Activator uPA Research Articles

Gene expression of fibrinolytic markers in coronary thrombi

BackgroundThe fibrinolytic system plays an important role in coronary artery atherothrombosis, and especially circulating plasminogen-activator inhibitor (PAI) type 1 (PAI-1) associates with increased mortality, infarct size and heart failure in patients with myocardial infarction (MI). In a cross-sectional study, we aimed to study whether genes encoding tissue plasminogen activator (tPA), urinary-type plasminogen activator (uPA), PAI-1 and PAI-2 are expressed in coronary thrombi from acute ST-elevation MI (STEMI) patients. Any relations to myocardial injury measured by peak troponin T, time from symptom onset to Percutaneous Coronary Intervention (PCI), and to different cell types present in the thrombi were also explored.MethodsIntracoronary thrombi were aspirated from 33 STEMI patients treated with primary PCI. The thrombi were snap-frozen for gene expression analyses, relatively quantified by RT PCR. Peripheral blood samples were drawn. Correlations were performed by Spearmans rho.ResultsThe genes were present in 74–94% of the thrombi. Median peak troponin T was 3434 μ/L and median ischemic time 152 min. There were no significant correlations between the measured genes and troponin T, or ischemic time. Genes encoding tPA, u-PA, PAI-1 and PAI-2 all correlated significantly to the presence of monocytes/macrophages (CD68) in the thrombi (p = 0.028, p < 0.001, p = 0.003, p < 0.001). PAI-1 and PAI-2 also correlated to endothelial cells (CD31) (p = 0.002, p = 0.016). uPA associated with neutrophil granulocytes (CD 66b) (p = 0.019).ConclusionGenes encoding tPA, uPA, PAI-1 and PAI-2 were highly expressed in human coronary thrombi from STEMI patients, indicating fibrinolytic regulators playing active roles in the thrombi, although not related to myocardial injury. All markers related to the presence of monocytes/macrophages, indicating connection to local inflammatory cells.Trial registrationThe study is registered at clinicaltrials.gov with identification number NCT02746822.

Inhibition of Human Urokinase-Type Plasminogen Activator (uPA) Enzyme Activity and Receptor Binding by DNA Aptamers as Potential Therapeutics through Binding to the Different Forms of uPA.

Urokinase-type plasminogen activator is widely discussed as a marker for cancer prognosis and diagnosis and as a target for cancer therapies. Together with its receptor, uPA plays an important role in tumorigenesis, tumor progression and metastasis. In the present study, systematic evolution of ligands by exponential enrichment (SELEX) was used to select single-stranded DNA aptamers targeting different forms of human uPA. Selected aptamers allowed the distinction between HMW-uPA and LMW-uPA, and therefore, presumably, have different binding regions. Here, uPAapt-02-FR showed highly affine binding with a KD of 0.7 nM for HMW-uPA and 21 nM for LMW-uPA and was also able to bind to pro-uPA with a KD of 14 nM. Furthermore, no cross-reactivity to mouse uPA or tissue-type plasminogen activator (tPA) was measured, demonstrating high specificity. Suppression of the catalytic activity of uPA and inhibition of uPAR-binding could be demonstrated through binding with different aptamers and several of their truncated variants. Since RNA aptamers are already known to inhibit uPA-uPAR binding and other pathological functions of the uPA system, these aptamers represent a novel, promising tool not only for detection of uPA but also for interfering with the pathological functions of the uPA system by additionally inhibiting uPA activity.

MicroRNA 449a can Attenuate Protective Effect of Urokinase Against Pulmonary Embolism.

