PLASMINOGEN ACTIVATION BY SINGLE-CHAIN UROKINASE (scu-PA) IN FUNCTIONAL ISOLATION - DEMONSTRATION OF A NOVEL MECHANISM

1987 ◽  
Author(s):  
V Ellis ◽  
M F Scully ◽  
V V Kakkar
Keyword(s):  
Human Kidney ◽  
Binding Pocket ◽  
Moderate Activity ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Irreversible Inhibitor ◽  
Peptide Substrates ◽  
Sds Page ◽  
Catalytic Constant ◽  
Kinetics Of

The kinetics of the activation of Glu- and Lys- plasminogen by single-chain urokinase (pro-urokinase) derived from the transformed human kidney cell line, TCL-598, has been studied and compared with two-chain urokinase (UK). Plasminogen activation was determined by the change in fluorescence polarization of fluorescein-labelled aprotinin (Trasylol), an essentially irreversible inhibitor of plasmin. This methodology allows plasmin production by scu-PA to be measured in functional isolation, with no interfering generation of two-chain UK. scu-PA was found to activate plasminogen to plasmin with Michaelis-Menten type kinetics. The Km for this reaction was determined as 70µM, with a catalytic constant of 2.25 min-l. The generation of two-chain plasmin was confirmed by reduced SDS-PAGE. Plasminogen activation by UK was found to have a similar Km but the kcat was 16-fold higher, at 36.0 min-l. This is in contrast to the amidolytic activity of scu-PA which was less than 0.2% that of UK. The activation of scu-PA to UK by plasmin was also characterized. Using these data it is possible to calculate the theoretical rate of plasminogen activation by scu-PA, in the absence of aprotinin when UK will be generated by plasmin action. The calculated rate was in good agreement with that determined experimentally when using the chromogenic substrate, S-2251. These data demonstrate that scu-PA has properties which distinguish it from conventional serine protease zymogens. There is a lack of activity against peptide substrates (and also DFP) demonstrating the inaccessibility of the substrate binding pocket. However, there is moderate activity against plasminogen suggesting that plasminogen may be acting as both an effector and a substrate for scu-PA.

Regulation of the single-chain urokinase–urokinase receptor complex activity by plasminogen and fibrin: novel mechanism of fibrin specificity

Blood ◽  
2005 ◽  
Vol 105 (3) ◽  
pp. 1021-1028 ◽  
Author(s):  
Abd Al-Roof Higazi ◽  
Feras Ajawi ◽  
Sa'ed Akkawi ◽  
Edna Hess ◽  
Alice Kuo ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Plasminogen Activator ◽  
Plasma Concentrations ◽  
Chain Molecule ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Michaelis Constant ◽  
Complex Activity ◽  
Catalytic Constant ◽  
Insight Into

AbstractActivation of plasminogen by urokinase plasminogen activator (uPA) plays important roles in several physiologic and pathologic conditions. Cells secrete uPA as a single-chain molecule (scuPA). scuPA can be activated by proteolytic cleavage to a 2-chain enzyme (tcuPA). scuPA is also activated when it binds to its receptor (uPAR). The mechanism by which the enzymatic activity of the scuPA/suPAR complex is regulated is only partially understood. We now report that the plasminogen activator activity of the scuPA/suPAR complex is inhibited by Glu- and Lys-plasminogen, but not by mini-plasminogen. In contrast, neither Glunor Lys-plasminogen inhibits the activation of plasminogen by 2-chain uPA. Inhibition of scuPA/suPAR activity was evident at a Glu-plasminogen concentration of approximately 100 nM, and at physiologic plasma concentrations inhibition was nearly complete. A plasminogen fragment containing kringles 1-3 inhibited the enzymatic activity of scuPA/suPAR with an inhibition constant (Ki) equal to 1.9 μM, increased the Michaelis constant (Km) of scuPA/suPAR from 18 nM to 49 nM, and decreased the catalytic constant (Kcat) approximately 3-fold from 0.035 sec—1 to 0.011 sec—1. Inhibition of scuPA/suPAR by plasminogen was completely abolished in the presence of fibrin clots. These studies provide insight into the regulation of uPA-mediated plasminogen activation and identify a novel mechanism for its fibrin specificity.

