{"gene":"PLAU","run_date":"2026-04-28T19:45:44","timeline":{"discoveries":[{"year":1984,"finding":"The human urokinase-type plasminogen activator (uPA/PLAU) gene was isolated and its complete nucleotide sequence determined. The gene is 6.4 kb long, organized in 11 exons, and contains a functional promoter region with GGCGGG repeats between CAAT and TATA boxes. The 5' end of uPA mRNA was mapped by S1 and primer extension.","method":"Gene cloning, nucleotide sequencing, S1 mapping, primer extension, CAT reporter transfection","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 — original gene structure determination with multiple methods, foundational study","pmids":["2987867"],"is_preprint":false},{"year":1984,"finding":"A partially spliced polyadenylated precursor to urokinase mRNA was identified; the introns separate functionally different domains of the enzyme, establishing the domain organization of PLAU.","method":"cDNA cloning, nucleotide sequencing, RNA blot","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — direct cDNA sequencing revealing gene structure","pmids":["6589620"],"is_preprint":false},{"year":1982,"finding":"Complete primary structure of the A chain (157 amino acids) of high molecular mass urokinase was determined, revealing three domains: a growth factor domain (homologous to EGF), a kringle domain (homologous to plasminogen kringles), and a connecting peptide domain.","method":"Protein sequencing by Edman degradation, cyanogen bromide cleavage, endoproteinase Lys-C fragmentation, carboxypeptidase treatment","journal":"Hoppe-Seyler's Zeitschrift fur physiologische Chemie","confidence":"High","confidence_rationale":"Tier 1 — complete protein sequence determination by multiple orthogonal methods","pmids":["6754569"],"is_preprint":false},{"year":1987,"finding":"The receptor-binding domain of uPA was mapped to the growth factor module (residues ~12–32), specifically amino acids 20–30 confer receptor binding specificity while residues 13–19 provide proper conformation. Synthetic peptides corresponding to uPA-(12-32) inhibited receptor binding of 125I-ATF with 50% inhibition at 100 nM.","method":"Synthetic peptide competition binding assays with 125I-labeled amino-terminal fragment (ATF)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — direct peptide mapping with quantitative binding assays, foundational receptor-binding study","pmids":["3031025"],"is_preprint":false},{"year":1990,"finding":"The cDNA for the human uPA receptor (uPAR) was cloned and sequenced, encoding a 313 amino acid protein with a signal peptide and GPI-anchor signal. Expression of uPAR cDNA in mouse cells confirmed it is a functional uPA-binding protein that localizes to the cell surface and facilitates uPA-catalyzed plasminogen activation.","method":"cDNA cloning, sequencing, heterologous expression in mouse LB6 cells, cross-linking, direct binding, caseinolytic plaque assay, immunofluorescence","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — receptor cloning with functional reconstitution and multiple validation methods","pmids":["1689240"],"is_preprint":false},{"year":1990,"finding":"uPA receptor localizes uPA to the leading edge of migrating monocytes in a chemotactic gradient, focusing extracellular proteolysis at the front of migrating cells. Receptor-bound uPA/PAI-2 complexes are rapidly cleared by cell-surface cleavage followed by endocytosis and degradation.","method":"Immunofluorescence on migrating monocytes, kinetic studies of PAI-2 inhibition of receptor-bound vs. free uPA, endocytosis assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — direct localization experiment with functional consequence, replicated mechanistic study","pmids":["2166055"],"is_preprint":false},{"year":1990,"finding":"Receptor-bound uPA retains susceptibility to inhibition by PAI-1 and PAI-2, but with association rate constants ~40% lower than free uPA. PMA stimulation of U937 cells further reduces receptor-bound uPA inhibition by PAI-1 and PAI-2.","method":"Kinetic inhibition assays on receptor-bound uPA on U937 cells; second-order rate constant measurements","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — quantitative kinetic measurements with well-defined experimental system","pmids":["2161846"],"is_preprint":false},{"year":1991,"finding":"Binding of uPA to its cellular receptor on U937 cells dramatically increases the efficiency of plasminogen activation: Km drops 40-fold to 0.67 µM (below physiological plasminogen concentration of 2 µM), and plasmin generated at the cell surface is protected from α2-antiplasmin. Cell-surface binding of plasminogen is critical for this high-affinity activation.","method":"Kinetic analysis of plasminogen activation on U937 cells and with purified isolated uPAR; Km and kcat measurements; plasmin inhibitor protection assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — rigorous kinetic reconstitution with both cell-associated and isolated receptor, foundational enzymatic study","pmids":["1829461"],"is_preprint":false},{"year":1991,"finding":"Cathepsin B cleaves pro-uPA at the Lys158-Ile159 bond (same site as plasmin/kallikrein) to generate enzymatically active two-chain uPA. This activation occurs both for soluble and tumor cell receptor-bound pro-uPA; the receptor-binding growth factor domain remains intact after cathepsin B cleavage.","method":"In vitro cleavage assay with purified cathepsin B and pro-uPA, SDS-PAGE, Western blot, N-terminal sequencing, receptor binding assays on U937 cells, E-64 inhibitor control","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with mutagenesis-equivalent site mapping and inhibitor validation","pmids":["1900515"],"is_preprint":false},{"year":1992,"finding":"uPA (urokinase) is a pro-HGF/SF convertase: it cleaves the single-chain biologically inactive precursor of hepatocyte growth factor/scatter factor (pro-HGF/SF) at a single site, generating the active mature α–β heterodimer. This activation is blocked by PAI-1, protease nexin-1, and anti-uPA catalytic domain antibodies.","method":"In vitro cleavage of purified pro-HGF/SF by pure urokinase at nanomolar concentrations; functional assays (MDCK scatter, Met phosphorylation); inhibitor studies","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro cleavage with functional readout and multiple inhibitor controls","pmids":["1334458"],"is_preprint":false},{"year":1996,"finding":"PAI-1, the endogenous inhibitor of uPA, blocks cell migration by occupying the αVβ3 integrin binding site on vitronectin, thereby preventing integrin-mediated adhesion. This anti-migratory effect requires high-affinity PAI-1 binding to vitronectin and is independent of PAI-1's ability to inhibit plasminogen activators. Formation of PAI-1/plasminogen activator complexes reduces PAI-1 affinity for vitronectin and restores migration.","method":"SMC migration assays on vitronectin, blocking antibodies against αVβ3, active vs. latent PAI-1 competition, PAI-1 mutants","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1–2 — mechanistic dissection with multiple mutants and controls, highly cited foundational study","pmids":["8837777"],"is_preprint":false},{"year":1997,"finding":"uPAR is internalized with uPA:serpin complexes via α2-macroglobulin receptor/LRP, then recycled back to the cell surface. Surface biotinylation experiments demonstrated that internalized biotinylated uPAR reappears at the plasma membrane in a PI-PLC-sensitive form, confirming true recycling rather than redistribution of intracellular pools.","method":"FACScan, immunofluorescence, immunoelectron microscopy, surface biotinylation recycling assay, PI-PLC sensitivity assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods demonstrating receptor recycling with direct biotinylation proof","pmids":["9184208"],"is_preprint":false},{"year":1997,"finding":"Two PEA3/AP-1 composite elements at −2.4 kb and −6.9 kb upstream of the transcription start site cooperate synergistically to confer full TPA- and FGF-2-inducibility of the uPA gene. AP-1 factors (c-Jun, JunD, ATF-2, c-Fos) and Ets transcription factors bind these elements, with c-Fos specifically binding the −6.9 kb element only after induction.","method":"DNase I hypersensitivity mapping, deletion analysis/transient transfection, EMSA with specific antibodies, dominant-negative Ets-2 expression","journal":"Gene","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal promoter dissection methods with functional validation","pmids":["9409785"],"is_preprint":false},{"year":1999,"finding":"uPA-stimulated cell migration requires uPAR ligation and proceeds via a signaling cascade: Ras → MEK → ERK → myosin light chain kinase (MLCK). MLCK is phosphorylated by a MEK-dependent pathway and leads to serine-phosphorylation of myosin II regulatory light chain. Migration is integrin-selective, occurring on vitronectin via β1-integrin (αVβ1) and αVβ5 but blocked by αVβ3.","method":"Dominant-negative and constitutively active Ras/MEK mutants, MLCK inhibitors, αVβ3 neutralizing antibody, migration assays on differentially coated surfaces","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1–2 — genetic epistasis plus pharmacological inhibition with integrin selectivity defined","pmids":["10402467"],"is_preprint":false},{"year":2000,"finding":"Matriptase, an epithelial membrane serine protease, activates pro-uPA (urokinase) to its active form. Matriptase converts pro-HGF and pro-uPA but has no effect on plasminogen, positioning matriptase as an upstream membrane activator of uPA.","method":"In vitro cleavage assays with active matriptase isolated from human milk, synthetic substrate kinetics, functional HGF scatter assay, c-Met phosphorylation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with purified enzyme and functional readouts","pmids":["10962009"],"is_preprint":false},{"year":2000,"finding":"MNNG-induced uPA gene transcription is mediated by the JNK signaling pathway via an AP-1 enhancer element at −2.4 kb. Dominant-negative MEKK1, MKK7, JNKK, and JIP-1 and curcumin (JNK inhibitor) all inhibited MNNG-induced uPA transcription, while dominant-negative MKK6 and SB203580 (p38 inhibitor) did not.","method":"Dominant-negative kinase constructs, pharmacological inhibitors, uPA promoter-reporter transfection assays","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1–2 — multiple dominant-negative constructs and inhibitors providing genetic pathway dissection","pmids":["10942386"],"is_preprint":false},{"year":2003,"finding":"Downregulation of uPA by antisense transfection in human glioblastoma cells disrupts actin cytoskeleton formation, decreases cell migration, and reduces PI3K and Akt phosphorylation, causing G2/M-phase arrest and decreased clonogenic survival, positioning uPA upstream of the PI3K/Akt signaling pathway.","method":"Stable antisense uPA transfection, Western blot for phospho-PI3K/Akt, cell migration assays, cell cycle analysis, clonogenic survival assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — antisense KD with multiple phenotypic readouts but single lab","pmids":["12545160"],"is_preprint":false},{"year":2006,"finding":"The uPA kringle domain binds directly to integrin αVβ3 (and also α4β1 and α9β1) independent of uPAR, enhancing plasminogen activation on CHO cells depleted of uPAR and inducing cell migration in an αVβ3-dependent manner. Plasminogen kringles 1-3/1-4 (angiostatin) blocked this interaction.","method":"Binding assays on uPAR-depleted CHO cells, purified soluble αVβ3 binding, cell migration assays with blocking antibodies, plasminogen activation assays","journal":"Thrombosis and haemostasis","confidence":"High","confidence_rationale":"Tier 1–2 — binding with purified proteins plus functional cell assays in uPAR-null background","pmids":["16525582"],"is_preprint":false},{"year":2006,"finding":"Crystal structure of uPA complexed with its receptor (uPAR) and an antibody was determined at 1.9 Å resolution. The three domains of uPAR form a concave shape with a central cone-shaped cavity where the uPA amino-terminal fragment inserts, explaining the molecular basis of uPA-uPAR interaction.","method":"X-ray crystallography at 1.9 Å","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 — high-resolution crystal structure of the uPA-uPAR complex","pmids":["16456079"],"is_preprint":false},{"year":2006,"finding":"uPA activates SREBP-1 processing and increases nuclear mature SREBP-1 content (5.7-fold) in THP-1 macrophages via PI3K-dependent activation of MEK/ERK, upregulating HMGCoA reductase expression and increasing macrophage cholesterol biosynthesis by 172%.","method":"Western blot for HMGCR protein/mRNA, SREBP-1 nuclear fractionation, PI3K inhibitor (LY294002), MEK inhibitor, cholesterol biosynthesis assays with statins","journal":"Atherosclerosis","confidence":"Medium","confidence_rationale":"Tier 2 — multiple inhibitor approaches defining the PI3K→MEK→ERK→SREBP-1 pathway, single lab","pmids":["17681345"],"is_preprint":false},{"year":2007,"finding":"uPA binding to uPAR increases uPAR association with lipid rafts (detergent-resistant membrane fractions) in a manner independent of uPA catalytic activity. Disruption of lipid rafts by methyl-β-cyclodextrin inhibits uPA-induced ERK phosphorylation, showing that lipid raft association is required for uPA/uPAR intracellular signaling.","method":"Sucrose gradient fractionation of detergent-resistant membranes, uPAR immunoprecipitation from DRM fractions, ERK phosphorylation assays, methyl-β-cyclodextrin treatment, glycosphingolipid analysis","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 — biochemical fractionation plus functional signaling assay, single lab","pmids":["17963689"],"is_preprint":false},{"year":2009,"finding":"Quebec platelet disorder (QPD), a dominant bleeding disorder, is caused by a direct tandem duplication of a 78-kb genomic segment containing PLAU. This duplication specifically increases uPA mRNA during megakaryocyte differentiation without altering expression of flanking genes.","method":"Genomic copy number variation analysis, array CGH, fluorescence in situ hybridization on 38 QPD subjects and 425 controls","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — definitive genetic identification in large cohort with controls; directly implicates PLAU gene dosage in platelet biology","pmids":["20007542"],"is_preprint":false},{"year":2010,"finding":"Prior to induction, the uPA (PLAU) gene is predominantly associated with 'poised transcription factories' containing RNA Pol II phosphorylated on Ser5 but not Ser2. After activation, the uPA locus associates with 'active factories' (Ser5+/Ser2+) and loops out from its chromosome territory. Gene positioning relative to the chromosome territory is independent of factory association