A Dual Role for the Plasminogen Activator Protease During the Preinflammatory Phase of Primary Pneumonic Plague.

Early after inhalation, Yersinia pestis replicates to high numbers in the airways in the absence of disease symptoms or notable inflammatory responses to cause primary pneumonic plague. The plasminogen activator protease (Pla) is a critical Y. pestis virulence factor that is important for early bacterial growth in the lung via an unknown mechanism. In this article, we define a dual role for Pla in the initial stages of pulmonary infection. We show that Pla functions as an adhesin independent of its proteolytic function to suppress early neutrophil influx into the lungs, and that Pla enzymatic activity contributes to bacterial resistance to neutrophil-mediated bacterial killing. Our results suggest that the fate of Y. pestis infection of the lung is decided extremely early during infection and that Pla plays a dual role to tilt the balance in favor of the pathogen.

Modeling Pneumonic Plague in Human Precision-Cut Lung Slices Highlights a Role for the Plasminogen Activator Protease in Facilitating Type 3 Secretion.

Pneumonic plague is the deadliest form of disease caused by Yersinia pestis Key to the progression of infection is the activity of the plasminogen activator protease Pla. Deletion of Pla results in a decreased Y. pestis bacterial burden in the lung and failure to progress into the lethal proinflammatory phase of disease. While a number of putative functions have been attributed to Pla, its precise role in the pathogenesis of pneumonic plague is yet to be defined. Here, we show that Pla facilitates type 3 secretion into primary alveolar macrophages but not into the commonly used THP-1 cell line. We also establish human precision-cut lung slices as a platform for modeling early host/pathogen interactions during pneumonic plague and solidify the role of Pla in promoting optimal type 3 secretion using primary human tissue with relevant host cell heterogeneity. These results position Pla as a key player in the early host/pathogen interactions that define pneumonic plague and showcase the utility of human precision-cut lung slices as a platform to evaluate pulmonary infection by bacterial pathogens.

On the contribution of S100A10 and annexin A2 to plasminogen activation and oncogenesis: an enduring ambiguity.

Plasminogen receptors are becoming increasingly relevant in regulating many diseases such as cancer, stroke and inflammation. However, controversy has emerged concerning the putative role of some receptors, in particular annexin A2, in binding plasminogen. Several reports failed to account for the effects of annexin A2 on the stability and conformation of its binding partner S100A10. This has created an enduring ambiguity as to the actual function of annexin A2 in plasmin regulation. Supported by a long line of evidence, we conclude that S100A10, and not annexin A2, is the primary plasminogen receptor within the annexin A2-S100A10 complex and contributes to the plasmin-mediated effects that were originally ascribed to annexin A2.

Three decades of research on plasminogen activator inhibitor-1: a multifaceted serpin.

Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of tissue-type (t-PA) and urokinase-type (u-PA) plasminogen activator and therefore plays an important role in the plasminogen-plasmin system. PAI-1 is involved in a variety of cardiovascular diseases (mainly through inhibition of t-PA) as well as in cell migration and tumor development (mainly through inhibition of u-PA and interaction with vitronectin). PAI-1 is a unique member of the serpin superfamily, exhibiting particular unique conformational and functional properties. Because of its involvement in various biologic and pathophysiologic processes, PAI-1 has been the subject of many studies, including extensive structural investigations, in vitro cell biologic studies, in vivo animal studies, and epidemiologic studies. The review provides an overview on the current knowledge on PAI-1.

Fibrin facilitates both innate and T cell-mediated defense against Yersinia pestis.

The Gram-negative bacterium Yersinia pestis causes plague, a rapidly progressing and often fatal disease. The formation of fibrin at sites of Y. pestis infection supports innate host defense against plague, perhaps by providing a nondiffusible spatial cue that promotes the accumulation of inflammatory cells expressing fibrin-binding integrins. This report demonstrates that fibrin is an essential component of T cell-mediated defense against plague but can be dispensable for Ab-mediated defense. Genetic or pharmacologic depletion of fibrin abrogated innate and T cell-mediated defense in mice challenged intranasally with Y. pestis. The fibrin-deficient mice displayed reduced survival, increased bacterial burden, and exacerbated hemorrhagic pathology. They also showed fewer neutrophils within infected lung tissue and reduced neutrophil viability at sites of liver infection. Depletion of neutrophils from wild-type mice weakened T cell-mediated defense against plague. The data suggest that T cells combat plague in conjunction with neutrophils, which require help from fibrin to withstand Y. pestis encounters and effectively clear bacteria.