CATALYTIC ACTIVITIES OF ONE-CHAIN tPA AS REVEALED BY AN ANALOGUE RESISTANT TO PLASMIN CLEAVAGE

1987 ◽  
Author(s):  
L C Petersen ◽  
M Johannessen ◽  
D Foster ◽  
A Kumar ◽  
E Mulvihill
Keyword(s):  
Enzymatic Activity ◽  
Plasminogen Activation ◽  
Activation Kinetics ◽  
Chromogenic Assay ◽  
Activation Rate ◽  
Sds Page ◽  
Activated State ◽  
The One ◽  
Kinetics Of ◽  
State Concentration

Substitution of Arg275 with Gly in the activation site of tPA provides a one-chain recombinant analogue, tPA-Gly275, which is very resistant to cleavage by plasmin. The amidolytic activity of tPA-Gly275 with simple synthetic substrates was investigated and compared to the kinetics obtained with authentic one- and two-chain tPA. Both one-chain (zymogen) forms possess enzymatic activity, however, in the absence of fibrin it is much lower than that of two-chain tPA. Fibrin enhances the activity of the one-chain tPA forms, but not of two-chain tPA.A chromogenic assay was developed for measurement of plasminogen activation. Due to the presence of a high affinity plasmin substrate (D-Val-Phe-Lys-pNA) and aprotinin in the reaction mixture, the assay ensures a low steady-state concentration of free plasmin during the measurement. With this assay and with tPA-Gly275 it is possible to measure plasminogen activation kinetics of one-chain tPA without any significant two-chain tPA formation even in the presence of fibrin. The intactness of tPA-Gly275 was confirmed by direct measurement of the one-/two-chain tPA content by means of reduced SDS-PAGE combined with Western blotting after exposure to plasmin digestion in the presence and absence of fibrin. The results suggest that one-chain tPA possesses enzymatic activity also with plasminogen as the substrate, however, the activity is much lower than that of two-chain tPA. Addition of fibrin profoundly enhances the plasminogen activation rate of both tPA-Gly275, one-chain, and two-chain authentic tPA to approx. the same maximal level. Taken together these observations indicate that fibrin binding can induce an activated state of the intact tPA ‘zymogen’

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 Activation of plasminogen by single-chain urokinase or by two-chain urokinase--a demonstration that single-chain urokinase has a low catalytic activity (pro-urokinase)

Blood ◽  
1987 ◽  
Vol 69 (1) ◽  
pp. 22-26 ◽  
Author(s):  
R Pannell ◽  
V Gurewich
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Reaction Rates ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Amidolytic Activity ◽  
Catalytic Activities ◽  
Synthetic Substrate ◽  
Sds Page ◽  
Secondary Reactions

Abstract Single-chain urokinase (SC-UK) has an intrinsic amidolytic activity, as measured with synthetic substrate (Kabi S-2444; pyro-Glu-Gly-Arg- pNitroanalide), which was found to be 0.1% to 0.2% that of its plasmin- activated derivative, two-chain UK (TC-UK). A study of the reaction of SC-UK with plasminogen is complicated by the effect of the reaction product, plasmin, on both reactants. The resultant generation of TC-UK and Lys-plasminogen produces secondary reactions which greatly augment plasminogen activation. To confine enzymatic activity to the primary reaction, after pretreatment to eliminate trace TC-UK contaminants, SC- UK was incubated with Glu- or Lys-plasminogen in the presence of aprotinin (500 KIU/mL) to inhibit generated plasmin and dansyl-glutamyl- glycyl-arginyl-chloromethylketone (20 mumol/L), which irreversibly inhibited TC-UK but not SC-UK. Analysis by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a plasminogen-activating activity for SC-UK that was approximately 0.4% that of TC-UK. Both SC-UK and TC-UK preferentially activated Lys- plasminogen over Glu-plasminogen. Similarly, Glu-plasminogen activation was augmented by lysine or soluble fibrin. The ratio of the reaction rates of SC-UK and TC-UK were comparable for Glu- and Lys-plasminogen. It is concluded that there is a major difference in the catalytic activities of SC-UK and TC-UK against plasminogen that is comparable to that against synthetic substrate.

