Plasmin Formation Research Articles

ARNTL2 and SERPINE1: potential biomarkers for tumor aggressiveness in colorectal cancer

Cathepsin and plasmin may favor cancer cell invasion degrading extracellular matrix. Plasmin formation from plasminogen is regulated by plasminogen activator inhibitor type-1 (PAI-1). ARNTL2 activates the promoters of the PAI-1 gene, officially called SERPINE1, driving the circadian variation in circulating PAI-1 levels. We evaluated ARNTL2 and SERPINE1 expression in 50 colorectal cancer specimens and adjacent normal tissue and in colon cancer cell lines. We found up-regulation of ARNTL2 (P = 0.004) and SERPINE1 (P = 0.002) in tumor tissue. A statistically significant association was found between high ARNTL2 mRNA levels and vascular invasion (P < 0.0001), and between high SERPINE1 mRNA levels and microsatellite instability (MSI-H and MSI-L, P = 0.025). Sorting the subjects into quartile groups, a statistically significant association was found between high ARNTL2 expression and lymph node involvement (P < 0.001), between high SERPINE1 expression and grading (P < 0.001) and between high SERPINE1 expression and MSI H-L (P < 0.0001). In SW480 cells, a more proliferative model compared to CaCo2 cells, there were higher mRNA levels of ARNTL2 (P < 0.001) and SERPINE1 (P = 0.001). ARNTL2 and SERPINE1 expression is increased in colorectal cancer and in a highly proliferative colon cancer cell line and is related to tumor invasiveness and aggressiveness.

A Plasmin Inhibitor Prevents Lethal Acute Graft-Versus-Host Disease in Mice

Abstract 1897Proinflammatory cytokines released upon cell damage can cause excessive tissue destruction in acute graft-versus-host disease (GVHD). Certain key cytokines for GVHD like TNF-α and Fas-ligand, are secreted by ectodomain shedding from matrix metalloproteinases (MMPs). Many MMP inhibitors have been developed and used over the last three or four decades, but its widespread use is hampered by severe side effects. We reported that the fibrinolytic system, above all plasmin formation promotes the activation of several MMPs. Therefore we hypothesized that plasmin inhibition might be a novel means to control acute GVHD in part by suppressing MMP and inflammatory cytokine secretion. We found that the fibrinolytic system is activated during the development of acute GVHD in mice and in humans. Here, we examined a novel plasmin inhibitor (YO-2) that inhibits MMP activation and inflammatory cytokines release for lethal acute GVHD model in mice. Administration of YO-2 into (BALB/c × C57BL/6) F1 that received C57BL/6 spleen cells showed markedly reduced mortality and weight loss in association with minimal signs of GVHD pathology in the liver, intestine, and hematopoietic tissues. Furthermore, our results suggest that administaration of YO-2 blocked the processing of inflammatory cytokines through MMP inhibition in vitro and in vivo. These data have major implications for transplantation medicine, as pharmacological inhibition of plasmin does not require the need for intensive immunosuppression. Our results suggest that YO-2 could be a novel molecular therapeutic agent for GVHD. Disclosures:No relevant conflicts of interest to declare.

Effects of Altered Plasminogen Activator Inhibitor-1 Expression on Cardiovascular Disease

Plasminogen Activator Inhibitor-1 (PAI-1) is a multifunctional protein with the ability to not only regulate fibrinolysis through inhibition of plasminogen activation, but also cell signaling events which have direct downstream effects on cell function. Elevated plasma levels of this protein have been shown to have profound effects on the development and progression of cardiovascular diseases. However, results from a number of studies, especially those using PAI-1 deficient mouse models, have demonstrated that its function is ambiguous, with evidence of both preventing and enhancing various disease states. A number of lifestyle changes and pharmacological reagents have been identified that can regulate PAI-1 levels or function. Those reagents that target function are focused on its ability to regulate plasmin formation, and have been studied in in vivo models of thrombosis. Further investigations involving regulation of cell function could potentially resolve paradoxical issues associated with the function of this protein in regulating cardiovascular disease.

