Plasminogen activator urokinase interacts with the fusion protein and antagonizes the growth of Peste des petits ruminants virus.

Peste des petits ruminants is an acute and highly contagious disease caused by the Peste des petits ruminants virus (PPRV). Host proteins play a crucial role in viral replication. However, the effect of fusion (F) protein-interacting partners on PPRV infection is poorly understood. In this study, we found that the expression of goat plasminogen activator urokinase (PLAU) gradually decreased in a time- and dose-dependent manner in PPRV-infected goat alveolar macrophages (GAMs). Goat PLAU was subsequently identified using co-immunoprecipitation and confocal microscopy as an F protein binding partner. The overexpression of goat PLAU inhibited PPRV growth and replication, whereas silencing goat PLAU promoted viral growth and replication. Additionally, we confirmed that goat PLAU interacted with a virus-induced signaling adapter (VISA) to antagonize F-mediated VISA degradation, increasing the production of type I interferon. We also found that goat PLAU reduced the inhibition of PPRV replication in VISA-knockdown GAMs. Our results show that the host protein PLAU inhibits the growth and replication of PPRV by VISA-triggering RIG-I-like receptors and provides insight into the host protein that antagonizes PPRV immunosuppression.IMPORTANCEThe role of host proteins that interact with Peste des petits ruminants virus (PPRV) fusion (F) protein in PPRV replication is poorly understood. This study confirmed that goat plasminogen activator urokinase (PLAU) interacts with the PPRV F protein. We further discovered that goat PLAU inhibited PPRV replication by enhancing virus-induced signaling adapter (VISA) expression and reducing the ability of the F protein to degrade VISA. These findings offer insights into host resistance to viral invasion and suggest new strategies and directions for developing PPR vaccines.

[Effect of plasminogen activator urokinase receptor gene on the activation and apoptosis of neutrophil].

Objective: To investigate the effect of plasminogen activator urokinase receptor (PLAUR) gene on neutrophil activation and apoptosis in neutrophil-like cell model. Methods: Human acute myeloid leukemia cell line HL60 was cultured in vitro and induced to differentiate into neutrophil-like cells by all-trans retinoic acid (ATRA). Lentiviral vectors interfering with human PLAUR gene was constructed and transfected into neutrophil-like cells (siRNA group). The phosphate buffer saline (PBS) group (untransfected neutrophil-like cells) and normal blank control group (NC group) (neutrophil-like cells transfected with blank plasmid) were used as controls (n=3). After starvation culture and addition of interleukin-17 afterwards in these 3 groups, the expression of CD11b on the cell membrane was detected by flow cytometry, and the levels of myeloperoxide (MPO) and extracellular neutrophil traps (NETs) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA) to investigate the activation of neutrophil-like cells. The apoptosis was detected by flow cytometry with annexin V/propidium iodide (PI) double staining and the expressions of apoptosis-related proteins caspase-3, bax and bcl-2 were detected by Western blotting. Results: The expression of CD11b in siRNA group (32.37±8.17) was lower than that in PBS group (46.27±1.54) and NC group (53.07±8.14) (P<0.05) by flow cytometry. The levels of MPO and NETs (33.37±1.11, 57.69±3.03) in the supernatant of siRNA group were significantly lower than those in PBS group (41.64±2.20, 77.60±4.33) and NC group (40.84±5.11, 76.15±2.10) (P<0.05). Flow cytometry with annexin V/PI showed that the expression of apoptosis in siRNA group (20.42%±2.45%) was significantly higher than that in PBS group (11.91%±2.23%) and NC group (11.13%±2.56%) (P<0.05). The relative expression of caspase-3 protein and bax protein (0.84±0.05, 0.83±0.04) in siRNA group was significantly higher than that in PBS group (0.68±0.02, 0.63±0.08) and NC group (0.71±0.01, 0.66±0.10) (P<0.05), and the relative expression of anti-apoptosis protein bcl-2 decreased in siRNA group (0.38±0.02) than in PBS group (0.73±0.05) and NC group (0.69±0.06) (P<0.05). Conclusion: PLAUR promotes the activation of neutrophil-like cells and inhibits the apoptosis.

GPR55 Inactivation Diminishes Splenic Responses and Improves Neurological Outcomes in the Mouse Ischemia/Reperfusion Stroke Model.

