Characterization of a recombinant chimeric plasminogen activator composed of a fibrin fragment-D-dimer-specific humanized monoclonal antibody and a truncated single-chain urokinase.

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-15CHu/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 micrograms u-PA-32k equivalent of the chimera/ml versus 5.4 +/- 0.3 micrograms/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 non-human origin. The chimera has intact antigen-binding capacity, u-PA enzymatic activity and a significantly increased fibrinolytic potency in a plasma medium in vitro.     

Characterization of a chimeric plasminogen activator consisting of a single-chain Fv fragment derived from a fibrin fragment D-dimer-specific antibody and a truncated single-chain urokinase

An Mr 57,000 single-chain chimeric plasminogen activator, K12G0S32, consisting of a variable region fragment (Fv) derived from the fibrin fragment D-dimer-specific monoclonal antibody MA-15C5 and of a 33-kDa (amino acids Ala132 to Leu411) recombinant single-chain urokinase-type plasminogen activator (rscu-PA-33k) was studied. K12G0S32, secreted by infected Spodoptera frugiperda insect cells at a rate of 1.5 micrograms/10(6) cells/48 h, was purified to homogeneity by ion-exchange chromatography and gel filtration. It was obtained essentially as a single-chain molecule with a Ka = 5.5 x 10(9) M-1 for immobilized fragment D-dimer, similar to that of MA-15C5. The specific activity of both its single-chain and two-chain forms on fibrin plates was 100,000 IU/mg of urokinase-type plasminogen activator (u-PA) equivalent. Activation of plasminogen by two-chain K12G0S32 obeyed Michaelis-Menten kinetics with Km = 2.9 +/- 0.6 microM and a k2 = 3.7 +/- 0.6 s-1 (mean +/- S.D.; n = 3), as compared to Km = 12 microM and k2 = 4.8 s-1 for rtcu-PA-32k (recombinant low Mr two-chain u-PA consisting of amino acids Leu144 to Leu411). Single-chain K12G0S32 induced a dose- and time-dependent lysis of a 125I-fibrin-labeled human plasma clot immersed in citrated human plasma; 50% lysis in 2 h was obtained with 0.70 +/- 0.07 micrograms/ml (mean +/- S.D.; n = 5), as compared with 8.8 +/- 0.1 micrograms/ml for rscu-PA-32k (recombinant low Mr single-chain u-PA consisting of amino acids Leu144 to Leu411) (mean +/- S.D.; n = 3). With two-chain K12G0S32, 50% clot lysis in 2 h required 0.25 +/- 0.03 micrograms/ml (mean +/- S.D.; n = 3), as compared with only 0.62 +/- 0.04 micrograms/ml (mean +/- S.D.; n = 2) for rtcu-PA-32k. These results indicate that low Mr single-chain u-PA can be targeted to a fibrin clot with a single-chain Fv fragment of a fibrin-specific antibody, resulting in a 13-fold increase of the fibrinolytic potency of the single-chain form and a 2.5-fold increase of the potency of the two-chain form.     

Biochemical properties of recombinant single-chain urokinase-type plasminogen activator mutants with deletion of Asn2 through Phe157 and/or substitution of Cys279 with Ala.

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 (rscu-PA 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 30,000, 54,000 and 30,000, and specific fibrinolytic activities on fibrin plates of (mean +/- SD; n = 3) 860 +/- 150 IU/mg, 43.0 +/- 2.5 IU/micrograms and 240 +/- 20 IU/mg, respectively, compared to 69.0 +/- 4.3 IU/micrograms 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 micrograms/ml [Ala279]rscu-PA or 3.4 micrograms/ml rscu-PA, whereas with both deletion mutants no significant clot lysis was observed with up to 16 micrograms/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/micrograms for [Ala279]rscu-PA as compared to an increase from about 0.3 IU/micrograms to 75.0 IU/micrograms for rscu-PA. In contrast, no significant amidolytic activity could be generated by treatment of des(Asn2-Phe157)rscu-PA or des(Asn2-Phe157)[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.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of recombinant human single chain urokinase-type plasminogen activator mutants produced by site-specific mutagenesis of lysine 158