Leishmania mexicana: LACK (Leishmania homolog of receptors for activated C-kinase) is a plasminogen binding protein

Leishmania mexicana is able to interact with the fibrinolytic system through its component plasminogen, the zymogenic form of the protease plasmin. In this study a new plasminogen binding protein of this parasite was identified: LACK, the Leishmania homolog of receptors for activated C-kinase. Plasminogen binds recombinant LACK with a Kd value of 1.6±0.4μM, and binding is lysine-dependent since it is inhibited by the lysine analog ε-aminocaproic acid. Inhibition studies with specific peptides and plasminogen binding activity of a mutated recombinant LACK have highlighted the internal motif 260VYDLESKAV268, similar to those found in several enolases, as involved in plasminogen binding. Recombinant LACK and secreted proteins, in medium conditioned by parasites, enhance plasminogen activation to plasmin by the tissue plasminogen activator (t-PA). In addition to its localization in the cytosol, in the microsomal fraction and as secreted protein in conditioned medium, LACK was also localized on the external surface of the membrane. The results presented here suggest that LACK might bind and enhance plasminogen activation in vivo promoting the formation of plasmin. Plasminogen binding of LACK represents a new function for this protein and might contribute to the invasiveness of the parasite.

Plasmin on adherent cells: from microvesiculation to apoptosis

Cell activation by stressors is characterized by a sequence of detectable phenotypic cell changes. A given stimulus, depending on its strength, induces modifications in the activity of membrane phospholipid transporters and calpains, which lead to phosphatidylserine exposure, membrane blebbing and the release of microparticles (nanoscale membrane vesicles). This vesiculation could be considered as a warning signal that may be followed, if the stimulus is maintained, by cell detachment-induced apoptosis. In the present study, plasminogen incubated with adherent cells is converted into plasmin by constitutively expressed tPA (tissue-type plasminogen activator) or uPA (urokinase-type plasminogen activator). Plasmin formed on the cell membrane then induces a unique response characterized by membrane blebbing and vesiculation. Hitherto unknown for plasmin, these membrane changes are similar to those induced by thrombin on platelets. If plasmin formation persists, matrix proteins are then degraded, cells lose their attachments and enter the apoptotic process, characterized by DNA fragmentation and specific ultrastructural features. Since other proteolytic or inflammatory stimuli may evoke similar responses in different types of adherent cells, the proposed experimental procedure can be used to distinguish activated adherent cells from cells entering the apoptotic process. Such a distinction is crucial for evaluating the effects of mediators, inhibitors and potential therapeutic agents.

Plasminogen activators in ischemic stroke: introduction.

A number of plasminogen activators (PAs), enzymes capable of converting the zymogen plasminogen to the active agent plasmin, have been studied as potential therapeutic agents in the settings of ischemic stroke, intracerebral parenchymal hemorrhage, and large vessel thrombosis. Several have found purpose in the acute management of focal cerebral ischemia under strictly limited conditions. All derive from naturally occurring PAs that can generate plasmin in the circulation or in fibrin clots, and all are known to lyse thrombi. This session examines potential modifications to accepted strategies for the treatment of ischemic stroke acutely. The principles for the acute use of PAs in ischemic stroke derive from the common understanding of plasminogen activation in the circulation and in thrombi known to target in situ thrombotic occlusions. Physiologically, the intrinsic (endogenous) thrombolytic system entails the secretion of single-chain urokinase (sc u-PA) and of single-chain tissue type plasminogen activator (sc t-PA) from endothelial cells.1–3 These precursor PAs are converted to urokinase (u-PA) and t-PA by locally derived plasmin. Plasmin is itself generated from the inactive zymogen plasminogen by u-PA and/or t-PA ( Figure ) . The formation of plasmin from plasminogen is the central reaction necessary for thrombus lysis. This conversion is highly regulated under ordinary circumstances. For instance, plasminogen activator inhibitor-1 and -2 directly inhibit the actions of u-PA and t-PA to convert plasminogen to plasmin.4,5 When t-PA is bound to fibrin and plasminogen (fibrin-bound plasminogen) in a ternary complex, both …

