scholarly journals The human fibrinolytic system is a target for the staphylococcal metalloprotease aureolysin

2008 ◽  
Vol 410 (1) ◽  
pp. 157-165 ◽  
Author(s):  
Nathalie Beaufort ◽  
Piotr Wojciechowski ◽  
Christian P. Sommerhoff ◽  
Grzegorz Szmyd ◽  
Grzegorz Dubin ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Active Form ◽  
Catalytic Efficiency ◽  
Plasminogen Activator Inhibitor ◽  
Opportunistic Pathogen ◽  
Fibrinolytic System ◽  
Growth Media ◽  
Single Chain ◽  
Plasminogen Activator Inhibitor 1 ◽  
Proteolytic Activation

The major opportunistic pathogen Staphylococcus aureus utilizes the human fibrinolytic system for invasion and spread via plasmin(ogen) binding and non-proteolytic activation. Because S. aureus secretes several proteases recently proposed as virulence factors, we explored whether these enzymes could add to the activation of the host's fibrinolytic system. Exposure of human pro-urokinase [pro-uPA (where uPA is urokinase-type plasminogen activator)] to conditioned growth media from staphylococcal reference strains results in an EDTA-sensitive conversion of the single-chain zymogen into its two-chain active form, an activity not observed in an aureolysin-deficient strain. Using purified aureolysin, we verified the capacity of this thermolysin-like metalloprotease to activate pro-uPA, with a 2.6×103 M−1·s−1 catalytic efficiency. Moreover, activation also occurs in the presence of human plasma, as well as in conditioned growth media from clinical isolates. Finally, we establish that aureolysin (i) converts plasminogen into angiostatin and mini-plasminogen, the latter retaining its capacity to be activated by uPA and to hydrolyse fibrin, (ii) degrades the plasminogen activator inhibitor-1, and (iii) abrogates the inhibitory activity of α2-antiplasmin. Altogether, we propose that, in parallel with the staphylokinase-dependent activation of plasminogen, aureolysin may contribute significantly to the activation of the fibrinolytic system by S. aureus, and thus may promote bacterial spread and invasion.

PURIFICATION AND CHARACTERIZATION OF REACTIVE AND NON-REACTIVE PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) FROM HUMAN PLACENTA

1987 ◽  
Author(s):  
M Philips ◽  
A G Juul ◽  
S Thorsen ◽  
J Selmer ◽  
L Thim
Keyword(s):  
Monoclonal Antibody ◽  
Plasminogen Activator ◽  
Human Placenta ◽  
Solid Phase ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Single Chain ◽  
Plasminogen Activator Inhibitor 1 ◽  
Sds Page ◽  
Pai 1

Reactive and non-reactive forms of PAI-1 have been identified in various biological materials. The structural differences between these forms remain to be determined.A monoclonal antibody specific for a non-reactive PAI-1 and a monoclonal antibody reacting with both the reactive and nonreactive form of the inhibitor were obtained by immunization with a tissue-type plasminogen activator (t-PA)-PAI-1 complex (Philips et al., Thromb Haemostas 1986; 55:213-7). These antibodies were used for the isolation of reactive and non-reactive PAI-1 by solid-phase immunoadsorption from extracts of human placenta. The inhibitor preparations were further purified by HPLC. Reactive and non-reactive PAI-1 both migrated with a Mr ∼ 52,000 when analyzed by SDS-PAGE. Furthermore, the two inhibitor forms were indistinguishable by N-terminal sequence analysis. Two N-terminal sequences were found in about equal ammounts for both the reactive and non-reactive PAI-1. They were Ser-Ala-Val-His-His-Pro-Pro- and a two residues shorter sequence (Val-His-His-Pro-Pro-). These sequences are in agreement with the published cDNA sequence of PAI-1 and shows that the inhibitor is N-terminally heterogeneously processed. The second order rate constant (ki) for the reaction between reactive PAI-1 and single-chain t-PA was about 6 106 M-1s-1. Treatment with 4 M guanidinium-HCl partially converted the non-reactive PAI-1 to a reactive form exhibiting a similar k1 for inhibition of single-chain t-PA. SDS-PAGE showed that the t-PA-PAI-1 complex could be dissociated by 1,5 M NH4OH/ 39 mM SDS resulting in the release of a PAI-1 with approximately the same Mr as native PAI-1. This indicates either that t-PA does not cleave the inhibitor or that it cleaves a peptide bond close to the C-terminus.In conclusion a non-reactive and a reactive form of PAI-1 can be purified from placenta. The two forms are distinguishable by monoclonal antibodies but they show similar Mr′ls and the same N-terminal sequences.

