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Fresh plasma was seeded with trace amounts of highly purified biologically intact iodine-labelled plasminogen and the plasmin · inhibitor complexes formed after activation with streptokinase or urokinase separated by gel filtration. Two radioactive peaks were observed, the first one eluted in the void volume and the second one just before the 7-S globulin peak. In incompletely activated samples, the second peak was always predominant over the first one. Both components were purified with high yield by a combination of affinity chromatography on lysine-agarose and gel filtration, and investigated by dodecylsulphate-polyacrylamide gel electrophoresis and immunoelectrophoresis. Neither component reacted with antisera against α1-antitrypsin, antithrombin III, C1-esterase inhibitor, inter-α-trypsin inhibitor or α1-antichymotrypsin. The component of the first peak appeared to be a complex between plasmin and α2-macroglobulin which reacted with antisera against human plasminogen and against α2-macroglobulin. The component of the second peak had a molecular weight (Mr) of 120000–140000 by dodecylsulphate-polyacrylamide gel electrophoresis and upon reduction displayed a doublet band with an Mr of 65000 – 70000 and a band with Mr 11000. It reacted with antisera against plasminogen and with antisera raised against this complex and absorbed with purified plasminogen. The latter antisera reacted with a single component in plasma which is different from the above-mentioned plasma protease inhibitors. Specific removal of this component from plasma by immuno-absorption resulted in disappearance of the fast-reacting antiplasmin activity whereas α2-macroglobulin was found to represent the slower-reacting plasmin-neutralizing activity. In the presence of normal plasma levels of these proteins, the specific removal or absence of α2-antitrypsin, antithrombin III or C1-esterase inhibitor did not alter the inactivation rate of plasmin when added to plasma in equimolar amounts to that of plasminogen. It is concluded that only two plasma proteins are important in the binding of plasmin generated by activation of the plasma plasminogen, namely a fast-reacting inhibitor which is different from the known plasma protease inhibitors and which we have provisionally named antiplasmin, and α2-macroglobulin, which reacts more slowly. [ABSTRACT FROM AUTHOR]

Two low-molecular-mass forms of human plasminogen, plasminogen-(543–791)-peptide (micro-plasminogen), comprising the serine protease domain, and plasminogen-(444–791)-peptide (mini-plasminogen), which in addition contains kringle 5, were displayed on filamentous phage by fusion to the N-terminus of the minor coat protein pIII, to levels of 0.5 molecules micro-plasminogen—pIII/phage particle and 0.1 molecules mini-plasminogen—pIII/phage particle. The proenzymes, quantitatively activated by urokinase, showed catalytic efficiencies that were virtually identical to their soluble counterparts, and activity remained associated with the phage as demonstrated by phage ELISA and biopanning with human a2-antiplasmin or the inhibitor Phe-Pro-Arg-CH2Cl. Micro-plasminogen—pIII was activated by streptokinase and staphylokinase, two non-enzymatic plasminogen activators, to the same extent as by urokinase. Activated forms of mini-plasminogen—pIII, micro-plasminogen—pIII and mini-plasminogen dissolved 125I-labelled fibrin films in a dose-dependent time-dependent manner, with 50% lysis in 20 h requiring 0.52, 3.2 and 0.46 nM active plasmin, respectively. Thus, proenzyme moieties derived from plasminogen can be successfully displayed on phage with maintenance of their enzymatic properties. The microplasminogen and mini-plasminogen phage-display systems may be useful to study mechanisms of plasminogen activation. [ABSTRACT FROM AUTHOR]