The cDNA encoding full-length single chain urokinase-type plasminogen activator (scu-PA) was cloned and sequenced, and the recombinant scu-PA (rscu-PA) was expressed in Chinese hamster ovary cells. Two mutants, constructed by in vitro site-specific mutagenesis of Lys158 in rscu-PA to Gly158 (rscu-PA-Gly158) or to Glu158 (rscu-PA-Glu158), were also expressed in Chinese hamster ovary cells. Wild type and mutant rscu-PAs were purified to homogeneity by immunoadsorption on an insolubilized monoclonal antibody raised against natural scu-PA (nscu-PA), followed by gel filtration. The specific activity of the mutant scu-PAs on fibrin plates is very low (less than 1,000 IU/mg) compared to that of the wild type rscu-PA (44,000 IU/mg). The mutants, in contrast to the wild type rscu-PA, are not converted to amidolytically active two chain u-PA (tcu-PA) by plasmin and do not cause lysis of a 125I-fibrin-labeled plasma clot immersed in citrated plasma. However, in a purified system, both rscu-PA-Gly158 and rscu-PA-Glu158 activate plasminogen following Michaelis-Menten kinetics, with a much lower affinity (Km = 60-80 microM) but with a higher turnover rate constant (k2 = 0.01 s-1) as compared to the wild type rscu-PA (Km = 1.0 microM, k2 = 0.002 s-1). We conclude that conversion of scu-PA to tcu-PA is not a prerequisite for the activation of plasminogen. Substitution of Lys158 by Gly158 or Glu158 does, however, markedly decrease the stability of the Michaelis complex.     

Biochemical characterization of single-chain chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin-specific antibody and single-chain urokinase.

K12G0S32 is a 57-kDa recombinant single-chain chimeric plasminogen activator consisting of scFv-K12Go, 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 microM 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 +/- 0.074 nmol.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 59-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.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biochemical properties of recombinant mutants of nonglycosylated single chain urokinase-type plasminogen activator.

The role of glycosylation on the enzymatic properties of single chain urokinase-type plasminogen activator (scu-PA) was investigated by site-specific mutagenesis of the glycosylated Asn-302 residu to Gln. In addition, the role of the NH2-terminal polypeptide chain and of the Cys-148 to Cys-279 interchain disulphide bond on the activity of non-glycosylated scu-PA was investigated. Therefore, variants of recombinant scu-PA (rscu-PA) were produced by transfecting Chinese hamster ovary cells with cDNA encoding rscu-PA N302Q (rscu-PA with Asn-302 to Gln mutation), rscu-PA C279A,N302Q (rscu-PA with Cys-279 to Ala and Asn-302 to Gln mutations) or rscu-PA del(N2-F157)C279A,N302Q (rscu-PA C279A,N302Q with deletion of Asn-2 through Phe-157). These mutants were purified to homogeneity from conditioned cell culture medium and were obtained essentially as single chain molecules with specific activities on fibrin plates of (mean +/- S.E.; n = 6) 45,000 +/- 5000. IU/mg, 19,000 +/- 800 IU/mg and < or = 100 IU/mg for rscu-PA N302Q, rscu-PA C279A,N302Q and rscu-PA del(N2-F157)C279A,N302Q, respectively, as compared to 64,000 +/- 2600 IU/mg for wild-type rscu-PA obtained in the same expression system. Plasmin quantitatively converts rscu-PA N302Q and rscu-PA C279A,N302Q to amidolytically active two-chain derivatives with a specific activity of 56,000 IU/mg and 32,000 IU/mg, respectively, as compared to 75,000 IU/mg for wild-type rscu-PA. Plasminogen activation as a function of time was comparable for rscu-PA N302Q and wild-type rscu-PA, and somewhat slower for rscu-PA C279A,N302Q. In a human plasma milieu in vitro, consisting of a 125I-fibrin labeled plasma clot submerged in plasma, 50 percent clot lysis in 2 h required 2.2 micrograms/ml rscu-PA N302Q and 6.0 micrograms/ml rscu-PA C279A,N302Q, as compared to 3.2 micrograms/ml wild-type rscu-PA. In contrast, rscu-PA del(N2-F157)C279A,N302Q was not converted to an amidolytically active two chain derivative by plasmin, and did not induce significant plasminogen activation in purified systems or clot lysis in a human plasma milieu. Following bolus injections in hamsters, the initial half-lives (1.8-2.6 min) and the plasma clearances (0.6-1.5 ml min-1) were comparable for wild-type rscu-PA and for the three rscu-PA mutants. These results suggest that the fibrinolytic activity in a plasma milieu in vitro and the in vivo turnover of rscu-PA are not markedly affected by the absence of carbohydrate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural and functional characterization of mutants of recombinant single-chain urokinase-type plasminogen activator obtained by site-specific mutagenesis of Lys158, Ile159 and Ile160

