The Chemotactic Action of Urokinase on Smooth Muscle Cells Is Dependent on Its Kringle Domain
Svetlana MukhinaVictoria StepanovaDmitri TraktouevAlexei PoliakovR.Sh. BeabealashvillyYaroslav GurskyMikhail MinashkinAlexander ShevelevTkachuk Va
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Urokinase plasminogen activator (uPA) is thought to exert its effects on cell growth, adhesion, and migration by mechanisms involving proteolysis and interaction with its cell surface receptor (uPAR). The functional properties of uPA and the significance of its various domains for chemotactic activity were analyzed using human airway smooth muscle cells (hAWSMC). The wild-type uPA (r-uPAwt), inactive urokinase with single mutation (His204 to Gln) (r-uPAH/Q), urokinase with mutation of His204to Gln together with a deletion of growth factor-like domain (r-uPAH/Q-GFD), the catalytic domain of urokinase (r-uPALMW), and its kringle domain (r-KD) were expressed inEscherichia coli. We demonstrate that glycosylated uPA, r-uPAwt, r-uPAH/Q, and r-uPAH/Q-GFD elicited similar chemotactic effects. Half-maximal chemotaxis (EC50) were apparent at approximately 2 nm with all the uPA variants. The kringle domain induced cell migration with an EC50 of about 6 nm, whereas the denaturated r-KD and r-uPALMW were without effect. R-uPAwt-induced chemotaxis was dependent on an association with uPAR and a uPA-kringle domain-binding site, determined using a monoclonal uPAR antibody to prevent the uPA-uPAR interaction, and a monoclonal antibody to the uPA-kringle domain. The binding of iodinated r-uPAwt with hAWSMC was due to interaction with a high affinity binding site on the uPAR, and a lower affinity binding site on an unidentified cell surface target, which was mediated exclusively through the kringle domain of urokinase. Specific binding of r-uPAH/Q-GFD to hAWSMC involved an interaction with a single site whose characteristics were similar to those of the low affinity site of r-uPAwt binding to hAWSMC. uPAR-deficient HEK 293 cells specifically bound r-uPAwt and r-uPAH/Q-GFD via a single, similar type of binding site. These cells migrated when stimulated by r-uPAH/Q-GFD and uPAwt, but not r-uPALMW. HEK 293 cells transfected with the uPAR cDNA expressed two classes of sites that bound r-uPAwt; however, only a single site was responsible for the binding of r-uPAH/Q-GFD. Together, these findings indicate that uPA-induced chemotaxis is dependent on the binding of the uPA-kringle to the membrane surface of cells and the association of uPA with uPAR. Urokinase plasminogen activator (uPA) is thought to exert its effects on cell growth, adhesion, and migration by mechanisms involving proteolysis and interaction with its cell surface receptor (uPAR). The functional properties of uPA and the significance of its various domains for chemotactic activity were analyzed using human airway smooth muscle cells (hAWSMC). The wild-type uPA (r-uPAwt), inactive urokinase with single mutation (His204 to Gln) (r-uPAH/Q), urokinase with mutation of His204to Gln together with a deletion of growth factor-like domain (r-uPAH/Q-GFD), the catalytic domain of urokinase (r-uPALMW), and its kringle domain (r-KD) were expressed inEscherichia coli. We demonstrate that glycosylated uPA, r-uPAwt, r-uPAH/Q, and r-uPAH/Q-GFD elicited similar chemotactic effects. Half-maximal chemotaxis (EC50) were apparent at approximately 2 nm with all the uPA variants. The kringle domain induced cell migration with an EC50 of about 6 nm, whereas the denaturated r-KD and r-uPALMW were without effect. R-uPAwt-induced chemotaxis was dependent on an association with uPAR and a uPA-kringle domain-binding site, determined using a monoclonal uPAR antibody to prevent the uPA-uPAR interaction, and a monoclonal antibody to the uPA-kringle domain. The binding of iodinated r-uPAwt with hAWSMC was due to interaction with a high affinity binding site on the uPAR, and a lower affinity binding site on an