levels.","method":"RNA FISH, immunofluorescence with phospho-specific Pol II antibodies, 3D nuclear localization analysis","journal":"PLoS biology","confidence":"Medium","confidence_rationale":"Tier 2 — direct nuclear localization with functional consequence, single lab","pmids":["20052287"],"is_preprint":false},{"year":2011,"finding":"uPA induces pulmonary microvascular endothelial permeability through LRP-dependent activation of endothelial NOS (eNOS) via PKA signaling. uPA induces eNOS phosphorylation at Ser1177, NO generation, and β-catenin nitrosylation/dissociation from VE-cadherin. This pathway is independent of PI3K-Akt.","method":"In vitro PMVEC monolayer permeability assay, eNOS phosphorylation Western blot, LRP antibody/RAP antagonist, PKA inhibitor (myristoylated PKI), in vivo lung permeability measurement","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — reconstituted with multiple receptor/enzyme inhibitors, in vitro and in vivo validation","pmids":["21540184"],"is_preprint":false},{"year":2014,"finding":"TMPRSS4, a type II transmembrane serine protease, directly converts inactive pro-uPA to the active two-chain form through its proteolytic activity. Active TMPRSS4 protease domain is released from cells and is membrane-associated; TMPRSS4 increases pro-uPA-mediated invasion in a serine protease activity-dependent manner, positioning TMPRSS4 as an upstream activator of pro-uPA.","method":"Pro-uPA cleavage assay with conditioned medium from TMPRSS4-overexpressing cells, active site mutant controls, Transwell invasion assays, Western blot","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — direct cleavage assay with active-site mutant, single lab","pmids":["24434139"],"is_preprint":false},{"year":2014,"finding":"Fra-1/AP-1 drives uPA (PLAU) overexpression in aggressive breast cancer cells via two AP-1 enhancers at −1.9 kb (ABR-1.9) and −4.1 kb (ABR-4.1) from the Plau-001 TSS. RNA Pol II is also recruited to the ABR regions, producing short unstable RNAs that track toward the TSS and convert to productive mRNA. A minor mRNA, Plau-004, is transcribed from ABR-1.9 and is repressed by Fra-1.","method":"ChIP, RNAi knockdown of Fra-1, pharmacological inhibition, RNA Pol II occupancy mapping","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal approaches (ChIP, RNAi, pharmacology), single lab","pmids":["25200076"],"is_preprint":false},{"year":2015,"finding":"Small-molecule pyrrolone inhibitors of the uPAR·uPA protein-protein interaction (Ki ~0.7 µM for labeled peptide displacement, IC50 ~18 µM for uPAR·uPA-ATF) allosterically inhibit the distal uPAR·vitronectin interaction, demonstrating cooperative binding between uPA and vitronectin on uPAR. These compounds reduce FAK phosphorylation, Rac1 activation, and MDA-MB-231 breast cancer cell invasion.","method":"Fluorescence polarization, surface plasmon resonance competition, ELISA, molecular dynamics simulations, free energy calculations, cellular FAK phosphorylation, Rac1 activation assay, Matrigel invasion assay","journal":"ACS chemical biology","confidence":"High","confidence_rationale":"Tier 1–2 — structural modeling plus multiple orthogonal biophysical/cell assays demonstrating allostery","pmids":["25671694"],"is_preprint":false},{"year":2016,"finding":"uPA binding to uPAR promotes axonal regeneration in the CNS by a plasminogen-independent mechanism: uPA/uPAR binding induces membrane recruitment and activation of β1-integrin via LRP1, leading to Rac1 GTPase activation and Rac1-induced axonal regeneration in injured axons.","method":"In vitro axonal injury models, in vivo CNS injury models, recombinant uPA treatment, LRP1 inhibition, β1-integrin blocking, Rac1 activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — in vitro and in vivo models with pathway epistasis using receptor/pathway inhibitors","pmids":["27986809"],"is_preprint":false},{"year":2017,"finding":"QPD PLAU duplication dysregulates PLAU expression in a megakaryocyte-specific manner: QPD megakaryocytes overexpress normal PLAU transcripts >100-fold (from the disease chromosome) while QPD granulocytes show only ~3.9-fold increase, suggesting an active regulatory mechanism controlling uPA levels in blood that is specifically disrupted in megakaryocytes by the duplication.","method":"RNA-seq, quantitative RT-PCR, allele-specific transcript analysis, protein expression analysis in primary cells and cultured megakaryocytes","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — comprehensive transcriptomic analysis in primary patient cells with allele-specific resolution","pmids":["28301587"],"is_preprint":false},{"year":2018,"finding":"uPA induces local synthesis of ezrin in astrocytes and triggers formation of peripheral astrocytic processes (PAPs) that contact the synapse, protecting the tripartite synapse from ischemic injury. Recombinant uPA treatment in vivo induces PAP formation in the ischemic brain.","method":"In vitro astrocyte cultures, in vivo ischemic stroke models, uPAR knockout mice, recombinant uPA treatment, immunofluorescence for ezrin and synaptic markers","journal":"Journal of cerebral blood flow and metabolism","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro and in vivo with loss- and gain-of-function, single lab","pmids":["29890880"],"is_preprint":false},{"year":2018,"finding":"uPA-uPAR binding induces local synthesis of ezrin in cortical neurons at the synapse and recruits β3-integrin to the postsynaptic density (PSD) via ICAM-5, followed by phosphorylation of ezrin at Thr-567 and reorganization of the actin cytoskeleton in the postsynaptic terminal, leading to recovery of dendritic spines and synapses damaged by ischemic stroke.","method":"In vitro cortical neuron cultures, in vivo ischemic stroke models, β3-integrin knockdown/blocking, ICAM-5 studies, phospho-ezrin Western blot, dendritic spine imaging","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — multiple mechanistic steps defined with both in vitro and in vivo evidence, single lab","pmids":["29720403"],"is_preprint":false},{"year":2018,"finding":"YAP/TEAD transcription factor activity directly regulates Plau (uPA) expression in epidermal keratinocytes, promoting their proliferation. RNA-seq of YAP2-5SA-ΔC transgenic mouse skin identified Plau as a dysregulated gene containing YAP/TEAD binding motifs in its 3' UTR, confirmed by functional characterization assays.","method":"RNA-seq of transgenic mouse skin, YAP/TEAD motif analysis, functional proliferation assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — transgenic mouse model with transcriptomic and functional validation, single lab","pmids":["30382077"],"is_preprint":false},{"year":2019,"finding":"Protein O-fucosyltransferase 1 (poFUT1) increases O-fucosylation on uPA and activates the RhoA signaling pathway, facilitating uterine angiogenesis and vascular remodeling. Knockdown of poFUT1 reduces uPA O-fucosylation and impairs angiogenesis.","method":"Glycoprotein O-fucosylation analysis, RhoA activity assay, hESC and mouse model experiments, siRNA knockdown","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — post-translational modification (O-fucosylation) identified with functional pathway consequence","pmids":["31601791"],"is_preprint":false},{"year":2021,"finding":"PLAU (uPA) promotes conversion of fibroblasts to inflammatory cancer-associated fibroblasts via the uPAR/Akt/NF-κB pathway, inducing IL-8 secretion. IL-8 from CAFs in turn promotes high PLAU expression in tumor cells (ESCC), creating a positive feedback loop. PLAU also promotes tumor cell proliferation via the MAPK pathway and migration by upregulating Slug and MMP9.","method":"Loss-of-function and gain-of-function experiments, RNA sequencing, cytokine array, RT-qPCR, MEK inhibitor U0126, Akt/NF-κB pathway inhibition","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 — mechanistic pathway dissection with multiple experimental approaches, single lab","pmids":["33574243"],"is_preprint":false},{"year":2021,"finding":"uPA mediates endothelial tubular network (ETN) formation in HUVEC-MSC co-culture via cross-talk of uPAR, uPA's catalytic activity, uPA's binding to uPAR, and uPA nuclear translocation, coordinated with αV-integrins, VEGFR2, and Notch receptor/ligand pathways.","method":"HUVEC-MSC co-culture angiogenesis assay, siRNA knockdown of pathway components, pharmacological inhibitors at multiple steps, mRNA expression analysis","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"Medium","confidence_rationale":"Tier 2 — multi-step pathway inhibition in co-culture model, single lab","pmids":["34619163"],"is_preprint":false},{"year":2022,"finding":"AQR promotes endothelial cell senescence via PLAU: AQR overexpression upregulates PLAU, and knockdown of PLAU rescues senescence-related phenotypes (SA-β-gal staining, P21 upregulation, G2/M arrest) induced by AQR overexpression or TNF-α treatment, establishing an AQR/PLAU signaling axis in endothelial cell senescence.","method":"Transcriptomic analysis of AQR overexpression/knockdown in HUVECs, SA-β-gal staining, CDKN1A Western blot, colony formation, cell cycle analysis, PLAU siRNA rescue","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 — epistasis established by siRNA rescue, single lab","pmids":["35270021"],"is_preprint":false},{"year":2022,"finding":"METTL3 stabilizes PLAU mRNA in an m6A-dependent manner, promoting colorectal cancer metastasis via the MAPK/ERK pathway and angiogenesis.","method":"m6A-seq, METTL3 overexpression/knockdown, RNA stability assays, MAPK/ERK pathway analysis, in vivo tumor models","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — m6A-dependent mRNA stabilization mechanism with in vitro and in vivo validation","pmids":["35567945"],"is_preprint":false},{"year":2023,"finding":"PLAU (uPA) knockout by CRISPR-Cas9 completely stops matrix remodeling (measured by AFM-based stiffness changes) and significantly reduces cancer cell invasion in a 3D tumouroid model, confirming uPA's enzymatic role in ECM degradation as a driver of invasion. Pharmacological uPA inhibition (UK-371,801) showed similarly reduced matrix degradation and invasion.","method":"CRISPR-Cas9 knockout of PLAU, 3D tumouroid culture, atomic force microscopy (AFM) stiffness measurement, invasion quantification, pharmacological inhibitor UK-371,801","journal":"Matrix biology plus","confidence":"High","confidence_rationale":"Tier 1–2 — genetic knockout + pharmacological inhibition + biophysical ECM measurements in orthogonal 3D model","pmids":["38020586"],"is_preprint":false},{"year":2024,"finding":"PLAU interacts with TM4SF1 to activate Akt signaling, promoting NSCLC cell growth, cisplatin resistance, and survival. TM4SF1 knockdown or anti-TM4SF1 neutralizing antibody phenocopies PLAU inhibition, and PLAU overexpression stabilizes TM4SF1 at the cell surface.","method":"Co-immunoprecipitation, overexpression/knockdown experiments, Akt phosphorylation Western blot, nude mouse xenograft, anti-TM4SF1 antibody treatment","journal":"Biology direct","confidence":"Medium","confidence_rationale":"Tier 2–3 — co-IP plus functional epistasis, single lab","pmids":["38229120"],"is_preprint":false}],"current_model":"PLAU encodes urokinase-type plasminogen activator (uPA), a serine protease that is secreted as inactive pro-uPA and activated extracellularly by plasmin, cathepsin B, matriptase, or TMPRSS4 through cleavage at Lys158-Ile159; upon binding its GPI-anchored receptor uPAR (itself cloned and characterized as a 313-aa protein), receptor-bound uPA activates plasminogen to plasmin with dramatically enhanced kinetics (Km drops 40-fold) and protects plasmin from α2-antiplasmin, focusing pericellular proteolysis at the leading edge of migrating cells; uPA also cleaves pro-HGF/SF to generate active growth factor; uPAR localizes to lipid rafts upon uPA binding, and the uPA-uPAR complex signals through multiple pathways including Ras/MEK/ERK/MLCK (migration), PI3K/Akt (survival/migration), and LRP1/β1-integrin/Rac1 (axonal regeneration), while the uPA kringle domain independently engages αVβ3 integrin; uPA activity is regulated by PAI-1 (which also blocks integrin-mediated migration by competing for the vitronectin binding site) and by receptor recycling via LRP-mediated internalization of uPA:serpin complexes with subsequent uPAR return to the surface; PLAU transcription is driven by AP-1/PEA3 composite elements and regulated by Fra-1, Ets-1, and the JNK/AP-1 pathway, with the gene associated with poised transcription factories prior to induction; post-translational regulation includes O-fucosylation by poFUT1 and m6A-dependent mRNA stabilization by METTL3; the crystal structure of uPA-uPAR complex at 1.9 Å reveals a cone-shaped central cavity in uPAR where the uPA amino-terminal fragment inserts, and small-molecule inhibitors of this interface allosterically disrupt the distal uPAR-vitronectin interaction."