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 plasminogen-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 microM-1 s-1 versus 0.28 microM-1 s-1), as well as its specific activity (48,000 IU/mg as compared to 60,000 IU/mg) and its fibrinolytic potential in a plasma medium (50% lysis in 2 h with 2.8 micrograms/ml versus 2.1 micrograms/ml). [Pro159]rscu-PA (Ile159 substituted with Pro) and [Gly159]rscu-PA (Ile159 converted to Gly) are virtually inactive towards plasminogen (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 Ile 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.     

Characterization of a fusion protein consisting of amino acids 1 to 263 of tissue-type plasminogen activator and amino acids 144 to 411 of urokinase-type plasminogen activator

A hybrid human cDNA was constructed by splicing of a cDNA fragment of tissue-type plasminogen activator (t-PA), encoding 5'-untranslated, the pre-pro region and amino acids Ser1-Thr263, with a cDNA fragment of urokinase-type plasminogen activator (u-PA), encoding amino acids Leu144-Leu411. The cDNA fragments were obtained from full length t-PA cDNA, cloned from Bowes melanoma poly(A)+ mRNA, and from full length u-PA cDNA, cloned from CALU-3 lung adenocarcinoma poly(A)+ mRNA. The hybrid (t-PA/u-PA) cDNA was expressed in Chinese hamster ovary cells and the translation product purified from the conditioned cell culture media. On SDS-gel electrophoresis under reducing conditions, the protein migrated as a single band with approximate Mr 70,000. On immunoblotting, it reacted both with rabbit antisera raised against human t-PA and against human u-PA. The urokinase-like amidolytic activity of the protein was only 320 IU/mg but increased to 43,000 IU/mg after treatment with plasmin, which resulted in conversion of the single-chain molecule (t-PA/scu-PA) to a two-chain molecule (t-PA/tcu-PA). The specific activity of the protein on fibrin plates was 57,000 IU/mg by comparison with the International Reference Preparation for Urokinase. Both the single-chain hybrid (t-PA/scu-PA) and the two-chain plasmin derivative (t-PA/tcu-PA) bound specifically to fibrin, albeit more weakly than t-PA. The t-PA/tcu-PA hybrid had a higher selectivity for fibrin than tcu-PA, measured in a system composed of a whole human 125I-fibrin-labeled plasma clot immersed in human plasma. Both hybrid proteins activated plasminogen directly with Km = 1.5 microM and k2 = 0.0058 s-1 for t-PA/scu-PA and with Km = 80 microM and k2 = 5.6 s-1 for t-PA/tcu-PA. CNBr-digested fibrinogen stimulated the activation of plasminogen with t-PA/tcu-PA (Km = 0.20 microM and k2 = 1.2 s-1). It is concluded that these t-PA/u-PA hybrid proteins combine, at least to some extent, the fibrin-affinity of t-PA with the enzymatic properties of u-PA (either scu-PA or tcu-PA), which in some assays result in improved fibrin-mediated plasminogen activation.     

