Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
 10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We studied the effects of fibrinogen degradation product (FDP) fragment D on endothelial monolayer integrity and the mechanisms of fragment D-induced endothelial cell detachment from the substratum. Incubation of bovine pulmonary artery endothelial cells (BPAEC) with fragment D caused concentration- and time-dependent cell detachment from the substratum. The optimal response occurred at fragment D concentrations of 2 microM and required an incubation time of 24 h. BPAEC challenged with fragment D increased the concentration and activity of urokinase-type plasminogen activator (uPA) in the conditioned medium within 2 to 4 h of incubation. Fragment D also induced the release of tissue-type plasminogen activator, but to a lesser extent than uPA. Fragment D concurrently increased plasminogen activator (PA) activity in a concentration-dependent manner. Increased PA activity was followed by augmentation of cell-associated plasmin activity and subsequent increase in the degradation of 125I-fibrinogen and 125I-vitronectin precoated in the subendothelial matrix. Pretreatment of BPAEC with anti-uPA antibody, and inhibitors of uPA (dansyl-GGACK) and plasmin (aprotinin) prevented approximately 60% of the fragment D-induced endothelial cell detachment. We conclude that FDP fragment D increases secretion of endothelial PAs and enhances the generation of plasmin, thereby contributing to proteolysis of extracellular matrix and endothelial cell detachment. Fragment D may be a critical mediator linking activation of fibrinolysis to vascular endothelial injury in inflammatory disorders.
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PMID:Fibrinogen degradation product fragment D induces endothelial cell detachment by activation of cell-mediated fibrinolysis. 128 36

In a previous study, it was shown that fibrin fragment E-2 selectively promotes the activation of plasminogen by pro-urokinase (pro-UK) [Liu, J., & Gurewich, V. (1991) J. Clin. Invest. 88, 2012-2017]. In this study, the kinetics of this promotion by fragment E-2 was studied. Alanine-158-rpro-UK (A-pro-UK), a recombinant plasmin-resistant mutant, was used in order to avoid interference by UK generation during the reaction. In some experiments, pro-UK was substituted in order to validate the mutant as a surrogate. In the presence of a range of concentrations (0-20 microM) of fragment E-2, a linear promotion of the catalytic efficiency of A-pro-UK against native Glu-plasminogen was seen which was 245.5-fold at the highest concentration of fragment E-2 and 450-fold at the highest ratio of E-2/plasminogen used. The promotion was largely a function of an increase in kcat, since fragment E-2 induced a less than 10-fold reduction in KM (8.50-1.40 microM). In contrast to this ligand, epsilon-aminocaproic acid (EACA) induced a biphasic promotion of the activation of Glu-plasminogen which was only 18-fold at maximum. Fragment E-2 did not promote the activation of Lys-plasminogen, but the catalytic efficiency of A-pro-UK was 19.7-fold greater against the open Lys-form than against the closed Glu- form of plasminogen. Fragment E-2 had no effect on the amidolytic activity of A-pro-UK or pro-UK, suggesting that the promotion of their activities was indirect and related to a fragment E-2-induced conformational change in Glu-plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fragment E-2 from fibrin substantially enhances pro-urokinase-induced Glu-plasminogen activation. A kinetic study using the plasmin-resistant mutant pro-urokinase Ala-158-rpro-UK. 138 27

The effects of fibrinogen and its plasmic cleavage fragments on the activation of Glu-, Lys-, and Val442- plasminogen by urokinase were investigated. A possible explanation for the large variations in the published steady state parameters for Glu-plasminogen activation is the undetected formation of Lys-plasminogen and its subsequent more rapid activation to plasmin. When Lys-plasminogen formation was avoided, the Km for Glu-plasminogen activation by urokinase was 2.5 microM with or without lysine present and the catalytic rate constant (kcat) was 3.4 min-1 in the absence of lysine, but increased to 49.0 min-1 in its presence. For Lys-plasminogen activation, both the Km of 2.7 microM and the kcat of 57.8 min-1 were only slightly increased by lysine. With Val442-plasminogen, the absence of the first 4 kringle structures of Lys-plasminogen resulted in a 6-fold higher Km and a 3-fold higher kcat, both of which were relatively unchanged by lysine. The specificity of urokinase for Val442-plasminogen, as measured by the quotient kcat/Km was thus half that for Lys-plasminogen. Fibrinogen, Fragment D, and Fragment E enhanced the rate of activation of Glu-plasminogen to Glu-plasmin as measured by the irreversible binding of plasmin to fluorescently labeled bovine pancreatic trypsin inhibitor. Both fibrinogen and Fragment D increased the value of kcat/Km about 4-fold whereas Fragment E caused a 2-fold enhancement. In contrast to Glu-plasminogen activation, the urokinase activation of Lys-plasminogen was not affected by fibrinogen or its fragments, yet a marked inhibition of Lys-plasmin autolysis occurred in their presence, with the half-life of plasmin being increased 13-fold by fibrinogen, 5-fold by Fragment D, and 3-fold by Fragment E. The K4 kringle region may be particularly involved in the plasmin-plasmin interaction that results in autolysis, since it significantly reduced degradation when incubated with Lys-plasmin. Val442-plasmin displayed essentially no autolysis, which further implicates the first 4 kringles in the autolytic reactions. In addition to these effects, the rate of Glu-plasminogen conversion to Lys-plasminogen by plasmin was increased 4-fold by fibrinogen or Fragment E, but only 2-fold by Fragment D. This augmentation was not merely due to inhibition of Lys-plasmin autolysis since Fragment D has a greater effect in that regard. The sum of these interactions indicates that Glu-plasminogen binds to the Fragment D region of fibrinogen/fibrin through its low affinity binding site(s) and, as when lysine binds at these sites, the activation to Glu-plasmin is then accelerated.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The effects of fibrinogen and its cleavage products on the kinetics of plasminogen activation by urokinase and subsequent plasmin activity. 622 55

Fragment-based lead discovery has been applied to urokinase-type plasminogen activator (uPA). The (R)-enantiomer of the orally active drug mexiletine 5 (a fragment hit from X-ray crystallographic screening) was the chemical starting point. Structure-aided design led to elaborated inhibitors that retained the key interactions of (R)-5 while gaining extra potency by simultaneously occupying neighboring regions of the active site. Subsequent optimization led to 15, a potent, selective, and orally bioavailable inhibitor of uPA.
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PMID:Fragment-based discovery of mexiletine derivatives as orally bioavailable inhibitors of urokinase-type plasminogen activator. 1816 48

Fragment-based drug discovery (FBDD) has evolved into an established approach for "hit" identification. Typically, most applications of FBDD depend on specialised cost- and time-intensive biophysical techniques. The substrate activity screening (SAS) approach has been proposed as a relatively cheap and straightforward alternative for identification of fragments for enzyme inhibitors. We have investigated SAS for the discovery of inhibitors of oncology target urokinase (uPA). Although our results support the key hypotheses of SAS, we also encountered a number of unreported limitations. In response, we propose an efficient modified methodology: "MSAS" (modified substrate activity screening). MSAS circumvents the limitations of SAS and broadens its scope by providing additional fragments and more coherent SAR data. As well as presenting and validating MSAS, this study expands existing SAR knowledge for the S1 pocket of uPA and reports new reversible and irreversible uPA inhibitor scaffolds.
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PMID:Repositioning the substrate activity screening (SAS) approach as a fragment-based method for identification of weak binders. 2515 78