Re-engineering of human urokinase provides a system for structure-based drug design at high resolution and reveals a novel structural subsite

Inhibition of urokinase has been shown to slow tumor growth and metastasis. To utilize structure-based drug design, human urokinase was re-engineered to provide a more optimal crystal form. The redesigned protein consists of residues Ile(16)-Lys(243) (in the chymotrypsin numbering system; for the urokinase numbering system it is Ile(159)-Lys(404)) and two point mutations, C122A and N145Q (C279A and N302Q). The protein yields crystals that diffract to ultra-high resolution at a synchrotron source. The native structure has been refined to 1.5 A resolution. This new crystal form contains an accessible active site that facilitates compound soaking, which was used to determine the co-crystal structures of urokinase in complex with the small molecule inhibitors amiloride, 4-iodo-benzo(b)thiophene-2-carboxamidine and phenylguanidine at 2. 0-2.2 A resolution. All three inhibitors bind at the primary binding pocket of urokinase. The structures of amiloride and 4-iodo-benzo(b)thiophene-2-carboxamidine also reveal that each of their halogen atoms are bound at a novel structural subsite adjacent to the primary binding pocket. This site consists of residues Gly(218), Ser(146), and Cys(191)-Cys(220) and the side chain of Lys(143). This pocket could be utilized in future drug design efforts. Crystal structures of these three inhibitors in complex with urokinase reveal strategies for the design of more potent nonpeptidic urokinase inhibitors.     

An inhibitor from placenta specifically binds urokinase and inhibits plasminogen activator released from ovarian carcinoma in tissue culture.

An inhibitor present in placenta and released in placental tissue culture forms specific complexes with each of two molecular forms of urokinase. Autoradiography demonstrated that the inhibitor shifted the electrophoretic position of 125I-labelled urokinase. It did not change the migration of diisopropyl-fluorophosphate-inactivated 125I-labelled urokinase, thereby indicating complex formation dependent on active serine site in urokinase. The inhibitor had a strong neutralizing effect on the plasminogen activators released from human ovarian carcinoma in tissue culture. The placental inhibitor might prove useful in inhibiting the fibrinolytic process necessary for proliferation of tumour vessels.     

Probing inhibitors binding to human urokinase crystals by Raman microscopy: implications for compound screening

Inhibition of urokinase activity represents a promising target for antimetastatic therapy for several types of tumor. The present study sets out to investigate the potential of Raman spectroscopy for defining the molecular details of inhibitor binding to this enzyme, with emphasis on single crystal studies. It is demonstrated that high quality Raman spectra from a series of five inhibitors bound individually to the active site of human urokinase can be obtained in situ from urokinase single crystals in hanging drops by using a Raman microscope. After recording the spectrum of the free crystal, a solution of inhibitor containing an amidine functional group on a naphthalene ring was added, and the spectrum of the crystal-inhibitor complex was obtained. The resulting difference Raman spectrum contained only vibrational modes due to bound inhibitor, originating from the protonated group, i.e., the amidinium moiety, as well as naphthalene ring modes and features from other functionalities that made up each inhibitor. The identification of the amidinium modes was placed on a quantitative basis by experimental and theoretical work on naphthamidine compounds. For the protonated group, -C-(NH2)(2)(+), the symmetric stretch occurs near 1520 cm(-1), and a less intense antisymmetric mode appears in the Raman spectra near 1680 cm(-1). The presence of vibrational modes near 1520 cm(-1) in each of the Raman difference spectra of the five complexes examined unambiguously identifies the protonated form of the amidinium group in the active site. Several advantages were found for single crystal experiments over solution studies of inhibitor-enzyme complexes, and these are discussed. The use of single crystals permits competitive binding experiments that cannot be undertaken in solution in any kind of homogeneous assay format. The Raman difference spectrum for a single crystal that had been exposed to equimolar amounts of all five inhibitors in the hanging drop showed only the Raman signature of the compound with the lowest K(i). These findings suggest that the Raman approach may offer a route in the screening of compounds in drug design applications as well as an adjunct to crystallographic analysis.     