The murine plasminogen/urokinase-type plasminogen-activator (u-PA) system was studied using puri fied proteins, plasma and endothelioma cells. Recombinant murine u-PA was obtained as a single chain molecule of 45 kDa which was converted to two-chain &mu-PA with plasmin by cleavage of the Lys159- Ile160 peptide bond. Murine plasminogen, purified from plasma as a single-chain protein of 95 kDa, was resistant to quantitative activation with murine recombinant two-chain μ-PA: only 15 % activation within 1 h at 37°C was obtained in mixtures of 1 μM plasminogen and 5 nM recombinant two-chain u-PA, whereas quantitative activation was observed in the autologous human system. Addition of 6-aminohexanoic acid to native murine plasminogen resulted in quantitative activation within 1 h. In murine plasma in vitro, plasminogen was also resistant to quantitative activation with &mu-PA (50% activation within 1 h at 37°C with 50 nM recombinant two chain u-PA, whereas in the human system nearly quantitative activation was obtained). Murine plasma clots submerged in murine plasma were resistant to lysis with μ-PA;≤2% clot lysis in 2 h was obtained with 80 nM recombinant two-chain μ-PA in the autologous murine system whereas 50% clot lysis in 2 h required only 15 nM recombinant two-chain μ-PA in the autologous human system. Saturable binding of murine recombinant two-chain μ-PA was observed to murine endothelioma cells that are genetically deficient in μ-PA (μ-PA −⁄− End cells). Binding was characterized by a Kd of 5.5 nM and 800000 binding sites/cell. However, μ-PA−⁄−. End cells did not significantly stimulate the activation rate of murine plasminogen by murine recombinant two-chain μ-PA and did not enhance the plasmin-mediated conversion rate of murine recombinant single-chain μ-PA to its two-chain derivative. Murine recombinant two-chain μ-PA bound to murine endothelioma cells was quantitatively inhibited by murine plasminogen-activator inhibitor-1 (PAI-l). Thus, the interactions between murine plasminogen, μ-PA and PAI-I are qualitatively similar to those between their human counterparts. However, quantitative differences were observed both in the presence of cells and in plasma which may contribute to a reduced μ-PA-mediated fibrinolytic activity in the murine systems. [ABSTRACT FROM AUTHOR]

Binding parameters |association-rate (kass) and dissociation-rate (kdiss) constants, and affinity constants (KA = kass/kdiss)] for the interaction between urokinase-type plasminogen activator (u-PA) and its substrate plasminogen, its inhibitor plasminogen activator inhibitor-1 (PAI-1) and its receptor (u-PAR), were determined by real-time biospecific interaction analysis (BIA). The KA values for the binding of [S741A]recombinant plasminogen (plasminogen with N-terminal Glu and with the active site Ser741 mutagenized to Ala) or of active site-blocked plasmin (D-ValPheLysCH2-plasmin) to the 54-kDa or 32-kDa molecular forms of recombinant single-chain u-PA (rscu-PA) ranged between 0.57×106M-1 and 1.7×106M-1, coinpared to 14-22×106 M-1 for binding to the corresponding active site-blocked recombinant two-chain u-PA (rtcu-PA) moieties. KA values for binding of these plasmin(ogen) moieties to [Ser356deHAla]rtcu-PA (rtcu-A with the active site Ser356 converted to dehydroAla) were 81 × 106M-1 and 670 × 106 M-1. respectively. Binding of active site-blocked LMM-plasmin (a low-molecular-mass plasmin derivative lacking kringles 1-4) and of the plasmin B chain to [Ser356deHA1a]rtcu-PA occurred with KA values of 3.7×106M-1 and 0.33×106 M-1, compared to 670×106 M-1 for the binding of intact D-ValPheLysCH2-plasmin to [Ser356deHA1a]rtcu-PA. The KA values for binding of latent PAI-1 to 54-kDa or 32-kDa molecular forms of rscu-PA and rtcu-PA were in the range 0.34-2.1 × 106 M-1. Reactivated PAI-1 bound to 54-kDa and 32-kDa rtcu-PA moieties with KA walues of 26 × 106 M-1 and 28×106 M-1, compared to 0.77 × 106M-1 and 3.2×0106M-1 for binding to the corresponding single-chain u-PA species, and 450 × 10106M-1 for binding to [Ser356deHAla]rtcu-PA. KA values for binding of plasmin(ogen) to the covalent rtcu-PA/PAI-1 complex were similar or somewhat higher than those for binding to uncomplexed rtcu-PA. Single-chain and two-chain 54-kDa u-PA moieties bound with a 1:1 stoichiometry and with very high affinity to u-PAR (KA of 4.6-8.5×10109M-1), whereas no significant binding of 32-kDa u-PA moieties was observed (KA ≥ 0.2×10106M-1). Binding of the covaleni rtcu-PA/PAI-1 complex to u-PAR was indistinguishable from that of rtcu-PA. These results indicate that plasminogen and plasmin bind with higher affinity to two-chain u-PA than to single-chain u-PA moieties, whereas binding requires the presence of both the A and the B chain: reactivated PAI-1 binds to rtcu-PA moieties with a higher affinity than latent PAI-1. but has a very low affinity for rscu-PA; 54-kDa u-PA moieties bind in a 1:1 stoichiometry to uPAR with high affinity which does not require the u-PA active site: and the binding sites of u-PA for PAI-1 and for plasminogen are within the 32-kDa u-PA moiety (Leu144-Leu411), they do not overlap and are fully accessible when 54-kDa u-PA is bound to its receptor. [ABSTRACT FROM AUTHOR]

