Influence of cyanogen-bromide-digested fibrinogen on the kinetics of plasminogen activation by urokinase

Lijnen, H.R.; Hoef, Berthe Van; Collen, D.

doi:10.1111/j.1432-1033.1984.tb08499.x

Cited by 45 publications

(19 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Clearly, there was little effect of 6-AHA binding to tPA as full-length tPA and protease were identical (overall, a slight inhibition of 14% was observed in both cases). A different mechanism operates in the case of uPA where the conformational change in Glu-plasminogen at around 2 mM 6-AHA, previously found (29), did produce an enhanced activation rate (3.2-fold stimulation in this case), in agreement with earlier findings (30). Activation by tPA was much less sensitive to this conformational change.…”

Section: And Equation 17)supporting

confidence: 80%

“…Previous studies have also suggested that the protease domain acts independently of the N-terminal, A-chain domains (34,44). It is interesting that the uPA system is stimulated in this way and is more sensitive to the conformation of plasminogen as affected by 6-AHA, fibrinogen fragments, or monoclonal antibodies to the 6-AHA binding site (30,45). Model 3 also assumes that only free plasminogen is available for reaction with cell-bound tPA if the correct "template" profile is to be obtained.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Regulation of Tissue Plasminogen Activator Activity by Cells

Sinniger

Merton

Fàbregas

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

A number of cell types have previously been shown to bind tissue plasminogen activator (tPA), which in some cases can remain active on the cell surface resulting in enhanced plasminogen activation kinetics. We have investigated several cultured cell lines, U937, THP1, K562, Molt4, and Nalm6 and shown that they bind both tPA and plasminogen and are able to act as promoters of plasminogen activation in kinetic assays. To understand what structural features of tPA are involved in cell surface interactions, we performed kinetic assays with a range of tPA domain deletion mutants consisting of fulllength glycosylated and nonglycosylated tPA (F-G-K1-K2-P), ⌬FtPA (G-K1-K2-P), K2-P tPA (BM 06.022 or Reteplase), and protease domain (P). Deletion variants were made in Escherichia coli and were nonglycosylated. Plasminogen activation rates were compared with and without cells, over a range of cell densities at physiological tPA concentrations, and produced maximum levels of stimulation up to 80-fold with full-length, glycosylated tPA. Stimulation for nonglycosylated full-length tPA dropped to 45-60% of this value. Loss of N-terminal domains as in ⌬FtPA and K2P resulted in a further loss of stimulation to 15-30% of the full-length glycosylated value. The protease domain alone was stimulated at very low levels of up to 2-fold. Thus, a number of different sites are involved in cell interactions especially within finger and kringle domains, which is similar to the regulation of tPA activity by fibrin. A model was developed to explain the mechanism of stimulation and compared with actual data collected with varying cell, plasminogen, or tPA concentrations and different tPA variants. Experimental data and model predictions were generally in good agreement and suggest that stimulation is well explained by the concentration of reactants by cells.

show abstract

Section: And Equation 17)supporting

confidence: 80%

mentioning

confidence: 99%

Regulation of Tissue Plasminogen Activator Activity by Cells

Sinniger

Merton

Fàbregas

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Activation occurs with a comparable affinity for Glu-plasminogen, Lys-plasminogen and low-M, plasminogen, while the catalytic rate constant is comparable for Glu-plasminogen and low-M, plasminogen but somewhat higher for Lysplasminogen. We have previously studied the activation of Giu-plasminogen and Lys-plasminogen by two-chain highmolecular-weight urokinase [22], and observed that K, for Lys-plasminogen is about tenfold lower as compared to Gluplasminogen (5.4 pM and 53 pM respectively) while the k,,, is not significantly different (1.5 s-' and 1.7 respectively). In the presence of 1 mM Ahx, a concentration that saturates the high-affinity lysine-binding site of plasminogen [23], the affinity of pro-UK for Glu-plasminogen is not significantly influenced while the k,,, increases threefold.…”

Section: Discussionmentioning

confidence: 99%

“…Both pro-UK and plasminogen were carefully rechromatographed in order to eliminate traces of active enzymes, ensuring that the observed kinetics are not influenced by contaminating urokinase or plasmin. In all experiments involving Glu-plasminogen, initial activation rates are obtaincd under conditions where the amount of plasmin generated is small compared to the concentration of plasminogen ( < 0.1 5 % ) and conversion of Glu-plasminogen to Lys-plasminogen is not observed, as shown by a linear generation of plasmin in time [22].…”

