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Svenningsen P, Uhrenholt TR, Palarasah Y, Skjodt K, Jensen BL, Skott O. Prostasin-dependent activation of epithelial [Na.sup.+] channels by low plasmin concentrations. Am J Physiol Regul Integr Comp Physiol 297: RI733-R1741, 2009. First published September 30, 2009; doi: I0.1152/ajpregu.00321.2009.--Several pathophysiological conditions, including nephrotic syndrome, are characterized by increased renal activity of the epithelial [Na.sup.+] channel (ENaC). We recently identified plasmin in nephrotic urine as a stimulator of ENaC activity and undertook this study to investigate the mechanism by which plasmin stimulates ENaC activity. Cy3-1abeled plasmin was found to bind to the surface of the mouse cortical collecting duct cell line, M-1. Binding depended on a glycosylphosphatidylinositol (GPI)anchored protein. Biotin-label transfer showed that plasmin interacted with the GPI-anchored protein prostasin on M-1 cells and that plasmin cleaved prostasin. Prostasin activates ENaC by cleavage of the 'y-subunit, which releases an inhibitory peptide from the extracellular domain. Removal of GPI-anchored proteins from the M-1 cells with phosphatidylinositol-specific phospholipase C (PI-PLC) inhibited plasmin-stimulated ENaC current in monolayers of M-1 cells at low plasmin concentration (1-4 [micro]g/ml). At a high plasmin concentration of 30 [micro]g/ml, there was no difference between cell layers treated with or without PI-PLC. Knockdown of prostasin attenuated binding of plasmin to M1 cells and blocked plasmin-stimulated ENaC current in single M-1 cells, as measured by whole-cell patch clamp. In M-1 cells expressing heterologous FLAG-tagged prostasin, '[gamma]ENaC and prostasin were colocalized. A monoclonal antibody directed against the inhibitory peptide of [gamma]ENaC produced specific immunofluorescence labeling of M-1 cells. Pretreatment with plasmin abolished labeling of M-1 cells in a prostasin-dependent way. We conclude that, at low concentrations, plasmin interacts with GPI-anchored prostasin, which leads to cleavage of the [gamma]-subunit and activation of ENaC, while at higher concentrations, plasmin directly activates ENaC. serine protease; sodium retention; kidney; M-1 cells; nephrotic syndrome doi: 10.1152/ajpregu.00321.2009

The outer membrane plasminogen activator Pla of Yersinia pestis is a central virulence factor in plague. The primary structure of the Pla [beta]-barrel is conserved in Y. pestis biovars Antiqua, Medievalis, and Orientalis, which are associated with pandemics of plague. The Pla molecule of the ancestral Y. pestis lineages Microtus and Angola carries the single amino acid change T259I located in surface loop 5 of the [beta]-barrel. Recombinant Y. pestis KIM D34 or Escherichia coli XL1 expressing Pla T259I was impaired in fibrinolysis and in plasminogen activation. Lack of detectable generation of the catalytic light chain of plasmin and inactivation of plasmin enzymatic activity by the Pla T259I construct indicated that Microtus Pla cleaved the plasminogen molecule more unspecifically than did common Pla. The isoform pattern of the Pla T259I molecule was different from that of the common Pla molecule. Microtus Pla was more efficient than wild-type Pla in [[alpha].sub.2]-antiplasmin inactivation. Pla of Y. pestis and PgtE of Salmonella enterica have evolved from the same omptin ancestor, and their comparison showed that PgtE was poor in plasminogen activation but exhibited efficient antiprotease inactivation. The substitution [sup.259]IIDKT/TIDKN in PgtE, constructed to mimic the L5 region in Pla, altered proteolysis in favor of plasmin formation, whereas the reverse substitution [sup.259]TIDKN/IIDKT in Pla altered proteolysis in favor of [[alpha].sub.2]-antiplasmin inactivation. The results suggest that Microtus Pla represents an ancestral form of Pla that has evolved into a more efficient plasminogen activator in the pandemic Y. pestis lineages.