An inhibitor from placenta specifically binds urokinase and inhibits plasminogen activator released from ovarian carcinoma in tissue culture

An inhibitor present in placenta and released in placental tissue culture forms specific complexes with each of two molecular forms of urokinase. Audoradiography demonstrated that the inhibitor shifted the electrophoretic position of ¹²⁵I-labelled urokinase. It did not change the migration of diisopropyl-fluorophosphate-inactivated ¹²⁵I-labelled urokinase, thereby indicating complex formation dependent on active serine site in urokinase. The inhibitor had a strong neutralizing effect on the plasminogen activators released from human ovarian carcinoma in tissue culture. The placental inhibitor might prove useful in inhibiting the fibrinolytic process necessary for proliferation of tumour vessels.     

Detection and partial characterization of a specific plasminogen activator inhibitor in human chondrocyte cultures

Serum-free culture medium collected from primary monolayer cultures of human articular chondrocytes was found to inhibit human urokinase [EC 3.4.21.31] activity. Although chondrocyte culture medium contained a small amount of endothelial-type plasminogen activator inhibitor which could be demonstrated by reverse fibrin autography, most of the urokinase inhibitory activity of chondrocyte culture medium was shown to be due to a different molecule from endothelial-type inhibitor, since it did not react with a specific antibody to this type of inhibitor. The dominant urokinase inhibitor in chondrocyte culture medium was partially purified by concanavalin A-Sepharose affinity chromatography. The partially purified inhibitor inhibited high-Mr urokinase more effectively than low-Mr urokinase, but no obvious inhibition was detected against tissue-type plasminogen activator, plasmin, trypsin, and thrombin. The inhibitor had an apparent Mr of 43,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and it was unstable to sodium dodecyl sulfate, acid, and heat treatments. Inhibition of urokinase by the inhibitor was accompanied with the formation of a sodium dodecyl sulfate-stable high-Mr complex between them. Inhibition and complex formation required the active site of urokinase. The partially purified inhibitor was thought to be immunologically different from the known classes of plasminogen activator inhibitors, including endothelial-type inhibitor, macrophage/monocyte inhibitor, and protease nexin, since it did not react with specific antibodies to these inhibitors.     

Purification and characterization of a novel inhibitor of urokinase from human urine. Quantitation and preliminary characterization in plasma

Urokinase-related proteins in human urine occur mainly as a 1:1 complex of urokinase with an inhibitor (Stump, D. C., Thienpont, M., and Collen, D. (1986) J. Biol. Chem. 261, 1267-1273). BALB/c mice were immunized with this urokinase-urokinase inhibitor complex and spleen cells fused with mouse myeloma cells, resulting in hybridomas producing monoclonal antibodies. Three antibodies reacting with the complex but not with urokinase were utilized to develop a sensitive (0.5 ng/ml) enzyme-linked immunosorbent assay for the urokinase inhibitor, which was used for monitoring its purification by chromatography on zinc chelate-Sepharose, concanavalin A-Sepharose, SP-Sephadex C-50, and Sephadex G-100. A homogenous glycoprotein of apparent Mr 50,000 was obtained with a yield of 40 micrograms/liter urine and a purification factor of 320. One mg of the purified protein inhibited 35,000 IU of urokinase within 30 min at 37 degrees C. This protein was immunologically related to both the purified urokinase-urokinase inhibitor complex and to the inhibitor portion dissociated from it by nucleophilic dissociation. It was immunologically distinct from all known protease inhibitors, including the endothelial cell-derived fast-acting inhibitor of tissue-type plasminogen activator, the placental inhibitor of urokinase and protease nexin. In electrophoresis the protein migrated with beta-mobility. Inhibition of urokinase occurred with a second order rate constant (k) of 8 X 10(3) M-1 s-1 in the absence and of 9 X 10(4) M-1 s-1 in the presence of 50 IU of heparin/ml. The urokinase inhibitor was inactive towards single-chain urokinase-type plasminogen activator and plasmin, but it inhibited two-chain tissue-type plasminogen activator with a k below 10(3) M-1 s-1 and thrombin with a k of 4 X 10(4) M-1 s-1 in the absence and 2 X 10(5) M-1 s-1 in the presence of heparin. The concentration of this urokinase inhibitor in plasma from normal subjects determined by immunoassay was 2 +/- 0.7 micrograms/ml (mean +/- S.D., n = 25). The protein purified from plasma by immunoabsorption had the same Mr, amino acid composition, and immunoreactivity as the urinary protein. Furthermore, when urokinase was added to plasma, time-dependent urokinase-urokinase inhibitor complex formation was observed at a rate similar to that observed for the inhibition of urokinase by the purified inhibitor from urine. This urokinase inhibitor, purified from human urine, most probably represents a new plasma protease inhibitor.     

