SINGLE-CHAIN UROKINASE TYPE PLASMINOGEN ACTIVATOR (SCU-PA) FROM HT-1080 HUMAN FIBROSARCOMA CELLS IS A GENUINE PROENZYME

1987 ◽  
Author(s):  
L Skriver ◽  
L C Petersen ◽  
L R Lund ◽  
L S Nielsen ◽  
K Danø
Keyword(s):  
Limited Proteolysis ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Single Polypeptide Chain ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Pancreatic Trypsin Inhibitor ◽  
Human Fibrosarcoma Cells ◽  
Single Polypeptide ◽  
Hydrolysis Of

U-PA is released from many cells as a single polypeptide chain (scu-PA) that is converted into its active two-chain form (tcu-PA) by limited proteolysis with plasmin. There is general agreement that scu-PA has an extremely low amidolytic activity, but different oppinions exist, as to whether scu-PA itself can activate plasminogen. We have reinvestigated the plasminogen activating activity of our scu-PA preparations by means of a direct [125]I-plasminogen conversion assay and two amidolytic assays for plasmin and u-PA activity. In the [125]I-plasminogen conversion assay in the presence of bovine pancreatic trypsin inhibitor (BPTI) the subsequent plasmin catalyzed conversion of scu-PA is blocked while the plasminogen activation is unaffected. In this assay with 3oo nM Glu-plasminogen and 15 pM BPTI, 4o nM scu-PA caused a low but significant plasminogen conversion, which could be fully inhibited by pretreatment of scu-PA with diisopropylfluorophos-phate (DFP). DFP-treated scu-PA was convertible to fully active tcu-PA. Rates of plasminogen activation in this type of assay for scu-PA activity was at least 4oo fold slower than that measured for tcu-PA. A coupled amidolytic assay with Lys-plasminogen, scuPA or tcu-PA, BPTI, and the high affinity plasmin substrate H-D-Val-Phe-LyspNA (S2390) was performed under conditions that ensures a low steady state concentration of free plasmin. In this assay the initial rate of Lys-plasminogen activation by DFP-treated scu-PA was at least 25o fold slower than that measured for tcu-PA. Finally, u-PA activity was measured in an assay with the chromogenic substrate <Glu-Gly-ArgpNA (S2444) (o.8mM) in the presence of highly purified Glu-plasminogen (3oonM) and DFP-treated scu-PA (2nM) in the absence of BPTI. Within the initial 15 min of incubation no detectable hydrolysis of S2444 occurred. Addition of tcu-PA (2pM) or plasmin (o.lnM) to the scu-PA/Glu-plasminogen mixture caused a significant reduction of the lag period before onset of the cascade reaction leading to scu-PA conversion and subsequent hydrolysis of S2444. We conclude that the low rates of plasminogen activation measured in these assays by scu-PA might be accounted for by the presence of trace amounts of tcu-PA in the scu-PA preparations, and that scu-PA meets the requirements for a genuine proenzyme

Thrombin Stimulation of Platelets Induces Plasminogen Activation Mediated by Endogenous Urokinase-Type Plasminogen Activator

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3579-3586 ◽  
Author(s):  
Catherine Lenich ◽  
Jian-Ning Liu ◽  
Victor Gurewich
Keyword(s):  
Plasminogen Activator ◽  
Gene Knockout ◽  
Direct Action ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Platelet Surface ◽  
Physiologic Mechanism ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Gene Knockout Mice

Abstract Gene knockout mice studies indicate that urokinase-type plasminogen activator (u-PA) is importantly involved in fibrinolysis, but its physiologic mechanism of action remains poorly understood. We postulated that platelets may be involved in this mechanism, as they carry a novel receptor for u-PA and a portion of the single-chain u-PA (scu-PA) intrinsic to blood is tightly associated with platelets. Therefore, plasminogen activation by platelet-associated u-PA was studied. When washed platelets were incubated with plasminogen, no plasmin was generated as detected by plasmin synthetic substrate (S2403) hydrolysis; however, after the addition of thrombin, but not other agonists, platelet-dependent plasminogen activation occurred. Plasminogen activation was surface-related, being inhibited by blocking platelet fibrinogen receptors or by preventing plasminogen binding to the thrombin-activated platelet surface. U-PA was identified as the only plasminogen activator responsible and enrichment of platelets with exogenous scu-PA significantly augmented plasminogen activation. These findings appeared paradoxical because thrombin inactivates scu-PA. Indeed, zymograms showed inactivation of scu-PA during the first hour of incubation with even the lowest dose of thrombin used (1 u/mL). However, this was followed by a thrombin dose-dependent (1 to 10 u/mL) partial return of u-PA activity. Reactivation of u-PA was not due to the direct action of thrombin, but required platelets and was found to be related to a platelet lysosomal thiol protease, consistent with cathepsin C. In conclusion, a new pathway of plasminogen activation by platelet-associated endogenous or exogenous scu-PA was demonstrated, which is specifically triggered by thrombin activation of platelets. These findings may help explain u-PA–mediated physiological fibrinolysis and have implications for therapeutic thrombolysis with scu-PA.