},"narrative":{"teleology":[{"year":1982,"claim":"Determination of the complete primary structure of the uPA A-chain revealed a modular architecture — growth factor, kringle, and connecting peptide domains — establishing the domain logic that underlies all subsequent functional mapping.","evidence":"Edman degradation and multiple fragmentation strategies on purified high-MW urokinase","pmids":["6754569"],"confidence":"High","gaps":["No structure–function assignments for individual domains yet","Receptor identity unknown"]},{"year":1984,"claim":"Isolation and sequencing of the PLAU gene (6.4 kb, 11 exons) showed that intron–exon boundaries separate functional domains, providing the genomic framework for understanding splicing, transcriptional regulation, and evolutionary relationships.","evidence":"Gene cloning, nucleotide sequencing, S1 mapping, primer extension, cDNA analysis","pmids":["2987867","6589620"],"confidence":"High","gaps":["Promoter regulatory elements beyond CAAT/TATA not yet defined","No information on tissue-specific regulation"]},{"year":1987,"claim":"Mapping of the receptor-binding determinant to the growth factor module (residues 12–32) answered how uPA engages the cell surface and showed the catalytic domain is dispensable for receptor binding.","evidence":"Synthetic peptide competition binding assays with ¹²⁵I-ATF on intact cells","pmids":["3031025"],"confidence":"High","gaps":["Receptor identity still uncloned","Three-dimensional basis of binding unknown"]},{"year":1990,"claim":"Cloning of uPAR as a 313-aa GPI-anchored protein and demonstration that receptor-bound uPA localizes to the leading edge of migrating cells established the paradigm of receptor-focused pericellular proteolysis.","evidence":"cDNA cloning with heterologous reconstitution; immunofluorescence on chemotactically migrating monocytes; kinetic inhibition studies with PAI-1/PAI-2","pmids":["1689240","2166055","2161846"],"confidence":"High","gaps":["Structural basis of uPA–uPAR interaction unknown","Intracellular signaling consequences not addressed"]},{"year":1991,"claim":"Quantitative enzymology revealed that uPAR binding drops the Km for plasminogen activation 40-fold (to 0.67 µM, below physiological [plasminogen]) and protects product plasmin from α2-antiplasmin, explaining why cell-surface localization matters for in vivo fibrinolysis and invasion.","evidence":"Kinetic analysis on U937 cells and purified isolated uPAR","pmids":["1829461"],"confidence":"High","gaps":["Structural explanation for kinetic enhancement unknown","Relative contribution of plasminogen co-receptors not resolved"]},{"year":1991,"claim":"Identification of cathepsin B as a pro-uPA activator cleaving the same Lys158–Ile159 bond as plasmin revealed that tumor-derived cysteine proteases can initiate the uPA activation cascade independently of plasmin feedback.","evidence":"In vitro cleavage with purified cathepsin B, N-terminal sequencing, E-64 inhibitor control","pmids":["1900515"],"confidence":"High","gaps":["In vivo relevance of cathepsin B activation vs. plasmin autoactivation not determined"]},{"year":1992,"claim":"Discovery that uPA converts pro-HGF/SF to active HGF/SF expanded the substrate repertoire beyond plasminogen, linking uPA to receptor tyrosine kinase signaling (c-Met) and cell scattering.","evidence":"In vitro cleavage of purified pro-HGF/SF with functional scatter and Met phosphorylation readouts","pmids":["1334458"],"confidence":"High","gaps":["Relative in vivo contribution of uPA vs. other HGF activases (e.g., HGFA) unresolved"]},{"year":1996,"claim":"Demonstration that PAI-1 blocks migration by competing with αVβ3 integrin for vitronectin — independent of its protease-inhibitory activity — revealed a non-canonical regulatory mechanism where serpin–protease complex formation restores migration by lowering PAI-1 affinity for vitronectin.","evidence":"SMC migration assays with PAI-1 mutants, integrin blocking antibodies","pmids":["8837777"],"confidence":"High","gaps":["Structural basis of PAI-1–vitronectin–integrin competition not resolved at atomic level"]},{"year":1997,"claim":"Elucidation of composite PEA3/AP-1 enhancer elements at −2.4 kb and −6.9 kb, combined with later JNK/AP-1 pathway mapping and Fra-1 studies, defined the transcriptional logic driving PLAU induction in response to growth factors and genotoxic stress.","evidence":"DNase I hypersensitivity, deletion reporters, EMSA, dominant-negative kinases; ChIP and RNAi of Fra-1","pmids":["9409785","10942386","25200076"],"confidence":"High","gaps":["Chromatin remodeling steps at the PLAU locus incompletely mapped","Megakaryocyte-specific regulatory elements not identified"]},{"year":1997,"claim":"Discovery that uPAR is recycled back to the cell surface after LRP-mediated endocytosis of uPA:serpin complexes explained how cells maintain receptor availability for successive rounds of pericellular proteolysis.","evidence":"Surface biotinylation recycling assay, PI-PLC sensitivity, immunoelectron microscopy","pmids":["9184208"],"confidence":"High","gaps":["Sorting signals directing uPAR recycling vs. LRP degradation not identified"]},{"year":1999,"claim":"Genetic epistasis with dominant-negative Ras/MEK and MLCK inhibitors established the first complete intracellular signaling cascade (Ras→MEK→ERK→MLCK→myosin II) downstream of uPA–uPAR ligation driving cell migration, demonstrating that uPA is a bona fide signaling ligand beyond its protease role.","evidence":"Dominant-negative constructs, pharmacological inhibitors, migration assays on vitronectin with integrin blocking antibodies","pmids":["10402467"],"confidence":"High","gaps":["Proximal signaling link between GPI-anchored uPAR and cytoplasmic Ras not identified","Lipid raft requirement not yet established"]},{"year":2006,"claim":"The 1.9 Å crystal structure of the uPA–uPAR complex revealed uPAR's three-domain concave architecture with a central cone-shaped cavity accommodating the uPA amino-terminal fragment, providing the atomic framework for understanding receptor engagement and enabling structure-based drug design.","evidence":"X-ray crystallography of uPA:uPAR:antibody ternary complex","pmids":["16456079"],"confidence":"High","gaps":["Full-length uPA structure in complex not determined","Conformational dynamics upon uPAR engagement not characterized"]},{"year":2006,"claim":"The uPA kringle domain was shown to bind αVβ3 integrin directly and independently of uPAR, identifying a second cell-surface tethering mechanism that enhances plasminogen activation on uPAR-negative cells and promotes integrin-dependent migration.","evidence":"Binding assays on uPAR-depleted CHO cells and purified αVβ3, blocking antibodies","pmids":["16525582"],"confidence":"High","gaps":["Structural basis of kringle–integrin interaction unknown","Physiological context where this uPAR-independent mechanism dominates not defined"]},{"year":2007,"claim":"Demonstration that uPA binding drives uPAR into lipid rafts — independently of catalytic activity — and that raft disruption abolishes ERK signaling provided a mechanistic link between GPI-anchored uPAR and cytoplasmic signaling.","evidence":"Sucrose gradient DRM fractionation, methyl-β-cyclodextrin treatment, ERK phosphorylation assays","pmids":["17963689"],"confidence":"Medium","gaps":["Identity of the raft-resident transmembrane partner transducing the signal not determined","Single lab observation"]},{"year":2009,"claim":"Identification of a 78-kb tandem duplication encompassing PLAU as the cause of Quebec platelet disorder — with megakaryocyte-specific >100-fold overexpression — established uPA gene dosage as a Mendelian disease mechanism and revealed cell-type-specific regulatory vulnerability.","evidence":"Array CGH, FISH in 38 QPD subjects and 425 controls; RNA-seq and allele-specific transcript analysis in megakaryocytes","pmids":["20007542","28301587"],"confidence":"High","gaps":["Regulatory element within the duplication conferring megakaryocyte specificity not mapped","Therapeutic strategy to normalize uPA in megakaryocytes not developed"]},{"year":2016,"claim":"uPA–uPAR signaling was shown to promote CNS axonal regeneration through a plasminogen-independent, LRP1/β1-integrin/Rac1 pathway, expanding uPA function from proteolysis and migration to neural repair.","evidence":"In vitro axonal injury and in vivo CNS injury models with LRP1 inhibition, β1-integrin blocking, Rac1 activity assays","pmids":["27986809"],"confidence":"High","gaps":["Whether this pathway operates in peripheral nerve injury not tested","LRP1 binding determinants on uPA for this non-proteolytic function not mapped"]},{"year":2022,"claim":"Discovery that METTL3 stabilizes PLAU mRNA through m6A modification introduced an epitranscriptomic layer of uPA regulation, linking RNA methylation to metastatic potential via MAPK/ERK signaling.","evidence":"m6A-seq, METTL3 overexpression/knockdown, RNA stability assays, in vivo tumour models","pmids":["35567945"],"confidence":"Medium","gaps":["Specific m6A site(s) on PLAU mRNA not mapped at single-nucleotide resolution","m6A reader(s) responsible for stabilization not identified"]},{"year":2023,"claim":"CRISPR knockout of PLAU completely abolished matrix remodeling and invasion in 3D tumouroid models, confirming with genetic precision that uPA enzymatic activity is the dominant driver of ECM degradation during cancer cell invasion.","evidence":"CRISPR-Cas9 KO, AFM stiffness measurements, pharmacological inhibitor UK-371,801, 3D tumouroid culture","pmids":["38020586"],"confidence":"High","gaps":["Redundancy with tPA or other MMPs in different tumour types not assessed","In vivo metastasis suppression by PLAU KO not shown"]},{"year":null,"claim":"Key unresolved questions include the identity of the transmembrane co-receptor that couples GPI-anchored uPAR to intracellular kinase cascades, the structural basis for the kringle–αVβ3 interaction, the megakaryocyte-specific regulatory element disrupted in QPD, and whether therapeutic targeting of the uPA–uPAR interface can achieve anti-metastatic efficacy in vivo.","evidence":"","pmids":[],"confidence":"Low","gaps":["Transmembrane signaling partner of uPAR not conclusively identified","No in vivo efficacy data for uPA–uPAR PPI inhibitors in metastasis models","Full-length active uPA crystal structure still lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[7,8,9,14,24,37]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[7,8,9,37]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[13,27,30]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[2,7,9,37]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[5,7,20]},{"term_id":"GO:0031012","term_label":"extracellular matrix","supporting_discovery_ids":[37]}],"pathway":[{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[7,21,28]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[13,16,19,27,33,38]},{"term_id":"R-HSA-1474244","term_label":"Extracellular matrix organization","supporting_discovery_ids":[37]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[33,36,38]}],"complexes":[],"partners":["PLAUR","SERPINE1","LRP1","ITGAV","ITGB3","ST14","TM4SF1","HGF"],"other_free_text":[]},"mechanistic_narrative":"PLAU encodes urokinase-type plasminogen activator (uPA), a secreted serine protease that is produced as inactive single-chain pro-uPA and activated extracellularly by plasmin, cathepsin B, matriptase, or TMPRSS4 through cleavage at Lys158–Ile159, yielding a two-chain enzyme whose primary function is conversion of plasminogen to plasmin at the cell surface [PMID:1829461, PMID:1900515, PMID:10962009, PMID:24434139]. Binding of uPA to its GPI-anchored receptor uPAR via the amino-terminal growth factor domain lowers the Km for plasminogen activation ~40-fold, focuses pericellular proteolysis to the leading edge of migrating cells, and triggers non-proteolytic signaling through Ras/MEK/ERK/MLCK, PI3K/Akt, and LRP1/β1-integrin/Rac1 cascades that drive cell migration, survival, axonal regeneration, and angiogenesis [PMID:2166055, PMID:10402467, PMID:27986809, PMID:16456079]. uPA also cleaves pro-HGF/SF to its active heterodimer, degrades extracellular matrix in 3D tumour models, and its kringle domain independently engages αVβ3 integrin to promote migration [PMID:1334458, PMID:38020586, PMID:16525582]. Tandem duplication of the PLAU locus causes Quebec platelet disorder, a dominant bleeding disorder driven by megakaryocyte-specific >100-fold overexpression of uPA [PMID:20007542, PMID:28301587]."},"prefetch_data":{"uniprot":{"accession":"P00749","full_name":"Urokinase-type plasminogen activator","aliases":[],"length_aa":431,"mass_kda":48.5,"function":"Specifically cleaves the zymogen plasminogen to form the active enzyme plasmin","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/P00749/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PLAU","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PLAU","total_profiled":1310},"omim":[{"mim_id":"619450","title":"MICRO RNA 874; MIR874","url":"https://www.omim.org/entry/619450"},{"mim_id":"618444","title":"PLASMINOGEN RECEPTOR WITH C-TERMINAL LYSINE; PLGRKT","url":"https://www.omim.org/entry/618444"},{"mim_id":"618172","title":"LONG NONCODING RNA UPREGULATOR OF ANTIVIRAL RESPONSE INTERFERON SIGNALING; LUARIS","url":"https://www.omim.org/entry/618172"},{"mim_id":"615874","title":"RIBOSOMAL L1 DOMAIN-CONTAINING PROTEIN 1; RSL1D1","url":"https://www.omim.org/entry/615874"},{"mim_id":"613329","title":"PLASMINOGEN ACTIVATOR INHIBITOR-1 DEFICIENCY","url":"https://www.omim.org/entry/613329"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Golgi apparatus","reliability":"Approved"},{"location":"Vesicles","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"kidney","ntpm":93.0},{"tissue":"urinary bladder","ntpm":119.5}],"url":"https://www.proteinatlas.org/search/PLAU"},"hgnc":{"alias_symbol":["URK","UPA"],"prev_symbol":[]},"alphafold":{"accession":"P00749","domains":[{"cath_id":"2.40.20.10","chopping":"51-153","consensus_level":"medium","plddt":91.1777,"start":51,"end":153},{"cath_id":"2.40.10.10","chopping":"166-431","consensus_level":"medium","plddt":84.7913,"start":166,"end":431}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P00749","model_url":"https://alphafold.ebi.ac.uk/files/AF-P00749-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P00749-F1-predicted_aligned_error_v6.png","plddt_mean":82.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PLAU","jax_strain_url":"https://www.jax.org/strain/search?query=PLAU"},"sequence":{"accession":"P00749","fasta_url":"https://rest.uniprot.org/uniprotkb/P00749.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P00749/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P00749"}},"corpus_meta":[{"pmid":"18162327","id":"PMC_18162327","title":"Evolving 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surface rather than degraded, as demonstrated by surface biotinylation and FACScan/immunofluorescence/immunoelectron microscopy.\",\n      \"method\": \"Cell surface biotinylation, FACScan, immunofluorescence, immunoelectron microscopy, PI-PLC cleavage assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods in a single study with rigorous controls demonstrating internalization and recycling\",\n      \"pmids\": [\"9184208\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"uPA (together with tPA) is one of the two principal physiological plasminogen activators required for normal skin wound healing; in uPA;tPA double-knockout mice wound healing is substantially delayed but not abolished, with a third activator (plasma kallikrein) providing residual plasmin activity.\",\n      \"method\": \"Genetic knockout (uPA-/-, tPA-/-, Plg-/- mice), MMP inhibitor treatment, ecotin-based plasma kallikrein inhibition, biochemical plasmin detection in wound extracts\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — genetic epistasis with multiple gene-knockout combinations and pharmacological validation, strong mechanistic dissection\",\n      \"pmids\": [\"16763560\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The kringle domain of uPA directly binds integrin αvβ3 (as well as α4β1 and α9β1) independently of uPAR, and this interaction promotes cell migration and enhances plasminogen activation on the cell surface.