Bifidobacteria represent one of the most important health-promoting bacterial groups of the intestinal microbiota. The binding of plasminogen to species of Bifidobacterium has been recently reported. To further explore the interaction between bifidobacteria and plasminogen, we investigated the role of Bifidobacterium lactis BI07 plasminogen-dependent proteolytic activity in the degradation of host-specific substrates. Our experimental data demonstrate that the recruitment of plasminogen on the bacterial cell surface and its subsequent conversion into plasmin by host-derived plasminogen activators provide B. lactis BI07 with a surface-associated plasmin activity effective in degradation of physiological substrates such as extracellular matrix, fibronectin and fibrinogen. The ability of bifidobacteria to intervene in the host plasminogen/ plasmin system may contribute to facilitating colonization of the host gastrointestinal tract.

Ischemic cardiovascular events represent the leading cause of mortality and morbidity worldwide, and platelet aggregation and thrombus formation are the main effectors of acute arterial ischemic events. Although antiplatelet therapy is the cornerstone of antithrombotic treatment of ischemic cardiovascular disorders, available antiplatelet agents have less than satisfactory efficacy; thus, the identification of novel potential target candidates for antiplatelet therapy is highly warranted. Recent evidence suggests that several molecules that amplify the aggregation response of platelets to activating stimuli, which are either released by platelets (potentiating molecules) or present in the milieu before platelets get activated (primers), play a major role in pathologic thrombus formation without being significantly involved in primary haemostasis. These molecules appear to be a particularly appealing novel potential pharmacologic target for antiplatelet therapy. Here, we review the present knowledge on some molecules acting as potentiators or primers of platelet activation and discuss their possible pharmacologic modulation for antithrombotic purposes.

Aldosterone is thought to modulate renal fibrosis, in part, through increasing plasminogen activator inhibitor type 1 (PAI-1), a major inhibitor of ECM degradation. The present study investigated aldosterone effects on PAI-1 and transforming growth factor (TGF)-[[beta].sub.1] and asked whether PAI-1 effects were TGF-[beta] mediated and whether aldosterone and TGF-[[beta].sub.1] acted synergistically to increase PAI-1 and decrease ECM degradation. Rat mesangial cells (MCs) and fibroblast cells [normal rat kidney (NRK)-49F] were used. [sup.3]H-labeled ECM was produced by MCs. The effect of aldosterone and TGF-[beta] on ECM degradation by newly plated MCs or NRK-49F was measured by the release of 3H into medium. Aldosterone markedly increased PAI-1 mRNA and protein in both cell types, increases completely blocked by spironolactone and partially blocked by TGF-[beta] neutralizing antibody. Adding both aldosterone and TGF-[[beta].sub.1] produced PAI-1 mRNA and protein increases higher than the sum of increases seen with either compound alone. Aldosterone or TGF-[[beta].sub.1] alone inhibited matrix degradation by 39 and 49% in MCs and 21 and 23% in NRK-49F, respectively. When both compounds were added, matrix degradation was further decreased by 93% in MCs and 61% in NRK-49F. The results indicate that aldosterone-induced PAI-1 increases are partially mediated by TGF-[[beta].sub.1] and lead to decreased ECM degradation. While aldosterone alone induced TGF-[[beta].sub.1] weakly, aldosterone and TGF-[[beta].sub.1] added together produced dramatic synergistic effects on PAI-1 production and subsequent ECM accumulation. Thus the elevated aldosterone induced by renin-angiotensin-aldosterone system activation may amplify renin-angiotensin-aldosterone system profibrotic actions. transforming growth factor-[beta]; plasminogen activator inhibitor-1

Based on their localization at the boundary of the bacterial cell and its environment, outer-membrane proteins (Omps) are important determinants for interaction of bacteria with their host cell. Therefore, they can be considered as important determinants for virulence. Looking for Legionella pneumophila Omps potentially involved in virulence, we identified a gene encoding a homologue of the plasminogen activator (Pla) of Yersinia pestis. Pla belongs to the class of omptins, a family of surface proteases/adhesins that exhibit different virulence-associated functions. In this report we describe the cloning and identification of the plasminogen activator homologue Lpa of L. pneumophila and demonstrate its outer-membrane localization. Transcriptional analysis of the Lpa region revealed expression of the gene in both exponential and stationary growth phase and showed that transcription of the Ipa gene is directed by its own promoter. We also show, to our knowledge for the first time, that L. pneumophila has the capacity to convert plasminogen into plasmin by the action of the outer-membrane Lpa protein.

The characterization of urea-induced unfolding of holo- and apo-forms of human ceruloplasmin (CP) reveal that in vivo equilibrium unfolding of CP involves intermediates and that the copper ions are removed in stages.