unidentified cell surface target, which was mediated exclusively through the kringle domain of urokinase. Specific binding of r-uPAH/Q-GFD to hAWSMC involved an interaction with a single site whose characteristics were similar to those of the low affinity site of r-uPAwt binding to hAWSMC. uPAR-deficient HEK 293 cells specifically bound r-uPAwt and r-uPAH/Q-GFD via a single, similar type of binding site. These cells migrated when stimulated by r-uPAH/Q-GFD and uPAwt, but not r-uPALMW. HEK 293 cells transfected with the uPAR cDNA expressed two classes of sites that bound r-uPAwt; however, only a single site was responsible for the binding of r-uPAH/Q-GFD. Together, these findings indicate that uPA-induced chemotaxis is dependent on the binding of the uPA-kringle to the membrane surface of cells and the association of uPA with uPAR. urokinase-type plasminogen activator growth factor-like domain of uPA kringle domain of uPA proteolytic domain of uPA recombinant single-chain urokinase with wild type structure proteolytically inactive urokinase with substitution of His204 in active center for Gln proteolytically inactive urokinase with substitution of His204 in active center for Gln, lacking GFD low molecular weight form of uPA, containing mainly PD uPA deletion mutant lacking kringle domain uPA receptor tissue-type plasminogen activator human airway smooth muscle cells human embryonic kidney cells monoclonal antibody phosphate-buffered saline Dulbecco's modified Eagle's medium polyacrylamide gel electrophoresis bovine serum albumin Plasminogen activators are directly involved in inflammation, angiogenesis, tissue remodeling, and tumor growth and invasion (for review, see Refs. 1.Blasi F. 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Chem. 1997; 272: 13390-13396Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Others have demonstrated that uPA-induced cell migration is dependent on its proteolytic activity, interaction with uPAR, as well as interactions with low density lipoprotein receptor-related protein/α2-macroglobulin receptor (LRP/α2-MR) or very low density lipoprotein receptor (34.Okada S.S. Grobmeyer S.R. Barnathan E.S. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 1269-1276Crossref PubMed Scopus (90) Google Scholar, 35.Wijnberg M.J. Quax P.H. Nieuwenbroek N.M. Verheijen J.H. Thromb. Haemost. 1997; 78: 880-886Crossref PubMed Scopus (60) Google Scholar). Together these studies indicate that signal transduction pathways utilized by uPA in initiating chemotaxis exhibit marked diversity depending upon cell type. The importance of uPA in cell migration and tissue remodeling was recently demonstrated in knockout mice (36.Carmeliet P. Moons L. Herbert J.M. Crawley J. Lupu F. Lijnen R. Collen D. Circ. Res. 1997; 81: 829-839Crossref PubMed Scopus (191) Google Scholar), where there was marked reduction in the size of the neointima which forms after intra-vessel injury in the uPA-deficient mice, when compared with tPA-deficient and normal animals. In contrast, when mice deficient in uPAR were similarly injured neointima size was unaffected (37.Carmeliet P. Moons L. Dewerchin M. Rosenberg S. Herbert J.M. Lupu F. Collen D. J. Cell Biol. 1998; 140: 233-245Crossref PubMed Scopus (117) Google Scholar). Together these observations suggest that mechanisms, distinct or additional to uPA/uPAR interaction might mediate processes of the uPA-dependent tissue remodeling. Since uPAR is attached to the cell membrane via glycosylphosphatidylinositol anchor and lacks transmembrane and cytoplasmic regions, alone it is not capable of initiating a chemotactic signal. An obligatory partner of yet unknown nature is probably required for uPAR to act as a signaling receptor (13.Blasi F. 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Science. 