Interaction of single-chain urokinase-type plasminogen activator with human endothelial cells

The interaction of urokinase-type plasminogen activators with receptors on the surface of endothelial cells may play an important role in the regulation of fibrinolysis and cell migration. Therefore, we investigated whether human umbilical vein endothelial cells (HUVEC) express receptors for single-chain urokinase (scu-PA) on the cell surface and examined the effect of such binding on plasminogen activator activity. Binding of 125I-labeled scu-PA to HUVEC, performed at 4 degrees C, was saturable, reversible, and specific (k+1 4 +/- 1 X 10(6) min-1 M-1, k-1 6.2 +/- 1.4 X 10(-3) min-1, Kd 2.8 +/- 0.1 nM; Bmax 2.2 +/- 0.1 X 10(5) sites/cell; mean +/- S.E.). Binding of radiolabeled scu-PA was inhibited by both natural and recombinant wild-type scu-PA, high molecular weight two-chain u-PA (tcu-PA), catalytic site-inactivated tcu-PA, an amino-terminal fragment of u-PA (amino acids 1-143), and a smaller peptide (amino acids 4-42) corresponding primarily to the epidermal growth factor-like domain. Binding was not inhibited by low molecular weight urokinase or by a recombinant scu-PA missing amino acids 9-45. Cell-bound scu-PA migrated at its native molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of plasminogen, scu-PA bound to endothelial cells generated greater plasmin activity than did scu-PA in the absence of cells. In contrast, when tcu-PA was added directly to HUVEC, sodium dodecyl sulfate-stable complexes formed with cell or matrix-associated plasminogen activator inhibitors with a loss of plasminogen activator activity. These studies suggest that endothelial cells in culture express high affinity binding sites for the epidermal growth factor domain of scu-PA. Interaction of scu-PA with these receptors may permit plasminogen activator activity to be expressed at discrete sites on the endothelial cell membrane.     

Identification of the epidermal growth factor-like domains of thrombomodulin essential for the acceleration of thrombin-mediated inactivation of single-chain urokinase-type plasminogen activator.

Single-chain urokinase-type plasminogen activator (scu-PA) can be cleaved by thrombin into a virtually inactive form called thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T), a process accelerated by thrombomodulin, which contains six epidermal growth factor (EGF)-like domains. In this study, we identified the EGF-like domains of thrombomodulin required for the acceleration of the inactivation of scu-PA by thrombin using various forms of thrombomodulin (TM). scu-PA was treated with thrombin in the absence and presence of full-length rabbit TM (containing EGF1-6), recombinant TM comprising all of the extracellular domains including EGF1-6 (TMLEO) and recombinant TM comprising EGF4-6 plus the interconnecting region between EGF3 and EGF4 (TMEi4-6), and the tcu-PA/T generated was quantitated in each case. Rabbit TM accelerated the inactivation of scu-PA approximately 35-fold, while both recombinant forms accelerated it only threefold due to the absence of a critical chondroitin sulfate moiety. Subsequently, TME5-6 was prepared by cyanogen bromide digestion of TMEi4-6. TME5-6 bound to thrombin but did not accelerate the activation of protein C. In contrast, the inactivation of scu-PA by thrombin was accelerated to the same extent as that induced by TMLEO and TMEi4-6. This study demonstrates that, in addition to the chondroitin sulfate moiety, only EGF-like domains 5 and 6 are essential for the acceleration of the inactivation of scu-PA by thrombin. This differs from the domains that are critical for activation of protein C (EGF-like domains i4-6) and thrombin activatable fibrinolysis inhibitor (EGF-like domains 3-6).     

Two distinct urokinase-serpin interactions regulate the initiation of cell surface-associated plasminogen activation.