Serine protease and ovarian paracrine factors in regulation of ovulation.

The controlled target extracellular matrix (ECM) degradation generated by serine protease and regulated by serine protease inhibitor and ovarian paracrine/autocrine factors is an event that affects a wide variety of physiological and pathological processes in the ovary. Evidence cumulated in the past decade clearly showed that the hormone-induced coordinated expression of the tissue-type PA (tPA) produced mainly by granulosa cells and oocyte, and its inhibitor PAI-1 secreted by theca cells in the preovulatory follicles may be responsible for a controlled and directed proteolysis leading to the rupture of selected follicles in the rat, monkey and other mammals. In recent years increasing evidence further demonstrated that oocyte maturation and ovulation may also be modulated by other serine protease and inhibitor, as well as endogenously- produced ovarian paracrine/autocrine factors. Thus, it is important to identify the interrelationship between the serine protease system and the multiple factors, and to know how they regulate the ovarian physiological and pathological processes during oocyte maturation and ovulation.

[Significance of urokinase and its inhibitors in the invasiveness and metastasing of malignant tumors]. / Význam urokinázy a jejích inhibitoru pro invazi a metastazování zhoubných nádoru

Fibrinolysis is process, which leads to the degradation of fibrin to fibrin monomers. Fibrinolysis helps to regulate hemostasis and prevents the creation of inappropriately large thrombus, which could reduce blood flow to the bloodstream. The main enzyme involved in fibrinolysis is plasmin. Tissue plasminogen activator (tPA) and urokinase (uPA) are agents converting plasminogen into active plasmin, together with urokinase receptor (uPAR) and urokinase inhibitors (PAI 1, PAI 2, PAI 3 and protease nexin) form plasminogen activator system (PAS) which is among others also part of the metastatic cascade and significantly contributes to invasive growth and angiogenesis of malignant tumours. In contrast to tPA that is fundamental in fibrinolysis, uPA plays an essential role in tissue degradation as part of physiological and pathological processes. uPAR is a GPI (glycosylphosphatidylinositol)-anchored protein. The binding of uPA to uPAR results in activation of protein tyrosine kinase, protein kinase C and MAP kinase. At the same time, direct signalling pathway via Jak/STAT cascade utilising signalling transduction of Scr-like protein tyrosine kinase have also been described. uPAR expression is regulated by many growth factors, e.g. EGF, FGF-2 and HGF. It seems that individual PAS factors are involved in the process of rendering malignant tumors invasive. To what degree this influence is essential to specific malignancies, should be answered by further research. In the article the authors present a summary of findings about the interaction of fibrinolysis and tumor process, especially on the effects of urokinase and other activators and their inhibitors in metastasis of malignant tumors. The text contains information on the factors theirs introduction into practice is still the subject of numerous discussions, but in the future, individual PAS factors could play an important role in planning treatment strategies and also could become targets of targeted therapy.

Fibrin(ogen)-independent role of plasminogen activators in acetaminophen-induced liver injury.

Hepatic fibrin(ogen) has been noted to occur after acetaminophen (APAP)-induced liver injury in mice. Deficiency in plasminogen activator inhibitor-1 (PAI-1), an endogenous inhibitor of fibrinolysis, increases APAP-induced liver injury in mice. However, the roles of fibrinogen and fibrinolysis in APAP-induced liver injury are not known. We tested the hypothesis that hepatic fibrin(ogen) deposition reduces severity of APAP-induced liver injury. APAP-induced (300 mg/kg) liver injury in mice was accompanied by thrombin generation, consumption of plasma fibrinogen, and deposition of hepatic fibrin. Neither fibrinogen depletion with ancrod nor complete fibrinogen deficiency [via knockout of the fibrinogen alpha chain gene (Fbg(-/-))] affected APAP-induced liver injury. PAI-1 deficiency (PAI-1(-/-)) increased APAP-induced liver injury and hepatic fibrin deposition 6 hours after APAP administration, which was followed by marked hemorrhage at 24 hours. As in PAI-1(-/-) mice, administration of recombinant tissue plasminogen activator (tenecteplase, 5 mg/kg) worsened APAP-induced liver injury and hemorrhage in wild-type mice. In contrast, APAP-induced liver injury was reduced in both plasminogen-deficient mice and in wild-type mice treated with tranexamic acid, an inhibitor of plasminogen activation. Activation of matrix metalloproteinase 9 (MMP-9) paralleled injury, but MMP-9 deficiency did not affect APAP-induced liver injury. The results indicate that fibrin(ogen) does not contribute to development of APAP-induced liver injury and suggest rather that plasminogen activation contributes to APAP-induced liver injury.