Genetic and environmental influences on the fibrinolytic system: a twin study

2004 ◽  
Vol 92 (08) ◽  
pp. 344-351 ◽  
Author(s):  
Anja Victor ◽  
Petra Adams ◽  
Hilde Erbes ◽  
Gerd Hafner ◽  
Karl Lackner ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Single Gene ◽  
Environmental Influences ◽  
Plasminogen Activator Inhibitor ◽  
Genetic Influence ◽  
Fibrinolytic System ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activation Markers ◽  
Interaction Terms ◽  
Pai 1

SummaryThe determination of heritability is a key issue to assess the predictive power of polymorphisms for disease in clinical studies. The aim of this study was to determine the heritability of proteins and activation markers of the fibrinolytic system in a large cohort of healthy twins. Heritability was calculated as 0.76 for thrombin activatable fibrinolysis inhibitor (TAFI), 0.44 for plasminogen activator inhibitor-1 (PAI-1), and 0.43 for tissue plasminogen activator. No significant genetic influence was observed for α2-antiplasmin-plasmin-complex and D-dimer. Heritability explained by single gene polymorphisms was 25.2% for TAFI 505G>A, 31.5% for 1542C>G, and 50.0% for combination of both. The influence on TAFI levels of 1542C>G (CC→GG, median: −280.5%) was considerably stronger than that of 505G>A (GG→AA, median: +49.3%) and in both cases there seems to be a dose-response relationship. Significant environmental influences on TAFI levels were observed for combined interaction terms (age*sex and bmi*sex). The PAI-1 4G/5G polymorphism explained 56.4% of the calculated heritability. The genetic variables accounting for the 43% heritability of tPA remain unknown. Our data show that the production of several key components of the fibrinolytic system is strongly genetically determined. This genetic influence is accounted for in large part but not completely by a limited number of polymorphisms within the respective genes associated with plasma levels of the gene products.

Plasminogen Activator Inhibitor-1 Is the Primary Inhibitor of Tissue-Type Plasminogen Activator in Pregnancy Plasma

1987 ◽  
Vol 58 (03) ◽  
pp. 872-878 ◽  
Author(s):  
Maja Jørgensen ◽  
Malou Philips ◽  
Sixtus Thorsen ◽  
Johan Selmer ◽  
Jesper Zeuthen
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Single Chain ◽  
Plasminogen Activator Inhibitor 1 ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Activator Inhibitor ◽  
Pai 1 ◽  
In Pregnancy

SummaryThe aim of the present work was to clarify to what extent plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) contribute to the increase in plasma inhibition of tissue-type plasminogen activator (t-PA) observed during pregnancy. It was demonstrated that a monoclonal antibody against PAI-1 almost completely quenched inhibition of single-chain t-PA and most of the inhibition of two-chain t-PA in plasma during the third trimester of piegnancy. The remaining inhibition of two-chain t-PA was to a great extent abolished by a PAI-2 antibody. The second order rate constant (k1) for inhibition of single-chain t-PA by the inhibitor neutralized by the PAI-1 antibody was about 4.8 · 106 M-1 · s-1. The conversion of singlechain t-PA to the two-ehain form increased the reaction rate with the inhibitor about 3-fold. These kinetic data are compaiable with those obtained with TAI-l in non-pregnancy plasma oi with purified PAI-1. From the above results it is concluded that PAI-1 is the primary inhibitor of both single-chain and two chain t PA and that PAI-2 is the secondary inhibitor of two-chain t-PA in pregnancy plasma. The concentration of reactive PAI-1 versus gestation age was assayed in plasma from 6 women by binding of PAI-1 to 125I-labelled single-chain t-PA followed by quantitation of the labelled t-PA-PAI-1 complex after separation by SDS- polyacrylamide gel electrophoresis. It was found that the concentration of PAI-1 increased 4 to 8-fold during the gestation period reaching a level of about 1.4 nM at term. Post partum the plasma concentration declined abruptly within 24 h to the level observed in age-matched non-pregnant women.