Acute pulmonary embolism (APE) is a disabling diseases with high incidence rate and mortality rate. Although with high specificity, D-Dimer lacks specificity to assess APE, hence additional diagnostic and prognostic biomarkers are necessary. APE is widely treated with serine protease urokinase or urokinase-type plasminogen activator (uPA), which act as a catalyst for conversion of plasminogen to plasmin to resolve blood clots. However, it is unknown the role of differential expression of microRNAs (miRNAs) in protective effect of uPA against APE. Hence, we performed miRNA profiling in a hypoxia/reoxygenation (H/R) model of bronchial epithelial BEAS-2B cells in vitro and a APE mice model in vivo. Our analysis revealed that miR-34a-5p, miR-324-5p, miR-331-3p are upregulated with H/R or APE induction, whereas miR-429, miR-491-5p, and miR-449a are downregulated. The differential expression of the miRNAs was attenuated to levels comparable to control by treatment with uPA both in vitro and in vivo. In situ target prediction and analysis of potential functions of the target genes showed that the enrichment of biological processes and pathways were related to cell growth, proliferation, and inflammation. Ectopic overexpression of miR-449a using a mimic completely attenuated the protective effect of uPA in the H/R model in vitro. These results provide a group of miRNAs that could be used as markers, and the modulation of these miRNAs might have potential therapeutic benefits in patients with APE, which need to be validated in additional studies in humans.

Insitu hybridization for the assessment of urokinase plasminogen activator and plasminogen activator inhibitor type‑1 informalin‑fixed paraffin‑embedded breast cancer specimens.

Urokinase plasminogen activator (uPA) and its inhibitor, plasminogen activator inhibitor type1 (PAI‑1), have been reported as prognostic and predictive biomarkers in breast cancer, particularly in patients with node‑negative tumors. uPA and PAI‑1 expression levels classify patients into a poor‑prognosis subgroup, requiring adjuvant chemotherapy and a favorable‑prognosis subgroup, which can be considered for de‑escalation. However, the clinical use of these two biomarkers remains limited, since fresh‑frozen/fresh tumor samples are currently required for their quantification. The aim of the present study was to compare PLAU and SERPINE1 mRNA expression levels (corresponding to uPA and PAI‑1 proteins, respectively), assessed using insitu hybridization in 83 formalin‑fixed paraffin‑embedded (FFPE) breast tumor samples, with uPA and PAI‑1 protein expression assessed using immunometric assay with paired fresh‑frozen breast cancer samples. The results from the two methods significantly correlated as regards uPA quantification; however, >30% of the samples were discordant, according to the clinically validated threshold. Concordance between the two analytical methods was less prominent for PAI‑1 protein and SERPINE1 mRNA. Taken together, the results of the present study indicate that although PLAU and SERPINE1 mRNA may be reliably detected in FFPE samples using insitu hybridization, this technology cannot be used as a substitute for the replacement of the immunometric assay‑derived quantification on fresh‑frozen samples.

Nasal symbiont Staphylococcus epidermidis restricts the cellular entry of influenza virus into the nasal epithelium

Our recent study presented that human nasal commensal Staphylococcus epidermidis could potentiate antiviral immunity in the nasal mucosa through interferon-related innate responses. Here, we found that human nasal commensal S. epidermidis promoted protease–protease inhibitor balance in favor of the host and prevented influenza A virus (IAV) replication in the nasal mucosa and lungs. A relatively higher induction of Serpine1 exhibited in S. epidermidis-inoculated nasal epithelium and S. epidermidis-induced Serpine1 significantly decreased the expression of serine proteases. Furthermore, the transcription of urokinase plasminogen activator (uPA) and Serpine1 was biologically relevant in S. epidermidis-inoculated nasal epithelium, and the induction of uPA might be related to the sequential increase of Serpine1 in human nasal epithelium. Our findings reveal that human nasal commensal S. epidermidis manipulates the cellular environment lacking serine proteases in the nasal epithelium through Serpine1 induction and disturbs IAV spread to the lungs at the level of the nasal mucosa.

Antimetastatic Potential of Quercetin Analogues with Improved Pharmacokinetic Profile: A Pharmacoinformatic Preliminary Study.