Purification and Characterization of Single-Chain Urokinase-Type Plasminogen Activator (Pro-Urokinase) from Human A431 Cells

1986 ◽  
Vol 56 (02) ◽  
pp. 219-224 ◽  
Author(s):  
Angelo Corti ◽  
Maria Luisa Nolli ◽  
Adolfo Soffientini ◽  
Giovanni Cassani
Keyword(s):  
Plasminogen Activator ◽  
Specific Activity ◽  
Active Form ◽  
Human Kidney ◽  
Single Chain ◽  
A431 Cells ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Sds Page ◽  
Aminoacid Sequence

SummaryA single-chain urokinase-type plasminogen activator (A431sc-uPA) was purified ˜18,000-fold from A431 human epidermoid carcinoma cell supernatants by monoclonal antibody immunoaffinity chromatography on 5B4-agarose and ion-exchange FPLC (overall yield 63%). More than 100 jig of A431sc-uPA can be recovered per liter of supernatant. The product is homogeneous by SDS-PAGE and reverse phase FPLC analysis while two main isoelectric forms of pi 9.05 and pi 9.20 were observed by IEF. SDS-PAGE in reducing and non-reducing conditions, Western blot analysis and zymography showed that A431sc-uPA is a single-chain protein of about 50,000 Mr immunologically related to urokinase (uPA) and distinct from tissue plasminogen activator (tPA). The N-terminal aminoacid sequence of A431sc-uPA (27 residues) is identical to that of human kidney single-chain uPA. A431sc-uPA does not incorporate 3H-diisopropylfluorophosphate and is virtually inactive on the synthetic substrate S-2444. Plasmin treatment converts A431sc-uPA into a two-chain active form with a fibrinolytic specific activity of 123,000 I.U./mg.

scholarly journals Activation of plasminogen by single-chain urokinase or by two-chain urokinase--a demonstration that single-chain urokinase has a low catalytic activity (pro-urokinase)

Blood ◽  
1987 ◽  
Vol 69 (1) ◽  
pp. 22-26 ◽  
Author(s):  
R Pannell ◽  
V Gurewich
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Reaction Rates ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Amidolytic Activity ◽  
Catalytic Activities ◽  
Synthetic Substrate ◽  
Sds Page ◽  
Secondary Reactions

Single-chain urokinase (SC-UK) has an intrinsic amidolytic activity, as measured with synthetic substrate (Kabi S-2444; pyro-Glu-Gly-Arg- pNitroanalide), which was found to be 0.1% to 0.2% that of its plasmin- activated derivative, two-chain UK (TC-UK). A study of the reaction of SC-UK with plasminogen is complicated by the effect of the reaction product, plasmin, on both reactants. The resultant generation of TC-UK and Lys-plasminogen produces secondary reactions which greatly augment plasminogen activation. To confine enzymatic activity to the primary reaction, after pretreatment to eliminate trace TC-UK contaminants, SC- UK was incubated with Glu- or Lys-plasminogen in the presence of aprotinin (500 KIU/mL) to inhibit generated plasmin and dansyl-glutamyl- glycyl-arginyl-chloromethylketone (20 mumol/L), which irreversibly inhibited TC-UK but not SC-UK. Analysis by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a plasminogen-activating activity for SC-UK that was approximately 0.4% that of TC-UK. Both SC-UK and TC-UK preferentially activated Lys- plasminogen over Glu-plasminogen. Similarly, Glu-plasminogen activation was augmented by lysine or soluble fibrin. The ratio of the reaction rates of SC-UK and TC-UK were comparable for Glu- and Lys-plasminogen. It is concluded that there is a major difference in the catalytic activities of SC-UK and TC-UK against plasminogen that is comparable to that against synthetic substrate.