[Clinical and etiopathogenetic role of plasminogen and metaloproteinase systems in the tumor growth. Pericellular proteolysis of extracellular matrix and tumor growth]. / Klinicka i etiopatogenetska uloga plazminogenskoga i metaloproteinaznoga sustava u tumorskome rastu. Pericelularna razgradnja medustanicnoga matriksa i tumorski rast.

Pericellular proteolysis is a cascade process involved in degradation of extracellular matrix. This process is included in various physiological and pathological processes. Pericellullar proteolysis has major functions like degradation of tissue stroma and weakening of intercellular connections but it also has a function in the synthesis of bioactive molecules (cytokines, growth factors and inhibitory factors). Plasminogen system is involved in fibrinolysis and starts metalloproteinase activation. Activity of proteolytic molecules is controlled by the rate of zymogenic activation, half-life of molecules, and action of inhibitory molecules. Inhibition is achieved through direct binding of inhibitor and enzyme and takes a few steps. Pericellular proteolysis is involved in tumor invasion and metastasis, inflammatory reaction, degenerative diseases and other diseases. Pathophysiological regulation of pericellular proteolysis in mentioned diseases contributes to clinical properties of diseases and has diagnostic and therapeutic importance.

Longistatin, a plasminogen activator, is key to the availability of blood-meals for ixodid ticks.

Ixodid ticks are notorious blood-sucking ectoparasites and are completely dependent on blood-meals from hosts. In addition to the direct severe effects on health and productivity, ixodid ticks transmit various deadly diseases to humans and animals. Unlike rapidly feeding vessel-feeder hematophagous insects, the hard ticks feed on hosts for a long time (5-10 days or more), making a large blood pool beneath the skin. Tick's salivary glands produce a vast array of bio-molecules that modulate their complex and persistent feeding processes. However, the specific molecule that functions in the development and maintenance of a blood pool is yet to be identified. Recently, we have reported on longistatin, a 17.8-kDa protein with two functional EF-hand Ca(++)-binding domains, from the salivary glands of the disease vector, Haemaphysalis longicornis, that has been shown to be linked to blood-feeding processes. Here, we show that longistatin plays vital roles in the formation of a blood pool and in the acquisition of blood-meals. Data clearly revealed that post-transcriptional silencing of the longistatin-specific gene disrupted ticks' unique ability to create a blood pool, and they consequently failed to feed and replete on blood-meals from hosts. Longistatin completely hydrolyzed α, ß and γ chains of fibrinogen and delayed fibrin clot formation. Longistatin was able to bind with fibrin meshwork, and activated fibrin clot-bound plasminogen into its active form plasmin, as comparable to that of tissue-type plasminogen activator (t-PA), and induced lysis of fibrin clot and platelet-rich thrombi. Plasminogen activation potentiality of longistatin was increased up to 4 times by soluble fibrin. Taken together, our results suggest that longistatin may exert potent functions both as a plasminogen activator and as an anticoagulant in the complex scenario of blood pool formation; the latter is critical to the feeding success and survival of ixodid ticks.

From Rous sarcoma virus to plasminogen activator, src oncogene and cancer management.

Plasminogen activator (PLAU) is a serine protease that converts plasminogen to plasmin, a general protease, which promotes fibrinolysis and degradation of extracellular matrix. PLAU was reported in 1970s as one of the robustly induced enzymatic activities in Rous sarcoma virus (RSV)-transformed chicken cells. More than three decades later, with the completion of the sequencing of the chicken genome and the subsequent availability of Affymetrix GeneChip genome arrays, several laboratories have surveyed the transcriptional program affected by the RSV transformation. Interestingly, the PLAU gene was shown to be the most highly upregulated transcript. The induction of PLAU was a transformation-dependent process because viruses with deleted Src gene did not induce the transcription of the PLAU gene. Both Src and PLAU genes are associated with and contribute to the complex phenotype of human cancer. Although the activity and structures of these two enzymes are well characterized, the precise molecular function of these gene products in signaling networks is still not fully understood. Yet, the knowledge of their association with cancer is already translated into the clinical setting. Src kinase inhibitors are being tested in clinical trials of cancer therapy, and PLAU gene and its inhibitor have been included as biomarkers with strong prognostic and therapeutic predictive values. This vignette reviews the history of PLAU and Src discovery, and illuminates the complexity of their relationship, but also points to their emerging impact on public health.