K12G0S32 is a 57-kDa recombinant single-chain chimeric plasminogen activator consisting of scFv-K12G0, a single-chain variable-region antigen-binding fragment (Fv) of the monoclonal antibody MA-15C5, which is specific for fragment D-dimer of human cross-linked fibrin, and a low-molecular-mass (33 kDa) urokinase-type plasminogen activator (u-PA-33k) containing amino acids Ala132-Leu411 (Holvoet, P., Laroche, Y., Lijnen, H. R., Van Cauwenberghe, R., Demarsin, E., Brouwers, E., Matthyssens, G. & Collen D. (1991) J. Biol. Chem. 266, 19717–19724). In addition, the Arg156-Phe157 thrombin-cleavage site in the u-PA moiety of K12G0S32 is removed by substitution of Phe157 with Asp. In the present study, the fibrinolytic potency of K12G0S32, determined in a system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated plasma, was found to be only twofold higher than that of intact single-chain u-Pa (rscu-PA), but 17-fold higher than that of rscu-PA(M), a variant of rscu-PA in which the thrombin-cleavage site was removed by substitution of Phe157 with Asp. The fibrinolytic potency of K12G0S32T, with an intact thrombin-cleavage site, was 6–15-fold higher than that of rscu-PA. Conversion of 1 μM single-chain K12G0S32 or rscu-PA(M) into their two-chain derivatives with plasmin occurred at a rate of 1.0±0.15 nmol · min-1 · nmol plasmin-1 and 0.85±0.074nmol · min-1 · nmol plasmin-1, compared to 14±2.3 nmol · min-1 · nmol plasmin-1 and 18±2.6 nM · min-1 · nmol plasmin-1 for K12G0S32T and rscu-PA, respectively. Purified fragment D-dimer of human cross-linked fibrin inhibited the fibrinolytic potency of single-chain K12G0S32T, but not of two-chain K12G0S32T, in a dose-dependent manner. Furthermore, the fibrinolytic potencies of two-chain K12G0S32 and K12G0S32T were not significantly higher than those of recombinant two-chain u-PA (rtcu-PA) or of rtcu-PA(M). These findings suggest that the 50-fold increase in fibrinolytic potency of K12G0S32T, relative to that of rscu-PA(M), is due both to targeting of the activator to the clot via the single-chain Fv fragment (sixfold increase) and to a more efficient conversion of single-chain K12G0S32T to its two-chain derivative (eightfold increase). Thus, targeting to clots by means of fibrin-specific antibodies results in a significant increase of the fibrinolytic potency of single-chain but not of two-chain u-PA. K12G0S45 which contains the scu-PA kringle domain, and K12G0S54 which contains intact scu-PA, had a similar fibrinolytic potency as K12G0S32, indicating that the fibrin-targeting in the Fv domain of K12G0S32 is not hampered by spatial constraints in the molecule. [ABSTRACT FROM AUTHOR]