1996; 273: 1551-1555Crossref PubMed Scopus (697) Google Scholar, 42.Kindzelskii A.L. Eszes M.M. Todd 3 rd, R.F. Petty H.R. Biophys. J. 1997; 73: 1777-1784Abstract Full Text PDF PubMed Scopus (77) Google Scholar, 43.Xue W. Mizukami I. Todd 3 rd, R.F. Petty H.R. Cancer Res. 1997; 57: 1682-1689PubMed Google Scholar, 44.May A.E. Kanse S.M. Lund L.R. Gisler R.H. Imhof B.A. Preissner K.T. J. Exp. Med. 1998; 188: 1029-1037Crossref PubMed Scopus (266) Google Scholar). Most investigating uPA-dependent chemotaxis has utilized full-length uPA, DFP-inactivated uPA, or its ATF. However, the ATF includes the growth factor-like and kringle domains, which behave as independently folded domains (45.Novokhatny V. Medved L. Mazar A. Marcotte P. Henkin J. Ingham K. J. Biol. Chem. 1992; 267: 3878-3885Abstract Full Text PDF PubMed Google Scholar). Urokinase PA-kringle is highly homologous to the kringle structure-containing fragments of blood plasma proteins, which affect cell motility and/or proliferation (46.Ji W.-R. Barrientos L.G. Llinas M. Gray H. Villarreal X. DeFord M.E. Castellino F.J. Kramer R.A. Trail P. Biochem. Biophys. Res. Commun. 1998; 247: 414-419Crossref PubMed Scopus (94) Google Scholar,47.Cao Y. Ji R.W. Davidson D. Schaller J. Marti D. Söhndel S. McCance S.G. O'Reilly M.S. Llinás M. Folkman J. J. Biol. Chem. 1996; 271: 29461-29467Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). Despite these observations, the precise mechanism mediating the uPA-dependent effects on cell migration and the impact of the different domains of uPA on cell migration remain unclear. To elucidate the role of uPA structural domains in cell migration, we have constructed and produced recombinant uPA forms deficient in the GFD, as well as the individual domains of urokinase. Recently we reported that a form of uPA lacking proteolytic activity and the growth factor-like domain is capable of inducing cell migration to a similar extent as uPA. It exhibited the atypical binding of urokinase, which was unrelated to any interaction with uPAR via GFD (48.Poliakov A.A. Mukhina S.A. Traktouev D.O. Bibilashvily R. Sh Gursky Y.G. Minashkin M.M. Stepanova V.V. Tkachuk V.A. J. Recept. Signal Transduct. Res. 1999; 19: 939-951Crossref PubMed Scopus (26) Google Scholar). Here we report that besides the growth factor-like domain, which interacts with uPAR, the kringle domain of urokinase can also be involved in the induction of cell chemotaxis by uPA. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, and LipofectinTM were purchased from Life Technologies, Inc.. Chromogenic substrate S-2444 was obtained from Chromogenix, Mölndal, Sweden. Glycosylated high molecular weight urokinase purified from urine (glycosylated uPA) was from Medac, Hamburg, Germany, and tPA (Actilyse) was from Roche Molecular Biochemicals. A mouse monoclonal antibody clone 3 (R-3-01) which reacts with domain I of human uPAR was from Monozyme, Denmark. The goat anti-mouse IgG conjugated with horseradish peroxidase and chemiluminescent substrate "Super Signal Substrate" were from Pierce. Plasminogen, a mouse IgG, and two different murine monoclonal antibodies of the IgG1 subtype (UIG-1 and UNG-5) raised against human urinary uPA (49.Kratasyuk G.A. Jakubov L.Z. Sinitsyn V.V. Domogatsky S.P. Rohklin O.V. Koltsova S.V. Bynyaeva N.A. Fedorova Z.D. Samsonov G.V. Biopolim. Kletka. 1989; 5: 95-101Google Scholar) were kindly provided by Dr. S. P. Domogatsky. An anti-uPA mAb UIG-1 detected high and low molecular forms of uPA by Western blots. An anti-uPA mAb UNG-5 detected urokinase forms containing the kringle domain, (specifically, glycosylated uPA, r-uPAwt, r-uPAH/Q, r-uPAH/Q-GFD, and r-KD); it does not interact with r-uPALMW, lacking the kringle domain, tPA, and plasminogen. UNG-5 did not interact with denaturated r-KD, uPAwt, and r-uPAH/Q-GFD. Recombinant uPA with wild-type structure (r-uPAwt), proteolytically inactive urokinase with substitution of His204 in active center for Gln (r-uPAH/Q), and proteolytically inactive urokinase with deletion of NH2-terminal 1–43 amino acids called "growth factor-like domains" (r-uPAH/Q-GFD) were expressed