The interaction of Bacteroides fragilis with components of the human fibrinolytic system

Bacteroides fragilis is a minor component of the intestinal microbiota and the most frequently isolated from intra-abdominal infections and bacteremia. Previously, our group has shown that molecules involved in laminin-1 (LMN-1) recognition were present in outer membrane protein extracts of B. fragilis MC2 strain. One of these proteins was identified and showed 98% similarity to a putative B. fragilis plasminogen-binding protein precursor, deposited in the public database. Thus, the objective of this work was to overexpress and further characterize this novel adhesin. The ability of B. fragilis MC2 strain and purified protein to convert plasminogen into plasmin was tested. Our results showed that B. fragilis strain MC2 strain adhered to both LMN-1 and plasminogen and this adhesion was inhibited by either LMN-1 or plasminogen. Regarding the plasminogen activation activity, both the whole bacterial cell and the purified protein converted plasminogen into plasmin similar to streptokinase used as a positive control. Bacterial receptors that recognize plasminogen bind to it and enhance its activation, transforming a nonproteolytic bacterium into a proteolytic one. We present in vitro evidence for a pathogenic function of the plasminogen receptor in promoting adherence to laminin and also the formation of plasmin by B. fragilis .     

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

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 x 10(3) M(-1) x 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 alpha(2)-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.     

The major fibrinolytic proteases of human leukocytes

The major fibrinolytic enzymes present in leukocyte granules and active at physiological pH have been identified. The fibrinolytic activity in extracts of leukocyte granules was bound to fibrinogen-Sepharose and eluted with 8.0 M urea. Two distinct zones of fibrinolytic activity were detected upon electrophoresis of leukocyte extracts on fibrinogen polyacrylamide gels, and both were qualitatively recovered in the 8.0 M urea eluate. Quantitatively, greater than 95% of the fibrinolytic activity was recovered in the urea eluate. Two major leukocyte proteases, elastase (EC 3.4.21.11) and cathepsin G (EC 3.4.21.-), were quantitatively recovered in the urea eluate. Both enzymes, when purified separately by affinity chromatography, were shown to: (a) possess fibrinolytic activity; (b) coincide in mobility and generate the two zones of fibrinolytic activity on fibrinogen polyacrylamide gels; and (c) quantitatively reconstitute the fibrinolytic activity of the leukocyte granules when combined at activity levels present in granular extracts. A highly significant correlation (r = 0.98) was found between the fibrinolytic activity and the sum of elastase and cathepsin G activity in leukocytes from five donors. Thus, elastase and cathepsin G are the major enzymes of the leukocyte fibrinolytic pathway, and fibrinogen-Sepharose chromatography may be used to obtain these enzymes.     

Gelatinolytic and fibrinolytic activity in fresh-frozen plasma

Fresh-frozen plasma (FFP) was evaluated for gelatinolytic and fibrinolytic activity. Gelatin zymography revealed that gelatinase A (MMP-2) was constitutively present in FFP whereas gelatinase B (MMP-9) was present at variable levels. The presence of MMP-9 likely represents differential release from neutrophils during FFP collection or processing. Although fibrin matrices generated from FFP or freshly prepared plasma contained characteristic crosslinked - dimers and -monomers, matrices generated from FFP were resistant to spontaneous plasmin-dependent fibrinolysis. This observation likely stems from the plasminogen activator instability and could potentially lead to a hypofibrinolytic state. The impact of these in vitro findings to protease balance in patients receiving multiple FFP doses remains to be determined.     

Peroxynitrite and fibrinolytic system: The effect of peroxynitrite on plasmin activity

We have shown that peroxynitrite (ONOO-) inhibits streptokinase-induced conversion of plasminogen to plasmin in a concentration-dependent manner and reduces both amidolytic (IC5o approximately 280 microM at 10 microM concentration of enzyme) and proteolytic activity of plasmin. Spectrophotometric and immunoblot analysis of peroxynitrite-treated plasminogen demonstrates a concentration-dependent increase in its nitrotyrosine residues that correlates with a decreased generation of active plasmin. Peroxynitrite (1 mM) causes the nitration of 2.9 tyrosines per plasminogen molecule. Glutathione, like deferoxamine, partially protects plasminogen from peroxynitrite-induced inactivation and reduces the extent of tyrosine nitration. These data suggest that nitration of plasminogen tyrosine residues by peroxynitrite might play an important role in the inhibition of plasmin catalytic activity.     

The effect of pentosan polysulphate (SP54) on the human fibrinolytic system

In human volunteers the oral and subcutaneous (s.c.) administration of SP54 enhanced the fibrinolytic potential of plasma. When the effect was compared between exercise and s.c. administration of SP54 the latter was found to "peak" at about 3 hours and to have a more sustained effect than the exercise. Oral versus s.c. administration suggests that only about 10% of the SP54 reaches the circulation by the oral route. However, repeat SP54 oral dosage did not elicit any resistance to the daily response over a period of five days and suggests that the volunteers used in this study tolerated repeated use of the drug with no ill-effects. The source of the fibrinolytic enhancement is as yet not fully explained and a hypothesis is presented which suggests that very small elevations of t-PA in plasma can affect the fibrinolytic potential in the presence of forming fibrin but may remain undetected by conventional immunometric assays.     

Laboratory control of fibrinolytic therapy

The mode of action of the different thrombolytic substances which are presently available is described. Alterations of the clotting mechanism which are to be expected during thrombolytic therapy as well as possible side effects of the fibrinolytic drugs are listed. Laboratory tests necessary for the control of thrombolytic therapy as well as the different test methods and their normal ranges are mentioned in detail.