Neutralisation of uPA with a Monoclonal Antibody Reduces Plasmin Formation and Delays Skin Wound Healing in tPA-Deficient Mice

BackgroundProteolytic degradation by plasmin and metalloproteinases is essential for epidermal regeneration in skin wound healing. Plasminogen deficient mice have severely delayed wound closure as have mice simultaneously lacking the two plasminogen activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). In contrast, individual genetic deficiencies in either uPA or tPA lead to wound healing kinetics with no or only slightly delayed closure of skin wounds.Methodology/Principal FindingsTo evaluate the therapeutic potential in vivo of a murine neutralizing antibody directed against mouse uPA we investigated the efficacy in skin wound healing of tPA-deficient mice. Systemic administration of the anti-mouse uPA monoclonal antibody, mU1, to tPA-deficient mice caused a dose-dependent delay of skin wound closure almost similar to the delayed kinetics observed in uPA;tPA double-deficient mice. Analysis of wound extracts showed diminished levels of plasmin in the mU1-treated tPA-deficent mice. Immunohistochemistry revealed that fibrin accumulated in the wounds of such mU1-treated tPA-deficent mice and that keratinocyte tongues were aberrant. Together these abnormalities lead to compromised epidermal closure.Conclusions/SignificanceOur findings demonstrate that inhibition of uPA activity with a monoclonal antibody in adult tPA-deficient mice mimics the effect of simultaneous genetic ablation of uPA and tPA. Thus, application of the murine inhibitory mU1 antibody provides a new and highly versatile tool to interfere with uPA-activity in vivo in mouse models of disease.

Plasminogen Activation by Airway Smooth Muscle Is Regulated by Type I Collagen

Plasmin, the activated protease product of plasminogen, is involved in collagen remodeling, and is strongly implicated in asthma pathophysiology by recent genome-wide association studies. This study examines plasminogen "activation" by airway smooth muscle cells, and its regulation in a fibrotic environment created by culture on type I collagen and incubation with transforming growth factor (TGF)-β. Urokinase plasminogen activator (uPA) activity was detected in the supernatants of human airway smooth muscle cell cultures maintained in serum-free conditions. Incubation with plasminogen (1.5-50.0 μg/ml, 24 h) increased plasmin activity in a concentration-dependent manner (P < 0.001). uPA activity was higher in cultures maintained on fibrillar type I collagen substrata than in those on plastic, as was plasmin activity after incubation with plasminogen (20 μg/ml). Pretreatment with TGF-β (100 pM) for 18 hours inhibited plasminogen activation by airway smooth muscle cells maintained on plastic, but not on collagen. TGF-β stimulated an increase in the level of uPA mRNA in airway smooth muscle cells grown on collagen, but not on plastic. Reducing the levels of β1-integrin collagen receptor, using interference RNA, attenuated plasmin formation by airway smooth muscle cells grown on collagen, and restored the inhibitory effect of TGF-β. This study shows that airway smooth muscle activation of plasminogen by uPA is accelerated in a collagen-rich environment in which the inhibitory effect of TGF-β is attenuated in association with greater uPA expression induced via β1-integrin signaling. These findings suggest that the plasminogen-activation system involving uPA has the potential to contribute to airway wall remodeling in asthma.

Thrombin-activatable fibrinolysis inhibitor is degraded by Salmonella enterica and Yersinia pestis.

Pathogenic bacteria modulate the host coagulation system to evade immune responses or to facilitate dissemination through extravascular tissues. In particular, the important bacterial pathogens Salmonella enterica and Yersinia pestis intervene with the plasminogen/fibrinolytic system. Thrombin-activatable fibrinolysis inhibitor (TAFI) has anti-fibrinolytic properties as the active enzyme (TAFIa) removes C-terminal lysine residues from fibrin, thereby attenuating accelerated plasmin formation. Here, we demonstrate inactivation and cleavage of TAFI by homologous surface proteases, the omptins Pla of Y. pestis and PgtE of S. enterica. We show that omptin-expressing bacteria decrease TAFI activatability by thrombin-thrombomodulin and that the anti-fibrinolytic potential of TAFIa was reduced by recombinant Escherichia coli expressing Pla or PgtE. The functional impairment resulted from C-terminal cleavage of TAFI by the omptins. Our results indicate that TAFI is degraded directly by the omptins PgtE of S. enterica and Pla of Y. pestis. This may contribute to the ability of PgtE and Pla to damage tissue barriers, such as fibrin, and thereby to enhance spread of S. enterica and Y. pestis during infection.