Although strokes are frequent and severe, treatment options are scarce. Plasminogen activators, the only FDA-approved agents for clot treatment (tissue plasminogen activators (tPAs)), are used in a limited patient group. Moreover, there are few approaches for handling the brain's inflammatory reactions to a stroke. The orphan G protein-coupled receptor 55 (GPR55)'s connection to inflammatory processes has been recently reported; however, its role in stroke remains to be discovered. Post-stroke neuroinflammation involves the central nervous system (CNS)'s resident microglia activation and the infiltration of leukocytes from circulation into the brain. Additionally, splenic responses have been shown to be detrimental to stroke recovery. While lymphocytes enter the brain in small numbers, they regularly emerge as a very influential leukocyte subset that causes secondary inflammatory cerebral damage. However, an understanding of how this limited lymphocyte presence profoundly impacts stroke outcomes remains largely unclear. In this study, a mouse model for transient middle cerebral artery occlusion (tMCAO) was used to mimic ischemia followed by a reperfusion (IS/R) stroke. GPR55 inactivation, with a potent GPR55-specific antagonist, ML-193, starting 6 h after tMCAO or the absence of the GPR55 in mice (GPR55 knock out (GPR55ko)) resulted in a reduced infarction volume, improved neurological outcomes, and decreased splenic responses. The inhibition of GPR55 with ML-193 diminished CD4+T-cell spleen egress and attenuated CD4+T-cell brain infiltration. Additionally, ML-193 treatment resulted in an augmented number of regulatory T cells (Tregs) in the brain post-tMCAO. Our report offers documentation and the functional evaluation of GPR55 in the brain-spleen axis and lays the foundation for refining therapeutics for patients after ischemic attacks.

MicroRNA-181b attenuates lipopolysaccharide-induced inflammatory responses in pulpitis via the PLAU/AKT/NF-κB axis.

OBJECTIVE: This study aimed to investigate the role and underlying mechanisms of microRNA (miRNA)-181b in the inflammatory response in pulpitis. METHODS: Quantitative reverse-transcription polymerase chain reaction (qRT-PCR), fluorescence in situ hybridization (FISH), and immunofluorescence techniques were used to determine the miRNA-181b and urokinase-type plasminogen activator (PLAU) expression levels in inflamed human dental pulp tissues (HDPTs) and lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs). The targets of miRNA-181b were identified and confirmed using a bioinformatics analysis, RNA sequencing, and dual-luciferase gene reporter assays. The effect of miRNA-181b or PLAU on proinflammatory cytokine expression in hDPCs was examined using qRT-PCR and western blotting. RNA sequencing was conducted to examine the signaling pathways implicated in miRNA-181b-mediated pulpitis. Western blotting and qRT-PCR were used to determine the miRNA-181b /PLAU/AKT/NF-κB signaling axis in pulpitis. A rat pulpitis model was created to observe the histopathological changes in the dental pulp tissue after the topical application of miRNA-181b agomir. RESULTS: A significant decrease in miRNA-181b and an increase in PLAU were observed in HDPTs compared to the healthy controls, and these two factors showed a negative correlation. MiRNA-181b directly targeted PLAU. The miRNA-181b inhibitor resulted in a significant upregulation of IL-1ß, IL-6 and TNF-α, whereas the knockdown of PLAU reversed this proinflammatory effect. Conversely, PLAU overexpression prevented the anti-inflammatory effects of the miRNA-181b mimics. Mechanistically, miRNA-181b inhibited the AKT/NF-κB pathway by targeting PLAU. In vivo application of the miRNA-181b agomir to inflamed pulp tissue alleviated inflammation. CONCLUSION: MiRNA-181b targets PLAU, negatively regulating pro-inflammatory cytokine expression via the AKT/NF-κB signaling pathway.

The fibrinolysis renaissance.