Urokinase-type plasminogen activator (uPA) system is a crucial pathway for tumor invasion and metastasis. Recently, multiple anticancer effects of quercetin have been described, including inhibitory activity against uPA. However, the clinical use of this flavonoid has been limited due to its low oral bioavailability. The objectives of the study were to assess the antimetastatic potential of quercetin analogues by analyzing their binding affinity for uPA, and to select the compounds with improved pharmacological profiles. Binding affinities of structural analogues of quercetin to uPA receptor were determined by molecular docking analysis using Molegro Virtual Docker software, and molecular descriptors relevant for estimating pharmacological profile were calculated from ligand structures using computational models. Among 44 quercetin analogues, only one quercetin analogue (3,6,2',4',5'-pentahydroxyflavone) was found to possess higher aqueous solubility and membrane permeability, and stronger affinity for uPA than quercetin, which makes it a potential lead compound for anticancer drug development. Like quercetin, this compound has five hydroxyl groups, but arranged differently, which contributes to the higher aqueous solubility and higher amphiphilic moment in comparison to quercetin. Since membrane permeability is not recognized as the limiting factor for quercetin absorption, analogues with higher aqueous solubility and retained or stronger uPA inhibitory activity should also be further experimentally validated for potential therapeutic use. Identified quercetin analogues with better physicochemical and pharmacological properties have a high potential to succeed in later stages of research in biological systems as potential anticancer agents with antimetastatic activity.

In Vitro and In Vivo Effects of the Urokinase Plasminogen Activator Inhibitor WX-340 on Anaplastic Thyroid Cancer Cell Lines.

Increased expression of the urokinase-type plasminogen activator (uPA) system is associated with tumor invasion, neo-angiogenesis, and metastatic spread, and has been shown to positively correlate with a poor prognosis in several cancer types, including thyroid carcinomas. In recent years, several uPA inhibitors were found to have anticancer effects in preclinical studies and in some phase II clinical trials, which prompted us to evaluate uPA as a potential therapeutic target for the treatment of patients affected by the most aggressive form of thyroid cancer, the anaplastic thyroid carcinoma (ATC). In this study, we evaluated the in vitro and in vivo effects of WX-340, a highly specific and selective uPA inhibitor, on two ATC-derived cell lines, CAL-62 and BHT-101. The results obtained indicated that WX-340 was able to reduce cell adhesion and invasiveness in a dose-dependent manner in both cell lines. In addition, WX-340 increased uPA receptor (uPAR) protein levels without affecting its plasma membrane concentration. However, this compound was unable to significantly reduce ATC growth in a xenograft model, indicating that uPA inhibition alone may not have the expected therapeutic effects.

Renal and Inflammatory Proteins as Biomarkers of Diabetic Kidney Disease and Lupus Nephritis.

Current methods for differentiation of kidney disease types are unspecific and may be invasive. Thus, there is a need for development of new biomarkers of kidney disorders that are specific and less invasive. In this study, we analyzed serum samples of diabetic kidney disease (DKD) and lupus nephritis (LN) patients to identify biomarkers of these two disorders. Serum samples were analyzed by Simple Plex assays. We calculated the area under the curve (AUC) as well as receiver operating characteristics (ROC) to obtain the sensitivity and specificity and other biomarker-related variables of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), interleukin- (IL-) 18, Lipocalin-2/NGAL, epidermal growth factor (EGF), u-Plasminogen Activator (uPA), and C-reactive protein (CRP) as potential biomarkers. Protein levels of ASC, IL-18, EGF, and Lipocalin-2/NGAL were higher in DKD and LN patients when compared to controls, whereas only uPA was elevated in DKD patients and CRP in LN patients. As determined by the AUC, of the six analytes studied, EGF (AUC = 0.9935), Lipocalin-2/NGAL (0.9554), ASC (0.7666), and uPA (0.7522) are reliable biomarkers of DKD, whereas EGF (1.000), Lipocalin-2/NGAL (0.9412), uPA (0.7443), and IL-18 (0.7384) are more reliable for LN. The biomarkers analyzed can differentiate between healthy and affected individuals. However, there was no difference between the levels of these biomarkers in DKD vs LN. Thus, although these biomarkers cannot be used to categorize patients between DKD and LN, they are useful as biomarkers of renal pathology.