Structure-directed discovery of potent non-peptidic inhibitors of human urokinase that access a novel binding subsite

BACKGROUND: Human urokinase-type plasminogen activator has been implicated in the regulation and control of basement membrane and interstitial protein degradation. Because of its role in tissue remodeling, urokinase is a central player in the disease progression of cancer, making it an attractive target for design of an anticancer clinical agent: Few urokinase inhibitors have been described, which suggests that discovery of such a compound is in the early stages. Towards integrating structural data into this process, a new human urokinase crystal form amenable to structure-based drug design has been used to discover potent urokinase inhibitors. RESULTS: On the basis of crystallographic data, 2-naphthamidine was chosen as the lead scaffold for structure-directed optimization. This co-crystal structure shows the compound binding at the primary specificity pocket of the trypsin-like protease and at a novel binding subsite that is accessible from the 8-position of 2-napthamidine. This novel subsite was characterized and used to design two compounds with very different 8-substituents that inhibit urokinase with K(i) values of 30-40 nM. CONCLUSIONS: Utilization of a novel subsite yielded two potent urokinase inhibitors even though this site has not been widely used in inhibitor optimization with other trypsin-like proteases, such as those reported for thrombin or factor Xa. The extensive binding pockets present at the substrate-binding groove of these other proteins are blocked by unique insertion loops in urokinase, thus necessitating the utilization of additional binding subsites. Successful implementation of this strategy and characterization of the novel site provides a significant step towards the discovery of an anticancer clinical agent.     

Urokinase-related proteins in human urine. Isolation and characterization of single-chain urokinase (pro-urokinase) and urokinase-inhibitor complex

Urokinase-related proteins were purified from 60-liter batches of human urine collected into the protease inhibitor aprotinin to prevent proteolytic degradation. Three homogeneous species were obtained by chromatography on zinc chelate-Sepharose, SP-Sephadex C-50, Sephadex G-100, benzamidine-Sepharose, and immunoadsorption on a murine anti-human urokinase monoclonal antibody. One urokinase-related protein with Mr 95,000 representing a complex of two-chain urokinase with an inhibitor accounts for about 70% of the total urokinase-related antigen in urine. Nucleophilic agents dissociate the complex into active two-chain urokinase and a protein with Mr 45,000-50,000 which is immunologically distinct from urokinase. Approximately 25% of the urinary urokinase-related antigen represents a single-chain molecule with Mr 54,000. This highly purified single-chain molecule was obtained with a yield of 5 micrograms/liter of urine. Only trace amounts (less than 5%) of the urokinase-related antigen were recovered as free two-chain urokinase. The urinary single-chain urokinase-related protein has no specific affinity for fibrin. It has a very low activity on Pyroglu-Gly-Arg-p-nitroanilide, a urokinase-specific synthetic substrate, but directly activates plasminogen following Michaelis-Menten kinetics with Km = 0.7 microM and kcat = 0.0011 S-1. The single-chain molecule is rapidly converted to active two-chain urokinase by plasmin. Active two-chain urinary urokinase has a very high amidolytic activity and activates plasminogen with Km = 60 microM and kcat = 1.4 S-1. It is concluded that the urokinase-related proteins in human urine consist of about 25% of single-chain urokinase (10-20 micrograms/liter) and of about 75% two-chain urokinase (40-50 micrograms/liter), the bulk of which is complexed to an inhibitor. Because even in freshly voided urine most of the urokinase-related antigen is already converted to two-chain urokinase, urine does not seem to be a suitable source for the large-scale purification of single-chain urokinase. In view of the very significant intrinsic plasminogen-activating properties of single-chain urokinase, it should not be considered to be a proenzyme form of urokinase. The dramatic differences of its kinetic constants from those of urokinase render the designation single-chain urokinase equally inadequate. Consequently, the designation "single-chain urokinase-type plasminogen activator" was recently adopted by the International Committee on Thrombosis and Haemostasis (Annual Meeting, San Diego, CA, July 13-14, 1985).     