Fibrinolysis is the system primarily responsible for removal of fibrin deposits and blood clots in the vasculature. The terminal enzyme in the pathway, plasmin, is formed from its circulating precursor, plasminogen. Fibrin is by far the most legendary substrate, but plasmin is notoriously prolific and is known to cleave many other proteins and participate in the activation of other proteolytic systems. Fibrinolysis is often overshadowed by the coagulation system and viewed as a simplistic poorer relation. However, the primordial plasminogen activators evolved alongside the complement system, approximately 70 million years before coagulation saw the light of day. It is highly likely that the plasminogen activation system evolved with its roots in primordial immunity. Almost all immune cells harbor at least one of a dozen plasminogen receptors that allow plasmin formation on the cell surface that in turn modulates immune cell behavior. Similarly, numerous pathogens express their own plasminogen activators or contain surface proteins that provide binding sites for host plasminogen. The fibrinolytic system has been harnessed for clinical medicine for many decades with the development of thrombolytic drugs and antifibrinolytic agents. Our refined understanding and appreciation of the fibrinolytic system and its alliance with infection and immunity and beyond are paving the way for new developments and interest in novel therapeutics and applications. One must ponder as to whether the nomenclature of the system hampered our understanding, by focusing on fibrin, rather than the complex myriad of interactions and substrates of the plasminogen activation system.

Management of Acute Cerebral Infarction by Intravenous Thrombolysis with Recombinant T Cell Receptor and Plasminogen Activator and Association of Emergency Nursing Route in the Prognosis.

The purpose of this study is to determine the impact and prognosis of the emergency nursing approach in conjunction with the use of recombinant T cell receptors and plasminogen activators in patients who have just had an acute stroke. In this study, 100 patients were randomly selected that were equally divided into experimental and control groups. The period of hospital admission, the results of the Montreal Cognitive Assessment (MoCA) and the Mini-mental State Examination (MMSE), the results of the Glasgow Outcome Scale (GOS), and the results of the Activities of Daily Living were all analysed before and after the intervention.. Both the amount of time it took to get a diagnosis after being admitted and the amount of time it took to receive specialised therapy after receiving a diagnosis were significantly reduced in the observation group (both P values less than 0.05). At one month after discharge, the scores of ADL, MoCA, MMSE, and GOS rose in both groups, with more significant changes occurring in the observation group (all P<0.05). This was due to the fact that ADL scores declined while scores for MoCA, MMSE, and GOS increased. The percentage of people who were disabled in the observation group was significantly lower than the percentage in the control group (P<0.05).  Including emergency, nursing might drastically reduce the time it takes for patients with acute stroke to be admitted and begin receiving specialised care.

Fibrinolytic drugs induced hemorrhage: mechanisms and solutions.

Thrombosis has been emerging as a major global life-threatening issue with high mortality and serious complications, especially in the post-COVID-19 era. Compared with the commonly used plasminogen activators thrombolytic drugs, fibrinolytic drugs are no longer heavily dependent on the patients' own plasminogen, which are poorly expressed in most patients. As a novel "direct acting" thrombolytic agent, fibrinolytic drugs are considered to have stronger thrombolytic efficacy and safety than the widely used plasminogen activators. However, the risk of their hemorrhage remains a major concern. Herein, the molecular mechanisms and solutions were summarized for the first time based on the systematic review of the latest developments, which could shed new light on the development of novel safety fibrinolytic drugs in the future.

C-terminal lysine residues enhance plasminogen activation by inducing conformational flexibility and stabilization of activator complex of staphylokinase with plasmin.

Staphylokinase (SAK), a potent fibrin-specific plasminogen activator secreted by Staphylococcus aureus, carries a pair of lysine at the carboxy-terminus that play a key role in plasminogen activation. The underlaying mechanism by which C-terminal lysins of SAK modulate its function remains unknown. This study has been undertaken to unravel role of C-terminal lysins of SAK in plasminogen activation. While deletion of C-terminal lysins (Lys135, Lys136) drastically impaired plasminogen activation by SAK, addition of lysins enhanced its catalytic activity 2-2.5-fold. Circular dichroism analysis revealed that C-terminally modified mutants of SAK carry significant changes in their beta sheets and secondary structure. Structure models and RING (residue interaction network generation) studies indicated that the deletion of lysins has conferred extensive topological alterations in SAK, disrupting vital interactions at the interface of SAK.plasmin complex, thereby leading significant impairment in its functional activity. In contrast, addition of lysins at the C-terminus enhanced its conformational flexibility, creating a stronger coupling at the interface of SAK.plasmin complex and making it more efficient for plasminogen activation. Taken together, these studies provided new insights on the role of C-terminal lysins in establishment of precise intermolecular interactions of SAK with the plasmin for the optimal function of activator complex.