Discovery of a novel inhibitor against urokinase-type plasminogen activator, a potential enzyme with a role in atherosclerotic plaque instability

The buildup of lipids, cholesterol, and other substances in and on the walls of the arteries is known as atherosclerosis and deposition is known as atherosclerotic plaque. Urokinase-type plasminogen activator (uPA) has multiple roles in the atherosclerotic plaque formation and even work simultaneously in making the atherosclerotic plaque unstable. Extracellular matrix plays a major role in the plaque remodeling and rapture. In this study, we have accessed that a higher interaction was observed in the molecular interaction score for uPA with ZINC380065722 having a GOLD fitness score of about 67.60, which is much higher as compared to the known standard inhibitor UK 122 which has reported an interaction score of 59.14. Ser217 and Asp192 are found to be the key amino acid residues in almost all the interactions. Protein frustration analysis has shown that these amino acid residues play a crucial role in the retention of the active pocket conformation and any mutation of these two residues can causes serious decrease in the overall function of the protein. It was observed that the molecule ZINC380065722 remained bound to the protein till 100 ns of simulation time. The average SASA for the apo-uPA and uPA-ligand complex was found to be stable. The network of hydrogen bonds for the intramolecular protein secondary structure and with the solvent system for the apo-protein and the uPA-ligand complex was found to be consistent. Communicated by Ramaswamy H. Sarma

Urokinase plasminogen activator induces epithelial-mesenchymal and metastasis of pancreatic cancer through plasmin/MMP14/TGF-β axis, which is inhibited by 4-acetyl-antroquinonol B treatment

BackgroundThe current standard therapy for metastatic pancreatic cancer is ineffective, necessitating a new treatment approach for prognosis improvement. The urokinase-plasmin activator (uPA) is a critical factor in epithelial-mesenchymal transition (EMT) and cancer metastasis, but its underlying mechanisms in pancreatic cancer remains elusive. MethodsWe investigated uPA expression in our pancreatic cancer cohort. A bioinformatics approach was used to further determine the role of uPA in pancreatic cancer. We employed MiaPaCa-2 and PANC-1 cell lines to investigate how uPA regulates EMT and metastasis in pancreatic cancer and present a novel approach aimed at inhibiting uPA in pancreatic cancer. ResultsWe observed that higher uPA mRNA expression was significantly associated with overall-poor survival and progression-free survival in pancreatic cancer. uPA was highly expressed in tumor tissue. Gene set enrichment analysis revealed a positive association between uPA mRNA expression and EMT and transforming growth factor β (TGF-β) signaling pathways. Moreover, shRNA-mediated uPA gene knockdown reduced plasmin, MMP14, and TGF-β activation, leading to the inhibition of PANC-1 cells’ EMT marker expression, migration, invasion, and cell viability. Notably, 4-acetyl-antroquinonol B (4-AAQB) treatment suppressed MiaPaCa-2 and PANC-1 cell migratory and invasive abilities by inhibiting the uPA/MMP14/TGF-β axis through upregulation of miR-181d-5p. In the xenograft mouse model of orthotropic pancreatic cancer, 4-AAQB treatment has reduced tumor growth and metastasis rate by deactivating uPA and improving the survival of the mice model. ConclusionAccordingly, to extent of our knowledge and previous studies, we demonstrated that 4-AAQB is an anti Pan-Cancer drug, and may inhibit pancreatic cancer EMT and metastasis and serve as a new therapeutic approach for patients with late-stage pancreatic cancer.

The uPA System Differentially Alters Fibroblast Fate and Profibrotic Ability in Skin Fibrosis.

Skin fibrosis is a common pathological feature of various diseases, and few treatment strategies are available because of the molecular pathogenesis is poorly understood. The urokinase-type plasminogen activator (uPA) system is the major serine protease system, and its components uPA, urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1(PAI-1) are widely upregulated in fibrotic diseases, including hypertrophic scars, keloids, and scleroderma. Here, we found that the successful binding of uPA and uPAR activates the downstream peroxisome proliferator-activated receptor (PPAR) signalling pathway to reduce the proliferation, migration, and contraction of disease-derived fibroblasts, contributing to the alleviation of skin fibrosis. However, increased or robust upregulation of the inhibitor PAI-1 inhibits these effects, suggesting of the involvement of PAI-1 in skin fibrosis. Subsequent in vivo studies showed that uPAR inhibitors increased skin fibrosis in mouse models, while uPA agonists and PAI-1 inhibitors reversed these effects. Our findings demonstrate a novel role for the uPA system and highlights its relationships with skin fibrosis, thereby suggesting new therapeutic approaches targeting the uPA system.