scholarly journals The mechanism of plasminogen activation and fibrin dissolution by single chain urokinase-type plasminogen activator in a plasma milieu in vitro

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1864-1872 ◽  
Author(s):  
HR Lijnen ◽  
B Van Hoef ◽  
F De Cock ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Conformational Transition ◽  
Resistant Mutant ◽  
Fibrin Clot ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Plasma Clot ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

Abstract The relative contribution of several mechanisms to plasminogen activation and fibrin dissolution by urokinase-type plasminogen activator (u-PA) in vitro was quantitated. The activation of plasminogen by recombinant single chain u-PA (rscu-PA), by its two chain derivative (rtcu-PA) and by a plasmin-resistant mutant, rscu-PA- Glu158, obeys Michaelis-Menten kinetics with catalytic efficiencies of 0.00064, 0.046, and 0.00005 L/mumol.s for native plasminogen (Glu- plasminogen) and of 0.0061, 1.21, and 0.0004 L/mumol.s for partially degraded plasminogen (Lys-plasminogen). In a purified system consisting of a fibrin clot submerged in a plasminogen solution, the equi- effective doses (50% lysis in one hour) for rscu-PA, rtcu-PA, and rscu- PA-Glu158 were 16, 6.5, and 32,000 ng/mL for Glu-plasminogen and two- to fourfold lower for Lys-plasminogen. In a plasma milieu, 50% lysis in two hours was obtained for a plasma clot with 2.1 micrograms/mL rscu- PA, 0.5 micrograms/mL rtcu-PA, and greater than 200 micrograms/mL rscu- PA-Glu158 and for a purified fibrin clot with 1.3 micrograms/mL rscu-PA and 0.27 microgram/mL rtcu-PA. After predigestion of a purified fibrin clot with plasmin, the apparent potency of rscu-PA and rtcu-PA increased by 40% and 20%, respectively. In conclusion, rscu-PA has an intrinsic plasminogen activating potential that is only about 1% of that of rtcu-PA and that is 13 times higher than that of rscu-PA- Glu158. Conformational transition of Glu-plasminogen to Lys-plasminogen enhances its sensitivity to activation by all u-PA moieties ten- to 20- fold. Predigestion of fibrin clots with associated increased binding of plasminogen results in a minor apparent increase of the fibrinolytic potency of rscu-PA and rtcu-PA. The relative fibrinolytic potency of rtcu-PA is two to three orders of magnitude higher than that of rscu-PA- Glu158 but only two- to five-fold higher than that of rscu-PA, both in purified systems and in a plasma milieu. These results indicate that conversion of rscu-PA to rtcu-PA constitutes the primary mechanism of fibrin dissolution.

Depression of Tissue-Type Plasminogen Activator and Enhancement of Urokinase-Type Plasminogen Activator as an Expression of Local Inflammation

1992 ◽  
Vol 68 (02) ◽  
pp. 180-184 ◽  
Author(s):  
Emile J P Brommer ◽  
Gerard Dooijewaard ◽  
Ben A C Dijkmans ◽  
Ferdinand C Breedveld
Keyword(s):  
Plasminogen Activator ◽  
Tissue Type ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Healthy Donors ◽  
Urokinase Type Plasminogen Activator ◽  
Knee Trauma ◽  
Fibrinolytic Parameters