Mechanism of the synergistic effect between oversulfated chondroitin-6-sulfate and lysine or 6-aminohexanoic acid in enhancing the in-vitro activation of glutamic plasminogen by tissue plasminogen activator or urokinase

Earlier studies of addition of naturally sulfated polysaccharides including unfractionated heparin showed a significant enhancement of the in-vitro activation of glutamic plasminogen (Glu-Plg) by tissue plasminogen activator (t-PA) or urokinase plasminogen activator (u-PA). However, supplementing of physiological concentration of NaCl (0.9%) to the buffer reversed the enhancement. To overcome this reversal attempts were made to oversulfate the compounds and re-evaluate their biological properties. Chondroitin-6-sulfate (N-2) was oversulfated using chlorosulfonic acid-pyridine complex and isolated as the sodium salt. Infrared and H-NMR studies of the oversulfated compound showed introduction of new sulfate groups with the formation of 60% of chondroitin-4-6-disulfate. In-vitro studies were conducted on comparing the effect of oversulfated chondroitin-6-sulfate (S-2) with native compound (N-2) and unfractionated heparin in enhancing the activation of Glu-Plg by t-PA or u-PA using 0.05 mol/l Tris buffer (pH 7.3) containing 0.9% of NaCl. The enhancement of activation of Glu-Plg by t-PA or u-PA was measured by formation of plasmin using H-D-Glu-Phe-Lys-pNA (S-2403) as the substrate. The activation by t-PA was enhanced two-fold by 2.86 microg/ml of S-2, 4-6-fold by addition of 32.4 mmol/l of lysine or 5.4 mmol/l of 6-aminohexanoic acid (6-AH) and 14-16-fold enhancement by addition of both S-2 and lysine or S-2 and 6-AH showing a synergistic effect, whereas unfractionated heparin alone gave no enhancement and in conjunction with lysine or 6-AH gave no additional enhancement. Similar studies using u-PA in place of t-PA gave identical results. During the activation of Glu-Plg to plasmin, lysine plasminogen (Lys-Plg) is reported to be an intermediate. Therefore we investigated the role of S-2, lysine and 6-AH in the activation of Lys-Plg to plasmin. The results showed that S-2 enhanced this activation, whereas lysine or 6-AH which were active in enhancing the activation of Glu-Plg were not active using Lys-Plg indicating that the site of enhancement by lysine or 6-AH was during the initial phase. Double reciprocal plot of Glu-Plg activation by t-PA with or without S-2 and lysine showed no change in Km but a 10-fold increase of Kcat suggesting a template mechanism for the attenuation when both cofactors are used.

Efficacy of Plasmin, Microplasmin, and Streptokinase-Plasmin Complex for the In Vitro Degradation of Fibronectin and Laminin- Implications for Vitreoretinal Surgery

Purpose: Plasmin enzyme generates vitreoretinal separation by degradation of laminin and fibronectin in the vitreoretinal interface. It can be activated from plasminogen by urokinase, tissue plasminogen activator, or by formation of a 1:1 complex with streptokinase. The latter is then converted into a streptokinase-plasmin-complex (SK-P), which displays fibrinolytic activity and can generate free plasmin by proteolysis of plasminogen. We compared the efficacy of SK-P, SK-P activated plasmin, urokinase activated plasmin (UK-P), and microplasmin, a truncated form of plasmin, in cleaving laminin and fibronectin.Methods: Streptokinase (SK) was added to human plasminogen in molar ratios between 1:100 and 2:1, generating SK-P at ratios > 1:1, and mixtures of SK-P and free plasmin (SK-P/plasmin) at lower ratios. SK-PL, SK-P/plasmin, UK-P, and microplasmin were added to laminin and fibronectin, incubated at 37°C for 30 min–22 hr and processed for SDS-PAGE.Results: Proteolysis using SK-activated plasminogen increased when the SK/plasminogen ratio was decreased, generating increasing amounts of free plasmin. Microplasmin and urokinase-activated plasmin displayed similar proteolysis of both laminin and fibronectin as SK/plasminogen at ratios of 1:10 or lower.Conclusion: The mode of plasminogen activation influences the efficacy of proteolysis for laminin and fibronectin and should be considered when plasmin is used in vitreoretinal surgery.