Induction of IDO1 and Kynurenine by Serine Proteases Subtilisin, Prostate Specific Antigen, CD26 and HtrA: A New Form of Immunosuppression?

Several serine proteases have been linked to autoimmune disorders and tumour initiation although the mechanisms are not fully understood. Activation of the kynurenine pathway enzyme indoleamine-2,3-dioxygenase (IDO1) modulates cellular activity in the brain, tolerogenesis in the immune system and is a major checkpoint in cancer development. We now report that IDO1 mRNA and IDO1 protein expression (generating kynurenine) are induced in human monocyte-derived macrophages by several chymotryptic serine proteases with direct links to tumorigenesis, including Prostate Specific Antigen (PSA), CD26 (Dipeptidyl-peptidase-4, CD26/DPP-4), High Temperature Requirement protein-A (HtrA), and the bacterial virulence factor subtilisin. These proteases also induce expression of the pro-inflammatory cytokine genes IL1B and IL6. Other serine proteases tested: bacterial glu-C endopeptidase and mammalian Pro-protein Convertase Subtilase-Kexin-3 (PCSK3, furin), urokinase plasminogen activator (uPA), cathepsin G or neutrophil elastase, did not induce IDO1, indicating that the reported effects are not a general property of all serine proteases. The results represent a novel mechanism of activating immunosuppressive IDO1 and inducing kynurenine generation which, together with the production of inflammatory cytokines, would contribute to tumour initiation and progression, providing a new target for drug development. In addition, the proteasomal S20 serine protease inhibitor carfilzomib, used in the treatment of myeloma, prevented the induction of IDO1 and cytokine gene expression, potentially contributing to its clinical anti-cancer activity.

Elevated expression of urokinase plasminogen activator in rodent models and patients with cerebral amyloid angiopathy.

The aim of this work is to study the association of urokinase plasminogen activator (uPA) with development and progression of cerebral amyloid angiopathy (CAA). We studied the expression of uPA mRNA by quantitative polymerase chain reaction (qPCR) and co-localisation of uPA with amyloid-β (Aβ) using immunohistochemistry in the cerebral vasculature of rTg-DI rats compared with wild-type (WT) rats and in a sporadic CAA (sCAA) patient and control subject using immunohistochemistry. Cerebrospinal fluid (CSF) uPA levels were measured in rTg-DI and WT rats and in two separate cohorts of sCAA and Dutch-type hereditary CAA (D-CAA) patients and controls, using enzyme-linked immunosorbent assays (ELISA). The presence of uPA was clearly detected in the cerebral vasculature of rTg-DI rats and an sCAA patient but not in WT rats or a non-CAA human control. uPA expression was highly co-localised with microvascular Aβ deposits. In rTg-DI rats, uPA mRNA expression was highly elevated at 3months of age (coinciding with the emergence of microvascular Aβ deposition) and sustained up to 12 months of age (with severe microvascular CAA deposition) compared with WT rats. CSF uPA levels were elevated in rTg-DI rats compared with WT rats (p = 0.03), and in sCAA patients compared with controls (after adjustment for age of subjects, p = 0.05 and p = 0.03). No differences in CSF uPA levels were found between asymptomatic and symptomatic D-CAA patients and their respective controls (after age-adjustment, p = 0.09 and p = 0.44). Increased cerebrovascular expression of uPA in CAA correlates with increased quantities of CSF uPA in rTg-DI rats and human CAA patients, suggesting that uPA could serve as a biomarker for CAA.

PET in vivo generators 134Ce and 140Nd on an internalizing monoclonal antibody probe

The in vivo-generator radionuclides 140Nd (t1/2 = 3.4 d) and 134Ce (t1/2 = 3.2 d) were used to trace a urokinase-type plasminogen activator (uPA)-targeting mouse monoclonal antibody, ATN-291, in U87 MG xenograft tumor-bearing mice. ATN-291 is known to internalize on the uPA/uPA-receptor pair, making it an appropriate targeting vector for investigating the fate of in vivo generator daughters on internalizing probes. Ante-mortem and post-mortem PET imaging at 120 h post-injection gave no indication of redistribution of the positron emitting daughter nuclides 134La and 140Pr from tumor tissue (p > 0.5). The lack of redistribution indicates that the parent radionuclides 134Ce and 140Nd could be considered as long-lived PET-diagnostic matches to therapeutic radionuclides like 177Lu, 161Tb and 225Ac when internalizing bioconjugates are employed.