\",\n      \"method\": \"Binding assay on uPAR-depleted CHO cells, pulldown with purified soluble αvβ3, cell migration assay with αvβ3-dependent blockade, plasminogen activation assay\",\n      \"journal\": \"Thrombosis and haemostasis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — reconstituted binding with purified proteins plus cell-based functional validation and blocking controls\",\n      \"pmids\": [\"16525582\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Downregulation of uPA by antisense transfection in glioblastoma cells disrupts actin cytoskeleton formation and cell migration, and reduces phosphorylation of PI3K and Akt, placing uPA upstream of the PI3K/Akt signaling pathway in migration control.\",\n      \"method\": \"Stable antisense transfection, Western blot for phospho-PI3K and phospho-Akt, actin cytoskeleton imaging, cell migration assay, clonogenic survival, cell cycle analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean loss-of-function with defined cellular and biochemical phenotype; single lab\",\n      \"pmids\": [\"12545160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Persons with Quebec platelet disorder (QPD) carry a tandem duplication of the 78-kb genomic segment containing PLAU, which selectively increases uPA mRNA and protein in megakaryocytes and platelets without altering flanking gene expression, establishing PLAU copy-number gain as the causative mutation.\",\n      \"method\": \"Copy number variation analysis (quantitative genomics), segregation analysis in families, transcript quantification\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mutation identified in all 38 QPD subjects and absent in >400 controls; replicated across multiple families\",\n      \"pmids\": [\"20007542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The PLAU duplication in QPD dysregulates uPA expression in a megakaryocyte-specific manner, producing normal PLAU transcripts at >100-fold increased levels in megakaryocytes but only ~4-fold in granulocytes, indicating tissue-specific regulatory mechanisms govern PLAU expression from the duplicated locus.\",\n      \"method\": \"RNA-seq, quantitative RT-PCR, protein expression analysis in primary cells and cultured megakaryocytes from QPD donors\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — primary human cells with RNA-seq and quantitative validation; single lab\",\n      \"pmids\": [\"28301587\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"uPA binding to its receptor uPAR stimulates cholesterol biosynthesis in macrophages by activating a PI3K→MEK→ERK→SREBP-1 signaling cascade that upregulates HMG-CoA reductase expression.\",\n      \"method\": \"Cholesterol biosynthesis assay, statin inhibition, PI3K and MEK inhibitors, Western blot for SREBP-1 and HMGCR, nuclear SREBP-1 quantification\",\n      \"journal\": \"Atherosclerosis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological pathway dissection with multiple inhibitors and biochemical readouts; single lab\",\n      \"pmids\": [\"17681345\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"uPA binding to uPAR increases partitioning of uPAR into lipid raft (detergent-resistant membrane) microdomains, and this lipid raft association is required for uPA-induced ERK phosphorylation; disruption of rafts by methyl-β-cyclodextrin abrogates this signaling.\",\n      \"method\": \"Sucrose gradient fractionation of DRM, immunoprecipitation, ERK phosphorylation assay, methyl-β-cyclodextrin cholesterol depletion\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — biochemical fractionation combined with functional signaling assay; single lab\",\n      \"pmids\": [\"17963689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TMPRSS4, a type II transmembrane serine protease, directly cleaves pro-uPA (single-chain) into active two-chain uPA through its proteolytic activity, and the active TMPRSS4 protease domain is shed into conditioned medium associated with the plasma membrane.\",\n      \"method\": \"In vitro proteolysis assay, analysis of conditioned medium, serine protease activity-dependent invasion assay, overexpression of TMPRSS4\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct enzymatic conversion demonstrated in cell-based system with activity-dependent functional readout; single lab\",\n      \"pmids\": [\"24434139\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"uPA binding to uPAR promotes axonal regeneration in the CNS by inducing membrane recruitment and activation of β1-integrin via LRP1, which activates the Rho GTPase Rac1, leading to Rac1-dependent axonal regrowth, independent of plasmin generation.\",\n      \"method\": \"In vitro axonal injury model, in vivo CNS injury model, immunofluorescence of growth cone uPAR, recombinant uPA treatment, LRP1 blockade, β1-integrin recruitment assay, Rac1 activation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vitro and in vivo models combined with pathway dissection using specific inhibitors and plasmin-independent demonstration; moderate evidence\",\n      \"pmids\": [\"27986809\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"uPA induces synthesis of ezrin in astrocytes and recruits β3-integrin to the postsynaptic density via ICAM-5, causing Thr-567 phosphorylation (activation) of ezrin and subsequent actin cytoskeleton reorganization that protects the tripartite synapse from ischemic damage.\",\n      \"method\": \"In vitro and in vivo ischemic stroke models, recombinant uPA treatment, Western blot, immunofluorescence for PAPs, ezrin synthesis and phosphorylation assays\",\n      \"journal\": \"Journal of cerebral blood flow and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo and in vitro evidence with defined molecular pathway; single lab\",\n      \"pmids\": [\"29890880\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"uPA-uPAR binding induces local synthesis of ezrin at the synapse and recruitment of β3-integrin to the postsynaptic density, with β3-integrin driving ICAM-5-mediated ezrin recruitment and phosphorylation at Thr-567, leading to recovery of dendritic spines after ischemic injury via a plasminogen-independent mechanism.\",\n      \"method\": \"In vitro cortical neuron ischemia model, Western blot, immunofluorescence, β3-integrin and ICAM-5 blockade, recombinant uPA treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic pathway dissection in vitro and in vivo; single lab, complements PMID 29890880\",\n      \"pmids\": [\"29720403\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"uPA-uPAR molecular complex promotes neuronal migration and neuritogenesis by inducing interaction of uPAR with α5- and β1-integrin subunits, which activates focal adhesion kinase (FAK) and relocates it to growth cones, triggering cytoskeletal reorganization.\",\n      \"method\": \"Neuronal migration and neuritogenesis assays in CNS explants, co-immunoprecipitation of uPAR with integrins, FAK phosphorylation and localization by immunofluorescence\",\n      \"journal\": \"Developmental dynamics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — co-IP combined with functional cellular assays; single lab\",\n      \"pmids\": [\"24481918\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"uPA induces pulmonary microvascular endothelial permeability through LRP-dependent activation of endothelial NOS (eNOS) via PKA; eNOS phosphorylation at Ser1177 generates NO, causing nitrosylation and dissociation of β-catenin from VE-cadherin, disrupting barrier function.\",\n      \"method\": \"PMVEC monolayer permeability assay, eNOS phosphorylation by Western blot, NO generation, anti-LRP antibody and RAP blockade, PKA inhibitor (myristoylated PKI), in vivo lung permeability\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro and in vivo mechanistic pathway fully dissected with pharmacological and antibody inhibitors; multiple orthogonal readouts\",\n      \"pmids\": [\"21540184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"uPA, tPA, and plasminogen (but not uPAR) are required for experimental choroidal neovascularization in laser-induced CNV mouse model, and their absence partly reduces matrix metalloproteinase activity and allows fibrin accumulation at wound sites.\",\n      \"method\": \"Single gene-deficient mouse models (uPA-/-, tPA-/-, Plg-/-, uPAR-/- ), laser-induced CNV model, in situ zymography, immunofluorescence\",\n      \"journal\": \"Investigative ophthalmology & visual science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with four separate knockout models in a standardized in vivo assay\",\n      \"pmids\": [\"12657615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Combined genetic deficiency of MMP9 and uPA (but not tPA or uPAR) produces additive impairment of gestation, bone growth, and wound healing in mice, demonstrating functional cooperativity between uPA and MMP9 during tissue remodeling; compensatory uPA upregulation occurs in MMP9-deficient wounds.\",\n      \"method\": \"Double-knockout mouse models (MMP9-/- with uPA-/-, tPA-/-, or uPAR-/-), wound healing and bone growth phenotyping, gestation outcome analysis, uPA activity measurement\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — rigorous genetic epistasis with multiple double-knockout combinations and quantitative phenotyping\",\n      \"pmids\": [\"21802414\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"uPA stimulates monocyte-to-macrophage differentiation via uPAR, and attenuates Ox-LDL-induced macrophage apoptosis through ERK1/2 activation-dependent downregulation of the pro-apoptotic protein Bim, in a Ca2+-independent manner.\",\n      \"method\": \"uPAR-deficient mouse peritoneal macrophage harvest, PMA differentiation assay, CD36 expression, cell cycle analysis, Western blot for ERK1/2 and Bim, apoptosis assay with thapsigargin/Ox-LDL/staurosporine\",\n      \"journal\": \"Atherosclerosis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — uPAR-KO cells plus pharmacological inhibitors and multiple apoptotic stimuli; single lab\",\n      \"pmids\": [\"24125407\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Ets-1 transcription factor positively regulates uPA gene expression in astrocytic tumors; dominant-negative Ets-1 abolishes uPA expression and invasive growth in collagen gel, placing Ets-1 upstream of uPA in the transcriptional control of glioma invasion.\",\n      \"method\": \"Quantitative RT-PCR, dominant-negative Ets-1 transfection, 3D collagen gel invasion assay, uPA inhibitor (aprotinin) treatment\",\n      \"journal\": \"Journal of neuropathology and experimental neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function with dominant-negative combined with pharmacological inhibition and functional invasion assay; single lab\",\n      \"pmids\": [\"10218628\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Two PEA3/AP1 composite elements at -2.4 kb and -6.9 kb upstream of the uPA transcription start site cooperate synergistically to mediate full TPA- and FGF-2-induced uPA gene transcription via binding of c-Jun, JunD, ATF-2, c-Fos, and Ets-2 transcription factors.\",\n      \"method\": \"DNase I hypersensitive site analysis, deletion analysis of uPA promoter, transient transfection reporter assays, EMSA with specific antibodies, dominant-negative Ets-2 overexpression\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (footprinting, deletion analysis, EMSA with supershift, dominant-negative) in a single study\",\n      \"pmids\": [\"9409785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The JNK/SAPK signaling pathway (MEKK1→MKK7→JNK) mediates transcriptional induction of uPA by the alkylating agent MNNG through an AP1 element at -2.4 kb in the uPA promoter; dominant-negative JNK pathway components and JNK inhibitors block this induction, while p38 inhibition does not.\",\n      \"method\": \"Transient transfection with uPA promoter-reporter constructs, dominant-negative kinase overexpression (MEKK1, MKK7, JNKK), JIP-1 overexpression, curcumin treatment, SB203580 (p38 inhibitor)\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — systematic dissection with multiple dominant-negatives and pharmacological tools targeting separate kinase branches; single lab but rigorous\",\n      \"pmids\": [\"10942386\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The inducible uPA gene is pre-associated with 'poised' transcription factories (RNA Pol II phosphorylated at Ser5 but not Ser2) prior to activation, and upon induction shifts to 'active' factories (Ser5+Ser2+) with concomitant looping out from its chromosome territory.\",\n      \"method\": \"RNA FISH, immunofluorescence for phospho-Pol II (Ser5, Ser2), chromosome conformation/CT positioning, quantitative factory-association assay in HepG2 cells\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct imaging-based localization linked to transcriptional mechanism; single lab with multiple imaging approaches\",\n      \"pmids\": [\"20052287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Plau (uPA) transcription in metastatic breast cancer cells is controlled by two AP-1 enhancers at -1.9 kb (ABR-1.9) and -4.1 kb (ABR-4.1) that bind Fra-1; RNA Pol II tracks from these sites toward the TSS producing unstable short RNAs that are converted to productive Plau-001 mRNA, and Fra-1 additionally suppresses a minor transcript (Plau-004) initiated from ABR-1.9.\",\n      \"method\": \"ChIP (Fra-1 and RNA Pol II), pharmacological inhibitors, RNAi knockdown, RNA analysis in MDA-MB231 cells\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP combined with RNAi and pharmacological approaches; single lab\",\n      \"pmids\": [\"25200076\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Plau (uPA) is a direct transcriptional target of YAP/TEAD signaling in keratinocytes and promotes keratinocyte proliferation downstream of YAP activation.\",\n      \"method\": \"RNA-seq of YAP2-5SA-ΔC transgenic mouse skin, YAP/TEAD binding motif analysis of dysregulated genes, functional validation of Plau in proliferation assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — RNA-seq plus bioinformatic identification of YAP/TEAD motif and functional assays; single lab\",\n      \"pmids\": [\"30382077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"O-fucosylation of uPA by poFUT1 activates the RhoA signaling pathway and promotes uterine angiogenesis and vascular remodeling.\",\n      \"method\": \"poFUT1 overexpression in hESCs and mouse model, O-fucosylation detection on uPA, RhoA pathway activation assay, vascular morphology analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — post-translational modification of uPA identified with downstream signaling readout in cell and mouse models; single lab\",\n      \"pmids\": [\"31601791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Small-molecule pyrrolone inhibitors of the uPAR·uPA protein-protein interaction bind the uPA pocket on uPAR and allosterically inhibit the distal uPAR·vitronectin interaction; molecular dynamics simulations show uPA stabilizes uPAR conformation to cooperatively favor VTN binding, and disruption of uPAR·uPA reduces FAK phosphorylation, Rac1 activation, and cancer cell invasion.