SummaryInflammatory processes are accompanied by extravascular deposition and breakdown of fibrin. We measured fibrinolytic parameters in synovial fluid (SF) and in plasma of 36 patients with rheumatoid arthritis (RA). As a control, SF of 13 patients with blunt knee trauma, and plasma of 17 healthy volunteers were studied. In RA patients, extravascular t-PA mediated plasminogen activation was depressed: mean SF tissue-type plasminogen activator (t-PA:Ag) concentration (2.1 ± 1.6 ng/ml) was four-fold lower, and plasminogen activator inhibitor (PAI) activity (284 ± 212%) four-fold higher than the plasma values of the same patients or of healthy donors. In contrast, u-PA related plasminogen activation was strongly enhanced: urokinase-type plasminogen activator (u-PA) antigen (23.1 ±12.4 ng/ml) was more than four-fold higher, single-chain u-PA (scu-PA) (5.3 ± 1.9 ng/ml) three-fold higher than in plasma of the same patients or of healthy donors, and active two-chain u-PA (tcu-PA) was detected in 14 of the 36 SF samples of RA patients. All of these changes in extravascular fibrinolytic parameters correspond with those induced by inflammatory mediators in cell cultures. In joint effusions of patients with a blunt knee trauma, the effects were intermediate: u-PA related parameters showed moderate changes in the same direction as in arthritis; t-PA antigen was also decreased. The only exception was that PAI was not increased. We conclude that the findings in traumatic effusions reflect transient effects as a reaction to trauma. In joint inflammation, the depressed t-PA mediated plasminogen activation, although more than compensated by the enhanced u-PA mediated plasminogen activation, results in protraction of fibrin removal. Besides, the enhanced u-PA activation might lead to proteolytic damage of the cartilage.

THE BIOCHEMISTRY AND CELL BIOLOGY OF SINGLE CHAIN UROKINASE TYPE PLASMINOGEN ACTIVATOR

1987 ◽  
Author(s):  
W D Schleuning
Keyword(s):  
Signal Transduction ◽  
Extracellular Matrix ◽  
Plasminogen Activator ◽  
Gene Transcription ◽  
Synthetic Peptide ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Peptide Substrates ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

Urokinase was discovered in the late nineteenth century, as an enzymatic principle in urine, that initiates the dissolution of blood clots. The basis of this phenomenon was recognized more than fifty years ago as the activation of plasminogen, the precursor of a tryptic protease, then known as profibrinolysin. Despite this long history, detailed data on the biochemistry of plasminogen activation have only become available recently. Urokinase (now designated urokinase-type plasminogen activator : u-PA) is synthesized and secreted as a single chain polypeptide (Mr-: 53,000) by many cell types. Single chain u-PA (scu-PA) is with equal justification called prourokinase (pro-u-PA), notwithstanding its low catalytic activity for synthetic peptide substrates and plasminogen, as most proenzymes of proteases display a certain degree of activity. The structure of pro-u-PA has been elucidated by protein and cDNA sequencing. It consists of three domains, exhibiting characteristic homology to other proteins: a serine protease domain, homologous to trypsin, chymotrypsin and elastase; a kringle domain, likewise found in prothrombin, plasminogen, tissue-type plasminogen activator (t-PA) and Factor XII; and an epidermal growth factor (EGF)-like domain, found in many other proteins, including certain clotting factors. Pro-u-PA is activated by the cleavage of its LYS158-Ile159 h1 bY either plasmin or kallikrein. This cleavage leads to a high increase of Kcat values with respect to both plasminogen and synthetic peptide substrates, but apparently to a reduction of its affinity to plasminogen. Thrartoin inactivates pro-u-PA irreversibly by the cleavage of the Arg156-Phe157 bond. U-PA but not pro-u-PA rapidly forms ccnplexes with plasminogen activator inhibitors (PAI)-l and PAI-2: second order rate constants Kass are respectively > 107 and 0.9xl06 (M-11sec-1). Unknown enzymes process pro-u-PA and u-PA to low molecular weight (LMW) pro-u-PA and LMW u-PA (Mr: 33,000) by cutting off a fragment consisting of the kr ingle and the EGF—like region. Pro—u—PA mediated plasminogen activation is fibrin dependent in vivo, and to a certain degree in vitro. Hie biochemical basis of this fibrin specificity is at present uncertain, although there are reports indicating that it may require polyvalent cations. Through its EGF-like region HMW pro-u-PA and HMW u-PA are capable of binding to specific membrane protein receptors which are found on many cells. Thus, u-PA activity may be restricted to the cell surface. According to a recent report, binding of u—PA to the receptor may also mediate signal transduction in auto- or paracrine growth control. In cells permissive for the respective pathways, pro-u-PA gene transcription is stimulated by mechanisms of signal transduction, that include the cAMP, the tyrosine specific kinase and the protein kinase C dependent pathways. Glucocorticoid hormones downregulate pro-u-PA gene transcription in cells where the gene is canstitutively expressed. Although different cells vary greatly in their response to agents that stimulate urokinase biosynthesis, growth factors and other mitogens are in many cases effective inducers. Significantly elevated levels of u-PA are also found in many malignant tissues. These findings and many others suggest that plasminogen activation by u-PA provides localized extracellular matrix degradation which is required for invasive growth, cell migration and other forms of tissue remodelling. Fibrin represents in this view only a variant of an extracellular matrix, which is provided through the clotting system in the case of an emergency.

