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.

scholarly journals Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

2009 ◽  
Vol 297 (4) ◽  
pp. L568-L577 ◽  
Author(s):  
Andrey A. Komissarov ◽  
Andrew P. Mazar ◽  
Kathy Koenig ◽  
Anna K. Kurdowska ◽  
Steven Idell
Keyword(s):  
Enzymatic Activity ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Receptor Complexes ◽  
Pleural Injury ◽  
Pai 1 ◽  
Formation Of Complexes

The proenzyme single-chain urokinase plasminogen activator (scuPA) more effectively resolved intrapleural loculations in rabbits with tetracycline (TCN)-induced loculation than a range of clinical doses of two-chain uPA (Abbokinase) and demonstrated a trend toward greater efficacy than single-chain tPA (Activase) (Idell S et al., Exp Lung Res 33: 419, 2007.). scuPA more slowly generates durable intrapleural fibrinolytic activity than Abbokinase or Activase, but the interactions of these agents with inhibitors in pleural fluids (PFs) have been poorly understood. PFs from rabbits with TCN-induced pleural injury treated with intrapleural scuPA, its inactive Ser195Ala mutant, Abbokinase, Activase, or vehicle, were analyzed to define the mechanism by which scuPA induces durable fibrinolysis. uPA activity was elevated in PFs of animals treated with scuPA, correlated with the ability to clear pleural loculations, and resisted (70–80%) inhibition by PAI-1. α-macroglobulin (αM) but not urokinase receptor complexes immunoprecipitated from PFs of scuPA-treated rabbits retained uPA activity that resists PAI-1 and activates plasminogen. Conversely, little plasminogen activating or enzymatic activity resistant to PAI-1 was detectable in PFs of rabbits treated with Abbokinase or Activase. Consistent with these findings, PAI-1 interacts with scuPA much slower than with Activase or Abbokinase in vitro. An equilibrium between active and inactive scuPA (kon= 4.3 h−1) limits the rate of its inactivation by PAI-1, favoring formation of complexes with αM. These observations define a newly recognized mechanism that promotes durable intrapleural fibrinolysis via formation of αM/uPA complexes. These complexes promote uPA-mediated plasminogen activation in scuPA-treated rabbits with TCN-induced pleural injury.

Lysis of Plasma Clots by Urokinase-Soluble Urokinase Receptor Complexes

Blood ◽  
1998 ◽  
Vol 92 (6) ◽  
pp. 2075-2083 ◽  
Author(s):  
Abd Al-Roof Higazi ◽  
Khalil Bdeir ◽  
Edna Hiss ◽  
Shira Arad ◽  
Alice Kuo ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Plasma Concentrations ◽  
Plasminogen Activator Inhibitor ◽  
Chain Molecule ◽  
Type I ◽  
Single Chain ◽  
Receptor Complexes ◽  
Pai 1 ◽  
Fibrin Clots

Single-chain urokinase plasminogen activator (scuPA), the unique form secreted by cells, expresses little intrinsic plasminogen activator activity. scuPA can be activated by proteolytic cleavage to form a two-chain enzyme (tcuPA), which is susceptible to inhibition by plasminogen activator inhibitor type I (PAI-1). scuPA is also activated when it binds to its cellular receptor (uPAR), in which case the protein remains as a single chain molecule with less susceptibility to PAIs. Fibrin clots are invested with PAI-1 derived from plasma and from activated platelets. Therefore, we compared the fibrinolytic activity of complexes between scuPA and recombinant soluble uPAR (suPAR) to that of scuPA, tcuPA, and tcuPA/suPAR complexes. scuPA/suPAR complexes mediated the lysis of plasma-derived fibrin clots 14-fold more extensively than did equimolar concentrations of scuPA and threefold more extensively than did tcuPA or tcuPA/suPAR, respectively. The enhanced catalytic activity of scuPA/suPAR required that all three domains of the receptor be present, correlated with its PAI-1 resistance, was not dependent on fibrin alone, and required a plasma cofactor that was identified as IgG. Human IgG bound specifically to suPAR and scuPA/suPAR as determined by using affinity chromatography and immunoprecipitation. Plasma depleted of IgG lost most of its capacity to promote the fibrinolytic activity of scuPA/suPAR, and the activity of the complex was restored by adding plasma concentrations of purified IgG. These studies indicate that scuPA/suPAR can function as a plasminogen activator in a physiological milieu. © 1998 by The American Society of Hematology.