A structural model for the inhibition of calpain by calpastatin: crystal structures of the native domain VI of calpain and its complexes with calpastatin peptide and a small molecule inhibitor

The Ca(2+)-dependent cysteine protease calpain along with its endogenous inhibitor calpastatin is widely distributed. The interactions between calpain and calpastatin have been studied to better understand the nature of calpain inhibition by calpastatin, which can aid the design of small molecule inhibitors to calpain. Here we present the crystal structure of a complex between a calpastatin peptide and the calcium-binding domain VI of calpain. DIC19 is a 19 residue peptide, which corresponds to one of the three interacting domains of calpastatin, which is known to interact with domain VI of calpain. We present two crystal structures of DIC19 bound to domain VI of calpain, determined by molecular replacement methods to 2.5A and 2.2A resolution. In the process of crystallizing the inhibitor complex, a new native crystal form was identified which had the homodimer 2-fold axis along a crystallographic axis as opposed to the previously observed dimer in the asymmetric unit. The crystal structures of the native domain VI and its inhibitor PD150606 (3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid) complex were determined with the help of molecular replacement methods to 2.0A and 2.3A resolution, respectively. In addition, we built a homology model for the complex between domain IV and DIA19 peptide of calpastatin. Finally, we present a model for the calpastatin-inhibited calpain.     

Changes in interactions in complexes of hirudin derivatives and human alpha-thrombin due to different crystal forms.

The three-dimensional structures of D-Phe-Pro-Arg-chloromethyl ketone-inhibited thrombin in complex with Tyr-63-sulfated hirudin (ternary complex) and of thrombin in complex with the bifunctional inhibitor D-Phe-Pro-Arg-Pro-(Gly)4-hirudin (CGP 50,856, binary complex) have been determined by X-ray crystallography in crystal forms different from those described by Skrzypczak-Jankun et al. (Skrzypczak-Jankun, E., Carperos, V.E., Ravichandran, K.G., & Tulinsky, A., 1991, J. Mol. Biol. 221, 1379-1393). In both complexes, the interactions of the C-terminal hirudin segments of the inhibitors binding to the fibrinogen-binding exosite of thrombin are clearly established, including residues 60-64, which are disordered in the earlier crystal form. The interactions of the sulfate group of Tyr-63 in the ternary complex structure explain why natural sulfated hirudin binds with a 10-fold lower K(i) than the desulfated recombinant material. In this new crystal form, the autolysis loop of thrombin (residues 146-150), which is disordered in the earlier crystal form, is ordered due to crystal contacts. Interactions between the C-terminal fragment of hirudin and thrombin are not influenced by crystal contacts in this new crystal form, in contrast to the earlier form. In the bifunctional inhibitor-thrombin complex, the peptide bond between Arg-Pro (P1-P1') seems to be cleaved.     

Isolation and characterization of alpha2-plasmin inhibitor from human plasma. A novel proteinase inhibitor which inhibits activator-induced clot lysis.