inEscherichia coli and purified as described previously (14.Stepanova V. Bobik A. Bibilashvily R. Belogurov A. Rybalkin I. Domogatsky S. Little P.J. Goncharova E. Tkachuk V. FEBS Lett. 1997; 414: 417-474Google Scholar,48.Poliakov A.A. Mukhina S.A. Traktouev D.O. Bibilashvily R. Sh Gursky Y.G. Minashkin M.M. Stepanova V.V. Tkachuk V.A. J. Recept. Signal Transduct. Res. 1999; 19: 939-951Crossref PubMed Scopus (26) Google Scholar). Two-chain low molecular weight urokinase with the catalytic domain r-uPALMW was prepared by proteolytic cleavage of r-uPAwt by plasmin, followed by purification with affinity chromatography, using monoclonal antibody against urokinase catalytic domain coupled to CNBr-Sepharose 4B (Amersham Pharmacia Biotech). The recombinant kringle domain (r-KD) was made in E. coli as follows. The region of uPA cDNA corresponding to amino acids 42–210 (50.Riccio A. Grimaldi G. Verde P. Sebastio G. Boast S. Blasi F. Nucleic Acids Res. 1985; 13: 2759-2771Crossref PubMed Scopus (172) Google Scholar, 51.Holmes W.E. Pennica D. Blaber M. Rey M.W. Guenzler W.A. Steffens G.J. Heyneker H.L. Bio/Technology. 1985; 3: 923-929Crossref Scopus (199) Google Scholar) was amplified using primers: M3, 5′-CTGTGATCTAGATAAGTCAAAAACCTGCTATGAGGG-3′, and M7, 5′-AGCACTGTGTGGCGCTGATCACCCAGCAAGGGCTG-3′. Primer M3 was designed to introduce an XbaI site (underlined) between GFD and KD of uPA without changing the amino acid sequence. The PCR product was digested with XbaI andEcoRI enzymes, generating a fragment coding urokinase amino acids 42–166. The expression vector was constructed by modifying a pTZ19 plasmid between HindIII and XbaI sites. This modification was done using a synthetic fragment containing a new translation initiation region and coding for the first 5 amino acids (MKSTL). This plasmid allowed the cloning of coding inserts between theXbaI and BamHI sites. TheXbaI-EcoRI fragment of uPA and theEcoRI and BamHI ending synthetic duplex: 5′-AATTCACCACCCTGCACCATCACCATCACCATTAATAG-3′; 3′-GTGGTGGGACGTGGTAGTGGTAGTGGTAATTATCCTAG-5′ were ligated between the XbaI and BamHI sites; the duplex sequence contains a hexahistidine tag and stop codon after theEcoRI site. The structure of the resulting construct was confirmed by sequencing. The cloning procedures outlined above resulted in a pKR-his6 plasmid coding for a polypeptide identical to r-uPAwt amino acids 43–166 as shown:MKSTLEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDA- LQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVKECMVHDCADGK- KPSSPPEELKFQCGQKTLRP RF KIIGGEFTTLHHHHHH. The non-urokinase amino acids acquired from the vector are underlined. E. coli strain JM109 was transformed by the resulting pKR-his6 plasmid. Purification of r-KD was carried out as follows: inclusion bodies were isolated and the proteins denatured in buffer containing 6 m guanidine chloride. Subsequent reconstitution was carried out by gradually removing the denaturing agents while maintaining appropriate redox conditions with a glutathione-containing buffer (52.Marston F.A.O. Biochem. J. 1986; 240: 1-12Crossref PubMed Scopus (752) Google Scholar). The primary structure of r-KD allowed us to use nickel-chelate affinity chromatography for its initial purification. Subsequent purification to homogeneity was achieved by affinity chromatography on Sepharose coupled with the monoclonal antibody UNG-5. Then the kringle polypeptide was treated with thrombin, to cleave the peptide bond (Arg156-Phe157) in urokinase (53.Ichinose A. Fujikawa K. Suyama T. J. Biol. Chem. 1986; 261: 3486-3489Abstract Full Text PDF PubMed Google Scholar) (shown in bold); affinity chromatography using anti-kringle mAb removed the hexahistidine tag, any uncleaved peptides and thrombin. The resulting purified r-KD corresponded to urokinase amino acids Glu43-Arg156. The homogeneity of recombinant urokinase forms was confirmed by SDS-PAGE and Western blotting using monoclonal antibodies directed against the urokinase catalytic and kringle domains. The amidolytic activities of the glycosylated uPA and recombinant uPA forms were determined using the chromogenic urokinase substrate S-2444, as described previously (54.Stepanova V. Mukhina S. Köhler E. Resink T.J. Erne P. Tkachuk V.A. Mol. Cell. Biochem. 