Single-chain urokinase-type plasminogen activator (scu-PA) possesses enzymatic activity that increases by 2-3 orders of magnitude upon binding to its cellular cofactor, the u-PA receptor (u-PAR), hence activating an enzymatic cascade initially composed of zymogens. The present study demonstrates that plasminogen activator inhibitor type 3 (PAI-3) reversibly inhibits scu-PA in solution, maintaining the system "off." Because the scu-PA/PAI-3 interaction is reversible, cellular expression of u-PAR allows partitioning of scu-PA from PAI-3 to u-PAR with resultant expression of full enzymatic activity. PAI-3 that was originally complexed to scu-PA remains in solution, retaining its functional activity. Importantly, the scu-PA on cell surface u-PAR is protected from PAI-3 inhibition, remaining an effective activator in a PAI-rich environment. Plasmin formed as a result of scu-PA activity then cleaves scu-PA to the mature protease, two-chain u-PA (tcu-PA), which is efficiently and irreversibly inhibited by PAI-3 via the standard serpin mechanism, even on u-PAR. This data generates a new hypothesis which, in contrast to the previous paradigm, holds that receptor bound scu-PA is the initiating enzyme and that tcu-PA is generated not to augment enzymatic activity but rather to allow for inhibition and therefore appropriate regulation of the process.     

Activation with plasmin of tow-chain urokinase-type plasminogen activator derived from single-chain urokinase-type plasminogen activator by treatment with thrombin

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 43,000 IU/mg) by hydrolysis of the Lys-158--Ile-159 peptide bond. This conversion follows Michaelis-Menten kinetics with a Michaelis constant Km of 37 microM 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.     

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.     

Comparative kinetic analysis of the activation of human plasminogen by natural and recombinant single-chain urokinase-type plasminogen activator

Single-chain urokinase-type plasminogen activator (scu-PA) may be obtained from conditioned cell culture media (natural scu-PA) or by expression of the cDNA encoding human scu-PA in Escherichia coli (recombinant scu-PA). The activation of Glu-plasminogen by natural and recombinant scu-PA can be described by a sequence of three reactions, each of which obeys Michaelis-Menten kinetics. Initial activation of plasminogen to plasmin by scu-PA (reaction I) occurs with a high affinity (Km below 0.8 microM) for both scu-PAs, while the catalytic rate constant (k2) is 0.017 s-1 for recombinant scu-PA but only 0.0009 s-1 for natural scu-PA. Subsequent conversion of scu-PA to urokinase (two-chain urokinase-type plasminogen activator, tcu-PA) by generated plasmin (reaction II) occurs with a comparable affinity (Km about 5 microM) for natural and recombinant scu-PA and with a k2 of 0.23 s-1 for natural and 1.2 s-1 for recombinant scu-PA. Finally, activation of plasminogen by tcu-PA (reaction III) occurs with low affinity (Km 30-50 microM) but with a high catalytic rate constant (k2 about 5 s-1) for both natural and recombinant tcu-PA. The differences in the kinetic parameters of the activation of plasminogen by natural or recombinant scu-PA are thus mainly due to differences in turnover rate in the first reaction. Indeed, the catalytic rate constant of the first reaction is about 20-times higher for recombinant scu-PA than for natural scu-PA. Thus, surprisingly, the artificial, unglycosylated recombinant scu-PA molecule has a better catalytic efficiency than its natural glycosylated counterpart.     

Activation of pro-urokinase and plasminogen on human sarcoma cells: a proteolytic system with surface-bound reactants

Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serum-containing medium, bound plasmin activity could be eluted from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited by an anticatalytic monoclonal antibody to u-PA, indicating that this enzyme was responsible for the activation. Preincubation of the cells with diisopropyl fluorophosphate-inhibited u-PA led to a decrease in surface-bound plasmin, indicating that a large part, if not all, of the cell surface plasminogen activation was catalyzed by surface-bound u-PA. In the absence of plasminogen, most of the cell surface u-PA was present in its single-chain proenzyme form, while addition of plasminogen led to formation of cell-bound two-chain u-PA. The latter reaction was catalyzed by cell-bound plasmin. Cell-bound u-PA was accessible to inhibition by endogenous PAI-1 and by added PAI-2, while the cell-bound plasmin was inaccessible to serum inhibitors, but accessible to added aprotinin and an anticatalytic monoclonal antibody. A model for cell surface plasminogen activation is proposed in which plasminogen binding to cells from serum medium is followed by plasminogen activation by trace amounts of bound active u-PA, to form bound plasmin, which in turn serves to produce more active u-PA from bound pro-u-PA. This exponential process is subject to regulation by endogenous PAI-1 and limited to the pericellular space.     