The contribution of the NH2-terminal polypeptide chain and of the Cys148-Cys279 interchain disulphide bond to the enzyme activity of urokinase-type plasminogen activator (u-PA) was studied using site-specific mutagenesis. Recombinant single-chain u-PA (rscu-PA) variants were produced by transfecting Chinese hamster ovary cells with cDNA encoding des(Asn2-Phe157)rscu-PA (rscuPA with deletion of Asn2-Phe157), [Ala279]rscu-PA (rscu-PA with Cys279→Ala mutation) or des(Asn2-Phe157)[Ala279]rscu-PA [des(Asn2-Phe157)rscu-PA with Cys279→Ala mutation]. Des(Asn2-Phe157)rscu-PA, [Ala279]rscu-PA and des(Asn2-Phe157)[Ala279]rscu-PA, purified from conditioned cell culture medium, were obtained as nearly homogeneous single-chain molecules with Mr approximately 30000, 54000 and 30000, and specific fibrinolytic activities on fibrin plates of (mean ± SD; n = 3) 860 ± 150 IU/mg, 43.0 ± 2.5 IU/µg and 240 ± 20 IU/mg, respectively, compared to 69.0 ± 4.3 IU/µg for wild-type rscu-PA obtained in the same expression system. The plasminogen activating potential in a buffer milieu of [Ala279]rscu-PA was somewhat lower than that of rscu-PA, but that of both deletion mutants was virtually abolished. In a human plasma milieu in vitro, consisting of a radiolabelled human plasma clot submerged in plasma, 50% clot lysis in 2 h required 6.5 µg/ml [Ala279]rscu-PA or 3.4 µg/ml rscu-PA, whereas with both deletion mutants no significant clot lysis was observed with up to 16 µg/ml. Treatment of [Ala279]rscu-PA or rscu-PA with plasmin resulted in quantitative conversion to two-chain molecules and was associated with an increase in specific amidolytic activity from about 600 IU/mg to 62.5 IU/µg for [Ala279]rscu-PA as compared to an increase from about 0.3 IU/µg to 75.0 IU/µg for rscu-PA. In contrast, no significant amidolytic activity could be generated by treatment of des(Asn2-Phe157)rscu-PA or des(Asn2Phe157)[Ala279]rscu-PA with plasmin. The u-PA B-chain, isolated from plasmin-treated [Ala279]rscu-PA, had enzymic properties which were comparable to those of rtcu-PA, with respect to specific fibrinolytic activity, amidolytic activity, kinetics of plasminogen activation and clot-lysis activity in a human plasma milieu in vitro. Following bolus injection into hamsters, the plasma clearances were comparable (0.7-1.1 ml/min) for wild-type rscu-PA and for the three truncated rscu-PA mutants. These results indicate that (a) deletion of residues Asn2-Phe 157 results in abolition of the enzyme activity of rscu-PA, (b) the interchain disulphide bond in u-PA is not required for the enzymic activity of scu-PA, (c) all the determinants required for the enzymic activity of two-chain u-PA are contained within the B-chain, and (d) the region comprising residues Asn2-Phe157 of u-PA is not required for the rapid in vivo clearance. [ABSTRACT FROM AUTHOR]