\",\n      \"method\": \"Fluorescence polarization displacement assay, ELISA, surface plasmon resonance, cell adhesion assay, FAK phosphorylation Western blot, Rac1 GTPase activation assay, Matrigel invasion assay, molecular dynamics simulations\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple biophysical, biochemical, and cell-based orthogonal methods defining allosteric mechanism and downstream signaling consequences\",\n      \"pmids\": [\"25671694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"uPA binding to uPAR increases localization of uPAR to lipid raft (DRM) microdomains; this raft association, independent of uPA catalytic activity, is required for uPA-induced intracellular ERK signaling (phosphorylation), as demonstrated by cholesterol depletion with methyl-β-cyclodextrin.\",\n      \"method\": \"Sucrose gradient DRM fractionation, immunoprecipitation with anti-uPAR antibody, ERK phosphorylation assay, methyl-β-cyclodextrin treatment, glycosphingolipid compositional analysis\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — biochemical fractionation and functional signaling; single lab\",\n      \"pmids\": [\"17963689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Blocking the uPA-uPAR interaction (genetically via CRISPR/siRNA/KO or pharmacologically) protects intestinal epithelial barrier integrity by enhancing EGF/EGFR signaling; uPAR-deficient mice show improved barrier function and reduced DSS-induced colitis.\",\n      \"method\": \"CRISPR KO, siRNA, uPAR-KO mice, TEER and FITC-dextran permeability assay, tight junction assessment, DSS colitis model, intestinal organoids\",\n      \"journal\": \"EBioMedicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic and pharmacological approaches with in vitro and in vivo functional readouts; rigorous multi-method study\",\n      \"pmids\": [\"34933179\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Antibody-mediated neutralization of uPA proteolytic activity significantly reduces arthritis progression in CIA and DTH arthritis mouse models, with efficacy comparable to TNF-α blockade; pharmacokinetics revealed target-mediated drug disposition consistent with high endogenous uPA turnover.\",\n      \"method\": \"Neutralizing anti-uPA monoclonal antibody treatment in CIA and DTH arthritis models, pharmacokinetic analysis, double immunofluorescence for uPA/uPAR cell types in human RA synovium\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo proof-of-concept in two independent mouse models with pharmacological and histological validation; replicated across models\",\n      \"pmids\": [\"29282305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"uPA is responsible for converting aberrantly filtered plasminogen to plasmin in the tubular lumen in vivo (nephrotic syndrome), as shown in uPA-/- mice where urinary plasmin formation from plasminogen is suppressed; however, uPA-dependent plasmin generation is not essential for ENaC-mediated sodium retention.\",\n      \"method\": \"uPA-/- mice in doxorubicin-induced nephrotic syndrome model, amiloride treatment, urinary uPA activity and plasmin measurement, ENaC current recording in Xenopus oocytes\",\n      \"journal\": \"Acta physiologica\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — genetic KO combined with in vitro reconstitution in oocytes and pharmacological inhibition; dual mechanistic conclusions\",\n      \"pmids\": [\"31006168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"METTL3 promotes colorectal cancer metastasis by stabilizing PLAU mRNA in an m6A-dependent manner, with PLAU then participating in the MAPK/ERK pathway to promote angiogenesis and metastasis.\",\n      \"method\": \"METTL3 overexpression/knockdown, m6A methylation assay, PLAU mRNA stability assay, in vitro and in vivo metastasis models, MAPK/ERK pathway analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — m6A-dependent mRNA stabilization of PLAU demonstrated with functional pathway readout; single lab\",\n      \"pmids\": [\"35567945\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PLAU (uPA) secreted by esophageal squamous cell carcinoma tumor cells promotes conversion of fibroblasts to inflammatory cancer-associated fibroblasts via the uPAR/Akt/NF-κB pathway, leading to IL-8 upregulation in CAFs, which in turn further increases PLAU expression in tumor cells.\",\n      \"method\": \"Loss- and gain-of-function experiments, cytokine detection, RNA sequencing, RT-qPCR, MEK1/2 inhibitor U0126, siRNA knockdown, co-culture systems\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal signaling loop established with multiple functional and molecular assays; single lab\",\n      \"pmids\": [\"33574243\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PLAU (uPA) interacts with TM4SF1 to promote Akt signaling activation, conferring growth, survival, and cisplatin resistance in ARID1A-depleted NSCLC cells; TM4SF1 knockdown or anti-TM4SF1 neutralizing antibody reverses these effects.\",\n      \"method\": \"Co-immunoprecipitation (PLAU-TM4SF1 interaction), TM4SF1 knockdown, anti-TM4SF1 neutralizing antibody, Akt signaling Western blot, xenograft tumorigenesis, in vitro proliferation and invasion assays\",\n      \"journal\": \"Biology direct\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus functional loss-of-function and in vivo validation; single lab\",\n      \"pmids\": [\"38229120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ECRG2 (an esophageal cancer-related gene with KAZAL-type serine protease inhibitor similarity) directly binds uPA (both 55 kDa and 33 kDa forms) via co-immunoprecipitation and reduces uPA/plasmin proteolytic activity, inhibiting cancer cell migration and invasion.\",\n      \"method\": \"Binding affinity assay, co-immunoprecipitation, plasmin substrate chromogenic assay (D-Val-Phe-Lys-p-nitroanilide), Matrigel invasion assay, in vivo metastasis (tail vein injection in nude mice)\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — direct binding plus enzymatic inhibition assay; single lab\",\n      \"pmids\": [\"17602171\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PLAU (urokinase-type plasminogen activator, uPA) is a serine protease that converts plasminogen to plasmin on the cell surface upon binding to its GPI-anchored receptor uPAR; this interaction—regulated by inhibitors PAI-1/PAI-2 and LRP1-mediated internalization/recycling—drives pericellular ECM proteolysis and simultaneously activates multiple intracellular signaling cascades (PI3K/Akt, ERK/MEK, Rac1, FAK, eNOS, SREBP-1) through uPAR's association with co-receptors (integrins αvβ3/β1, LRP1) in lipid raft microdomains, controlling cell migration, invasion, angiogenesis, axonal regeneration, synaptic repair, macrophage differentiation, and epithelial barrier integrity, while its transcription is governed by AP-1/PEA3 enhancer elements responding to JNK, ERK, and Ets-1 pathways.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1984,\n      \"finding\": \"The human urokinase-type plasminogen activator (uPA/PLAU) gene was isolated and its complete nucleotide sequence determined. The gene is 6.4 kb long, organized in 11 exons, and contains a functional promoter region with GGCGGG repeats between CAAT and TATA boxes. The 5' end of uPA mRNA was mapped by S1 and primer extension.\",\n      \"method\": \"Gene cloning, nucleotide sequencing, S1 mapping, primer extension, CAT reporter transfection\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — original gene structure determination with multiple methods, foundational study\",\n      \"pmids\": [\"2987867\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1984,\n      \"finding\": \"A partially spliced polyadenylated precursor to urokinase mRNA was identified; the introns separate functionally different domains of the enzyme, establishing the domain organization of PLAU.\",\n      \"method\": \"cDNA cloning, nucleotide sequencing, RNA blot\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct cDNA sequencing revealing gene structure\",\n      \"pmids\": [\"6589620\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1982,\n      \"finding\": \"Complete primary structure of the A chain (157 amino acids) of high molecular mass urokinase was determined, revealing three domains: a growth factor domain (homologous to EGF), a kringle domain (homologous to plasminogen kringles), and a connecting peptide domain.\",\n      \"method\": \"Protein sequencing by Edman degradation, cyanogen bromide cleavage, endoproteinase Lys-C fragmentation, carboxypeptidase treatment\",\n      \"journal\": \"Hoppe-Seyler's Zeitschrift fur physiologische Chemie\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — complete protein sequence determination by multiple orthogonal methods\",\n      \"pmids\": [\"6754569\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1987,\n      \"finding\": \"The receptor-binding domain of uPA was mapped to the growth factor module (residues ~12–32), specifically amino acids 20–30 confer receptor binding specificity while residues 13–19 provide proper conformation. Synthetic peptides corresponding to uPA-(12-32) inhibited receptor binding of 125I-ATF with 50% inhibition at 100 nM.\",\n      \"method\": \"Synthetic peptide competition binding assays with 125I-labeled amino-terminal fragment (ATF)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct peptide mapping with quantitative binding assays, foundational receptor-binding study\",\n      \"pmids\": [\"3031025\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"The cDNA for the human uPA receptor (uPAR) was cloned and sequenced, encoding a 313 amino acid protein with a signal peptide and GPI-anchor signal. Expression of uPAR cDNA in mouse cells confirmed it is a functional uPA-binding protein that localizes to the cell surface and facilitates uPA-catalyzed plasminogen activation.\",\n      \"method\": \"cDNA cloning, sequencing, heterologous expression in mouse LB6 cells, cross-linking, direct binding, caseinolytic plaque assay, immunofluorescence\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — receptor cloning with functional reconstitution and multiple validation methods\",\n      \"pmids\": [\"1689240\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"uPA receptor localizes uPA to the leading edge of migrating monocytes in a chemotactic gradient, focusing extracellular proteolysis at the front of migrating cells. Receptor-bound uPA/PAI-2 complexes are rapidly cleared by cell-surface cleavage followed by endocytosis and degradation.\",\n      \"method\": \"Immunofluorescence on migrating monocytes, kinetic studies of PAI-2 inhibition of receptor-bound vs. free uPA, endocytosis assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct localization experiment with functional consequence, replicated mechanistic study\",\n      \"pmids\": [\"2166055\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"Receptor-bound uPA retains susceptibility to inhibition by PAI-1 and PAI-2, but with association rate constants ~40% lower than free uPA. PMA stimulation of U937 cells further reduces receptor-bound uPA inhibition by PAI-1 and PAI-2.\",\n      \"method\": \"Kinetic inhibition assays on receptor-bound uPA on U937 cells; second-order rate constant measurements\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — quantitative kinetic measurements with well-defined experimental system\",\n      \"pmids\": [\"2161846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"Binding of uPA to its cellular receptor on U937 cells dramatically increases the efficiency of plasminogen activation: Km drops 40-fold to 0.67 µM (below physiological plasminogen concentration of 2 µM), and plasmin generated at the cell surface is protected from α2-antiplasmin. Cell-surface binding of plasminogen is critical for this high-affinity activation.\",\n      \"method\": \"Kinetic analysis of plasminogen activation on U937 cells and with purified isolated uPAR; Km and kcat measurements; plasmin inhibitor protection assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — rigorous kinetic reconstitution with both cell-associated and isolated receptor, foundational enzymatic study\",\n      \"pmids\": [\"1829461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"Cathepsin B cleaves pro-uPA at the Lys158-Ile159 bond (same site as plasmin/kallikrein) to generate enzymatically active two-chain uPA. This activation occurs both for soluble and tumor cell receptor-bound pro-uPA; the receptor-binding growth factor domain remains intact after cathepsin B cleavage.\",\n      \"method\": \"In vitro cleavage assay with purified cathepsin B and pro-uPA, SDS-PAGE, Western blot, N-terminal sequencing, receptor binding assays on U937 cells, E-64 inhibitor control\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with mutagenesis-equivalent site mapping and inhibitor validation\",\n      \"pmids\": [\"1900515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"uPA (urokinase) is a pro-HGF/SF convertase: it cleaves the single-chain biologically inactive precursor of hepatocyte growth factor/scatter factor (pro-HGF/SF) at a single site, generating the active mature α–β heterodimer. This activation is blocked by PAI-1, protease nexin-1, and anti-uPA catalytic domain antibodies.\",\n      \"method\": \"In vitro cleavage of purified pro-HGF/SF by pure urokinase at nanomolar concentrations; functional assays (MDCK scatter, Met phosphorylation); inhibitor studies\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro cleavage with functional readout and multiple inhibitor controls\",\n      \"pmids\": [\"1334458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"PAI-1, the endogenous inhibitor of uPA, blocks cell migration by occupying the αVβ3 integrin binding site on vitronectin, thereby preventing integrin-mediated adhesion. This anti-migratory effect requires high-affinity PAI-1 binding to vitronectin and is independent of PAI-1's ability to inhibit plasminogen activators. Formation of PAI-1/plasminogen activator complexes reduces PAI-1 affinity for vitronectin and restores migration.\",\n      \"method\": \"SMC migration assays on vitronectin, blocking antibodies against αVβ3, active vs. latent PAI-1 competition, PAI-1 mutants\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mechanistic dissection with multiple mutants and controls, highly cited foundational study\",\n      \"pmids\": [\"8837777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"uPAR is internalized with uPA:serpin complexes via α2-macroglobulin receptor/LRP, then recycled back to the cell surface. Surface biotinylation experiments demonstrated that internalized biotinylated uPAR reappears at the plasma membrane in a PI-PLC-sensitive form, confirming true recycling rather than redistribution of intracellular pools.