免疫亲和层析法纯化单链尿激酶型纤溶酶原激活剂

尿激酶原 (Ｐｒｏ ｕｒｏｋｉｎａｓｅ ,ｐｒｏ ＵＫ) ,也称单链尿激酶型纤溶酶原激活剂 (Ｓｉｎｇｌｅ ｃｈａｉｎｕｒｏｋｉｎａｓｅ ｔｙｐｅｐｌａｓｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒ,ｓｃｕ ＰＡ) ,与ｔ ＰＡ一样是第二代溶栓药物。给药时 ,保持无活性的酶原状态 ,只激活被纤维蛋白吸附的纤溶酶原 ,而对游离的纤溶酶原没有作用 ,即只在血栓表面才能活化转变为双链尿激酶 (Ｔｗｏ ｃｈａｉｎｕｒｏｋｉｎａｓｅ ｔｙｐｅｐｌａｓｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒ,ｔｃｕ ＰＡ或ＵＫ) ,因而具有较高的特异性溶血栓作用[1 ] 。尿激酶原…     

Thrombin increases proliferation and decreases fibrinolytic activity of kidney glomerular epithelial cells.

Human glomerular epithelial cells (GECs) in culture synthesize single-chain, urokinase-type plasminogen activator (SC-uPA), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor 1 (PAI-1) and possess specific membrane-binding sites for u-PA. Using purified 125I-alpha thrombin, we demonstrate here the presence of two populations of specific binding sites for thrombin on GECs (1.Kd = 4.3 +/- 1.0 x 10(-10) M, 5.4 +/- 1.4 x 10(4) M sites per cell, 2. Kd = 1.6 +/- 0.5 x 10(-8) M, 7.9 +/- 1.8 x 10(5) sites per cell). Purified human alpha thrombin promoted the proliferation of GECs and induced a time- and dose-dependent increase of SC-uPA, t-PA, and PAI-1 antigens released by GECs. Thrombin-mediated increase in antigen was paralleled by an increase in the levels of corresponding u-PA and PAI-1 messenger RNA. In contrast, thrombin decreased u-PA activity in conditioned medium. This discrepancy between u-PA antigen and u-PA activity was explained by a limited proteolysis of SC-uPA by thrombin, leading to a two-chain form detected by immunoblotting and that could not be activated by plasmin. Thrombin also decreased the number of u-PA binding sites on GECs (p less than 0.05) without changing receptor affinity. Hirudin inhibited the binding and the cellular effects of thrombin, whereas thrombin inactivated by diisopropylfluorophosphate had no effect, indicating that both membrane binding and catalytic activity of thrombin were required. We conclude that thrombin, through specific membrane receptors, stimulates proliferation of GECs and decreases the fibrinolytic activity of GECs both at the cell surface and in the conditioned medium. These results suggest that thrombin could be involved in the pathogenesis of extracapillary proliferation and persistency of fibrin deposits in crescentic glomerulonephritis.     

Synthesis of recombinant human single-chain urokinase-type plasminogen activator variants resistant to plasmin and thrombin

Single-chain urokinase-type plasminogen activator (scu-PA), a potential therapeutic reagent for thrombosis, is activated in plasma by plasmin. The activated enzyme is further digested by plasmin to generate low-molecular-weight urokinase (LMW-UK), which has no affinity for fibrin. To circumvent this dual effect of plasmin, we synthesized in Escherichia coli a variant of scu-PA, which is not converted to LMW-UK on treatment with plasmin. In another variant, the activation cleavage site was modified such that activation by plasmin was slowed down and that inactivation by thrombin was greatly diminished. The combination of these variants may be applicable as an effective thrombolytic reagent for clinical use.     

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.     

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.     

Single chain urokinase. Augmentation of enzymatic activity upon binding to monocytes

Cellular migration typically requires cell surface-associated urokinase-type plasminogen activator (u-PA) and plasminogen, both of which are present as proenzymes. Because each active enzyme can activate the other zymogen, the mechanism by which the initial proteolytic event of this two-zymogen system occurs is unclear. A mutant of single chain u-PA that could not be cleaved to the more active two-chain u-PA was used to demonstrate that (i) u-PA in its single-chain form exhibits a reactive active site serine, (ii) the enzymatic activity of this molecule is augmented 100-fold upon binding to the u-PA receptor on monocytes as compared with the enzymatic activity of the same number of molecules in the fluid phase, and (iii) the molecule thus bound and active remains in the single-chain form. This is likely an important mechanism for the initiation and control of cell surface-associated fibrinolysis.     