Detection of both Type 1 and Type 2 Plasminogen Activator Inhibitors in Human Monocytes

1990 ◽  
Vol 63 (01) ◽  
pp. 067-071 ◽  
Author(s):  
Joan C Castellote ◽  
Enric Grau ◽  
Maria A Linde ◽  
Nuria Pujol-Moix ◽  
Miquel LI Rutllant
Keyword(s):  
Molecular Mass ◽  
Plasminogen Activator ◽  
Human Peripheral Blood ◽  
Direct Interaction ◽  
Apparent Molecular Weight ◽  
Fibrinolytic System ◽  
Human Monocytes ◽  
Blood Monocytes ◽  
Single Chain ◽  
Sds Page

SummaryIncreasing evidence suggests the involvement of leukocytes in the fibrinolytic system. Monocytes secrete pro-urokinase (Grau, Thromb Res 1989; 53: 145) and it has been shown that these cells have specific receptors for urokinase and plasminogen (Miles, Thromb Haemostas 1987; 58: 936). The aim of this study was to analyse the presence of plasminogen activator inhibitor(s) in platelet-free suspensions of human peripheral blood monocytes and polymorphonuclear leukocytes (PMN). SDS-PAGE and reverse fibrin autography showed an inhibitory band of 50 kDa in the monocyte extracts (Triton X-100) but not in the PMN extracts. Urokinase (u-PA) was mixed with increasing amounts of monocyte extract for 10 min and the mixtures were added to 125Ifibrin coated wells containing plasminogen. A dose-dependent decrease in the u-PA fibrinolytic activity was observed. The amount of inhibition increased when the monocyte releasates were preincubated with u-PA (40% inhibition after 5 min preincubation and 80% after 15 min), indicating a direct interaction between this activator and an inhibitor(s). After SDS-PAGE of monocyte extracts, immunoblotting and peroxidase staining identified both PAI1 and PAI2, with an apparent molecular weight of 47-50 kDa. Monocyte-associated PAI1 formed complexes with single chain t-PA with a molecular mass 50 kDa higher than the molecular mass of the free PAI1. However, a significant amount of PAI remained unbound to t-PA. This inactive PAI1 could have come from a rapid inactivation of the primary active PAI1. These PAI1 and PAI2 detected in human monocytes may be transcendent in the regulation of the fibrinolytic system.

In Vitro Stability of a Tissue-Type Plasminogen Activator Mutant, BM 06.022, in Human Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 906-911 ◽  
Author(s):  
D C Rijken ◽  
E Groeneveld ◽  
M M Barrett-Bergshoeff
Keyword(s):  
Plasminogen Activator ◽  
Half Life ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tissue Type ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Sds Page ◽  
Chain Form

SummaryBM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated.Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, α2-antiplasmin and α1-antitrypsin.During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of a2-anti-plasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i. e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher.In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, a2-antiplasmin and a j-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form.

Molecular Forms of Human Protein C: Comparison and Distribution in Human Adult Plasma

1989 ◽  
Vol 62 (03) ◽  
pp. 902-905 ◽  
Author(s):  
Brian S Greffe ◽  
Marilyn J Manco-Johnson ◽  
Richard A Marlar
Keyword(s):  
Heavy Chain ◽  
Protein C ◽  
Molecular Forms ◽  
Post Translational Modification ◽  
Single Chain ◽  
Beta Form ◽  
Sds Page ◽  
Dependent Protein ◽  
Adult Plasma ◽  
The Difference

SummaryProtein C (PC) is a vitamin K-dependent protein which functions as both an anticoagulant and profibrinolytic. It is synthesized as a single chain protein (SC-PC) and post-transla-tionally modified into a two chain form (2C-PC). Two chain PC consists of a light chain (LC) and a heavy chain (HC). The present study was undertaken to determine the composition of the molecular forms of PC in plasma. PC was immunoprecipitated, subjected to SDS-PAGE and Western blotting. The blots were scanned by densitometry to determine the distribution of the various forms. The percentage of SC-PC and 2C-PC was found to be 10% and 90% respectively. This is in agreement with previous work. SC-PC and the heavy chain of 2C-PC consisted of three molecular forms (“alpha”, “beta”, and “gamma”). The “alpha” form of HC is the standard 2C form with a MW of 40 Kd. The “beta” form of HC has also been described and has MW which is 4 Kd less than the “alpha” form. The “gamma” species of the SC and 2C-PC has not been previously described. However, its 3 Kd difference from the “beta” form could be due to modification of the “beta” species or to a separate modification of the alpha-HC. The LC of PC was shown to exist in two forms (termed form 1 and form 2). The difference between these two forms is unknown. The molecular forms of PC are most likely due to a post-translational modification (either loss of a carbohydrate or a peptide) rather than from plasma derived degradation.

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