1999; 195: 199-206Crossref PubMed Scopus (44) Google Scholar). The proteolytic activities of glycosylated uPA, r-uPAwt, and r-uPALMW were ∼5000 units/nmol; the other urokinase forms with substitution of His204 in active center for Gln (r-uPAH/Q and r-uPAH/Q-GFD) as well as r-KD were proteolytically inactive. R-uPAwt and r-uPAH/Q coupled to CNBr-Sepharose 4B (Amersham Pharmacia Biotech) specifically precipitated uPAR from cell lysates with similar efficiency, while r-uPAH/Q-GFD and r-uPALMW did not. This was performed as described previously (46.Ji W.-R. Barrientos L.G. Llinas M. Gray H. Villarreal X. DeFord M.E. Castellino F.J. Kramer R.A. Trail P. Biochem. Biophys. Res. Commun. 1998; 247: 414-419Crossref PubMed Scopus (94) Google Scholar, 47.Cao Y. Ji R.W. Davidson D. Schaller J. Marti D. Söhndel S. McCance S.G. O'Reilly M.S. Llinás M. Folkman J. J. Biol. Chem. 1996; 271: 29461-29467Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). Briefly, r-KD (110 μg, 1 ml) was reduced by dithiothreitol (0.5m, 15 min at room temperature). The reduced r-KD was alkylated by the addition of 0.25 m iodoacetamide for 1 h at 4 °C and then dialyzed against PBS. Both the denatured r-KD and the intact r-KD were analyzed by SDS-PAGE and Western blotting using anti-kringle mAb UNG-5. Human airway smooth muscle cells (hAWSMC) were isolated from trachea and characterized as previously described (55.Panettieri R.A. Murray R.K. DePalo L.R. Yadvish P.A. Kotlikoff M.I. Am. J. Physiol. 1989; 256: C329-C355Crossref PubMed Google Scholar). Human embryonic kidney cells HEK 293 were from Cardiology Research Center Cell Collection, Moscow. The cell lines were cultured in DMEM supplemented with 10% fetal bovine serum, 10 mm HEPES, 5 mm glutamine, 100 units/ml penicillin, and 100 units/ml streptomycin at 37 °C and 5% CO2. The XbaI-EcoRI fragment from pB SK-(uPAR) plasmid containing human uPAR-cDNA (GenBank accession number X51675), was ligated into pUC19 (New England Biolabs) producing an intermediate plasmid pUC-uPAR. Then theHindIII-EcoRI fragment from pUC-uPAR was inserted into the pcDNA3 expression vector (Invitrogen). The resultant plasmid pcDNA-uPAR transfected into HEK 293 cells using Lipofectin, according to the manufacturers protocol. Two days later the cells were plated onto 96-well plates for clone selection. The transfected cells were cultured in DMEM with 10% fetal bovine serum containing 1.5 mg/ml G418 (Life Technologies, Inc.) for 25 days and G418-resistant clones were analyzed. Recombinant-uPAR expressed by these HEK 293 cells specifically bound recombinant uPAwt, assessed using an affinity uPAR precipitation method from cell lysates and immobilized r-uPAwt. Cells HEK 293 transfected by plasmid pcDNA3-anti-βGAL bearing a 3-kilobase fragment of E. coli β-galactosydase gene inserted in the antisense orientation (HEK-control cells) were used as controls. Cells were scraped into 10 mm HEPES buffer (pH 7.2), containing 150 mmNaCl, 1 mm EDTA, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 1 mm phenylmethylsulfonyl fluoride, and centrifuged at 1500 × g for 3 min at 4 °C. The pellet was resuspended in 10 mm Tris-HCl buffer (pH 8.0), containing 150 mm NaCl, 2 mm EDTA, 1% Triton X-100, 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 1 mmphenylmethylsulfonyl fluoride. After incubation for 20 min at 4 °C, the suspension was centrifuged at 5,000 × g for 15 min at 4 °C. The proteins in the supernatant were mixed with SDS sample buffer and subjected to 10% SDS-PAGE/Western blotting onto polyvinylidene difluoride membrane. The polyvinylidene difluoride membrane was immersed overnight at 4 °C in PBS containing 1% casein, 0.05% Tween 20, and 1 μg/ml