\",\n      \"method\": \"FACScan, immunofluorescence, immunoelectron microscopy, surface biotinylation recycling assay, PI-PLC sensitivity assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods demonstrating receptor recycling with direct biotinylation proof\",\n      \"pmids\": [\"9184208\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Two PEA3/AP-1 composite elements at −2.4 kb and −6.9 kb upstream of the transcription start site cooperate synergistically to confer full TPA- and FGF-2-inducibility of the uPA gene. AP-1 factors (c-Jun, JunD, ATF-2, c-Fos) and Ets transcription factors bind these elements, with c-Fos specifically binding the −6.9 kb element only after induction.\",\n      \"method\": \"DNase I hypersensitivity mapping, deletion analysis/transient transfection, EMSA with specific antibodies, dominant-negative Ets-2 expression\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal promoter dissection methods with functional validation\",\n      \"pmids\": [\"9409785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"uPA-stimulated cell migration requires uPAR ligation and proceeds via a signaling cascade: Ras → MEK → ERK → myosin light chain kinase (MLCK). MLCK is phosphorylated by a MEK-dependent pathway and leads to serine-phosphorylation of myosin II regulatory light chain. Migration is integrin-selective, occurring on vitronectin via β1-integrin (αVβ1) and αVβ5 but blocked by αVβ3.\",\n      \"method\": \"Dominant-negative and constitutively active Ras/MEK mutants, MLCK inhibitors, αVβ3 neutralizing antibody, migration assays on differentially coated surfaces\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — genetic epistasis plus pharmacological inhibition with integrin selectivity defined\",\n      \"pmids\": [\"10402467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Matriptase, an epithelial membrane serine protease, activates pro-uPA (urokinase) to its active form. Matriptase converts pro-HGF and pro-uPA but has no effect on plasminogen, positioning matriptase as an upstream membrane activator of uPA.\",\n      \"method\": \"In vitro cleavage assays with active matriptase isolated from human milk, synthetic substrate kinetics, functional HGF scatter assay, c-Met phosphorylation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with purified enzyme and functional readouts\",\n      \"pmids\": [\"10962009\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"MNNG-induced uPA gene transcription is mediated by the JNK signaling pathway via an AP-1 enhancer element at −2.4 kb. Dominant-negative MEKK1, MKK7, JNKK, and JIP-1 and curcumin (JNK inhibitor) all inhibited MNNG-induced uPA transcription, while dominant-negative MKK6 and SB203580 (p38 inhibitor) did not.\",\n      \"method\": \"Dominant-negative kinase constructs, pharmacological inhibitors, uPA promoter-reporter transfection assays\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple dominant-negative constructs and inhibitors providing genetic pathway dissection\",\n      \"pmids\": [\"10942386\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Downregulation of uPA by antisense transfection in human glioblastoma cells disrupts actin cytoskeleton formation, decreases cell migration, and reduces PI3K and Akt phosphorylation, causing G2/M-phase arrest and decreased clonogenic survival, positioning uPA upstream of the PI3K/Akt signaling pathway.\",\n      \"method\": \"Stable antisense uPA transfection, Western blot for phospho-PI3K/Akt, cell migration assays, cell cycle analysis, clonogenic survival assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — antisense KD with multiple phenotypic readouts but single lab\",\n      \"pmids\": [\"12545160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The uPA kringle domain binds directly to integrin αVβ3 (and also α4β1 and α9β1) independent of uPAR, enhancing plasminogen activation on CHO cells depleted of uPAR and inducing cell migration in an αVβ3-dependent manner. Plasminogen kringles 1-3/1-4 (angiostatin) blocked this interaction.\",\n      \"method\": \"Binding assays on uPAR-depleted CHO cells, purified soluble αVβ3 binding, cell migration assays with blocking antibodies, plasminogen activation assays\",\n      \"journal\": \"Thrombosis and haemostasis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — binding with purified proteins plus functional cell assays in uPAR-null background\",\n      \"pmids\": [\"16525582\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Crystal structure of uPA complexed with its receptor (uPAR) and an antibody was determined at 1.9 Å resolution. The three domains of uPAR form a concave shape with a central cone-shaped cavity where the uPA amino-terminal fragment inserts, explaining the molecular basis of uPA-uPAR interaction.\",\n      \"method\": \"X-ray crystallography at 1.9 Å\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution crystal structure of the uPA-uPAR complex\",\n      \"pmids\": [\"16456079\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"uPA activates SREBP-1 processing and increases nuclear mature SREBP-1 content (5.7-fold) in THP-1 macrophages via PI3K-dependent activation of MEK/ERK, upregulating HMGCoA reductase expression and increasing macrophage cholesterol biosynthesis by 172%.\",\n      \"method\": \"Western blot for HMGCR protein/mRNA, SREBP-1 nuclear fractionation, PI3K inhibitor (LY294002), MEK inhibitor, cholesterol biosynthesis assays with statins\",\n      \"journal\": \"Atherosclerosis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple inhibitor approaches defining the PI3K→MEK→ERK→SREBP-1 pathway, single lab\",\n      \"pmids\": [\"17681345\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"uPA binding to uPAR increases uPAR association with lipid rafts (detergent-resistant membrane fractions) in a manner independent of uPA catalytic activity. Disruption of lipid rafts by methyl-β-cyclodextrin inhibits uPA-induced ERK phosphorylation, showing that lipid raft association is required for uPA/uPAR intracellular signaling.\",\n      \"method\": \"Sucrose gradient fractionation of detergent-resistant membranes, uPAR immunoprecipitation from DRM fractions, ERK phosphorylation assays, methyl-β-cyclodextrin treatment, glycosphingolipid analysis\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — biochemical fractionation plus functional signaling assay, single lab\",\n      \"pmids\": [\"17963689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Quebec platelet disorder (QPD), a dominant bleeding disorder, is caused by a direct tandem duplication of a 78-kb genomic segment containing PLAU. This duplication specifically increases uPA mRNA during megakaryocyte differentiation without altering expression of flanking genes.\",\n      \"method\": \"Genomic copy number variation analysis, array CGH, fluorescence in situ hybridization on 38 QPD subjects and 425 controls\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — definitive genetic identification in large cohort with controls; directly implicates PLAU gene dosage in platelet biology\",\n      \"pmids\": [\"20007542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Prior to induction, the uPA (PLAU) gene is predominantly associated with 'poised transcription factories' containing RNA Pol II phosphorylated on Ser5 but not Ser2. After activation, the uPA locus associates with 'active factories' (Ser5+/Ser2+) and loops out from its chromosome territory. Gene positioning relative to the chromosome territory is independent of factory association levels.\",\n      \"method\": \"RNA FISH, immunofluorescence with phospho-specific Pol II antibodies, 3D nuclear localization analysis\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct nuclear localization with functional consequence, single lab\",\n      \"pmids\": [\"20052287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"uPA induces pulmonary microvascular endothelial permeability through LRP-dependent activation of endothelial NOS (eNOS) via PKA signaling. uPA induces eNOS phosphorylation at Ser1177, NO generation, and β-catenin nitrosylation/dissociation from VE-cadherin. This pathway is independent of PI3K-Akt.\",\n      \"method\": \"In vitro PMVEC monolayer permeability assay, eNOS phosphorylation Western blot, LRP antibody/RAP antagonist, PKA inhibitor (myristoylated PKI), in vivo lung permeability measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — reconstituted with multiple receptor/enzyme inhibitors, in vitro and in vivo validation\",\n      \"pmids\": [\"21540184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TMPRSS4, a type II transmembrane serine protease, directly converts inactive pro-uPA to the active two-chain form through its proteolytic activity. Active TMPRSS4 protease domain is released from cells and is membrane-associated; TMPRSS4 increases pro-uPA-mediated invasion in a serine protease activity-dependent manner, positioning TMPRSS4 as an upstream activator of pro-uPA.\",\n      \"method\": \"Pro-uPA cleavage assay with conditioned medium from TMPRSS4-overexpressing cells, active site mutant controls, Transwell invasion assays, Western blot\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct cleavage assay with active-site mutant, single lab\",\n      \"pmids\": [\"24434139\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Fra-1/AP-1 drives uPA (PLAU) overexpression in aggressive breast cancer cells via two AP-1 enhancers at −1.9 kb (ABR-1.9) and −4.1 kb (ABR-4.1) from the Plau-001 TSS. RNA Pol II is also recruited to the ABR regions, producing short unstable RNAs that track toward the TSS and convert to productive mRNA. A minor mRNA, Plau-004, is transcribed from ABR-1.9 and is repressed by Fra-1.\",\n      \"method\": \"ChIP, RNAi knockdown of Fra-1, pharmacological inhibition, RNA Pol II occupancy mapping\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal approaches (ChIP, RNAi, pharmacology), single lab\",\n      \"pmids\": [\"25200076\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Small-molecule pyrrolone inhibitors of the uPAR·uPA protein-protein interaction (Ki ~0.7 µM for labeled peptide displacement, IC50 ~18 µM for uPAR·uPA-ATF) allosterically inhibit the distal uPAR·vitronectin interaction, demonstrating cooperative binding between uPA and vitronectin on uPAR. These compounds reduce FAK phosphorylation, Rac1 activation, and MDA-MB-231 breast cancer cell invasion.\",\n      \"method\": \"Fluorescence polarization, surface plasmon resonance competition, ELISA, molecular dynamics simulations, free energy calculations, cellular FAK phosphorylation, Rac1 activation assay, Matrigel invasion assay\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — structural modeling plus multiple orthogonal biophysical/cell assays demonstrating allostery\",\n      \"pmids\": [\"25671694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"uPA binding to uPAR promotes axonal regeneration in the CNS by a plasminogen-independent mechanism: uPA/uPAR binding induces membrane recruitment and activation of β1-integrin via LRP1, leading to Rac1 GTPase activation and Rac1-induced axonal regeneration in injured axons.\",\n      \"method\": \"In vitro axonal injury models, in vivo CNS injury models, recombinant uPA treatment, LRP1 inhibition, β1-integrin blocking, Rac1 activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vitro and in vivo models with pathway epistasis using receptor/pathway inhibitors\",\n      \"pmids\": [\"27986809\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"QPD PLAU duplication dysregulates PLAU expression in a megakaryocyte-specific manner: QPD megakaryocytes overexpress normal PLAU transcripts >100-fold (from the disease chromosome) while QPD granulocytes show only ~3.9-fold increase, suggesting an active regulatory mechanism controlling uPA levels in blood that is specifically disrupted in megakaryocytes by the duplication.\",\n      \"method\": \"RNA-seq, quantitative RT-PCR, allele-specific transcript analysis, protein expression analysis in primary cells and cultured megakaryocytes\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — comprehensive transcriptomic analysis in primary patient cells with allele-specific resolution\",\n      \"pmids\": [\"28301587\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"uPA induces local synthesis of ezrin in astrocytes and triggers formation of peripheral astrocytic processes (PAPs) that contact the synapse, protecting the tripartite synapse from ischemic injury. Recombinant uPA treatment in vivo induces PAP formation in the ischemic brain.\",\n      \"method\": \"In vitro astrocyte cultures, in vivo ischemic stroke models, uPAR knockout mice, recombinant uPA treatment, immunofluorescence for ezrin and synaptic markers\",\n      \"journal\": \"Journal of cerebral blood flow and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro and in vivo with loss- and gain-of-function, single lab\",\n      \"pmids\": [\"29890880\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"uPA-uPAR binding induces local synthesis of ezrin in cortical neurons at the synapse and recruits β3-integrin to the postsynaptic density (PSD) via ICAM-5, followed by phosphorylation of ezrin at Thr-567 and reorganization of the actin cytoskeleton in the postsynaptic terminal, leading to recovery of dendritic spines and synapses damaged by ischemic stroke.\",\n      \"method\": \"In vitro cortical neuron cultures, in vivo ischemic stroke models, β3-integrin knockdown/blocking, ICAM-5 studies, phospho-ezrin Western blot, dendritic spine imaging\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple mechanistic steps defined with both in vitro and in vivo evidence, single lab\",\n      \"pmids\": [\"29720403\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"YAP/TEAD transcription factor activity directly regulates Plau (uPA) expression in epidermal keratinocytes, promoting their proliferation. RNA-seq of YAP2-5SA-ΔC transgenic mouse skin identified Plau as a dysregulated gene containing YAP/TEAD binding motifs in its 3' UTR, confirmed by functional characterization assays.\",\n      \"method\": \"RNA-seq of transgenic mouse skin, YAP/TEAD motif analysis, functional proliferation assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — transgenic mouse model with transcriptomic and functional validation, single lab\",\n      \"pmids\": [\"30382077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Protein O-fucosyltransferase 1 (poFUT1) increases O-fucosylation on uPA and activates the RhoA signaling pathway, facilitating uterine angiogenesis and vascular remodeling. Knockdown of poFUT1 reduces uPA O-fucosylation and impairs angiogenesis.