A recombinant chimeric plasminogen activator, MA-15C5Hu/scu-PA-32k, composed of a humanized fibrin fragment-D-dimer-specific monoclonal antibody (MA-15C5Hu) and a recombinant low-molecular-mass single-chain urokinase-type plasminogen activator, comprising amino acids Leu144-Leu411 (scu-PA-32k), was produced by cotransfecting Chinese hamster ovary (CHO) cells with the cDNA encoding the MA-15C5Hu light-chain sequence and the cDNA encoding the MA-15C5Hu heavy-chain sequence fused with the cDNA encoding scu-PA-32k. Purified MA-15C5Hu/scu-PA-32k migrated as a 215-kDa band on non-reducing SDS/PAGE, which is consistent with a molecule composed of one antibody and two scu-PA-32k moieties. However, the chimera was obtained as a mixture of single-chain u-PA-32k (37%) and amidolytically inactive (50%) and active (13%) two-chain u-PA-32k, the latter of which was removed by immunoadsorption on a monoclonal antibody specific for two-chain urokinase. The fragment-D-dimer affinity and enzymatic properties of MA-15C5Hu/scu-PA-32k were similar to those of MA-15C5Hu or of scu-PA-32k. In an in vitro system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated human plasma, MA-15C5Hu/scu-PA-32k had a 12-fold higher fibrinolytic potency than scu-PA-32k: 50% lysis in 2 h required 0.43 ± 0.12 μg u-PA-32k equivalent of the chimera/ml versus 5.4 ± 0.3 μg/ml of scu-PA-32k (mean ± SEM, n = 4). Addition of purified fibrin fragment-D dimer reduced the fibrinolytic potency of MA-15C5Hu/scu-PA-32k in a concentration-dependent way, indicating that the increased potency is the result of antibody targeting. Thus, a recombinant humanized antifibrin antibody/u-PA chimera has been obtained in which only the variable domains of the antibody moiety are of nonhuman origin. The chimera has intact antigen-binding capacity, u-PA enzymatic activity and a significantly increased fibrinolytic potency in a plasma medium in vitro. [ABSTRACT FROM AUTHOR]

Equimolar mixtures of recombinant single chain urokinase-type plasminogen activator (rscu-PA) and a murine monoclonal antibody (MA-15C5) directed against fragment-D dimer of human cross-linked fibrin were conjugated, using the cross-linking agent N-succinimidyl 3-(2-pyridyldithio)propionate (PySSProSu). The conjugate (rscu-PA/MA-15C5), purified by immunoadsorption on a urokinase antibody and affinity chromatography on fibrin fragment-D dimer with a yield of 42±15% (mean ± SD, n = 3), contained an average of 1.2 ± 0.3 IgG molecules/rscu-PA molecule. On non-reduced SDS/PAGE it migrated as a main band with apparent Mr of 200000. Specific amidolytic activities expressed/mass of u-PA were <250 lU/mg for rscu-PA/MA-15C5 and rtcu-PA, 140000 ± 13000 lU/mg and 100000 ± 17000 lU/mg for their plasmin-generated two chain derivatives rtcu- PA/MA-15C5 and rtcu-PA respectively. Specific activities on fibrin plates were 100000 ± 24000 lU/mg and 130000 ± 49000 lU/mg for rscu-PA/MA-15C5 and rtcu-PA/MA-15C5 respectively, as compared to 180000 ± 15000 lU/mg for both rscu-PA and rtcu-PA. Activation of plasminogen with rscu-PA/MA-15C5 (Km = 0.37 ± 0.16 μM, k2 = 0.0063 ± 0.0030 s-1) or rtcu-PA/MA-15C5 (Km = 19 ± 3.0 μM, k2 = 2.0 ± 0.10 s-1) purified systems followed Michaelis-Menten kinetics with Km and k2 values comparable to those of rscu-PA and rtcu-PA. In an in vitro system composed of a 125I-fibrin-labeled whole human plasma clot immersed in citrated human plasma, dose- and time-dependent lysis was obtained; 50% lysis in 2 h required 1.4 μg/ml of rscu-PA or 0.33 μg/ml of rtcu-PA, but only 0.22 μg u-PA/ml of rscu-PA/MA-15C5 or 0.15 μg u-PA/ml of rtcu-PA/MA-15C5. Addition of purified fragment-D dimer reversed the increased fibrinolytic potency of rscu-PA/MA-ISCS in a concentration-dependent way (50% inhibition at 7.2 μg fragment-D dimer/ml). Thus, conjugation of u-PA moieties with the fibrin-specific antibody MA-15C5 targets the plasminogen activator to the clot, resulting in a significant increase of their fibrinolytic potencies as compared to their unconjugated counterparts: 6.4-fold for rscu-PA and 2.2-fold for rtcu-PA. [ABSTRACT FROM AUTHOR]