Comprehensive analysis of the diagnostic and therapeutic value of the hypoxia-related gene PLAUR in the progression of atherosclerosis.

Atherosclerosis (AS) is a major contributor to a variety of negative clinical outcomes, including stroke and myocardial infarction. However, the role and therapeutic value of hypoxia-related genes in AS development has been less discussed. In this study, Plasminogen activator, urokinase receptor (PLAUR) was identified as an effective diagnostic marker for AS lesion progression by combining WGCNA and random forest algorithm. We validated the stability of the diagnostic value on multiple external datasets including humans and mice. We identified a significant correlation between PLAUR expression and lesion progression. We mined multiple single cell-RNA sequencing (sc-RNA seq) data to nominate macrophage as the key cell cluster for PLAUR mediated lesion progression. We combined cross-validation results from multiple databases to predict that HCG17-hsa-miR-424-5p-HIF1A, a competitive endogenous RNA (ceRNA) network, may regulate hypoxia inducible factor 1 subunit alpha (HIF1A) expression. The DrugMatrix database was used to predict alprazolam, valsartan, biotin A, lignocaine, and curcumin as potential drugs to delay lesion progression by antagonizing PLAUR, and AutoDock was used to verify the binding ability of drugs and PLAUR. Overall, this study provides the first systematic identification of the diagnostic and therapeutic value of PLAUR in AS and offers multiple treatment options with potential applications.

m6A Methyltransferase, WTAP, Promotes Cancer Progression in Laryngeal Squamous Cell Carcinoma by Regulating PLAU Stability.

OBJECTIVE: Laryngeal squamous cell carcinoma (LSCC) is a malignancy originating from laryngeal squamous cell lesions. Wilm's tumor 1-associated protein (WTAP)-mediated N6-methyladenosine (m6A) modification has been verified to stimulate the progression of numerous cancers, except for LSCC. This study was aimed at exploring the role of WTAP and its mechanism of action in LSCC. METHODS: The expression of WTAP and plasminogen activator urokinase (PLAU) mRNAs in LSCC tissues and cells was quantified using qRT-PCR. Western blotting was performed to estimate PLAU levels in LSCC cells. The relationship between WTAP and PLAU was ascertained using luciferase reporter and methylated-RNA immunoprecipitation (Me-RIP) assays. Functionally, the interaction of WTAP with PLAU in LSCC cells was investigated using CCK-8, EdU, and Transwell assays. RESULTS: The expression of WTAP and PLAU was increased in LSCC, and was positively correlated. WTAP regulated PLAU stability in an m6A-dependent manner. WTAP deficiency suppressed the migration, invasion, and proliferation of LSCC cells. Overexpression of PLAU rescued the phenotype induced by WTAP knockdown in vitro. CONCLUSIONS: These results indicate that WTAP mediates the m6A modification of PLAU to accelerate the growth, migration, and invasion of cells in LSCC. To our knowledge, this is the first report to clarify the functions of WTAP in LSCC and the underlying mechanisms in detail. Based on these findings, we suggest that WTAP may serve as a therapeutic target for LSCC.

PLAU contributes to the development of cholangiocarcinoma via activating NF-κB signaling pathway.

Cholangiocarcinoma (CCA) is a type of epithelial cancer with poor outcomes and late diagnosis. Accumulating evidence has demonstrated the promoting role of plasminogen activator, urokinase (PLAU) in several tumor types, while its function in CCA is largely unknown. The expression of PLAU in CCA was determined by data from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) database and further confirmed in human tissues using immunohistochemical (IHC) staining. Moreover, PLAU-silencing CCA cell models were constructed for subsequent functional assays in vitro and in vivo. PLAU expression in CCA was significantly higher than that in normal tissues. High PLAU expression was positively correlated with poor patients' survival. PLAU knockdown remarkably suppressed proliferation and migration of CCA cells, whereas enhanced apoptosis. Consistently, tumor growth in mice injected with PLAU-silencing CCA cells was also impaired. Furthermore, we revealed that the activation of NF-κB signaling was required for PLAU-induced malignant phenotypes of CCA cells. Inhibiting the high expression of PLAU in CCA may be a potential entry point for targeted therapy in CCA patient.