A procedure is presented for purifying a novel proteinase inhibitor in human plasma whose apparent unique biological property is to inhibit efficiently the lysis of fibrin clots induced by plasminogen activator. The final product is homogeneous as judged by disc gel electrophoresis, and immunoelectrophoresis. Its molecular weight estimated by sodium dodecyl sulfate gel electrophoresis or sedimentation equilibrium is 67,000 and 63,000, respectively. The inhibitor is a glycoprotein consisting polypeptide chain containing 11.7% carbohyrate. It migrates in the alpha2-globulin region in immunoelectrophoresis. The inhibitor is chemically and immunologically different from all the other known inhibitors in plasma. Inhibition of plasmin by the inhibitor is almost instantaneous even at 0 degrees, in contrast to the slow inhibition of urokinase (plasminogen activator in urine). Plasminogen activation by urokinase-induced clot lysis is inhibited by the inhibitor mainly through a mechanism of instantaneous inhibition of plasmin formed and not through the inhibition of urokinase. The inhibitor also inhibits trypsin. Consequently, it is suggested that this newly identified inhibitor is named alpha2-plasmin inhibitor or alpha2-proteinase inhibitor. A specific antibody directed against the inhibitor neutralizes virtually all inhibitory activity of plasma to activator-induced clot lysis. Immunochemical quantitation of the inhibitor was specific antiserum to the inhibitor and the purified inhibitor as a standard indicates that the concentration of the inhibitory in the serum of a healthy man is in or near the range of 5 to 7 mg/100 ml, which is the lowest concentration among the concentration of the proteinase inhibitors in plasma. The inhibitor and plasmin, trypsin, or urokinase form a complex which cannot be dissociated with denaturing and reducing agents. The formation of the enzyme-inhibitor complex occurs on a 1:1 molar basis and is associated with the cleavage of a unique peptide bone, which is most clearly demonstrated in the interaction of the inhibitor and beta-trypsin. In the complex formation between the inhibitor and plasmin, the inhibitor is cross-linked with the light chain which contains the active site of plasmin. It is suggested that, in a fashion analogous to complex formation between alpha1-antitrypsin and trypsin, the cross-links are formed between the active site serine of the enzyme and the newly formed COOH-terminal residue of the inhibitor, with cleavage of a peptide bond.     

Modulation of plasminogen activator and plasminogen activator inhibitor expression in the human U373 glioblastoma/astrocytoma cell line by inflammatory mediators.

The human U373 glioblastoma/astrocytoma cell line was found to constitutively produce and secrete a plasminogen activator and a plasminogen activator inhibitor. The plasminogen activator was identified as urokinase based on apparent molecular weight, immunoblotting with anti-urokinase antibodies, and Northern blotting with a human urokinase cDNA probe. The inhibitor secreted by U373 cells was found to be related to the PAI-1 molecule based on reactivity with anti-human PAI-1 antibodies, apparent molecular weight, and Northern blot analysis with a human PAI-1 cDNA probe. The expression of both urokinase and the PAI-1-like molecule by U373 cells could be modulated by phorbol myristate acetate or by inflammatory mediators such as interferon-gamma and interleukin-1. In the case of interleukin-1, the alpha form exhibited no detectable effect while the beta form not only elevated inhibitor levels, it also appeared to induce the production of tissue plasminogen activator. Thus, in these cells interleukin-1 beta induces alterations in PA and PAI expression and interleukin-1 alpha does not, even though the two forms are reported to utilize the same cellular receptor.     

Characterization of a macrophage-derived plasminogen-activator inhibitor. Similarities with placental urokinase inhibitor.

Human and mouse macrophages release a fibrinolytic inhibitor after stimulation by endotoxin in vitro. The released mouse inhibitor was indistinguishable in size by molecular-sieve chromatography from an intracellular form (approx. 50 kDa), and both inhibitors blocked urokinase directly as judged by a 125I-plasminogen conversion assay. The intracellular inhibitor was found mostly to dissociate from 125I-urokinase during sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reduced conditions, but a dodecyl sulphate-stable complex at 65-67 kDa was observed. Because of similarities in the reported size, stability and urokinase-binding properties of a placental urokinase inhibitor, the kinetic properties of the two inhibitors were compared. Under the reaction conditions employed (37 degrees C at pH7.4 in the presence of 0.2% Triton X-100), the association rate constants and equilibrium dissociation constants of the two inhibitors were indistinguishable, 3 X 10(5) M-1 X s-1 and 4 X 10(-10) M respectively. These data show that peritoneal macrophages contain a plasminogen-activator very similar to a previously recognized placental inhibitor. Although the inhibitor appears to be a trace protein in macrophages, placental macrophages may account for the accumulation of the inhibitor in placental tissue.     