\",\n      \"method\": \"Glycoprotein O-fucosylation analysis, RhoA activity assay, hESC and mouse model experiments, siRNA knockdown\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — post-translational modification (O-fucosylation) identified with functional pathway consequence\",\n      \"pmids\": [\"31601791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PLAU (uPA) promotes conversion of fibroblasts to inflammatory cancer-associated fibroblasts via the uPAR/Akt/NF-κB pathway, inducing IL-8 secretion. IL-8 from CAFs in turn promotes high PLAU expression in tumor cells (ESCC), creating a positive feedback loop. PLAU also promotes tumor cell proliferation via the MAPK pathway and migration by upregulating Slug and MMP9.\",\n      \"method\": \"Loss-of-function and gain-of-function experiments, RNA sequencing, cytokine array, RT-qPCR, MEK inhibitor U0126, Akt/NF-κB pathway inhibition\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic pathway dissection with multiple experimental approaches, single lab\",\n      \"pmids\": [\"33574243\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"uPA mediates endothelial tubular network (ETN) formation in HUVEC-MSC co-culture via cross-talk of uPAR, uPA's catalytic activity, uPA's binding to uPAR, and uPA nuclear translocation, coordinated with αV-integrins, VEGFR2, and Notch receptor/ligand pathways.\",\n      \"method\": \"HUVEC-MSC co-culture angiogenesis assay, siRNA knockdown of pathway components, pharmacological inhibitors at multiple steps, mRNA expression analysis\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multi-step pathway inhibition in co-culture model, single lab\",\n      \"pmids\": [\"34619163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"AQR promotes endothelial cell senescence via PLAU: AQR overexpression upregulates PLAU, and knockdown of PLAU rescues senescence-related phenotypes (SA-β-gal staining, P21 upregulation, G2/M arrest) induced by AQR overexpression or TNF-α treatment, establishing an AQR/PLAU signaling axis in endothelial cell senescence.\",\n      \"method\": \"Transcriptomic analysis of AQR overexpression/knockdown in HUVECs, SA-β-gal staining, CDKN1A Western blot, colony formation, cell cycle analysis, PLAU siRNA rescue\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epistasis established by siRNA rescue, single lab\",\n      \"pmids\": [\"35270021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"METTL3 stabilizes PLAU mRNA in an m6A-dependent manner, promoting colorectal cancer metastasis via the MAPK/ERK pathway and angiogenesis.\",\n      \"method\": \"m6A-seq, METTL3 overexpression/knockdown, RNA stability assays, MAPK/ERK pathway analysis, in vivo tumor models\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — m6A-dependent mRNA stabilization mechanism with in vitro and in vivo validation\",\n      \"pmids\": [\"35567945\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PLAU (uPA) knockout by CRISPR-Cas9 completely stops matrix remodeling (measured by AFM-based stiffness changes) and significantly reduces cancer cell invasion in a 3D tumouroid model, confirming uPA's enzymatic role in ECM degradation as a driver of invasion. Pharmacological uPA inhibition (UK-371,801) showed similarly reduced matrix degradation and invasion.\",\n      \"method\": \"CRISPR-Cas9 knockout of PLAU, 3D tumouroid culture, atomic force microscopy (AFM) stiffness measurement, invasion quantification, pharmacological inhibitor UK-371,801\",\n      \"journal\": \"Matrix biology plus\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — genetic knockout + pharmacological inhibition + biophysical ECM measurements in orthogonal 3D model\",\n      \"pmids\": [\"38020586\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PLAU interacts with TM4SF1 to activate Akt signaling, promoting NSCLC cell growth, cisplatin resistance, and survival. TM4SF1 knockdown or anti-TM4SF1 neutralizing antibody phenocopies PLAU inhibition, and PLAU overexpression stabilizes TM4SF1 at the cell surface.\",\n      \"method\": \"Co-immunoprecipitation, overexpression/knockdown experiments, Akt phosphorylation Western blot, nude mouse xenograft, anti-TM4SF1 antibody treatment\",\n      \"journal\": \"Biology direct\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — co-IP plus functional epistasis, single lab\",\n      \"pmids\": [\"38229120\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PLAU encodes urokinase-type plasminogen activator (uPA), a serine protease that is secreted as inactive pro-uPA and activated extracellularly by plasmin, cathepsin B, matriptase, or TMPRSS4 through cleavage at Lys158-Ile159; upon binding its GPI-anchored receptor uPAR (itself cloned and characterized as a 313-aa protein), receptor-bound uPA activates plasminogen to plasmin with dramatically enhanced kinetics (Km drops 40-fold) and protects plasmin from α2-antiplasmin, focusing pericellular proteolysis at the leading edge of migrating cells; uPA also cleaves pro-HGF/SF to generate active growth factor; uPAR localizes to lipid rafts upon uPA binding, and the uPA-uPAR complex signals through multiple pathways including Ras/MEK/ERK/MLCK (migration), PI3K/Akt (survival/migration), and LRP1/β1-integrin/Rac1 (axonal regeneration), while the uPA kringle domain independently engages αVβ3 integrin; uPA activity is regulated by PAI-1 (which also blocks integrin-mediated migration by competing for the vitronectin binding site) and by receptor recycling via LRP-mediated internalization of uPA:serpin complexes with subsequent uPAR return to the surface; PLAU transcription is driven by AP-1/PEA3 composite elements and regulated by Fra-1, Ets-1, and the JNK/AP-1 pathway, with the gene associated with poised transcription factories prior to induction; post-translational regulation includes O-fucosylation by poFUT1 and m6A-dependent mRNA stabilization by METTL3; the crystal structure of uPA-uPAR complex at 1.9 Å reveals a cone-shaped central cavity in uPAR where the uPA amino-terminal fragment inserts, and small-molecule inhibitors of this interface allosterically disrupt the distal uPAR-vitronectin interaction.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PLAU encodes urokinase-type plasminogen activator (uPA), a secreted serine protease that converts plasminogen to plasmin on the cell surface, driving pericellular extracellular matrix proteolysis essential for wound healing, tissue remodeling, angiogenesis, and fibrin clearance [PMID:16763560, PMID:21802414, PMID:12657615]. Pro-uPA is activated by cleavage (e.g., by TMPRSS4) and signals through its GPI-anchored receptor uPAR, which upon uPA binding partitions into lipid raft microdomains and engages co-receptors including integrins (αvβ3, α5β1, β1, β3) and LRP1 to activate PI3K/Akt, ERK/MEK, Rac1, FAK, and eNOS cascades that control cell migration, invasion, neuronal regeneration, synaptic repair, macrophage differentiation, and endothelial barrier permeability—many of these functions independent of plasmin generation [PMID:24434139, PMID:17963689, PMID:27986809, PMID:29720403, PMID:12545160, PMID:21540184, PMID:25671694]. Transcription of PLAU is governed by distal AP-1/PEA3 enhancer elements responsive to JNK, ERK, Fra-1, Ets-1, and YAP/TEAD pathways, and its mRNA is further stabilized by METTL3-mediated m6A modification [PMID:9409785, PMID:10942386, PMID:25200076, PMID:10218628, PMID:35567945]. Tandem duplication of the PLAU locus causes Quebec platelet disorder, a bleeding disorder driven by >100-fold megakaryocyte-specific uPA overexpression that results in intra-platelet plasmin-mediated degradation of α-granule proteins [PMID:20007542, PMID:28301587].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Defining how uPA is cleared from the cell surface resolved the question of receptor fate: uPAR internalizes uPA:PAI-1 complexes via LRP and recycles rather than being degraded, establishing a regenerative receptor trafficking cycle that sustains surface uPA signaling capacity.\",\n      \"evidence\": \"Surface biotinylation, FACS, immunoelectron microscopy in cultured cells with PI-PLC cleavage controls\",\n      \"pmids\": [\"9184208\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinetic parameters of uPAR recycling in vivo not defined\", \"Whether recycled uPAR re-acquires uPA on the surface was not tested\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Identification of two cooperative PEA3/AP-1 composite enhancers at -2.4 kb and -6.9 kb upstream of the PLAU TSS, bound by c-Jun, JunD, ATF-2, c-Fos, and Ets-2, established the core transcriptional architecture governing inducible uPA expression.\",\n      \"evidence\": \"DNase I hypersensitivity mapping, promoter deletion analysis, EMSA with supershift, dominant-negative Ets-2 in reporter assays\",\n      \"pmids\": [\"9409785\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Chromatin-level regulation (histone modifications, 3D looping) at these enhancers not addressed at this time\", \"Cell-type specificity of enhancer usage not resolved\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Demonstrating that Ets-1 drives uPA transcription in glioma placed an upstream transcription factor in the invasion program, linking oncogenic signaling to PLAU gene activation.\",\n      \"evidence\": \"Dominant-negative Ets-1 abolishes uPA mRNA and collagen gel invasion in astrocytic tumor cells\",\n      \"pmids\": [\"10218628\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Ets-1 acts through the same PEA3/AP-1 elements identified at -2.4/-6.9 kb was not directly tested\", \"Ets-1 occupancy at the PLAU locus not shown by ChIP\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Mapping the MEKK1→MKK7→JNK cascade to the AP-1 element at -2.4 kb resolved which MAPK branch induces PLAU transcription in response to genotoxic stress, distinguishing JNK from p38.\",\n      \"evidence\": \"Dominant-negative kinases, JIP-1 scaffold overexpression, curcumin, and SB203580 with uPA promoter-reporter constructs\",\n      \"pmids\": [\"10942386\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether JNK pathway also regulates the -6.9 kb enhancer not tested\", \"Post-transcriptional contributions to uPA induction not excluded\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Genetic knockout studies established uPA (with tPA) as required for choroidal neovascularization and showed uPA functions through plasminogen-dependent MMP activation and fibrin clearance in vivo, defining its non-redundant role in pathological angiogenesis.\",\n      \"evidence\": \"uPA−/−, tPA−/−, Plg−/−, and uPAR−/− mice in laser-induced CNV model with in situ zymography\",\n      \"pmids\": [\"12657615\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of uPA vs tPA to CNV not quantitatively separated\", \"Whether uPA's signaling functions (vs proteolysis) contribute to CNV not addressed\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Loss-of-function experiments first placed uPA upstream of PI3K/Akt signaling and actin cytoskeleton organization in glioblastoma, revealing a signaling role for uPA beyond proteolysis in controlling tumor cell migration.\",\n      \"evidence\": \"Stable antisense transfection reducing uPA, with Western blot for phospho-PI3K/Akt and actin imaging in glioblastoma cells\",\n      \"pmids\": [\"12545160\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PI3K activation requires uPA catalytic activity or only uPAR binding not distinguished\", \"Antisense approach does not exclude off-target effects\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Double- and triple-knockout mouse studies demonstrated that uPA and tPA are the two principal physiological plasminogen activators for skin wound healing, with plasma kallikrein providing a third, residual mechanism—quantifying the protease hierarchy in tissue repair.\",\n      \"evidence\": \"uPA−/−;tPA−/− and Plg−/− mice with wound healing kinetics, MMP inhibitors, and ecotin-based kallikrein inhibition\",\n      \"pmids\": [\"16763560\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative spatial and temporal contributions of uPA vs tPA during healing not resolved\", \"Role of uPA signaling (vs proteolysis) in wound repair not separated\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Discovery that the uPA kringle domain directly binds integrins αvβ3, α4β1, and α9β1 independently of uPAR established a receptor-independent mechanism by which uPA promotes cell migration and surface plasminogen activation.\",\n      \"evidence\": \"Binding assays on uPAR-depleted cells, pulldown with purified soluble αvβ3, migration and plasminogen activation assays with blocking antibodies\",\n      \"pmids\": [\"16525582\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of kringle-integrin interaction not determined\", \"In vivo significance of uPAR-independent integrin binding not established\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Showing that uPA binding drives uPAR into lipid raft microdomains and that this raft association is required for ERK phosphorylation (independent of uPA catalytic activity) revealed the membrane microdomain basis of uPA-initiated signaling.\",\n      \"evidence\": \"Sucrose gradient DRM fractionation, methyl-β-cyclodextrin cholesterol depletion, ERK phosphorylation assay\",\n      \"pmids\": [\"17963689\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the transmembrane signaling adapter linking GPI-anchored uPAR to ERK in rafts not identified\", \"DRM fractionation is an indirect proxy for raft association\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstration that uPA-uPAR activates SREBP-1 via PI3K→MEK→ERK to upregulate HMG-CoA reductase and cholesterol biosynthesis in macrophages expanded uPA's role beyond proteolysis and migration into metabolic reprogramming relevant to atherosclerosis.\",\n      \"evidence\": \"Cholesterol biosynthesis assays, PI3K and MEK inhibitors, SREBP-1 nuclear translocation, HMG-CoA reductase expression in macrophages\",\n      \"pmids\": [\"17681345\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether this metabolic effect occurs in vivo in atherosclerotic plaques not shown\", \"Contribution of plasmin-dependent vs -independent signaling not separated\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identification of a tandem 78-kb PLAU duplication as the causative mutation in Quebec platelet disorder resolved a decades-old genetic mystery and established that PLAU copy-number gain drives megakaryocyte-specific uPA overexpression causing intra-platelet fibrinolysis.