Extracellular vesicle-derived LINC00511 promotes glycolysis and mitochondrial oxidative phosphorylation of pancreatic cancer through macrophage polarization by microRNA-193a-3p-dependent regulation of plasminogen activator urokinase.

OBJECTIVE: The involvement of tumor-derived extracellular vesicles (EVs) in macrophage polarization has been reported. In our present study, we tried to discuss the regulatory role of LINC00511 encapsulated in pancreatic cancer (PCa) cell-derived EVs in the development and progression of PCa. METHODS: EVs from PCa cell line BxPC-3 culture medium were collected and subsequently identified by electron microscopy and nanoparticle tracking analysis. The expression pattern of LINC00511 in PCa cell-derived EVs was determined. The interaction among LINC00511, microRNA-193a-3p, and plasminogen activator urokinase (PLAU) was explored. After co-culture of PCa cell-derived EVs with macrophages, the regulatory roles of LINC00511 in macrophage polarization, PCa cell functions, glucose consumption, lactate production, glycolysis, and mitochondrial oxidative phosphorylation were investigated. RESULTS: PCa cell line BxPC-3 had highly expressed LINC00511 and LINC00511 could be internalized by macrophages. LINC00511 affected macrophage polarization through miR-193a-3p-dependent regulation of PLAU expression. Besides, EV-derived LINC00511 accelerated glycolysis and promoted mitochondrial oxidative phosphorylation of PCa cells through macrophage polarization, thus inducing invasion and migration of PCa cells. CONCLUSION: LINC00511 encapsulated in PCa cell-derived EVs facilitates glycolysis of PCa cells through regulation of macrophage polarization in the tumor microenvironment.

Proteomics analysis uncovers plasminogen activator PLAU as a target of the STING pathway for suppression of cancer cell migration and invasion.

The stimulator of interferon genes (STING) pathway is vital for immune defense against pathogen invasion and cancer. Although ample evidence substantiates that the STING signaling pathway plays an essential role in various cancers via cytokines, no comprehensive investigation of secretory proteins regulated by the STING pathway has been conducted hitherto. Herein, we identify 24 secretory proteins significantly regulated by the STING signaling pathway through quantitative proteomics. Mechanistic analyses reveal that STING activation inhibits the translation of urokinase-type plasminogen activator (PLAU) via the STING-PERK-eIF2α signaling axis. PLAU is highly expressed in a variety of cancers and promotes the migration and invasion of cancer cells. Notably, the activation of STING inhibits cancer cell migration and invasion by suppressing PLAU. Collectively, these results provide novel insights into the anticancer mechanism of the STING pathway, offering a theoretical basis for precision therapy for this patient population.

Immunohistochemical study of the plasminogen activator system in benign epithelial odontogenic lesions.

The aim of this study was to analyze and compare the immunohistochemical expression of plasminogen activator system (PAS) proteins (uPA, uPAR, and PAI-1) in ameloblastomas (AMBs), odontogenic keratocysts (OKCs), and dental follicles (DFs) representing normal odontogenic tissue, as well as to investigate possible correlations between these proteins. Twenty AMBs, 20 OKCs, and 10 DFs were selected for immunohistochemical analysis. In each case, the immunoexpression of uPA, uPAR, and PAI-1 was evaluated semiquantitatively based on the percentage of positivity in odontogenic epithelial and connective tissue cells. The epithelial immunoexpression of uPA was significantly lower in AMBs when compared to OKCs (p = 0.001) and DFs (p = 0.029). Significantly higher epithelial immunostaining for uPAR was observed in AMBs when compared to OKCs (p < 0.001). There were no significant differences in the epithelial immunoexpression of PAI-1 between AMBs and OKCs (p = 1.000). The correlations found for the expression of the studied proteins were not statistically significant (p > 0.05). However, the epithelial and connective tissue expressions of uPAR have a strong positive and statistically significant correlation in AMBs. The present results suggest that uPA is involved in the pathogenesis of OKCs and that uPAR may participate in tumorigenesis in AMBs. The high percentage of PAI-1-positive cells suggests a possible role for this protein in the development of AMBs and OKCs. Furthermore, the studied proteins do not seem to act synergistically in AMBs, OKCs, and DFs.

TCF7L2 promotes anoikis resistance and metastasis of gastric cancer by transcriptionally activating PLAUR.