Single-chain urokinase-type plasminogen activator (scu-PA) is converted to urokinase by hydrolysis of the Lys158-Ile159 peptide bond. Site-directed mutagenesis of Lys158 to Gly or Glu yields plasmin-resistant mutants with a 10–20-fold reduced catalytic efficiency for the activation of plasminogen [Nelles et al. (1987) J. Biol. Chem. 262, 5682–5689]. In the present study, we have further evaluated the enzymatic properties of derivatives of recombinant scu-PA (rscu-PA), produced by site-directed mutagenesis of Lys158, Ile159 or Ile160, in order to obtain additional information on the structure/function relations underlying the enzymatic properties of the single- and two-chain u-PA moieties. [Arg158]rscu-PA (rscu-PA with Lys158 substituted with Arg) appeared to be indistinguishable from wild-type rscu-PA with respect to plaminogen-activating potential (catalytic efficiency k2/Km=0.21 mM-1 s-1 versus 0.64 mM-1 s-1), conversion to active two-chain urokinase by plasmin (k2/Km=0.13 μM=0.21 mM-1 s-1 versus 0.28 μM-1 s-1), as well as its specific activity (48000 IU/mg as compared to 60000 IU/mg) and its fibrinolytic potential in a plasma medium (50% lysis in 2 h with 2.8 μg/ml versus 2.1 μg/ml). [Pro159]RSCU-pa (Ile159 substituted with Pro) and [Gly159]rscu-PA (Ile159 converted to Gly) are virtually inactive towards plaminogen (k2/Km less than 0.004 mM-1 s-1). They are however converted to inactive two-chain derivatives by plasmin following cleavage of the Arg156-Phe157 peptide bond in [Pro159]rscu-PA and of the Lys158-Gly159 peptide bond in [Gly159]rscu-PA. [Gly158, Lys160]rscu-PA (with Lys158 converted to Gly and Ile160 to Lys) has a low catalytic efficiency towards plasminogen both as a single-chain form (k2/Km-0.012 mM-1 s-1) and as the two-chain derivative (k2/Km=0.13 mM-1 s-1) generated by cleavage of both the Arg156-Phe157 and/or the Lys160-Gly161 peptide bonds by plasmin. These findings suggest that the enzymatic properties of rscu-PA are critically dependent on the amino acids in position 158 (requirement for Arg or Lys) and position 159 (requirement for Ile). Conversion of the basic amino acid in position 158 results in a 10–20-fold reduction of the catalytic efficiency of the single-chain molecule but yields a fully active two-chain derivative. The presence of He in position 159 is not only a primary determinant for the activity of the two-chain derivative, but also of the single-chain precursor. Cleavage of the Arg156-Phe157 or the Lys160-Gly161 peptide bonds by plasmin yields inactive two-chain derivatives. [ABSTRACT FROM AUTHOR]

Recombinant single-chain urokinase-type plasminogen activator (rscu-PA), in which the plasmin-sensitive peptide bond Lys158-Ile159 is destroyed by site-specific mutagenesis of Lys158 to Glu (rscu-PA-Glu158), is quantitatively converted to two-chain urokinase-type plasminogen activator (rtcu-PA-Glu158) by treatment with endoproteinase Glu-C (Staphylococcus aureus V8 proteinase). The catalytic efficiency (k2/Km) of rscu-PA-Glu158 for the activation of plasminogen is 20 times lower (0.0001 µM-1 s-1) than that of rscu-PA (0.002 µM-1 s-1). In contrast, rtcu-PA-Glu158 has very similar properties to rtcu-PA obtained by digestion of rscu-PA with plasmin, including binding to benzamidine-Sepharose, catalytic efficiency for the activation of plasminogen (0.035 µM-1 s-1 versus 0.046 µM-1 s-1) and fibrinolytic activity in an in vitro plasma clot lysis system. It is concluded that the amino acid in position 158 is a main determinant of the functional properties of single-chain urokinase-type plasminogen activator but not of the two-chain form. [ABSTRACT FROM AUTHOR]