Section: Discussionmentioning

confidence: 99%

Characterization of the high‐affinity interaction between human plasminogen and pro‐urokinase

Lijnen

Zamarron

Collen

1985

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

Activation of human Glu‐plasminogen, Lys‐plasminogen and low‐Mr plasminogen (lacking lysine‐binding sites) by pro‐urokinase (pro‐UK), obtained from a human lung adenocarcinoma cell line (Calu‐3, ATCC). obeys Michaelis‐Menten kinetics. Activation ocurs with a comparable affinity (Km 0.40–0.77 μM), while the catalytic rate constant (kcat) is comparable for Glu‐plasminogen (0.0022 s−1) and low‐Mr plasminogen (0.0034s−1), but is somewhat higher for Lys‐plasminogen (0.0106s−1). The rate of activation of plasminogen by pro‐UK is not significantly influenced by the presence of 6‐aminohexanoic acid, purified fragments LBS I or LBS II or histidinerich glycoprotein, indicating that the high affinity of pro‐UK for plasminogen is not mediated via the high‐affinity lysine‐binding site of plasminogen located in kringles 1–3 (LBS I) nor via the low‐affinity lysine‐binding site comprised within kringle 4 (LBS II). The site(s) in plasminogen involved in the high‐affinity interaction with pro‐UK thus appear to be located within the low‐Mr plasminogen moiety.

show abstract

“…29 Furthermore, the presence of CNBrfibrinogen accelerates tPA-mediated plasminogen activation more significantly than urokinase plasminogen activatormediated plasminogen activation. 30 The amidolytic assay, although lacking the ability to resolve activity in fine tissue locales, can provide information on net tPA activity within specific dissectible neural compartments in mice, and shows that tPA activity is significantly higher in the cortex when compared with the underlying sub-cortical structures and the cerebellum (Supplementary Figure S2).…”

Section: Discussionmentioning

confidence: 99%

Compartment- and context-specific changes in tissue-type plasminogen activator (tPA) activity following brain injury and pharmacological stimulation

Sashindranath

Samson

Downes

et al. 2011

Laboratory Investigation

View full text Add to dashboard Cite

Tissue-type plasminogen activator (tPA) is a major protease of the central nervous system. Most studies to date have used in situ-or gel-based zymographic assays to monitor in vivo changes in neural tPA activity. In this study, we demonstrate that the amidolytic assay can be adapted to accurately detect changes in net tPA activity in mouse brain tissues. Using the amidolytic assay, we examined differences in net tPA activity in the cerebral cortex, sub-cortical structures and cerebellum in wildtype (WT) and tPA À/À mice, and in transgenic mice selectively overexpressing tPA in neurons. In addition, we assessed changes in endogenous net tPA activity in WT mice following morphine administration, epileptic seizures, traumatic brain injury and ischaemic stroke-neurological settings in which tPA has a known functional role. Under these conditions, acute and compartment-specific regulation of tPA activity was observed. tPA also participates in various forms of chronic neurodegeneration. Accordingly, we assessed tPA activity levels in mouse models of Alzheimer's disease (AD) and spinocerebellar ataxia type-1 (SCA1). Decreased tPA activity was detected in the cortex and subcortex of AD mice, whereas increased tPA activity was found in the cerebellum of SCA1 mice. These findings extend the existing hypotheses that low tPA activity promotes AD, whereas increased tPA activity contributes to cerebellar degeneration. Collectively, our results exemplify the utility of the amidolytic assay and emphasise tPA as a complex mediator of brain function and dysfunction. On the basis of this evidence, we propose that alterations in tPA activity levels could be used as a biomarker for perturbations in brain homeostasis.

show abstract

Influence of cyanogen-bromide-digested fibrinogen on the kinetics of plasminogen activation by urokinase

Cited by 45 publications

References 27 publications

Regulation of Tissue Plasminogen Activator Activity by Cells

Regulation of Tissue Plasminogen Activator Activity by Cells

Characterization of the high‐affinity interaction between human plasminogen and pro‐urokinase

Compartment- and context-specific changes in tissue-type plasminogen activator (tPA) activity following brain injury and pharmacological stimulation

Contact Info

Product

Resources

About