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

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

Purification and properties of a single-chain urokinase-type plasminogen activator form produced by subcultured human umbilical vein endothelial cells

Single-chain Mr 54,000 u-PA (scu-PA) was isolated, in the presence of aprotinin, from 3-liter batches of 60-h serum-free conditioned media obtained from subcultured (4-6th passage) human umbilical vein endothelial cells (HUVECs, approximately 1.8 x 10(9) cells). In the presence of heparin and endothelial cell growth factor, subcultured human umbilical vein endothelial cells produced u-PA proteins consisting of about 85-90% Mr 54,000 scu-PA and 10-15% two-chain Mr 54,000. The major scu-PA form was purified to homogeneity by ion-exchange chromatography on CM-Sephadex C-50, immunoadsorption on purified anti-u-PA IgG-Sepharose and affinity chromatography on p-amino-benzamidine-Agarose. Typically, about 8-10 micrograms of purified scu-PA protein (antigen/protein ratio = 1) was isolated from 3-liter batches of heparin-containing serum-free conditioned media with a yield of about 41% of the total starting u-PA antigen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this purified u-PA protein showed a single Ag-stained band (nonreduced and reduced), with an estimated molecular weight of about 54,000, which exhibited very low fibrinolytic activity. Purified HUVEC-derived scu-PA did not incorporate 3H-labeled diisopropyl fluorophosphate. This protein did, however, exhibit very low amidolytic activity (approximately 5,000 IU/mg) on the u-PA-specific synthetic substrate pyroglu-Gly-Arg-p-nitroanilide, very low plasminogen-dependent fibrinolytic activity on 125I-labeled fibrin coated plates, and directly activated 125I-labeled plasminogen following Michaelis-Menten kinetics with high affinity, Km = 0.72 microM and low turnover number, kcat = 0.0005 s-1. Treatment with plasmin rapidly converted the HUVEC-derived scu-PA to the active two-chain Mr 54,000 u-PA form (approximately 90,000 IU/mg). Binding to fibrin clots, using antigen quantitation, indicated about 20, 10, and 90% binding for equimolar amounts of HUVEC-derived scu-PA, two-chain u-PA, and tissue plasminogen activator standards, respectively. These results indicate that subcultured HUVECs synthesize and secrete their u-PA protein as a single-chain molecule with low intrinsic amidolytic and fibrinolytic activity, high affinity for plasminogen and no specific affinity for fibrin. The role of scu-PA in endothelial cell-mediated vascular function has yet to be clearly defined.     

Enzymatic properties of single-chain and two-chain forms of a Lys158----Glu158 mutant of urokinase-type plasminogen activator

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 microM-1 s-1) than that of rscu-PA (0.002 microM-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 microM-1 s-1 versus 0.046 microM-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.     

Construction and characterization of a recombinant murine monoclonal antibody directed against human fibrin fragment-D dimer

cDNA libraries in lambda phage were generated from the murine hybridoma secreting mAb-15C5, a monoclonal antibody directed against fragment-D dimer of crosslinked human fibrin [Holvoet et al. (1989) Thromb. Haemostasis 61, 307-313], and clones encoding fragments of the heavy (gamma 1) and the light (kappa) chain were isolated. The kappa-chain cDNA was reconstructed from two overlapping clones encoding 20 amino acids of signal sequence and the 214 amino acids of the mature protein chain. The gamma 1-chain cDNA was reconstructed from the mAb-15C5 kappa-chain signal sequence, the mAb-15C5 gamma 1 variable-domain coding sequence and murine gamma 1-gene and gamma 1-chain cDNA fragments encoding the constant domains. These cDNAs were expressed in Chinese hamster ovary cells, selected cell lines were scaled up in roller bottle culture, and recombinant mAb-15C5 was purified from the conditioned medium by chromatography on Zn-chelate - Sepharose, protein-A - Sepharose and insolubilized fragment-D dimer, with a yield of 50 micrograms/l and a recovery of 20%. SDS-gel electrophoresis without reduction revealed a homogeneous band, and after reduction a light-chain band with identical and a heavy-chained band with a somewhat slower mobility than that of the natural mAb-15C5. Competitive binding revealed a comparable affinity of natural and recombinant mAb-15C5 for fibrin fragment-D dimer. Thus recombinant mAb-15C5, obtained by co-expression of the reconstructed cDNAs of the kappa and gamma 1 chain in Chinese hamster ovary cells, has very similar properties to natural mAb-15C5. These recombinant mAb-15C5 cDNAs may be useful for the construction of a humanized monoclonal antibody for thrombus imaging, and for targeting of thrombolytic agents to fibrin.     