Thrombin converts single-chain urokinase-type plasminogen activator (scu-PA) to an inactive two-chain derivative (thrombin-derived tcu-PA) by hydrolysis of the Arg-156—Phe-157 peptide bond. In the present study, we show that inactive thrombin-derived tcu-PA (specific activity 1000 IU/mg) can be converted with plasmin to active two-chain urokinase-type plasminogen activator (specific activity 43000 IU/mg) by hydrolysis of the Lys-158—I1e-159 peptide bond. This conversion follows Michaelis-Menten kinetics with a Michaelis constant Km of 37 μM and a catalytic rate constant k2 of 0.013 s-1. The catalytic efficiency (k2/Km) for the activation of thrombin- derived tcu-PA by plasmin is about 500-fold lower than that for the conversion of intact scu-PA to tcu-PA. tcu-PA, generated by plasmin treatment of thrombin-derived tcu-PA, has similar properties to tcu-PA obtained by digestion of intact scu-PA with plasmin (plasmin-derived tcu-PA); its plasminogen activating potential and fibrinolytic activity in an in vitro plasma clot lysis system appear to be unaltered. These observations confirm that the structure of the NH2-terminal region of the B chain of u-PA is an important determinant for its enzymatic activity, whereas that of the COOH-terminal region of the A chain is not. [ABSTRACT FROM AUTHOR]

Glu-Gly-Arg-CH2Cl, a synthetic inhibitor which alkylates the active-site histidine of urokinase with an apparent second-order rate constant of approximately 104 M-1 s-1, reacts differently with single-chain and two-chain forms of urokinase-type plasminogen activators (scu-PA and tcu-PA). Kinetic analysis indicated that the plasminogen activating potential of tcu-PA is irreversibly inhibited in a two-step reaction according to ... with Ki = 5.0 x 10-6 M and k2 = 0.05 s-1 The plasminogen-activating potential of scu-PA, however, is competitively inhibited by Glu-Gly-Arg-CH2Cl with Ki = 1.3 x 10-6 M. Reversibility of the interaction of Glu-Gly-Arg-CH2Cl with scu-PA was confirmed by the restoration of full enzymatic activity after removal of inhibitor. The differential interaction of Glu-Gly-Arg-CH2Cl with scu-PA and tcu-PA supports the hypothesis that these molecular forms of urokinase-type PA have intrinsically different enzymatic properties. [ABSTRACT FROM AUTHOR]

Activation of human Glu-plasminogen, Lys-plasminogen and low-Mr plasminogen (lacking lysine-binding sites) by pro-urokinase (pro-OK), obtained from a human lung adenocarcinoma cell line (Calu-3, ATCC), obeys Michaelis-Menten kinetics. Activation ocurs with a comparable affinity (Km 0.40–0.77 μM), while the catalytic rate constant (kcat) is comparable for Glu-plasminogen (0.0022 s-1) and low-Mr plasminogen (0.0034 s-1), but is somewhat higher for Lys-plasminogen (0.0106 s-1). The rate of activation of plasminogen by pro-UK is not significantly influenced by the presence of 6-aminohexanoic acid, purified fragments LBS I or LBS II or histidine-rich glycoprotein, indicating that the high affinity of pro-UK for plasminogen is not mediated via the high-affinity lysine-binding site of plasminogen located in kringles 1–3 (LBS I) nor via the low-affinity lysine-binding site comprised within kringle 4 (LBS II). The site(s) in plasminogen involved in the high-affinity interaction with pro-UK thus appear to be located within the low-Mr plasminogen moiety. [ABSTRACT FROM AUTHOR]