Abstract 2349: DLC1 interaction with S100A10 mediates inhibition of in vitro cell invasion and tumorigenicity of lung cancer cells

Abstract Deleted in liver cancer 1 (DLC1) is a multidomain Rho-GTPase-activating protein (RhoGAP) and a tumor suppressor in several human common cancers. DLC1's structure predicts its interaction with a number of other proteins with various cellular functions. Previously we have shown that S100A10 (also known as p11), a key plasminogen receptor on the surface of cell membrane, colocalizes and binds with DLC1 in cell cytoplasm. The binding sites were mapped to the central domain of the DLC1 and at the end of C-terminus of S100A10. The same terminus is also a binding site for Annexin A2, which is believed to regulate availability of extracellular S100A10 and, therefore, dynamics of plasmin formation. Now we demonstrate that DLC1 competes with Annexin A2 for interaction with S100A10. DLC1 binding to S100A10 did not affect DLC1's RhoGAP activity but did reduce availability of S100A10 for binding by Annexin A2. Furthermore, the expression of DLC1 also affected S100A10 expression in a dose dependent manner, and increased S100A10 ubiquitin-dependent degradation. All of the above interactions attenuated plasminogen activation and resulted in inhibition of in vitro cell migration, invasion, colony formation and anchorage independent growth of aggressive and tumorigenic lung tumor cells. Transduction of DLC1-negative breast tumor cells significantly reduced the ability of tumor cells to form pulmonary metastases as well as inhibited distant dissemination of liver tumor cells in athymic mice. Current observations provide a plausible mechanism for DLC1 antimetastatic function by negatively affecting plasminogen activation, which in turn lead to a significant reduction in invasiveness of tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2349.

Temperature-Induced Changes in the Lipopolysaccharide ofYersinia pestisAffect Plasminogen Activation by the Pla Surface Protease

The Pla surface protease of Yersinia pestis activates human plasminogen and is a central virulence factor in bubonic and pneumonic plague. Pla is a transmembrane beta-barrel protein and member of the omptin family of outer membrane proteases which require bound lipopolysaccharide (LPS) to be proteolytically active. Plasminogen activation and autoprocessing of Pla were dramatically higher in Y. pestis cells grown at 37 degrees C than in cells grown at 20 degrees C; the difference in enzymatic activity by far exceeded the increase in the cellular content of the Pla protein. Y. pestis modifies its LPS structure in response to growth temperature. We purified His(6)-Pla under denaturing conditions and compared various LPS types for their capacity to enhance plasmin formation by His(6)-Pla solubilized in detergent. Reactivation of His(6)-Pla was higher with Y. pestis LPSs isolated from bacteria grown at 37 degrees C than with LPSs from cells grown at 25 degrees C. Lack of O antigens and the presence of the outer core region as well as a lowered level of acylation in LPS were found to enhance the Pla-LPS interaction. Genetic substitution of arginine 138, which is part of a three-dimensional protein motif for binding to lipid A phosphates, decreased both the enzymatic activity of His(6)-Pla and the amount of Pla in Y. pestis cells, suggesting the importance of the Pla-lipid A phosphate interaction. The temperature-induced changes in LPS are known to help Y. pestis to avoid innate immune responses, and our results strongly suggest that they also potentiate Pla-mediated proteolysis.