anti-uPAR mAb (Monozyme). After multiple washings the membranes were incubated with a goat anti-mouse IgG conjugated to horseradish peroxidase and visualized using the chemiluminescent substrate "Super Signal Substrate." Migration of hAWSMC was determined as described previously, using a micro-Boyden chamber (14.Stepanova V. Bobik A. Bibilashvily R. Belogurov A. Rybalkin I. Domogatsky S. Little P.J. Goncharova E. Tkachuk V. FEBS Lett. 1997; 414: 417-474Google Scholar). Briefly, to determine the role of uPAR in uPA-induced cell migration, a hAWSMC cell suspension in DMEM supplemented with 0.1% BSA was preincubated with the anti-uPAR mAb (50 μg/ml) or a mouse Ig (50 μg/ml) for 1 h prior to seeding the cells into the upper wells of the Boyden chamber. When the role of the urokinase-kringle domain in uPA-induced cell migration was to be assessed, r-uPAwt, r-uPAH/Q-GFD, or r-KD and the anti-kringle mAb UNG-5 (20 μg/ml) or with total mouse Ig (20 μg/ml) were preincubated for 1 h prior to placement into the lower wells. HEK cell suspension was prepared in DMEM supplemented with 1% BSA. The extent of cell migration was evaluated after incubation for 3 h at 37 °C in the CO2 incubator. Data are presented as peak area on scanned fields with stained cells and expressed as percentage of cells that migrate across a filter compared with control chemotaxis. Comparisons between cell migration under the different conditions were analyzed using Student's t test. A p < 0.05 value was considered statistically significant. All results are expressed as mean ± S.E. Urokinase constructs (60 μg) were iodinated with 1 mCi of Na125I and 0.1 mg of IODO-GEN (Pierce) for 8 min at room temperature and the reaction was terminated with excessl-tyrosine. Purification of iodinated peptides was carried out by chromatography on PD-10 columns (Amersham Pharmacia Biotech) equilibrated with PBS. Iodinated peptides could be precipitated (95–98%) by trichloroacetic acid and had specific activities from 3.0 to 4.0 × 105 cpm/pmol. Binding studies with the iodinated peptides were carried out onKeywords:
Kringle domain
Objective To investigate the relationship between plasma level of urokinase-type plasminogen activator(uPA),urokinase-type plasminogen activator receptor(uPAR)and plasminogen activator inhibitor type 1(PAI-1)and ovarian cancer.Method The concentration of uPA,uPAR and PAI-1 in 52 patients with ovarian cancer and 30 healthy subjects were simultaneously determined by ELISA.Results There were significant differences for uPA and uPAR in different grades of the patients with ovarian cancer(P0.01).However,there was no significant difference for PAI-1 in ovarian cancer patients with different grades.There were significant differences in PAI-1,uPA,uPAR between the patients with ovarian cancer and healthy subjects(P0.01).Conclusion uPA,uPAR and PAI-1 may play important roles and be used as the parameters for progression and reimplantation of ovarian malignant cancer cells.
SuPAR
Plasminogen activator inhibitor-1
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Summary The kinetics of the activation of plasminogen into plasmin with urokinase and the inactivation rate of the plasmin formed are studied. As a first order reaction is obtained with low plasminogen concentrations and a zero-order reaction is obtained with high concentrations, the activation seems to follow the Michaelis-Menten’s law. The reaction does not go to completion, however. Different activity levels, which are dependent on the urokinase concentration, can be observed. The activation rate increases with temperature. A maximum can be seen at about 42° C. Between 4° C and 15° C, the inactivation of the plasmin formed is minimal, but it increases rapidly at higher temperatures. The inactivation follows approximately a first order reaction with respect to time. If the plasminogen concentration is low, the over-all reaction will be that of two consecutive first order reactions.