Lysis of Plasma Clots by Urokinase-Soluble Urokinase Receptor Complexes

Blood ◽  
1998 ◽  
Vol 92 (6) ◽  
pp. 2075-2083 ◽  
Author(s):  
Abd Al-Roof Higazi ◽  
Khalil Bdeir ◽  
Edna Hiss ◽  
Shira Arad ◽  
Alice Kuo ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Plasma Concentrations ◽  
Plasminogen Activator Inhibitor ◽  
Chain Molecule ◽  
Type I ◽  
Single Chain ◽  
Receptor Complexes ◽  
Pai 1 ◽  
Fibrin Clots

Abstract Single-chain urokinase plasminogen activator (scuPA), the unique form secreted by cells, expresses little intrinsic plasminogen activator activity. scuPA can be activated by proteolytic cleavage to form a two-chain enzyme (tcuPA), which is susceptible to inhibition by plasminogen activator inhibitor type I (PAI-1). scuPA is also activated when it binds to its cellular receptor (uPAR), in which case the protein remains as a single chain molecule with less susceptibility to PAIs. Fibrin clots are invested with PAI-1 derived from plasma and from activated platelets. Therefore, we compared the fibrinolytic activity of complexes between scuPA and recombinant soluble uPAR (suPAR) to that of scuPA, tcuPA, and tcuPA/suPAR complexes. scuPA/suPAR complexes mediated the lysis of plasma-derived fibrin clots 14-fold more extensively than did equimolar concentrations of scuPA and threefold more extensively than did tcuPA or tcuPA/suPAR, respectively. The enhanced catalytic activity of scuPA/suPAR required that all three domains of the receptor be present, correlated with its PAI-1 resistance, was not dependent on fibrin alone, and required a plasma cofactor that was identified as IgG. Human IgG bound specifically to suPAR and scuPA/suPAR as determined by using affinity chromatography and immunoprecipitation. Plasma depleted of IgG lost most of its capacity to promote the fibrinolytic activity of scuPA/suPAR, and the activity of the complex was restored by adding plasma concentrations of purified IgG. These studies indicate that scuPA/suPAR can function as a plasminogen activator in a physiological milieu. © 1998 by The American Society of Hematology.

Plasminogen Activation In Vivo upon Intravenous Infusion of DDAVP

1992 ◽  
Vol 67 (01) ◽  
pp. 111-116 ◽  
Author(s):  
Marcel Levi ◽  
Jan Paul de Boer ◽  
Dorina Roem ◽  
Jan Wouter ten Cate ◽  
C Erik Hack
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Plasma Levels ◽  
Fold Increase ◽  
Fibrinolytic System ◽  
Plasminogen Activation ◽  
Plasminogen Activator Activity ◽  
Insight Into

SummaryInfusion of desamino-d-arginine vasopressin (DDAVP) results in an increase in plasma plasminogen activator activity. Whether this increase results in the generation of plasmin in vivo has never been established.A novel sensitive radioimmunoassay (RIA) for the measurement of the complex between plasmin and its main inhibitor α2 antiplasmin (PAP complex) was developed using monoclonal antibodies preferentially reacting with complexed and inactivated α2-antiplasmin and monoclonal antibodies against plasmin. The assay was validated in healthy volunteers and in patients with an activated fibrinolytic system.Infusion of DDAVP in a randomized placebo controlled crossover study resulted in all volunteers in a 6.6-fold increase in PAP complex, which was maximal between 15 and 30 min after the start of the infusion. Hereafter, plasma levels of PAP complex decreased with an apparent half-life of disappearance of about 120 min. Infusion of DDAVP did not induce generation of thrombin, as measured by plasma levels of prothrombin fragment F1+2 and thrombin-antithrombin III (TAT) complex.We conclude that the increase in plasminogen activator activity upon the infusion of DDAVP results in the in vivo generation of plasmin, in the absence of coagulation activation. Studying the DDAVP induced increase in PAP complex of patients with thromboembolic disease and a defective plasminogen activator response upon DDAVP may provide more insight into the role of the fibrinolytic system in the pathogenesis of thrombosis.

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.