Characterization of functional domains in human tissue-type plasminogen activator with the use of monoclonal antibodies

Two murine monoclonal antibodies (MA-2G6 and MA-1C8), secreted by hybridomas obtained by fusion of myeloma cells with spleen cells from mice immunized with human tissue-type plasminogen activator (t-PA), inhibited the activity of t-PA on fibrin plates. MA-2G6 inhibited the amidolytic activity of t-PA and did not react with t-PA in which the active-site serine was blocked with diisopropylfluorophosphate nor with t-PA in which the active-site histidine was alkylated by reaction with D-Ile-Pro-Arg-CH2Cl. This indicated that MA-2G6 is directed against an epitope covering the active site of t-PA. MA-1C8 did not inhibit the amidolytic activity of t-PA, but abolished both the binding of t-PA to fibrin and the stimulatory effect of fibrin on the activation of plasminogen by t-PA. Thus MA-1C8 is directed against an epitope which covers the fibrin-binding site of t-PA. The A and B chains of partially reduced two-chain t-PA were separated by immunoadsorption on immobilized MA-1C8 and MA-2G6. The purified B chain reacted with MA-2G6 but not with MA-1C8 and activated plasminogen following Michaelis-Menten kinetics with kinetic constants similar to those of intact t-PA (Km = 100 microM and kcat = 0.02 s-1). However, fibrin or CNBr-digested fibrinogen did not stimulate the activation of plasminogen by the B chain. The purified A chain reacted with MA-1C8 but not with MA-2G6. It bound to fibrin with an affinity similar to that of intact t-PA but did not activate plasminogen. It is concluded that the active center of t-PA is located in the B chain and the fibrin-binding site in the A-chain. Both functional domains are required for the regulation by fibrin of the t-PA-mediated activation of plasminogen.     

Urokinase mutant with better fibrin-specificity

A 150 -156 amino acids-deleted single-chain urokinase-type plasminogen activator (dscu-PA) and its rccombinant wild-type counterpart (rscu-PA) were both expressed in Escherichia coli. After denaturation and renaturation in nitro, the expressed products were both purified lo a single silver-stained band by means of IgG affinity chromatography. After activation by plasrran, similar enzymatic constants based on the hydrolysis of synthetic substrate S2444 by the two-chain molecular forms of dscu-PA and rscu-PA, or native tcu-PA were observed, suggesting that no impairment had been exerted on the catalytic active site of dtcu-PA by the 150-156 amino acids deletion. In both in vitro fibrin-clot and 125I-fibrin sepharose lysis tests, dtcu-PA showed a significantly higher fibrinolytic activity than rtcu-PA or rscu-PA. Hardly any effect on the concentration of fibrinogen in plasma was found in dtcu-PA. It was concluded that dtcu-PA had a higher fibrin specificity and that tcu-PA could be provided with better fib     

Characterization of a chimeric plasminogen activator consisting of amino acids 1 to 274 of tissue-type plasminogen activator and amino acids 138 to 411 of single-chain urokinase-type plasminogen activator