scholarly journals The mechanism of plasminogen activation and fibrin dissolution by single chain urokinase-type plasminogen activator in a plasma milieu in vitro

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1864-1872
Author(s):  
HR Lijnen ◽  
B Van Hoef ◽  
F De Cock ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Conformational Transition ◽  
Resistant Mutant ◽  
Fibrin Clot ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Plasma Clot ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

The relative contribution of several mechanisms to plasminogen activation and fibrin dissolution by urokinase-type plasminogen activator (u-PA) in vitro was quantitated. The activation of plasminogen by recombinant single chain u-PA (rscu-PA), by its two chain derivative (rtcu-PA) and by a plasmin-resistant mutant, rscu-PA- Glu158, obeys Michaelis-Menten kinetics with catalytic efficiencies of 0.00064, 0.046, and 0.00005 L/mumol.s for native plasminogen (Glu- plasminogen) and of 0.0061, 1.21, and 0.0004 L/mumol.s for partially degraded plasminogen (Lys-plasminogen). In a purified system consisting of a fibrin clot submerged in a plasminogen solution, the equi- effective doses (50% lysis in one hour) for rscu-PA, rtcu-PA, and rscu- PA-Glu158 were 16, 6.5, and 32,000 ng/mL for Glu-plasminogen and two- to fourfold lower for Lys-plasminogen. In a plasma milieu, 50% lysis in two hours was obtained for a plasma clot with 2.1 micrograms/mL rscu- PA, 0.5 micrograms/mL rtcu-PA, and greater than 200 micrograms/mL rscu- PA-Glu158 and for a purified fibrin clot with 1.3 micrograms/mL rscu-PA and 0.27 microgram/mL rtcu-PA. After predigestion of a purified fibrin clot with plasmin, the apparent potency of rscu-PA and rtcu-PA increased by 40% and 20%, respectively. In conclusion, rscu-PA has an intrinsic plasminogen activating potential that is only about 1% of that of rtcu-PA and that is 13 times higher than that of rscu-PA- Glu158. Conformational transition of Glu-plasminogen to Lys-plasminogen enhances its sensitivity to activation by all u-PA moieties ten- to 20- fold. Predigestion of fibrin clots with associated increased binding of plasminogen results in a minor apparent increase of the fibrinolytic potency of rscu-PA and rtcu-PA. The relative fibrinolytic potency of rtcu-PA is two to three orders of magnitude higher than that of rscu-PA- Glu158 but only two- to five-fold higher than that of rscu-PA, both in purified systems and in a plasma milieu. These results indicate that conversion of rscu-PA to rtcu-PA constitutes the primary mechanism of fibrin dissolution.

scholarly journals Bound plasminogen is rate-limiting for cell-surface-mediated activation of plasminogen by urokinase

1995 ◽  
Vol 309 (3) ◽  
pp. 977-982 ◽  
Author(s):  
S Namiranian ◽  
Y Naito ◽  
V V Kakkar ◽  
M F Scully
Keyword(s):  
Cell Line ◽  
Cell Receptor ◽  
U937 Cells ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Rate Limiting ◽  
Rapid Transformation ◽  
Rate Of Activation