Gastric cancer (GC) is the most common gastrointestinal malignant tumor, and distant metastasis is a critical factor in the prognosis of patients with GC. Understanding the mechanism of GC metastasis will help improve patient prognosis. Studies have confirmed that urokinase-type plasminogen activator receptor (PLAUR) promotes GC metastasis; however, its relationship with anoikis resistance and associated mechanisms remains unclear. In this study, we demonstrated that PLAUR promotes the anoikis resistance and metastasis of GC cells and identified transcription Factor 7 Like 2 (TCF7L2) as an important transcriptional regulator of PLAUR. We also revealed that TCF7L2 is highly expressed in GC and promotes the anoikis resistance and metastasis of GC cells. Moreover, we found that TCF7L2 transcription activates PLAUR. Finally, we confirmed that TCF7L2 is an independent risk factor for poor prognosis of patients with GC. Our results show that TCF7L2 and PLAUR are candidate targets for developing therapeutic strategies for GC metastasis.

Plasminogen and plasmin can bind to human T cells and generate truncated CCL21 that increases dendritic cell chemotactic responses.

Plasmin is a broad-spectrum protease and therefore needs to be tightly regulated. Active plasmin is formed from plasminogen, which is found in high concentrations in the blood and is converted by the plasminogen activators. In the circulation, high levels of α2-antiplasmin rapidly and efficiently inhibit plasmin activity. Certain myeloid immune cells have been shown to bind plasmin and plasminogen on their cell surface via proteins that bind to the plasmin(ogen) kringle domains. Our earlier work showed that T cells can activate plasmin but that they do not themselves express plasminogen. Here, we demonstrate that T cells express several known plasminogen receptors and that they bind plasminogen on their cell surface. We show T cell-bound plasminogen was converted to plasmin by plasminogen activators upon T cell activation. To examine functional consequences of plasmin generation by activated T cells, we investigated its effect on the chemokine, C-C motif chemokine ligand 21 (CCL21). Video microscopy and Western blotting confirmed that plasmin bound by human T cells cleaves CCL21 and increases the chemotactic response of monocyte-derived dendritic cells toward higher CCL21 concentrations along the concentration gradient by increasing their directional migration and track straightness. These results demonstrate how migrating T cells and potentially other activated immune cells may co-opt a powerful proteolytic system from the plasma toward immune processes in the peripheral tissues, where α2-antiplasmin is more likely to be absent. We propose that plasminogen bound to migrating immune cells may strongly modulate chemokine responses in peripheral tissues.

PgtE Enzyme of Salmonella enterica Shares the Similar Biological Roles to Plasminogen Activator (Pla) in Interacting With DEC-205 (CD205), and Enhancing Host Dissemination and Infectivity by Yersinia pestis.

Yersinia pestis, the cause of plague, is a newly evolved Gram-negative bacterium. Through the acquisition of the plasminogen activator (Pla), Y. pestis gained the means to rapidly disseminate throughout its mammalian hosts. It was suggested that Y. pestis utilizes Pla to interact with the DEC-205 (CD205) receptor on antigen-presenting cells (APCs) to initiate host dissemination and infection. However, the evolutionary origin of Pla has not been fully elucidated. The PgtE enzyme of Salmonella enterica, involved in host dissemination, shows sequence similarity with the Y. pestis Pla. In this study, we demonstrated that both Escherichia coli K-12 and Y. pestis bacteria expressing the PgtE-protein were able to interact with primary alveolar macrophages and DEC-205-transfected CHO cells. The interaction between PgtE-expressing bacteria and DEC-205-expressing transfectants could be inhibited by the application of an anti-DEC-205 antibody. Moreover, PgtE-expressing Y. pestis partially re-gained the ability to promote host dissemination and infection. In conclusion, the DEC-205-PgtE interaction plays a role in promoting the dissemination and infection of Y. pestis, suggesting that Pla and the PgtE of S. enterica might share a common evolutionary origin.

Production, purification and characterization of a novel antithrombotic and anticoagulant serine protease from food grade microorganism Neurospora crassa.

