with tPA, uPA, and Lys-plasminogen. The stimulation of tPA activity by fibrin, however, is diminished by heparin. The ability of heparin to promote plasmin generation is destroyed by incubation of the heparin with heparinase, whereas incubation with chondroitinase ABC or AC has no effect. Also, stimulation of plasmin formation is not observed with dextran sulfate or chondroitin sulfate A, B, or C. Analyses of heparin fractions after separation on columns of antithrombin III-Sepharose suggest that both the high-affinity and the low-affinity fractions, which have dramatically different anticoagulant activity, have similar activity toward the fibrinolytic components.

Animals↗

PubMed1991

On the reversible interaction of plasminogen activator inhibitor-1 with tissue-type plasminogen activator and with urokinase-type plasminogen activator.

The reaction between plasminogen activators and plasminogen activator inhibitor-1 is characterized by an initial rapid formation of an inactive reversible complex. The second-order association rate constant (k1) of complex formation of recombinant two-chain tissue-type plasminogen activator (rt-PA) or recombinant two-chain urokinase-type plasminogen activator (rtcu-PA) by recombinant plasminogen activator inhibitor-1 (rPAI-1) is 2.9 +/- 0.4 x 10(7) M-1 s-1 (mean +/- S.D., n = 30) and 2.0 +/- 0.6 x 10(7) M-1 s-1 (n = 12), respectively. Different molecular forms of tissue- or urokinase-type plasminogen activator which do not form covalent complexes with rPAI-1, including rt-PA-Ala478 (rt-PA with the active-site Ser478 mutagenized to Ala) and anhydro-urokinase (rtcu-PA with the active-site Ser356 converted to dehydroalanine) reduced k1 in a concentration-dependent manner, compatible with 1:1 stoichiometric complex formation between rPAI-1 and these ligands. The apparent dissociation constant (KD) of the complex between rPAI-1 and rt-PA-Ala478, determined as the concentration of rt-PA-Ala478 which reduced k1 to 50% of its control value, was 3-5 nM. Corresponding concentrations of active-site-blocked two-chain rt-PA were 150-250-fold higher. The concentration of anhydro-urokinase which reduced k1 to 50% was 4-6 nM, whereas that of active-site-blocked rtcu-PA was 100-250-fold higher. Recombinant single-chain urokinase-type plasminogen activator had an apparent KD of about 2 microM. These results suggest that inhibition of rt-PA or rtcu-PA by rPAI-1 proceeds via a reversible high affinity interaction which does not require a functional active site but which is markedly reduced following inactivation of the enzymes with active-site titrants.

Animals↗