Crystal structures of the RNA-dependent RNA polymerase genotype 2a of hepatitis C virus reveal two conformations and suggest mechanisms of inhibition by non-nucleoside inhibitors

Crystal structures of the RNA-dependent RNA polymerase genotype 2a of hepatitis C virus (HCV) from two crystal forms have been determined. Similar to the three-dimensional structures of HCV polymerase genotype 1b and other known polymerases, the structures of the HCV polymerase genotype 2a in both crystal forms can be depicted in the classical right-hand arrangement with fingers, palm, and thumb domains. The main structural differences between the molecules in the two crystal forms lie at the interface of the fingers and thumb domains. The relative orientation of the thumb domain with respect to the fingers and palm domains and the beta-flap region is altered. Structural analysis reveals that the NS5B polymerase in crystal form I adopts a "closed" conformation that is believed to be the active form, whereas NS5B in crystal form II adopts an "open" conformation and is thus in the inactive form. In addition, we have determined the structures of two NS5B polymerase/non-nucleoside inhibitor complexes. Both inhibitors bind at a common binding site, which is nearly 35 A away from the polymerase active site and is located in the thumb domain. The binding pocket is predominantly hydrophobic in nature, and the enzyme inhibitor complexes are stabilized by hydrogen bonding and van der Waals interactions. Inhibitors can only be soaked in crystal form I and not in form II; examination of the enzyme-inhibitor complex reveals that the enzyme has undergone a dramatic conformational change from the form I (active) complex to the form II (inactive).     

A new apo-caspase-6 crystal form reveals the active conformation of the apoenzyme

Caspase-6 has been identified as a key component in the pathway of neurodegenerative diseases such as Alzheimer's disease and Huntington's disease. It has been the focus of drug development for some time, but only recently have structural data become available. The first study identified a novel noncanonical conformation of apo-caspase-6 contrasting with the typical caspase conformation. Then, the structures of both caspase-6 zymogen and the Ac-VEID-CHO peptide inhibitor complex described caspase-6 in the canonical conformation, raising the question of why the intermediate between these two structures (mature apo-caspase-6) would adopt the noncanonical conformation. In this study, we present a new crystal form of the apoenzyme in the canonical conformation by identifying the previous apostructure as a pH-inactivated form of caspase-6. Our new apostructure is further compared to the Ac-VEID-CHO caspase-6 inhibitor complex. The structural comparison allows us to visualize the organization of loops L2, L3, and L4 upon ligand binding and how the catalytic groove forms to accommodate the inhibitor.     

A glia-derived neurite-promoting factor with protease inhibitory activity.

Brain cells and glioma cells in culture release a protein which induces neurite outgrowth in neuroblastoma cells. This neurite-promoting factor (NPF), which has been purified from serum-free glioma conditioned medium, has an apparent mol. wt. of 43 000. NPF inhibits urokinase as well as plasminogen activator-dependent caseinolysis or fibrinolysis. NPF and urokinase form an SDS-resistant complex. The fact that this glia-derived NPF is a potent protease inhibitor indicates that glial cells modulate the proteolytic activity associated with neuronal cells and suggests that this phenomenon is one of the biochemical events involved in the regulation of neurite growth.     

An inhibitor of plasminogen activation from human placenta. Purification and characterization

Placental extracts contain inhibitors of human urinary urokinase. These extracts form a heterogeneous population of complexes with 125I-urokinase that are recognizable by changes in gel filtration profile and mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with reducing agents eliminated the size heterogeneity without loss of activity, thereby allowing the placental inhibitor to be purified. Active inhibitor has been isolated in apparently homogeneous form after an eight-step procedure that included salt extraction, ammonium sulfate fractionation, column chromatography on CM-cellulose, DEAE-Sepharose, and hydroxylapatite, chromatofocusing, preparative gel electrophoresis, and hydrophobic chromatography. The purified inhibitor has Mr = 47,000. The inhibitor is relatively specific for plasminogen activators since it does not inhibit the action of plasmin, factor XIIa, plasma kallikrein, or thrombin. The inhibitor forms complexes with 1:1 stoichiometry that block the active sites of urokinase (but not prourokinase) and both one- and two-chain forms of tissue plasminogen activator. The stability of these complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggest that they are based on covalently bonded structures. Although both types of plasminogen activator are inhibited, the rate of interaction is significantly faster with urokinase, tissue plasminogen activator being inhibited less efficiently. The complexes formed can be dissociated by mild alkali or hydroxylamine, thereby regenerating both enzymes and inhibitor at their original molecular weights. The results suggest that the complexes are stabilized by ester-like bonds; these might involve the hydroxyl of serine at the active site of the proteases and a carboxyl group in the inhibitor.     