A chimeric plasminogen activator (t-PA/scu-PA-s), consisting of amino acids 1-263 of tissue-type plasminogen activator (t-PA) and 144-411 of single-chain urokinase-type plasminogen activator (scu-PA), was previously shown to maintain the enzymatic properties of scu-PA but to have only partially acquired the fibrin affinity of t-PA, possibly as a result of steric interaction between the functional domains of t-PA and scu-PA (Nelles, L., Lijnen, H. R., Collen, D., and Holmes, W.E. (1987) J. Biol. Chem. 262, 10855-10862). Therefore, we now have constructed an extended chimeric t-PA/scu-PA protein, consisting of amino acids 1-274 of t-PA and 138-411 of scu-PA, which thus has an additional sequence of 17 residues in the region joining the two proteins. The highly purified extended chimeric protein (t-PA/scu-PA-e) was found to have similar specific activity on fibrin film (65,000 IU/mg), kinetic constants for the activation of plasminogen (Km = 1 microM, k2 = 0.0026 s-1), fibrin affinity (50% binding at a fibrin concentration of 3.3 g/liter), and fibrin specificity of clot lysis in a plasma environment (50% lysis in 2 h with 8 nM of the chimer) as the previously characterized chimeric protein (t-PA/scu-PA-s). Thus, unexpectedly, the fibrin affinity of t-PA is also only partially expressed in this extended chimeric protein. Therefore, the NH2-terminal chains (A-chains) of the plasmin-generated two-chain derivatives t-PA/tcu-PA-e, t-PA/tcu-PA-s, and of t-PA were isolated. These A-chain structures of the chimers were found to have lost most of their fibrin affinity, whereas the fibrin affinity of the A-chain of native t-PA was maintained. Differential reactivity of the A-chain structures of both chimeric molecules with monoclonal antibodies directed against the A-chain of t-PA suggested that they were conformationally altered. Sequential fibrin binding experiments with t-PA/scu-PA-e and t-PA/scu-PA-s yielded 45 +/- 8 (n = 11) and 43 +/- 5% (n = 8), respectively, binding in the first cycle and 44 +/- 7 (n = 11) and 27 +/- 10% (n = 8), respectively, binding in the second cycle. This suggests that the low affinity of the chimeric molecules for fibrin is not due to the occurrence of subpopulations of molecules with different fibrin affinity but, instead, to a uniformly decreased fibrin affinity in all molecules.     

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

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

Biochemical and thrombolytic properties of a low molecular weight form (comprising Leu144 through Leu411) of recombinant single-chain urokinase-type plasminogen activator

The cDNA encoding a low Mr derivative (residues 144-411) of human single-chain urokinase-type plasminogen activator was cloned, the recombinant low Mr single-chain urokinase-type plasminogen activator (rscu-PA-32k) was expressed in Chinese hamster ovary cells, and the translation product was purified to homogeneity from conditioned cell culture medium. rscu-PA-32k is very similar to intact recombinant single-chain urokinase-type plasminogen activator in terms of its very low activity (120 IU/mg) on a chromogenic substrate for urokinase (pyroglutamylglycylarginine p-nitroanilide), its plasminogen-dependent fibrinolytic activity on fibrin plates (specific activity = 170,000 IU/mg), its plasminogen activating potential, and the lack of specific binding to fibrin. In a rabbit jugular vein thrombosis model, comparable thrombolysis was obtained with rscu-PA-32k as compared to low molecular weight two-chain urokinase (50% lysis at 2.1 and 1.6 mg/kg infused over 4 h). Thrombolysis was associated with much less extensive systemic fibrinogen breakdown with rscu-PA-32k than with two-chain urokinase (residual fibrinogen at 50% lysis of 71 and 10%, respectively). It is concluded that the functional properties of rscu-PA-32k, expressed with a high efficiency, are similar to those of its previously characterized natural counterpart.     