Fibrinolytic cross-talk: a new mechanism for plasmin formation

AbstractFibrinolysis and pericellular proteolysis depend on molecular coassembly of plasminogen and its activator on cell, fibrin, or matrix surfaces. We report here the existence of a fibrinolytic cross-talk mechanism bypassing the requirement for their molecular coassembly on the same surface. First, we demonstrate that, despite impaired binding of Glu-plasminogen to the cell membrane by ϵ-aminocaproic acid (ϵ-ACA) or by a lysine-binding site–specific mAb, plasmin is unexpectedly formed by cell-associated urokinase (uPA). Second, we show that Glu-plasminogen bound to carboxy-terminal lysine residues in platelets, fibrin, or extracellular matrix components (fibronectin, laminin) is transformed into plasmin by uPA expressed on monocytes or endothelial cell–derived microparticles but not by tissue-type plasminogen activator (tPA) expressed on neurons. A 2-fold increase in plasmin formation was observed over activation on the same surface. Altogether, these data indicate that cellular uPA but not tPA expressed by distinct cells is specifically involved in the recognition of conformational changes and activation of Glu-plasminogen bound to other biologic surfaces via a lysine-dependent mechanism. This uPA-driven cross-talk mechanism generates plasmin in situ with a high efficiency, thus highlighting its potential physiologic relevance in fibrinolysis and matrix proteolysis induced by inflammatory cells or cell-derived microparticles.

A nonfibrin macromolecular cofactor for tPA-mediated plasmin generation following cellular injury

Tissue-type plasminogen activator (tPA) is an extracellular protease that converts plasminogen into plasmin. For tPA to generate plasmin under biologic conditions, a cofactor must first bring tPA and plasminogen into physical proximity. Fibrin provides this cofactor for tPA-mediated plasmin generation in blood. Despite being naturally devoid of fibrin(ogen), tPA-mediated plasmin formation also occurs in the brain. The fibrin-like cofactor(s) that facilitates plasmin formation in the injured brain has remained unknown. Here we show that protein aggregates formed during neuronal injury provide a macromolecular, nonfibrin cofactor that promotes tPA-mediated plasmin formation and subsequent cell breakdown. The binding of plasminogen and tPA to these protein aggregates occurs via distinct mechanisms. Importantly, nonneuronal cell types also exhibit this cofactor effect upon injury, indicating a general phenomenon. This novel cofactor identified in nonviable cells has ramifications for ischemic stroke where tPA is used clinically and where plasmin activity within the injured brain is unwanted. A means of selectively inhibiting the binding of tPA to nonviable cells while preserving its association with fibrin may be of benefit for the treatment of ischemic stroke.

The Single Substitution I259T, Conserved in the Plasminogen Activator Pla of PandemicYersinia pestisBranches, Enhances Fibrinolytic Activity

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(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 (259)IIDKT/TIDKN in PgtE, constructed to mimic the L5 region in Pla, altered proteolysis in favor of plasmin formation, whereas the reverse substitution (259)TIDKN/IIDKT in Pla altered proteolysis in favor of alpha(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.

The interaction ofBacteroides fragiliswith 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.

A pilot trial of microplasmin in patients with long-term venous access catheter thrombosis

Microplasmin, a truncated form of plasmin, degrades fibrin and reacts with the circulating inhibitor alpha(2)-antiplasmin. We investigated the safety and efficacy of intra-catheter microplasmin bolus administration for the restoration of catheter function in long-term venous access catheter thrombosis. This open-label, ascending-dose, pilot study enrolled 31 subjects. Two doses of microplasmin were evaluated, (5 mg and 8 mg) administered via a 2 ml intra-catheter bolus injection in 10 and 21 patients respectively. Catheter function was evaluated 30 min after the first bolus administration. In case of incomplete catheter function restoration, a second bolus was administered with reassessment of catheter function 30 min thereafter. After the first bolus, complete restoration of catheter withdrawal function was observed in 5 out of 10 (50%) and 14 of out 21 (66%) subjects treated with 5 mg and 8 mg respectively and in 8 out of 10 (80%) and 18 out of 21 (86%) subjects after a second administration of microplasmin. No bleeding complications nor other adverse events were related to microplasmin. In this pilot trial, microplasmin restored catheter function in a safe and effective way.