Reaction rate
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A bstract : A high plasma concentration of lipoprotein Lp(a) is now considered to be a major and independent risk factor for cerebro‐ and cardiovascular atherothrombosis. The mechanism by which Lp(a) may favour this pathological state may be related to its particular structure, a plasminogen‐like glycoprotein, apo(a), that is disulfide linked to the apo B100 of an atherogenic LDL‐like particle. Apo(a) exists in several isoforms defined by a variable number of copies of plasminogen‐like kringle 4 and single copies of kringle 5 and the catalytic region. At least one of the plasminogen‐like kringle 4 copies present in apo(a) (kringle IV type 10) contains a lysine binding site (LBS) that is similar to that of plasminogen. This structure allows binding of these proteins to fibrin and cell membranes. Plasminogen thus bound is cleaved at Arg 561 ‐Val 562 by plasminogen activators and transformed into plasmin. This mechanism ensures fibrinolysis and pericellular proteolysis. In apo(a) a Ser‐Ile substitution at the Arg‐Val plasminogen activation cleavage site prevents its transformation into a plasmin‐like enzyme. Because of this structural/functional homology and enzymatic difference, Lp(a) may compete with plasminogen for binding to lysine residues and impair, thereby, fibrinolysis and pericellular proteolysis. High concentrations of Lp(a) in plasma may, therefore, represent a potential source of antifibrinolytic activity. Indeed, we have recently shown that during the course of the nephrotic syndrome the amount of plasminogen bound and plasmin formed at the surface of fibrin are directly related to in vivo variations in the circulating concentration of Lp(a) ( Arterioscler. Thromb. Vasc. Biol. , 2000, 20: 575–584; Thromb. Hæmost. , 1999, 82: 121–127). This antifibrinolytic effect is primarily defined by the size of the apo(a) polymorphs, which show heterogeneity in their fibrin‐binding activity—only small size isoforms display high affinity binding to fibrin ( Biochemistry , 1995, 34: 13353–13358). Thus, in heterozygous subjects the amount of Lp(a) or plasminogen bound to fibrin is a function of the affinity of each of the apo(a) isoforms and of their concentration relative to each other and to plasminogen. The real risk factor is, therefore, the Lp(a) subpopulation with high affinity for fibrin. According to this concept, some Lp(a) phenotypes may not be related to atherothrombosis and, therefore, high Lp(a) in some individuals might not represent a risk factor for cardiovascular disease. In agreement with these data, it has been recently reported that Lp(a) particles containing low molecular mass apo(a) emerged as one of the leading risk conditions in advanced stenotic atherosclerosis ( Circulation , 1999, 100: 1154–1160). The predictive value of high Lp(a) as a risk factor, therefore, depends on the relative concentration of Lp(a) particles containing small apo(a) isoforms with the highest affinity for fibrin. Within this context, the development of agents able to selectively neutralise the antifibrinolytic activity of Lp(a), offers new perspectives in the prevention and treatment of the cardiovascular risk associated with high concentrations of thrombogenic Lp(a).