The catalytic efficiency (kcat/Km) of high-molecular-mass urokinase for the activation of Glu-plasminogen is increased about 10-fold in the presence of CNBr-digested fibrinogen. This stimulation is similar to that observed with 6-aminohexanoic acid, and yields kinetic parameters comparable to those for the activation of Lys- plasminogen by urokinase. The increase of the activation rate of Glu-plasminogen by urokinase in the presence of CNBr-Fg can thus be explained by a conformational change in the plasminogen molecule similar to that observed upon conversion of Glu-plasminogen to Lys-plasminogen and upon binding of 6-aminohexanoic acid to Glu-plasminogen. Stabilization of the Michaelis complex between urokinase and plasminogen by formation of a cyclic ternary complex with CNBr-Fg, which has been invoked to explain the dramatic stimulatory effect of CNBr-Fg on the activation of plasminogen by tissue-type plasminogen activator, does not appear to play a significant role in the increased activation rate. [ABSTRACT FROM AUTHOR]

Presents a study which discusses the role of urokinase-type plasminogen activator (u-PA). Revelation of the topographic analysis of arterial wound healing after electric injury; What formation of blood vessels during embryogenesis involves; Constituency of matrix metalloproteinases (MMPs).

Single-chain urokinase-type plasminogen activator (scu-PA) is converted to urokinase by hydrolysis of the Lys158-Ile159 peptide bond. Site-directed mutagenesis of Lys158 to Gly or Glu yields plasmin-resistant mutants with a 10–20-fold reduced catalytic efficiency for the activation of plasminogen [Nelles et al. (1987) J. Biol. Chem. 262, 5682–5689]. In the present study, we have further evaluated the enzymatic properties of derivatives of recombinant scu-PA (rscu-PA), produced by site-directed mutagenesis of Lys158, Ile159 or Ile160, in order to obtain additional information on the structure/function relations underlying the enzymatic properties of the single- and two-chain u-PA moieties. [Arg158]rscu-PA (rscu-PA with Lys158 substituted with Arg) appeared to be indistinguishable from wild-type rscu-PA with respect to plaminogen-activating potential (catalytic efficiency k2/Km=0.21 mM-1 s-1 versus 0.64 mM-1 s-1), conversion to active two-chain urokinase by plasmin (k2/Km=0.13 μM=0.21 mM-1 s-1 versus 0.28 μM-1 s-1), as well as its specific activity (48000 IU/mg as compared to 60000 IU/mg) and its fibrinolytic potential in a plasma medium (50% lysis in 2 h with 2.8 μg/ml versus 2.1 μg/ml). [Pro159]RSCU-pa (Ile159 substituted with Pro) and [Gly159]rscu-PA (Ile159 converted to Gly) are virtually inactive towards plaminogen (k2/Km less than 0.004 mM-1 s-1). They are however converted to inactive two-chain derivatives by plasmin following cleavage of the Arg156-Phe157 peptide bond in [Pro159]rscu-PA and of the Lys158-Gly159 peptide bond in [Gly159]rscu-PA. [Gly158, Lys160]rscu-PA (with Lys158 converted to Gly and Ile160 to Lys) has a low catalytic efficiency towards plasminogen both as a single-chain form (k2/Km-0.012 mM-1 s-1) and as the two-chain derivative (k2/Km=0.13 mM-1 s-1) generated by cleavage of both the Arg156-Phe157 and/or the Lys160-Gly161 peptide bonds by plasmin. These findings suggest that the enzymatic properties of rscu-PA are critically dependent on the amino acids in position 158 (requirement for Arg or Lys) and position 159 (requirement for Ile). Conversion of the basic amino acid in position 158 results in a 10–20-fold reduction of the catalytic efficiency of the single-chain molecule but yields a fully active two-chain derivative. The presence of He in position 159 is not only a primary determinant for the activity of the two-chain derivative, but also of the single-chain precursor. Cleavage of the Arg156-Phe157 or the Lys160-Gly161 peptide bonds by plasmin yields inactive two-chain derivatives. [ABSTRACT FROM AUTHOR]