Screening bioactive compounds from Danggui-shaoyao-san for treating sodium retention in nephrotic syndrome using bio-affinity ultrafiltration

Ethnopharmacological relevanceDanggui-shaoyao-san (DSS), a representative formula of Traditional Chinese Medicine (TCM) for promoting blood circulation and diuresis (Huo-Xue-Li-Shui) therapy, has been used to clinically nephrotic syndrome (NS) and relieve nephrotic edema. Aim of the studyTo explore the effects and mechanisms of DSS in improving sodium retention and to identify the bioactive compounds from DSS. Materials and methodsDSS prescriptions were disassembled into Yangxue-Huoxue (YXHX) and Jianpi-Lishui (JPLS). A nephrotic rat model was induced with puromycin aminonucleoside (PAN), and the effects on urinary sodium excretion, urinary plasmin(gen) content, and plasmin activity of DSS, YXHX, and JPLS extracts were assessed. The inhibitory effects on urokinase-type plasminogen activator (uPA) and plasmin activity of extracts were evaluated in vitro. Bio-affinity ultrafiltration and high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (BAU-UPLC-Q/TOF-MS) were used to rapidly screen and qualitatively analyze the uPA/plasmin affinity compounds from DSS extract. Additionally, uPA/plasmin inhibition assays and molecular docking were used to verify the activity and affinity mechanisms of the potential bioactive compounds. ResultsIn vivo, DSS, YXHX, and JPLS prevented sodium retention in nephrotic rats. DSS and YXHX treatment decreased urinary plasmin activity but did not alter urinary plasmin(ogen) concentration, and their extracts showed strong uPA and plasmin inhibitory activity in vitro. These results suggested that uPA and plasmin are direct targets of DSS and YXHX in intervening NS sodium retention. Using BAU-UPLC-Q/TOF-MS, gallic acids, methyl gallate, albiflorin, and 1,2,3,4,6-O-pentagalloylglucose (PGG) were screened as uPA or plasmin affinity compounds. Among them, PGG was found to be a uPA and plasmin dual inhibitor, with an IC50 of 6.861 μM against uPA and an IC50 of 149.0 μM against plasmin. The molecular docking results of PGG with uPA and plasmin were consistent with the verification results. ConclusionIntervening in sodium retention by inhibiting uPA-mediated plasmin generation and plasmin activity in the kidneys could be possible mechanisms for DSS, as indicated by the results in PAN-induced nephrotic rats. We conclude that PGG is a potential bioactive compound responsible for the effect of DSS on natriuresis.

Exploring the activity and inhibition of urokinase-type plasminogen activator (UPA)

Urokinase-type plasminogen activator (uPA) is a trypsin-like serine protease that activates plasminogen into the active serine protease, plasmin. Studying the catalytic activity of uPA gives insight to plasmin’s involvement in anticoagulation, cell adhesion, and degradation of the extracellular matrix, among others. The uPA’s catalytic activity is influenced by the orientation of the catalytic triad and the substrate binding affinity at the specificity pocket. This enzyme consists of an Amino-Terminal Fragment (ATF), a protease domain, and a disordered linker which connects the ATF and the protease domain.

A disordered linker with “something” on its N-terminus drives the allostery in the urokinase-type plasminogen activator (uPA)

The urokinase-type Plasminogen Activator (uPA) catalyzes the rate limiting step of fibrinolysis, the process by which blood clots are dissolved. This protein has three domains: an EGF-like, a Kringle and a Serine-protease domain. The first two constitute the N-terminal fragment (ATF) which is connected to the serine-protease domain through a disordered linker. Our lab previously showed that the ATF increases the protease activity of uPA towards its substrate Plasminogen by 6-fold. It also reduces the dynamics of the protease domain.