A novel thrombolytic enzyme was produced by food grade microorganism Neurospora crassa using agro-industrial by-products as substrates. Process parameters were optimized using Plackett-Berman and Box-Benhken design. Under the optimized fermentation conditions, high fibrinolytic activity of 403.59 U/mL was obtained. It was purified with a specific activity of 3572.4 U/mg by ammonium sulfate precipitation and SP Sepharose chromatography. The molecular weight of the enzyme was approximately 32 kDa. It exhibited maximum activity at 40 °C and pH 7.4. Its activity was enhanced by Cu2+, Na+, Zn2+, and completely inhibited by phenylmethanesulfonyl fluoride, soybean trypsin inhibitor, aprotinin, which indicates it could be a serine protease. The enzyme could degrade fibrin clot directly without the need of plasminogen activator, and effectively cleaved Aα, Bß, γ chains of fibrinogen. It could inhibit the formation of blood clots in vitro and acts as an anticoagulant. Compared to heparin the purified enzyme showed extended anticoagulant activity. Blood clots were dissolved effectively and dissolution rate was increased with time. Based on these results, this novel enzyme has the potential to be developed as a thrombolytic agent.

Identification, purification, and pharmacological activity analysis of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAα1) expressed in transgenic rabbit mammary glands.

Desmodus rotundus plasminogen activator alpha 1(DSPAα1) is a thrombolytic protein with advantages, such as a long half-life, high accuracy and specificity for thrombolysis, wide therapeutic window, and no neurotoxicity. To date, DSPAα1 has only been expressed in the Chinese hamster ovary, insect cells, transgenic tobacco plants, and Pichia pastoris. To the best of our knowledge, we are the first to report the expression of DSPAα1 in transgenic rabbit mammary glands, extract the product, and analyze its pharmacology activity. An efficient mammary gland-specific expression vector pCL25/DSPAα1 was transferred to prokaryotic zygotes in rabbits by microinjection to generate six DSPAα1 transgenic rabbits. The recombinant DSPAα1 (rDSPAα1) expression in transgenic rabbit milk was 1.19 ± 0.26 mg/mL. The rDSPAα1 purification protocol included pretreatment, ammonium sulfate precipitation, benzamidine affinity chromatography, cation exchange chromatography, and Cibacron blue affinity chromatography; approximately 98% purity was achieved using gel electrophoresis. According to sequencing results, the primary structure of rDSPAα1 was consistent with the theoretical design sequence, and its molecular weight was consistent with that of the natural protein. N-terminal sequencing results indicated rDSPAα1 to be a mature protein, as the goat signal peptide sequence of the expression vector was no longer detected. The fibrinolytic activity of rDSPAα1 was estimated to be 773,333 IU/mg. Fibrin-agarose plate assay and in vitro rat blood clot degradation assay showed that rDSPAα1 had strong thrombolytic activity. In conclusion, we report recombinant DSPAα1 with high thrombolytic activity expressed in transgenic rabbit mammary glands.

Astrocyte tissue plasminogen activator expression during brain development and its role in pyramidal neuron neurite outgrowth.

The serine protease tissue plasminogen activator (tPA), encoded by the gene Plat, exerts a wide range of proteolysis-dependent and proteolysis-independent functions. In the developing brain, tPA is involved in neuronal development via the modulation of the proteolytic degradation of the extracellular matrix (ECM). Both lack of and excessive tPA are associated with neurodevelopmental disorders and with brain pathology. Astrocytes play a major role in neurite outgrowth of developing neurons as they are major producers of ECM proteins and ECM proteases. In this study we investigated the expression of Plat in developing and mature hippocampal and cortical astrocytes of Aldh1l1-EGFP-Rpl10a mice in vivo following Translating Ribosome Affinity Purification (TRAP) and the role of tPA in modulating astrocyte-mediated neurite outgrowth in an in vitro astrocyte-neuron co-culture system. We show that Plat is highly enriched in astrocytes in the developing, but not in the mature, hippocampus and cortex. Both the silencing of tPA expression in astrocytes and astrocyte exposure to recombinant tPA reduce neuritogenesis in co-cultured hippocampal neurons. These results suggest that astrocyte tPA is involved in modulating neuronal development and that tight control of astrocyte tPA expression is important for normal neuronal development, with both experimentally elevated and reduced levels of this proteolytic enzyme impairing neurite outgrowth. These results are consistent with the hypothesis that the ECM, by serving as adhesive substrate, enables neurite outgrowth, but that controlled proteolysis of the ECM is needed for growth cone advancement.