On the mechanism of fibrin-specific plasminogen activation by staphylokinase

The mechanism of plasminogen activation by recombinant staphylokinase was studied both in the absence and in the presence of fibrin, in purified systems, and in human plasma. Staphylokinase, like streptokinase, forms a stoichiometric complex with plasminogen that activates plasminogen following Michaelis-Menten kinetics with Km = 7.0 microM and k2 = 1.5 s-1. In purified systems, alpha 2-antiplasmin inhibits the plasminogen-staphylokinase complex with k1(app) = 2.7 +/- 0.30 x 10(6) M-1 s-1 (mean +/- S.D., n = 12), but not the plasminogen-streptokinase complex. Addition of 6-aminohexanoic acid induces a concentration-dependent reduction of k1(app) to 2.0 +/- 0.17 x 10(4) M-1 s-1 (mean +/- S.D., n = 5) at concentrations greater than or equal to 30 mM, with a 50% reduction at a 6-aminohexanoic acid concentration of 60 microM. Staphylokinase does not bind to fibrin, and fibrin stimulates the initial rate of plasminogen activation by staphylokinase only 4-fold. Staphylokinase induces a dose-dependent lysis of a 0.12-ml 125I-fibrin-labeled human plasma clot submersed in 0.5 ml of citrated human plasma; 50% lysis in 2 h is obtained with 17 nM staphylokinase and is associated with only 5% plasma fibrinogen degradation. Corresponding values for streptokinase are 68 nM and more than 90% fibrinogen degradation. In the absence of a fibrin clot, 50% fibrinogen degradation in human plasma in 2 h requires 790 nM staphylokinase, but only 4.4 nM streptokinase. These results suggest the following mechanism for relatively fibrin-specific clot lysis with staphylokinase in a plasma milieu. In plasma in the absence of fibrin, the plasminogen-staphylokinase complex is rapidly neutralized by alpha 2-antiplasmin, thus preventing systemic plasminogen activation. In the presence of fibrin, the lysine-binding sites of the plasminogen-staphylokinase complex are occupied and inhibition by alpha 2-antiplasmin is retarded, thus allowing preferential plasminogen activation at the fibrin surface.     

Specific functional and immunologic assay of plasma plasminogen in hereditary angioedema, in hereditary angioedema treated with tranexamic acid, and in normal subjects

Plasma plasminogen levels were determined by a specific esterolytic assay and a radial immunodiffusion assay, both of which were standardized on a weight basis by using highly purified plasminogen diluted in plasminogen-free plasma. In thirteen normal individuals the functional and antigenic levels were 275 +/- 61 microgram/ml and 285 +/- 61 microgram/ml, respectively (r = 0.95), whereas in 58 plasmas from individuals with hereditary angioedema the levels were 272 +/- 58 microgram/ml and 265 +/- 64 microgram/ml, respectively (r = 0.89). In two patients treated with tranexamic acid either acutely or chronically, both the functional and antigenic plasminogen levels were diminished.     

Characterization of a recombinant chimeric plasminogen activator with enhanced fibrin binding

A recombinant chimeric plasminogen activator (GHRP-scu-PA-32K), consisting of the tetrapeptide Gly-His-Arg-Pro fused to the N-terminus of the low-molecular single-chain urokinase-type plasminogen activator (Leu144-Leu411), was produced by expression in CHO cells. The stable expression cell line was selected for large-scale expression. The product was purified by antibody-Sepharose affinity chromatography with a recovery of 67%. The apparent molecular weight of purified GHRP-scu-PA-32K was 33 kDa according to SDS-PAGE. Its specific activity was 150000 IU/mg protein according to fibrin plate determination. The conversion of single-chain to two-chain molecules mediated by plasmin was comparable for GHRP-scu-PA-32K (K(m)=4.9 microM, k(2)=0.35 s(-1)) and scu-PA-32K. The activation of plasminogen by GHRP-scu-PA-32K (K(m)=1.02 microM, k(2)=0.0028 s(-1)) was also similar to that of scu-PA-32K. The fibrin binding of GHRP-scu-PA-32K was 2.5 times higher than that of scu-PA-32K at a fibrin concentration of 3.2 mg/ml. In contrast to scu-PA-32K in vitro 125I-fibrin-labeled plasma clot lysis, GHRP-scu-PA had a higher thrombolytic potency, whereas it depleted less fibrinogen in plasma. These results show that GHRP-scu-PA-32K as expected is a potential thrombolytic agent.