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.     

Expression of an anchored urokinase in the apical endothelial cell membrane. Preservation of enzymatic activity and enhancement of cell surface plasminogen activation.

A mutant single chain urokinase plasminogen activator (scu-PA) was constructed by the addition of an apical membrane targeting signal from decay accelerating factor to the scu-PA carboxyl terminus. Bovine aortic endothelial cells (EC) were transduced with the mutant scu-PA. Metabolic labeling, immunoprecipitation, and gel electrophoresis revealed that the mutant scu-PA was present in a single-chain form at the EC surface. Immunohistochemistry and enzyme-linked immunosorbent assay before and after treatment of EC with phosphotidylinositol-specific phospholipase C confirmed that scu-PA was attached to the EC surface by a glycosyl-phosphotidylinositol anchor. Approximately 10(6) anchored scu-PA molecules/cell were present; however, anchoring was not 100% efficient, with scu-PA released into the medium as well. Selective biotinylation of the apical and basolateral surfaces revealed that anchored scu-PA was polarized to the apical surface. Apically anchored scu-PA could be converted by plasmin to two-chain urokinase, with a normal specific activity (140,000 IU/mg) as measured with the chromogenic substrate S-2444. Expression of anchored scu-PA resulted in an increase in EC surface plasminogen activator activity, as compared with the activity of either untransduced EC or EC transduced with a wild type scu-PA. These experiments demonstrate: 1) apical membrane targeting can be accomplished in EC; 2) scu-PA can be anchored to the EC surface with preservation of enzymatic activity; 3) EC surface plasminogen activator activity is significantly increased by the presence of anchored scu-PA. Cell surface targeted plasminogen activators may eventually be useful in the prevention and treatment of intravascular thrombosis.     

Mutant and chimeric recombinant plasminogen activators. Production in eukaryotic cells and preliminary characterization

Mutant urokinase-type plasminogen activator (u-PA) genes and hybrid genes between tissue-type plasminogen activator (t-PA) and u-PA have been designed to direct the synthesis of new plasminogen activators and to investigate the structure-function relationship in these molecules. The following classes of constructs were made starting from cDNA encoding human t-PA or u-PA: 1) u-PA mutants in which the Arg156 and Lys158 were substituted with threonine, thus preventing cleavage by thrombin and plasmin; 2) hybrid molecules in which the NH2-terminal regions of t-PA (amino acid residues 1-67, 1-262, or 1-313) were fused with the COOH-terminal region of u-PA (amino acids 136-411, 139-411, or 195-411, respectively); and 3) a hybrid molecule in which the second kringle of t-PA (amino acids 173-262) was inserted between amino acids 130 and 139 of u-PA. In all cases but one, the recombinant proteins, produced by transfected eukaryotic cells, were efficiently secreted in the culture medium. The translation products have been tested for their ability to activate plasminogen after in situ binding to an insolubilized monoclonal antibody directed against urokinase. All recombinant enzymes were shown to be active, except those in which Lys158 of u-PA was substituted with threonine. Recombination of structural regions derived from t-PA, such as the finger, the kringle 2, or most of the A-chain sequences, with the protease part or the complete u-PA molecule did not impair the catalytic activity of the hybrid polypeptides. This observation supports the hypothesis that structural domains in t-PA and u-PA fold independently from one to another.     

Purification and characterization of a novel low molecular weight form of single-chain urokinase-type plasminogen activator

A low Mr form (Mr 32,000) of single-chain urokinase-type plasminogen activator (scu-PA) was isolated from conditioned culture medium of a human lung adenocarcinoma cell line, CALU-3 (ATCC, HTB-55). The purified material (scu-PA-32k) consists of a single polypeptide chain and is immunologically similar to Mr 33,000 urokinase. Its NH2-terminal sequence is identical to that beginning at Leu-144 of Mr 54,000 urokinase. Whereas low Mr urokinase is derived from mature Mr 54,000 scu-PA by limited hydrolysis by plasmin first of the Lys-158-Ile-159 peptide bond and then of the Lys-136-Lys-137, scu-PA-32k is generated by specific hydrolysis of the Glu-143-Leu-144 peptide bond by an unidentified protease. scu-PA-32k resembles its Mr 54,000 scu-PA counterpart by its very low activity on chromogenic substrates for urokinase, by plasminogen-dependent fibrinolytic activity on fibrin plates, and by the lack of specific binding to fibrin. It activates plasminogen directly with high affinity, Km = 0.9 microM, but low turnover number, kcat = 0.0028 s-1. It is converted to fully active two-chain urokinase by plasmin with Km = 12 microM and kcat = 0.3 s-1. Like Mr 54,000 scu-PA, it causes significant lysis of a 125I-labeled fibrin clot in human plasma with relatively less fibrinogen breakdown as compared to urokinase. scu-PA-32k, which also has conserved fibrin specificity, represents a molecular variant which may be more suitable for large scale production as a fibrin-specific thrombolytic agent by recombinant DNA technology.     