[Plasminogen activator system and its clinical significance in patients with a malignant disease]. / Plazminogen aktivátor systém a jeho klinický význam u pacientu s nádorovým onemocnením.

Urokinase (uPA) plays an essential role in the activation of plasminogen to plasmin, a serine protease participating in the activation of matrixmetaloproteinases, latent elastases, growth factors and cytokines involved in the degradation of extracellular matrix elements. Together with its receptor (uPAR), tissue activator (tPA) and urokinase inhibitors (PAI-1, PAI-2, PAI-3 and protease nexin), it forms the plasminogen activator system (PAS), a component of metastatic cascade importantly contributing to the invasive growth and angiogenesis of malignant tumours. Plasminogen activator inhibitor 1 inhibits uPA-dependent invasiveness of some cancer cell lines. The vitronectin-PAI-1 complex inhibits migration of smooth muscle cells by binding alpha(v)beta3 integrin to vitronectin. PAI-1 or its deficiency interferes with signalling pathways such as PI3K/Akt and JAK/STAT and it is included in the processes of maintaining the integrity of the endothelial cells and thereby regulation of cell death. PAI-1 affects apoptosis by reducing cell adhesion and functioning of intracellular signalling pathways. The individual components of PAS undoubtedly play an important role in angiogenesis and metastasising of malignant tumours. In the near future, results of published studies with various types of cancer could be reflected in diagnostic and therapeutic algorithms and, at the same time, could serve as the goal for targeted therapies.

The plasmin-antiplasmin system: structural and functional aspects.

The plasmin-antiplasmin system plays a key role in blood coagulation and fibrinolysis. Plasmin and α(2)-antiplasmin are primarily responsible for a controlled and regulated dissolution of the fibrin polymers into soluble fragments. However, besides plasmin(ogen) and α(2)-antiplasmin the system contains a series of specific activators and inhibitors. The main physiological activators of plasminogen are tissue-type plasminogen activator, which is mainly involved in the dissolution of the fibrin polymers by plasmin, and urokinase-type plasminogen activator, which is primarily responsible for the generation of plasmin activity in the intercellular space. Both activators are multidomain serine proteases. Besides the main physiological inhibitor α(2)-antiplasmin, the plasmin-antiplasmin system is also regulated by the general protease inhibitor α(2)-macroglobulin, a member of the protease inhibitor I39 family. The activity of the plasminogen activators is primarily regulated by the plasminogen activator inhibitors 1 and 2, members of the serine protease inhibitor superfamily.

VGLUT2-dependent sensory neurons in the TRPV1 population regulate pain and itch.

The natural response to itch sensation is to scratch, which relieves the itch through an unknown mechanism. Interaction between pain and itch has been frequently demonstrated, and the selectivity hypothesis of itch, based on data from electrophysiological and behavioral experiments, postulates the existence of primary pain afferents capable of repressing itch. Here, we demonstrate that deletion of vesicular glutamate transporter (VGLUT) 2 in a subpopulation of neurons partly overlapping with the vanilloid receptor (TRPV1) primary afferents resulted in a dramatic increase in itch behavior accompanied by a reduced responsiveness to thermal pain. The increased itch behavior was reduced by administration of antihistaminergic drugs and by genetic deletion of the gastrin-releasing peptide receptor, demonstrating a dependence on VGLUT2 to maintain normal levels of both histaminergic and nonhistaminergic itch. This study establishes that VGLUT2 is a major player in TRPV1 thermal nociception and also serves to regulate a normal itch response.

Dysregulation of cellular signaling in gastric cancer.

The pathogenesis of gastric cancer is complex and related to multiple factors. Dysregulation of intracellular signaling pathways represents a common pathogenic mechanism and may be amenable to drug targeting. Multiple well-established oncogenic pathways, such as those mediated by cell cycle regulators, nuclear factor-kappaB, cyclooxygenase-2 and epidermal growth factor receptor are implicated in gastric carcinogenesis. Emerging evidence also underscores the importance of signaling pathways involved in the developmental process, including transforming growth factor-beta/bone morphogenetic protein signaling, Wnt/beta-catenin signaling, Hedgehog signaling and Notch signaling. Understanding their biological significance will provide a rational basis for drug development. Their relative importance and cross-talk in gastric carcinogenesis, however, are still not completely understood and warrant further investigation.

[In vitro function of outer membrane protease T of Escherichia coli K1 pathogenic strain].