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.

scholarly journals Enhancement of the Enzymatic Activity of Single-chain Urokinase Plasminogen Activator by Soluble Urokinase Receptor

1995 ◽  
Vol 270 (29) ◽  
pp. 17375-17380 ◽  
Author(s):  
Abd Al-Roof Higazi ◽  
Robert L. Cohen ◽  
Jack Henkin ◽  
Douglas Kniss ◽  
Bradford S. Schwartz ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Plasminogen Activator ◽  
Urokinase Plasminogen Activator ◽  
Urokinase Receptor ◽  
Single Chain

Demonstration of a Functionally Active tPA-Like Plasminogen Activator in Human Platelets

1994 ◽  
Vol 71 (04) ◽  
pp. 493-498 ◽  
Author(s):  
D L Wang ◽  
Y T Pan ◽  
J J Wang ◽  
C H Cheng ◽  
C Y Liu
Keyword(s):  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Protein Band ◽  
Human Platelets ◽  
Free Form ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Western Blots ◽  
A Minor ◽  
Pai 1

SummaryThe mechanism of platelet-enhanced fibrinolysis is unclear. We therefore investigated the fibrinolytic activity of human platelets and demonstrated that they contain a tissue plasminogen activator (tPA)- like plasminogen activator, abbreviated as tPA-like-PA. This activator was detected by ELISA in platelet incubation medium and in platelet Triton extracts. Plasminogen activation assays showed that this tPA- like-PA could induce plasminogen activation to form plasmin. Western blots of Triton extracts incubated with anti-tPA antibody demonstrated a major 64-kD protein band, compared to a 70-kD band for standard single chain tPA, plus a minor 118-kD band corresponding to a complex of tPA-like-PA and plasminogen activator inhibitor (PAI-1). Western blots of Triton extracts incubated with anti-PAI-1 antibody produced an approximately similar high-molecular-weight (118 kD) protein band. Fibrin zymographic analysis of affinity-purified tPA-like- PA demonstrated a major and a minor fibrin lysis zone, which approximately corresponded to the tPA-like-PA and its complex with PAI-1 observed by Western blots. Immunogold labelling and electron microscopy demonstrated that platelet activator, either as the free form or co-localized with PAI-1, was present in granules and in channels of the open canalicular system. We conclude that platelets contain a functionally active tPA-like-PA, whose low fibrinolytic activity might be due to its readily forming a complex with PAI-1. This functionally active tPA-like-PA might contribute to the enhanced fibrinolytic activity of platelets observed in platelet-rich thrombi.

Pharmacokinetics and Pharmacodynamics of Saruplase, an Unglycosylated Single-chain Urokinase-type Plasminogen Activator, in Patients with Acute Myocardial Infarction

1994 ◽  
Vol 72 (05) ◽  
pp. 740-744 ◽  
Author(s):  
Rudolph W Koster ◽  
Adam F Cohen ◽  
Gwyn R Hopkins ◽  
Horst Beier ◽  
Wolfgang A Gunzler ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Plasminogen Activator ◽  
Degradation Products ◽  
Plasma Concentrations ◽  
Significant Proportion ◽  
Single Chain ◽  
Clotting System ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

SummaryWe examined in patients with acute myocardial infarction (AMI) the pharmacokinetics of saruplase, an unglycosylated, single chain, urokinase-type plasminogen activator (rscu-PA) by measuring urokinase-type plasminogen activator (u-PA) antigen and total u-PA activity, its conversion to active two-chain urokinase-type plasminogen activator (tcu-PA) and evaluated its effect on haemostatic parameters.Twelve patients were studied during and after administration of 20 mg bolus plus 60 mg continuous 1 h i.v. infusion of saruplase. For u-PA antigen and total u-PA activity (expressed as protein equivalents), where 234 U corresponds to 1 μg, respectively, steady state plasma concentrations were 2.75 ± 8.3 and 2.50 ± 7.0 μg/ml (mean ± standard deviation) and were reached within 20min, t1/2M was 9.1 ± 1.8 and 7.8 ± 1.3 min, t1/2λ2 1.2 ± 0.2 and 1.9 ± 0.5 h, and the total clearance was 393 ±110 and 427 ± 113 ml/min. Inactivation of saruplase in plasma was negligible.After 15 min, tcu-PA was detected in plasma. From the ratio of the areas under the curve of tcu-PA and total u-PA activities it was calculated that 28 ± 9.3% of the saruplase dose is converted into active tcu-PA. Systemic plasminaemia occurs as shown by a decrease in α2-antiplas-min and fibrinogen and an increase in fibrinogen degradation products. Thrombin-antithrombin complex formation indicated activation of the clotting system.Saruplase is eliminated rapidly from plasma in AMI patients. A variable, but significant proportion of saruplase is converted into active tcu-PA. At the end of the infusion tcu-PA accounted for 55% of total u-PA activity. Systemic plasmin generation and activation of the clotting system occur during saruplase treatment for AMI.

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