Kringle domain
Lipoprotein(a)
Proteolysis
Antifibrinolytic
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Abstract Angiostatin4.5 (AS4.5) is the product of plasmin autoproteolysis and consists of kringles 1 to 4 and ∼85% of kringle 5. In culture, cancer cell surface globular β-actin mediates plasmin autoproteolysis to AS4.5. We now show that plasminogen binds to prostate cancer cells and that the binding colocalizes with surface β-actin, but AS4.5 does not bind to the cell surface. Plasminogen and plasmin bind to immobilized β-actin similarly, with a Kd of ∼140 nmol/L. The binding is inhibited by ε-aminocaproic acid (εACA), indicating the requirement for a lysine-kringle domain interaction. Using a series of peptides derived from β-actin in competitive binding studies, we show that the domain necessary for plasminogen binding is within amino acids 55 to 69 (GDEAQSKRGILTLKY). Substitution of Lys61 or Lys68 with arginine results in the loss of the ability of the peptide to block plasminogen binding, indicating that Lys61 and Lys68 are essential for plasminogen binding. Other actin peptides, including peptides with lysine, did not inhibit the plasminogen-actin interaction. AS4.5 did not bind actin at concentrations up to 40 μmol/L. Plasminogen, plasmin, and AS4.5 all contain kringles 1 to 4; however, kringle 5 is truncated in AS4.5. Isolated kringle 5 binds to actin, suggesting intact kringle 5 is necessary for plasminogen and plasmin to bind to cell surface β-actin, and the truncated kringle 5 in AS4.5 results in its release from β-actin. These data may explain the mechanism by which AS4.5 is formed locally on cancer cell surfaces and yet acts on distant sites. (Cancer Res 2006; 66(14): 7211-5)
Kringle domain
Angiostatin
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Kringle domain
Elaidic acid
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To investigate the pathogenesis of unknown nosebleed patients.The ELISA test were used to detected plasma Urokinase-type plasminogen activator (uPA) and Urokinase-type plasminogen activator receptor (uPAR) level in 19 cases unknown factor nosebleed patients and 36 health persons.The results showed uPAR and uPA level in nosebleed group (before treatment) uPAR (0.14 +/- 0.04) microg/L, uPA (0.24 +/- 0.09) microg/L; (after treatment) uPAR (0.08 +/- 0.02) microg/L, uPA (0.18 +/- 0.07) microg/L. And normal group uPAR (0.07 +/- 0.03) microg/L, uPA (0.17 +/- 0.05) microg/L. The uPAR and uPA level in nosebleed group before treatment is higher than that in normal group (P <0.05). There is no significant difference between nosebleed group after treatment and normal group (P>0.05).The reasons of uPAR and uPA level high in unknown factor nosebleed patients were not clear, maybe relation to vascular endothelial cell, smooth muscle cell and neutrophil-monocytic release more uPAR and uPA. So uPAR and uPA density of nostril accumulation is more high in its microenvironment, that fibrinolytic system activated increase and result in its hyperactivity, and happened nosebleed when blood be in hypocoagulable state.
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Human serum inhibitors of plasmin, urokinase and kallikrein were studied using chromogenic substrate assays,(Plasmin substrate, S-22 51, Kabi, Sweden, Urokinase substrate, Chromozyme -UK and Plasma Kallikrein substrate, Chromozyme -PK, Pentapharm, Basle, Switzerland). In whole serum both “immediate” and “time dependent” inhibition of plasmin and kallikrein was observed, whilst only very weak inhibition of urokinase was detected. When serum samples were fractionated by Sephadex G-200 gel-filtration the “immediate” plasmin inhibitors were identified as α2-macroglobulin and low molecular weight antiplasmin whilst α2-macroglobulin and C1-esterase inhibitorwere immediate inhibitors of kallikrein.“Time-dependent” inhibition of both enzymes was observed in the ai-antitrypsin containing fractions.
Chromogenic
Sephadex
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urokinasr를 치료목적으로 사용할 때 바람직한 urokinase의 혈중 농도를 알기 위하여 시험관 내에서 free form plasmin을 형성하는 urokinase의 농도를 다음과 같은 실험을 통하여 규명하였다. Normal pooled plasma (NPP)에 urokinase를 가하여 다양한 농도의 urokinase를 포함하는 NPP를 만들고 S-2444 및 S-2241를 이용하여 free form urokinase 및 plasminm을 측정하였고 동시에 FDP가 형성되는 양상을 관찰하였으며 SDS-PAGE and electrophoretic enzymography를 이용하여 free form plasmin이 형성되는 urokinase의 농도를 알아내고자 하였던 바 free form plasmin은 urokinase 5-10 unit/ml의 농도에서 검출되기 시작하였다. 따라서 urokinase 투여중 therapeutic window는 urokinase 10 unit/ml을 기준으로 설정되어야 하며 이를 기준으로 loading dose 및 maintenance dosage를 위한 연구가 있어야 할 것으로 사료된다.
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