One-chain urokinase-type plasminogen activator from human sarcoma cells is a proenzyme with little or no intrinsic activity

We have compared the plasminogen activating capacity of one- and two-chain urokinase-type plasminogen activator (u-PA). In a 125I-plasminogen conversion assay in the presence of high amounts of a plasmin inhibitor, one-chain u-PA pretreated with diisopropyl fluorophosphate had no detectable activity, the detection limit corresponding to the activity of a 400-fold lower amount of two-chain u-PA. In coupled assays in which generated plasmin was measured with a synthetic substrate, activity was clearly observed with the one-chain preparation, but the initial rate of plasminogen activation was lower than that of a 250-fold smaller concentration of two-chain u-PA. The coupled assays for one-chain u-PA are self-activating because plasmin catalyzes conversion of one- to two-chain u-PA, and it is not possible to decide whether the low activity of one-chain u-PA observed with this type of assay is intrinsic or due to contaminations. On the basis of these findings and a discussion of previous studies, it is concluded that one-chain u-PA has a variety of properties similar to the one-chain proenzyme forms of other serine proteases and that it should, therefore, be considered as a genuine proenzyme form of u-PA.     

Localization of urokinase-type plasminogen activator receptor on U937 cells: Phorbol ester PMA induces heterogeneity

The binding of human urokinase-type plasminogen activator (u-PA) to the surface of the human monocytic cell line U937 was studied by immunological detection of bound u-PA or binding of biotinylated diisopropyl fluorophosphate-inactivated human u-PA visualized by light or electron microscopy. Untreated U937 cells showed a characteristic binding pattern, with the majority of the u-PA bound to the microvillar-containing protruding pole of the cells. After treatment with the phorbol ester PMA, the resulting adherent cell population was very heterogeneous with respect to both cellular morphology and u-PA binding. The bound u-PA was distributed on both the dorsal and the substrate side of the cells, and the patches of bound u-PA could not be correlated to any typical membrane conformations or cell-cell or cell-substratum contacts. When a monoclonal antibody directed against the amino-terminal fragment (ATF) of u-PA was used, the results were identical regardless of whether intact u-PA or ATF was used for binding to the cells. In contrast, when a monoclonal antibody recognizing the non-receptor-binding protease domain of u-PA was used, bound ATF showed no staining, while bound intact u-PA was stained as efficiently as above. The alteration of u-PA receptor distribution following treatment with PMA could be related to the changes in glycosylation and ligand affinity of the purified u-PA receptor previously described following PMA treatment of U937 cells.     

Properties of the Urokinase-Type Plasminogen Activator Modified with Phenylglyoxal

A chemical modification of single-chain urokinase-type plasminogen activator (scu-PA) with phenylglyoxal under mild conditions resulted in scu-PA derivatives with various numbers of the modified Arg residues. The study of properties of the resulting derivatives demonstrated that the modification of 4–12 Arg residues did not cause any loss of the activator, fibrinolytic, and potential amidase activities of the activator. The scu-PA with four modified Arg residues was found to be the most stable derivative in human blood plasma; it causes a more efficient lysis of plasma clots than the native activator. Three of four modified Arg residues are supposed to be within the R178RHRGGS184 cluster, which was located in the superficial loop of the scu-PA globule and was shown to interact with a complementary series of negatively charged residues in the molecule of the main plasma inhibitor PAI-1. The neutralization of positively charged Arg residues in this cluster decreases the affinity of scu-PA and the two-chain urokinase-type plasminogen activator for PAI-1, which results in an enhancement of the stability in plasma and the fibrinolytic efficiency of the activator.