UNLABELLED: Plasminogen activation and antimicrobial peptide hydrolysis contribute to pathogens invasion and survival in vivo. OBJECTIVE: To demonstrate the expression of outer membrane protease T in E. coli K1 pathogenic strain E44, its activity of plasminogen activator and protamine hydrolysis. METHODS: After Benzamidine Sepharose Fast Flow and SOURCE 30Q chromatography, we got E44 outer membrane mixed fraction, and examined its activity of plasminogen activation with chromogenic substrate S-2251 method. An ompT deletion mutant of E44 was constructed by using the suicide vector pCVD442, termed as E44ompT. We examined 0.1 mg/mL cationic antimicrobial peptide protamine susceptibility of E44, ompT mutant strain E44ompT and E44ompT harboring pUCT, which was constructed by inserting complete ompT open reading frame into pUC13. RESULTS: We got about 37 kDa E44 membrane extract, which could activate plasminogen, and activation was membrane extract dose dependent. This confirmed the expression of outer membrane protease T in the outer membrane of E44. E44ompT was, more susceptible to 0.1 mg/mL protamine than E44, and E440mpT was partially complemented by pUCT. CONCLUSION: Outer membrane protease T is expressed in E. coli K1 pathogenic strain E44, and can activate plasminogen and hydrolyze protamine.

Plasmin induces apoptosis of aortic valvular myofibroblasts.

Previous studies have described remodelling of the extracellular substratum by matrix metalloproteinases (MMPs) in aortic valves. However, involvement of the fibrinolytic system has not yet been analysed. We hypothesized that plasminogen and plasminogen activator(s) are present in aortic valves and that plasminogen activation could induce the degradation of adhesive proteins and apoptosis of the valvular myofibroblasts. We employed ELISA, western blotting, fibrin-agar zymography, and immunochemistry to detect components of the plasminogen activation system, in samples of aortic valves and valvular myofibroblasts in primary culture. Using myofibroblast cultures, real-time measurement of plasminogen activation was performed in the absence and presence of inhibitors (amiloride, epsilon-aminocaproic acid, and an MMP inhibitor); the degradation of fibronectin was visualized on western blots; and the apoptotic process was assessed by detection of phosphatidylserine exposure (binding of FITC-annexin V) and DNA fragmentation (TUNEL and ELISA). We demonstrate that a time- and plasminogen concentration-dependent generation of plasmin occurs on the surface of cultured valvular myofibroblasts expressing both u-PA and t-PA. Only u-PA appears to activate plasminogen as t-PA is essentially found in complex with PAI-1. Plasmin-dependent degradation of pericellular proteins, such as fibronectin, leads to cell detachment and apoptosis. In conclusion, various proteins of the fibrinolytic system are synthesized in vitro by cultured myofibroblasts from aortic valves, leading to plasmin-dependent cell detachment-induced apoptosis, a biological process named anoikis. The presence of plasminogen in aortic valves suggests that this process may be operating in vivo and may participate in valvular tissue remodelling, as also suggested by the finding of apoptotic cells in valvular tissue. This is the first demonstration of the presence and potential role of enzymes of the fibrinolytic system in aortic valves.

Fibrin binding and the regulation of plasminogen activators during thrombolytic therapy.

First generation thrombolytics (streptokinase and urokinase) had no fibrin binding capabilities and caused systemic plasminogen activation with concomitant destruction of haemostatic proteins. A primary driving force behind the development of the second generation plasminogen activator tissue plasminogen activator (tPA or alteplase) was its ability to bind to fibrin and target thrombolysis. Although in vitro assays highlighted advantages of fibrin binding, clinical trials were disappointing, showing only small benefits in mortality with tPA versus streptokinase, but also with some increase in haemorrhagic stroke. Third generation thrombolytic agents (reteplase, tenecteplase and pamiteplase) are variants of tPA engineered to have improved structure/function, such as longer half life and resistance to inhibitors. However, clear therapeutic advantages of third generation thrombolytics in clinical trials have also been difficult to demonstrate. Although fibrin binding is critical in regulating the activity of tPA, it is not clear how important it is for thrombolytic treatment. Advances are needed in our understanding of the relationship between structure/binding and activity of PAs in vivo under normal conditions and when administered in pharmacological doses. Clearly the impact of fibrin structure and the other components in fibrin clots must also be considered. Ultimately these studies may lead to better engineered therapeutics or optimised mixtures of molecules. With a more detailed understanding of the regulation of plasminogen activation and fibrinolysis it might be possible to tailor thrombolytic therapy to different situations such as myocardial or cerebrovascular treatment or to the patient's age and sex and other characteristics.