Comparison of Recombinant Plasminogen Activator Inhibitor-1 and Epsilon Amino Caproic Acid in a Hemorrhagic Rabbit Model

1992 ◽  
Vol 67 (06) ◽  
pp. 692-696 ◽  
Author(s):  
Adrienne L Racanelli ◽  
Mark J Diemer ◽  
A Cathy Dobies ◽  
Joan R Dubin ◽  
Thomas M Reilly
Keyword(s):  
Blood Loss ◽  
Plasminogen Activator ◽  
Active Form ◽  
Rabbit Model ◽  
Plasminogen Activator Inhibitor ◽  
Caproic Acid ◽  
Tissue Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Type Plasminogen Activator ◽  
Activator Inhibitor

SummaryA rabbit ear model of blood loss was developed to compare the effects of an active form of recombinant plasminogen activator inhibitor-1 (rPAI-1) with epsilon amino caproic acid (EACA) in antagonizing tissue-type plasminogen activator (r-tPA)-induced blood loss. The antagonism of both rebleeding, which occurs as a result of hemostatic plug degradation, and r-tPA-induced hemorrhage, where rabbits lose approximately 30% of their blood volume, was studied. rPAI-1 (1 mg/kg i. v.) or EACA (70 mg/kg i. v.) antagonized the rebleeding induced by r-tPA (10 μg kg-1 min-1) to a similar extent. In the hemorrhagic studies, rPAI-1 effectively antagonized the r-tPA-induced hemorrhage with an ED50 of 3 mg/kg i. v., while the ED50 obtained for EACA was 230 mg/kg i. v. rPAI-1 may be of value in reversing r-tPA-induced blood loss during thrombolytic therapy or in clinical situations where excessive fibrinolysis contributes to bleeding.

scholarly journals Role of Shear Stress and tPA Concentration in the Fibrinolytic Potential of Thrombi

2021 ◽  
Vol 22 (4) ◽  
pp. 2115
Author(s):  
Claire S. Whyte ◽  
Hadj Ahmed. Mostefai ◽  
Kim M. Baeten ◽  
Andrew J. Lucking ◽  
David E. Newby ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Ex Vivo ◽  
Active Form ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
High Shear ◽  
Plasminogen Activator Inhibitor 1 ◽  
Fibrinolytic Potential ◽  
Pai 1 ◽  
Low Shear

The resolution of arterial thrombi is critically dependent on the endogenous fibrinolytic system. Using well-established and complementary whole blood models, we investigated the endogenous fibrinolytic potential of the tissue-type plasminogen activator (tPA) and the intra-thrombus distribution of fibrinolytic proteins, formed ex vivo under shear. tPA was present at physiologically relevant concentrations and fibrinolysis was monitored using an FITC-labelled fibrinogen tracer. Thrombi were formed from anticoagulated blood using a Chandler Loop and from non-anticoagulated blood perfused over specially-prepared porcine aorta strips under low (212 s−1) and high shear (1690 s−1) conditions in a Badimon Chamber. Plasminogen, tPA and plasminogen activator inhibitor-1 (PAI-1) concentrations were measured by ELISA. The tPA–PAI-1 complex was abundant in Chandler model thrombi serum. In contrast, free tPA was evident in the head of thrombi and correlated with fibrinolytic activity. Badimon thrombi formed under high shear conditions were more resistant to fibrinolysis than those formed at low shear. Plasminogen and tPA concentrations were elevated in thrombi formed at low shear, while PAI-1 concentrations were augmented at high shear rates. In conclusion, tPA primarily localises to the thrombus head in a free and active form. Thrombi formed at high shear incorporate less tPA and plasminogen and increased PAI-1, thereby enhancing resistance to degradation.

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