Characterisation of urokinase plasminogen activator receptor variants in human airway and peripheral cells

BackgroundExpression of the urokinase plasminogen activator receptor (UPAR) has been shown to have clinical relevance in various cancers. We have recently identified UPAR as an asthma susceptibility gene and there is evidence to suggest that uPAR may be upregulated in lung diseases such as COPD and asthma. uPAR is a key receptor involved in the formation of the serine protease plasmin by interacting with uPA and has been implicated in many physiological processes including proliferation and migration. The current aim was to determine key regulatory regions and splice variants of UPAR and quantify its expression in primary human tissues and cells (including lung, bronchial epithelium (HBEC), airway smooth muscle (HASM) and peripheral cells).ResultsUsing Rapid Amplification of cDNA Ends (RACE) a conserved transcription start site (-42 to -77 relative to ATG) was identified and multiple transcription factor binding sites predicted. Seven major splice variants were identified (>5% total expression), including multiple exon deletions and an alternative exon 7b (encoding a truncated, soluble, 229aa protein). Variants were differentially expressed, with a high proportion of E7b usage in lung tissue and structural cells (55–87% of transcripts), whereas classical exon 7 (encoding the GPI-linked protein) was preferentially expressed in peripheral cells (~80% of transcripts), often with exon 6 or 5+6 deletions. Real-time PCR confirmed expression of uPAR mRNA in lung, as well as airway and peripheral cell types with ~50–100 fold greater expression in peripheral cells versus airway cells and confirmed RACE data. Protein analysis confirmed expression of multiple different forms of uPAR in the same cells as well as expression of soluble uPAR in cell supernatants. The pattern of expression did not directly reflect that seen at the mRNA level, indicating that post-translational mechanisms of regulation may also play an important role.ConclusionWe have identified multiple uPAR isoforms in the lung and immune cells and shown that expression is cell specific. These data provide a novel mechanism for uPAR regulation, as different exon splicing may determine uPAR function e.g. alternative E7b results in a soluble isoform due to the loss of the GPI anchor and exon deletions may affect uPA (ligand) and/or integrin binding and therefore influence downstream pathways. Expression of different isoforms within the lung should be taken into consideration in studies of uPAR in respiratory disease.

Thrombomodulin Suppresses Invasiveness of HT1080 Tumor Cells by Reducing Plasminogen Activation on the Cell Surface through Activation of Thrombin-Activatable Fibrinolysis Inhibitor

Cell malignancy is negatively correlated with the expression of thrombomodulin (TM), a thrombin receptor expressed on the surface of various cells, including tumor cells. TM accelerates thrombin-activatable fibrinolysis inhibitor (TAFI) activation catalyzed by thrombin. The active form of TAFI (TAFIa) contributes to inhibition of plasmin formation through its carboxypeptidase B (CPB)-like activity. Here, we examined whether TM- and tumor cell-dependent TAFI activation participates in controlling pericellular fibrinolysis and cell invasion. Human fibrosarcoma HT1080 cells retained the ability to activate both prothrombin and plasminogen, but did not express TM. HT1080 cells mediated activation of TAFI in plasma in the presence of soluble-type TM (sTM) through cell-dependent prothrombin activation. HT1080 cells stably expressing TM (TM-HT1080) mediated plasma TAFI activation without added sTM, but HT1080 (wild-type) and Mock-transfected HT1080 cells (Mock) did not. Production of TAFIa suppressed cell-mediated plasminogen activation. Matrigel invasion by wild-type and Mock cells was enhanced two-fold by diluted plasma (10%), whereas the plasma-induced invasion of TM-HT1080 cells (65% of wild-type invasion) was lower than those of wild-type and Mock cells. Cell invasion by TM-HT1080 was partially enhanced by addition of a TAFIa/CPB inhibitor. These results suggest that TM suppresses pericellular fibrinolysis and plasma-induced tumor cell invasion, and that it is mediated, at least in part, by plasma TAFI activation.