The Urokinase Plasminogen Activation System in Pancreatic Cancer: Prospective Diagnostic and Therapeutic Targets.

Pancreatic cancer is a highly aggressive malignancy that features high recurrence rates and the poorest prognosis of all solid cancers. The urokinase plasminogen activation system (uPAS) is strongly implicated in the pathophysiology and clinical outcomes of patients with pancreatic ductal adenocarcinoma (PDAC), which accounts for more than 90% of all pancreatic cancers. Overexpression of the urokinase-type plasminogen activator (uPA) or its cell surface receptor uPAR is a key step in the acquisition of a metastatic phenotype via multiple mechanisms, including the increased activation of cell surface localised plasminogen which generates the serine protease plasmin. This triggers multiple downstream processes that promote tumour cell migration and invasion. Increasing clinical evidence shows that the overexpression of uPA, uPAR, or of both is strongly associated with worse clinicopathological features and poor prognosis in PDAC patients. This review provides an overview of the current understanding of the uPAS in the pathogenesis and progression of pancreatic cancer, with a focus on PDAC, and summarises the substantial body of evidence that supports the role of uPAS components, including plasminogen receptors, in this disease. The review further outlines the clinical utility of uPAS components as prospective diagnostic and prognostic biomarkers for PDAC, as well as a rationale for the development of novel uPAS-targeted therapeutics.

Bioresponsive Polyphenol-Based Nanoparticles as Thrombolytic Drug Carriers.

Thrombolytic (clot-busting) therapies with plasminogen activators (PAs) are first-line treatments against acute thrombosis and ischemic stroke. However, limitations such as narrow therapeutic windows, low success rates, and bleeding complications hinder their clinical use. Drug-loaded polyphenol-based nanoparticles (NPs) could address these shortfalls by delivering a more targeted and safer thrombolysis, coupled with advantages such as improved biocompatibility and higher stability in vivo. Herein, a template-mediated polyphenol-based supramolecular assembly strategy is used to prepare nanocarriers of thrombolytic drugs. A thrombin-dependent drug release mechanism is integrated using tannic acid (TA) to cross-link urokinase-type PA (uPA) and a thrombin-cleavable peptide on a sacrificial mesoporous silica template via noncovalent interactions. Following drug loading and template removal, the resulting NPs retain active uPA and demonstrate enhanced plasminogen activation in the presence of thrombin (1.14-fold; p < 0.05). Additionally, they display lower association with macrophage (RAW 264.7) and monocytic (THP-1) cell lines (43 and 7% reduction, respectively), reduced hepatic accumulation, and delayed blood clearance in vivo (90% clearance at 60 min vs 5 min) compared with the template-containing NPs. Our thrombin-responsive, polyphenol-based NPs represent a promising platform for advanced drug delivery applications, with potential to improve thrombolytic therapies.

Pharmacophore Modeling, Docking, and Molecular Dynamics Simulation of Flavonoids as Inhibitors of Urokinase-type Plasminogen Activator

The urokinase-type plasminogen activator (uPA) system plays a significant role in the invasion and metastasis of cancer cells. The present study was conducted to investigate natural product compounds as inhibitors and hit molecules of uPA using in-silico analysis. A pharmacophore model was built to screen the Indonesian Herbal Database (HerbalDB) to obtain inhibitors of different scaffolds. Based on the molecular docking score, four ligands were selected as potential uPA inhibitors. Subsequently, the stability of the ligand-uPA complex was analyzed using molecular dynamics (MD) simulation. An RMSD graph of the backbone protein and the RMSF values of the amino acid residues were also determined. In addition, the MM-PBSA method was applied to calculate the free binding energy. According to the results, Model_3, characterized by aromatic rings 23 (F1 and F2), cationic H-bond donor (F3), and metal ligator (F4) features, had an adequate goodness-of-hit score (GH). The four top-ranked ligands, isorhamnetin, rhamnetin, quercetin, and kaempferol, showed higher docking scores compared to the others. This study confirmed that isorhamnetin, rhamnetin, and kaempferol build stable complexes with uPA with lower binding energy than quercetin.