The ability of U937 monocyte-like cells and KATO III cells (a human gastric carcinoma line) to potentiate activation of plasminogen by single-chain urokinase-type plasminogen activator (scu-PA), as mediated by the cell receptor for urokinase (u-PAR), was compared. It was observed that, although the concentration of u-PAR on these cell lines differed considerably (U937 cells: 5000 receptors/cell, Kd 0.35 nM; KATO III cells: 400 receptors/cell, Kd 0.85 nM), the rate of activation of plasminogen by scu-PA in the presence of the same density of each cell line was equivalent. From data generated in the presence of increasing concentrations of scu-PA, the kcat, for plasminogen activation in the presence of each cell line was calculated and found to differ by 26-fold (0.36 s-1 on U937 cells; 9.25 s-1 on KATO III cells). However, the Km for plasminogen with respect to the rate of formation of plasmin was lower than the Kd for binding (0.2 microM compared with 0.5 microM on U937 cells; 0.34 microM compared with 1.6 microM on KATO III cells). A rapid transformation from Glu-plasminogen (native plasminogen with N-terminal Glu) to Lys-plasminogen (plasmin-degraded plasminogen with primarily N-terminal Lys-77) occurred on the surface of U937 cells (unlike KATO III cells), but this transition did not coincide with faster rates of plasminogen activation. From this evidence it is concluded that the accessibility of bound plasminogen acts to limit the rate of activation by cell-bound urokinase. The significance of this proposal is that the proteolytic potential of the cell-mediated activation of plasminogen would be controlled by the accessibility of plasminogen for activation rather than by the concentration of u-PAR (the latter may act to localize proteolysis to appropriate domains on the surface of the cell).

Expression and Characterization of Clustered Charge-to-Alanine Mutants of Low Mr Single-Chain Urokinase-Type Plasminogen Activator

1994 ◽  
Vol 71 (01) ◽  
pp. 134-140 ◽  
Author(s):  
S Ueshima ◽  
P Holvoet ◽  
H R Lijnen ◽  
L Nelles ◽  
V Seghers ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Solvent Accessibility ◽  
Site Directed Mutagenesis ◽  
Clot Lysis ◽  
Wild Type ◽  
Single Chain ◽  
Charged Amino Acids ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Pharmacokinetic Properties

SummaryIn an effort to modify the fibrinolytic and/or pharmacokinetic properties of recombinant low M r single-chain urokinase-type plasminogen activator (rscu-PA-32k), mutants were prepared by site-directed mutagenesis of clusters of charged amino acids with the highest solvent accessibility. The following mutants of rscu-PA-32k were prepared: LUK-2 (Lys 212, Glu 213 and Asp 214 to Ala), LUK-3 (Lys 243 and Asp 244 to Ala), LUK-4 (Arg 262, Lys 264, Glu 265 and Arg 267 to Ala), LUK-5 (Lys 300, Glu 301 and Asp 305 to Ala) and LUK-6 (Arg 400, Lys 404, Glu 405 and Glu 406 to Ala).The rscu-PA 32k moictic3 were expressed in High Five Ttichoplasiani cells, and purified to humugciicily from the conditioned cell culture medium, with recoveries of 0.8 to 3.7 mg/1. The specific fibrinolytic activities (220,000 to 300,000 IU/mg), the rates of plasminogen activation by the single-chain moieties and the rates of conversion In lwo chain moieties by plasmin were comparable for mutant and wild-type rscu PA 32k moieties, with the exception of LUK-5 which was virtually inactive. Equi-effective lysis (50% in 2 h) of 60 pi 125I-fibrin labeled plasma clots submerged in 0.5 ml normal human plasma was obtained with 0.7 to 0.8 μg/ml of wild-type or mutant rscu-PA-3?.k, except with LUK-5 (no significant lysis with 16 pg/ml). Following bolus injection in hamsters, all rscu-PA-32k moieties had a comparably rapid plasma clearance (1.3 to 2.7 ml/min), as a result of a short initial half-life (1.4 to 2.5 min). In hamsters with pulmonary embolism, continuous intravenous infusion over 60 min at a dose of 1 mg/kg, resulted in 53 to 72% clot lysis with the mutants, but only 23% with LUK-5, as compared to 36% for wild-type rscu-PA-32k.These data indicate that clustered charge-to-alanine mutants of rscu-PA-32k, designed to eliminate charged regions with the highest solvent accessibility, do not have significantly improved functional, fibrinolytic or pharmacokinetic properties.

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