Separation and characterization of nonphosphorylated and serine-phosphorylated urokinase. Catalytic properties and sensitivity to plasminogen activator inhibitor type 1.

Urokinase synthesized by human A431 epidermoid carcinoma cells is phosphorylated on serine (Mastronicola, M. R., Stoppelli, M. P., Migliaccio, A., Auricchio, F., and Blasi, F. (1990) FEBS Lett. 266, 109-114). To test the possibility that phosphorylation may have specific effects on urokinase function, the phosphorylated and nonphosphorylated forms of urokinase were separated by Fe(3+)-Sepharose chromatography. Both forms exhibit indistinguishable Km and kcat for plasminogen activation. On the other hand, their sensitivity toward the specific plasminogen activator inhibitor type 1 is different as assessed by measuring both the stability of the covalent complex and the residual enzymatic activity. Phosphorylated urokinase was 50% inhibited at a concentration of plasminogen activator inhibitor type 1 4-fold higher than nonphosphorylated urokinase (0.7 versus 0.15 nM). Furthermore about 10% of phosphorylated urokinase was resistant to plasminogen activator inhibitor type 1 at a concentration as high as 20 nM. Thus, phosphorylation affects urokinase sensitivity to plasminogen activator inhibitor type 1, therefore resulting in a net, although indirect, increase of urokinase activity. These results suggest the existence of a novel cellular regulatory mechanism of extracellular proteolysis.     

The primary inhibitor of plasmin in human plasma.

A complex between plasmin and an inhibitor was isolated by affinity chromatography from urokinase-activated human plasma. The complex did not react with antibodies against any of the known proteinase inhibitors in plasma. A rabbit antiserum against the complex was produced. It contained antibodies agianst plasminogen+plasmin and an alpha2 protein. By crossed immunoelectrophoresis the alpha2 protein was shown to form a complex with plasmin, when generated by urokinase in plasma, and with purified plasmin. The alpha2 protein was eluted by Sephadex G-200 gel filtration with KD approx. 0.35, different from the other inhibitors of plasmin in plasma, and corresponding to an apparent relative molecular mass (Mr) of about 75000. By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Mr of the complex was found to be approx. 130000. After reduction of the complex two main bands of protein were observed, with Mr, about 72000 and 66000, probably representing an acyl-enzyme complex of plasmin-light chain and inhibitor-heavy chain, and a plasmin-heavy chain. A weak band with Mr 9000 was possibly an inhibitor-light chain. The inhibitor was partially purified and used to titrate purified plasmin of known active-site concentration. The inhibitor bound plasmin rapidly and strongly. Assuming an equimolar combining ratio, the concentration of active inhibitor in normal human plasma was estimated to be 1.1 mumol/1. A fraction about 0.3 of the antigenic inhibitor protein appeared to be functionally inactive. In plasma, plasmin is primarily bound to the inhibitor. Only after its saturation does lysis of fibrinogen and fibrin occur and a complex between plasmin and alpha2 macroglobulin appear.     

Characterization of the dexamethasone-induced inhibitor of plasminogen activator in HTC hepatoma cells

Incubation of HTC rat hepatoma cells with the synthetic glucocorticoid dexamethasone rapidly inhibits plasminogen activator (PA) activity secondary to the induction of a specific acid-stable inhibitor of plasminogen activation (Cwikel, B. J., Barouski-Miller, P.A., Coleman, P.L., and Gelehrter, T.D. (1984) J. Biol. Chem. 259, 6847-6851). We have further characterized this inhibitor with respect to its interaction with both urokinase and tissue plasminogen activator, and its protease specificity. The HTC PA inhibitor rapidly inhibits urokinase and tissue plasminogen activator with an apparent second-order rate constant of 3-5 x 10(7) M-1 X s-1. The inhibitor forms stable covalent complexes with both urokinase and tissue plasminogen activator, with which plasmin, trypsin, and factor Xa apparently do not compete. Complex formation is saturable and requires the active site of the PA. The mass of the inhibitor-PA complex is 50,000 daltons greater than that of PA alone, consistent with an Mr for the PA inhibitor of 50,000 as demonstrated directly by reverse fibrin autography. The HTC PA inhibitor does not inhibit thrombin and differs in its kinetic and biochemical properties from protease nexin.