\",\n      \"evidence\": \"Copy-number analysis in 38 QPD subjects and >400 controls, family segregation, transcript quantification\",\n      \"pmids\": [\"20007542\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of megakaryocyte-specific dysregulation from the duplication not yet explained\", \"Whether the duplication alters chromatin architecture at the locus not tested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrating that the PLAU locus is pre-associated with poised (Ser5-phosphorylated Pol II) transcription factories before induction and shifts to active factories upon stimulation provided a chromatin-level explanation for the rapid inducibility of uPA expression.\",\n      \"evidence\": \"RNA FISH, phospho-Pol II (Ser5/Ser2) immunofluorescence, chromosome territory positioning in HepG2 cells\",\n      \"pmids\": [\"20052287\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the distal AP-1/PEA3 enhancers mediate the factory association not tested\", \"Generalizability beyond HepG2 cells not established\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Genetic epistasis showing additive impairment of wound healing, bone growth, and gestation in MMP9−/−;uPA−/− double knockouts established functional cooperativity between uPA-generated plasmin and MMP9 in tissue remodeling, with compensatory uPA upregulation in MMP9-deficient wounds.\",\n      \"evidence\": \"Double-knockout mice (MMP9−/− × uPA−/−, tPA−/−, or uPAR−/−), quantitative phenotyping of wounds, bone, and gestation\",\n      \"pmids\": [\"21802414\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether plasmin directly activates MMP9 in these tissues or acts in parallel not distinguished\", \"Molecular basis of compensatory uPA upregulation in MMP9−/− not identified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Dissecting uPA's endothelial permeability effect through LRP→PKA→eNOS→NO→β-catenin nitrosylation and VE-cadherin dissociation revealed a complete signaling axis by which uPA disrupts vascular barrier integrity, independent of plasmin.\",\n      \"evidence\": \"PMVEC permeability assays, eNOS Ser1177 phosphorylation, RAP/anti-LRP blockade, PKA inhibitor, in vivo lung permeability\",\n      \"pmids\": [\"21540184\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this pathway operates in all vascular beds not tested\", \"Contribution of uPA proteolytic activity vs receptor binding not fully excluded\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Showing that uPA-uPAR signaling promotes monocyte-to-macrophage differentiation and protects macrophages from Ox-LDL-induced apoptosis via ERK1/2-dependent Bim downregulation linked the uPA system to macrophage survival in atherogenesis.\",\n      \"evidence\": \"uPAR-KO mouse peritoneal macrophages, ERK and Bim Western blot, apoptosis assays with multiple stimuli\",\n      \"pmids\": [\"24125407\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo relevance to atherosclerotic plaque macrophage survival not demonstrated\", \"Whether uPA or another uPAR ligand drives this in vivo not distinguished\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identification of TMPRSS4 as a direct activator that cleaves pro-uPA to two-chain uPA provided the first evidence of a membrane-anchored serine protease converting pro-uPA in the tumor microenvironment.\",\n      \"evidence\": \"In vitro proteolysis assay, conditioned medium analysis, activity-dependent invasion assay\",\n      \"pmids\": [\"24434139\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo contribution of TMPRSS4 to pro-uPA activation relative to other activators (e.g., plasmin, kallikrein) not established\", \"Cleavage site specificity not mapped\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrating that uPA-uPAR engages α5β1-integrin to activate FAK at growth cones and promote neuronal migration/neuritogenesis established a plasmin-independent developmental function for uPA in the CNS.\",\n      \"evidence\": \"Co-immunoprecipitation of uPAR with integrins, FAK phosphorylation and localization in CNS explants\",\n      \"pmids\": [\"24481918\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether uPA catalytic activity contributes at all to neuritogenesis not tested\", \"Co-IP-based integrin interaction not validated by reciprocal pulldown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"ChIP mapping of Fra-1 and Pol II at -1.9 and -4.1 kb AP-1 enhancers in metastatic breast cancer cells resolved how PLAU transcription is regulated in invasion-competent cells and revealed Pol II tracking from enhancers to the TSS producing unstable short RNAs.\",\n      \"evidence\": \"ChIP for Fra-1 and Pol II, RNAi knockdown, pharmacological inhibitors in MDA-MB231 cells\",\n      \"pmids\": [\"25200076\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the enhancer-derived short RNAs have functional roles not determined\", \"Relationship of these enhancers to the -2.4/-6.9 kb elements defined in earlier studies not clarified\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Small-molecule pyrrolone inhibitors of the uPAR–uPA interface revealed allosteric coupling between the uPA-binding and vitronectin-binding sites on uPAR, mechanistically linking uPA occupancy to integrin co-signaling and providing pharmacological validation that this interaction drives FAK/Rac1 activation and invasion.\",\n      \"evidence\": \"Fluorescence polarization, SPR, ELISA, FAK/Rac1 activation, Matrigel invasion, molecular dynamics simulations\",\n      \"pmids\": [\"25671694\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo efficacy of pyrrolone inhibitors not demonstrated\", \"Whether allosteric coupling operates identically across uPAR-expressing cell types not tested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"In vivo and in vitro CNS injury models showed that uPA-uPAR promotes axonal regeneration via LRP1-mediated β1-integrin membrane recruitment and Rac1 activation, entirely independent of plasmin—establishing a proteolysis-independent neuroprotective signaling function.\",\n      \"evidence\": \"Recombinant uPA treatment in axonal injury models, LRP1 blockade, β1-integrin recruitment, Rac1 activation assays\",\n      \"pmids\": [\"27986809\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether uPA-driven axonal regeneration translates to functional recovery long-term not shown\", \"Specific LRP1 domain mediating β1-integrin recruitment not identified\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"The >100-fold megakaryocyte-specific upregulation (vs ~4-fold in granulocytes) from the QPD PLAU duplication revealed lineage-specific regulatory control of the duplicated locus, explaining the platelet-restricted phenotype of QPD.\",\n      \"evidence\": \"RNA-seq and quantitative RT-PCR in primary megakaryocytes and granulocytes from QPD donors\",\n      \"pmids\": [\"28301587\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The cis-regulatory mechanism underlying megakaryocyte-specific amplification not identified\", \"Whether epigenetic marks differ between the two PLAU copies not tested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Therapeutic neutralization of uPA proteolytic activity reduced arthritis progression comparably to TNF-α blockade in two mouse models, providing in vivo proof-of-concept that uPA enzymatic activity drives inflammatory joint disease.\",\n      \"evidence\": \"Anti-uPA neutralizing antibody in CIA and DTH arthritis models, pharmacokinetics, immunofluorescence in human RA synovium\",\n      \"pmids\": [\"29282305\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether uPA signaling (vs proteolysis) also contributes to arthritis pathology not separated\", \"Human clinical validation not performed\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Discovery that uPA induces ezrin synthesis and β3-integrin/ICAM-5-dependent ezrin phosphorylation at both astrocytic perisynaptic processes and postsynaptic densities established a plasmin-independent mechanism for tripartite synapse protection after ischemia.\",\n      \"evidence\": \"In vitro and in vivo ischemic stroke models, recombinant uPA, Western blot, immunofluorescence for ezrin Thr-567 phosphorylation, β3-integrin and ICAM-5 blockade\",\n      \"pmids\": [\"29890880\", \"29720403\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether endogenous uPA reaches sufficient concentrations at synapses during ischemia not established\", \"Upstream signal linking uPA-uPAR to ezrin mRNA translation not identified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identification of PLAU as a direct transcriptional target of YAP/TEAD signaling in keratinocytes added the Hippo pathway to the transcriptional inputs governing PLAU expression and linked uPA to epithelial proliferation control.\",\n      \"evidence\": \"RNA-seq of YAP2-5SA-ΔC transgenic mouse skin, YAP/TEAD motif analysis, functional proliferation assays\",\n      \"pmids\": [\"30382077\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct YAP/TEAD occupancy at the PLAU promoter not shown by ChIP\", \"Whether YAP-driven PLAU induction requires the known AP-1 enhancers not tested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstration that O-fucosylation of uPA by poFUT1 activates RhoA signaling and promotes uterine angiogenesis identified a post-translational modification that modulates uPA function in vascular remodeling.\",\n      \"evidence\": \"poFUT1 overexpression, O-fucosylation detection on uPA, RhoA pathway activation in hESCs and mouse uterus\",\n      \"pmids\": [\"31601791\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific O-fucosylation sites on uPA not mapped\", \"Whether O-fucosylation affects uPA-uPAR binding affinity not tested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Genetic ablation of uPA in nephrotic mice suppressed tubular plasminogen-to-plasmin conversion, establishing uPA as the principal activator of filtered plasminogen in the renal tubular lumen, though this was dispensable for ENaC-mediated sodium retention.\",\n      \"evidence\": \"uPA−/− mice with doxorubicin-induced nephrotic syndrome, urinary plasmin measurement, ENaC currents in Xenopus oocytes\",\n      \"pmids\": [\"31006168\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other tubular proteases compensate long-term not assessed\", \"Source of tubular uPA (luminal vs basolateral secretion) not determined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Blocking uPA-uPAR interaction enhanced EGF/EGFR signaling and protected intestinal epithelial barrier integrity, with uPAR-KO mice showing reduced DSS colitis—revealing that uPA-uPAR negatively regulates epithelial barrier function through EGFR competition.\",\n      \"evidence\": \"CRISPR KO, siRNA, uPAR-KO mice, TEER, FITC-dextran permeability, tight junction assessment, DSS colitis model, organoids\",\n      \"pmids\": [\"34933179\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism by which uPAR engagement suppresses EGFR signaling not fully elucidated\", \"Whether uPA proteolytic activity contributes to barrier disruption separately from receptor binding not distinguished\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"METTL3-dependent m6A modification was shown to stabilize PLAU mRNA and drive MAPK/ERK-mediated angiogenesis and metastasis in colorectal cancer, adding an epitranscriptomic layer to PLAU regulation.\",\n      \"evidence\": \"METTL3 overexpression/knockdown, m6A methylation assay, mRNA stability measurement, in vitro/in vivo metastasis models\",\n      \"pmids\": [\"35567945\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific m6A sites on PLAU mRNA not mapped\", \"m6A reader protein mediating stabilization not identified\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Discovery that uPA interacts with TM4SF1 to activate Akt and confer cisplatin resistance in ARID1A-depleted NSCLC identified a new transmembrane partner linking uPA to drug resistance signaling.\",\n      \"evidence\": \"Co-immunoprecipitation, TM4SF1 knockdown and neutralizing antibody, Akt signaling, xenograft assays\",\n      \"pmids\": [\"38229120\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reciprocal validation of PLAU-TM4SF1 interaction not shown\", \"Whether this interaction is direct or mediated by uPAR not distinguished\", \"Generalizability beyond ARID1A-depleted context not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: the structural basis of how uPAR transmits signals across the membrane via transmembrane co-receptors; the identity of the m6A reader stabilizing PLAU mRNA; the cis-regulatory mechanism underlying megakaryocyte-specific PLAU overexpression in QPD; and whether uPA's proteolytic versus signaling functions can be therapeutically separated in vivo.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of the uPAR–integrin–LRP1 signaling complex exists\", \"Therapeutic window between beneficial (wound healing) and pathological (invasion, inflammation) uPA activities not defined\", \"Complete post-translational modification map of uPA lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 2, 8, 14, 15, 27, 28]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 6, 9, 13, 24, 26]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [1, 2, 28, 30, 32]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 7, 25]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [1, 2, 14, 15]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 6, 7, 9, 13, 24, 26]},\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [4, 5, 28]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [16, 27]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [17, 18, 19, 20, 21, 22]}\n    ],\n    \"complexes\": [\n      \"uPA:uPAR complex\",\n      \"uPA:PAI-1:uPAR:LRP1 internalization complex\"\n    ],\n    \"partners\": [\n      \"PLAUR\",\n      \"LRP1\",\n      \"ITGAV\",\n      \"ITGB3\",\n      \"ITGB1\",\n      \"SERPINE1\",\n      \"TM4SF1\",\n      \"TMPRSS4\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"PLAU encodes urokinase-type plasminogen activator (uPA), a secreted serine protease that is produced as inactive single-chain pro-uPA and activated extracellularly by plasmin, cathepsin B, matriptase, or TMPRSS4 through cleavage at Lys158–Ile159, yielding a two-chain enzyme whose primary function is conversion of plasminogen to plasmin at the cell surface [PMID:1829461, PMID:1900515, PMID:10962009, PMID:24434139]. Binding of uPA to its GPI-anchored receptor uPAR via the amino-terminal growth factor domain lowers the Km for plasminogen activation ~40-fold, focuses pericellular proteolysis to the leading edge of migrating cells, and triggers non-proteolytic signaling through Ras/MEK/ERK/MLCK, PI3K/Akt, and LRP1/β1-integrin/Rac1 cascades that drive cell migration, survival, axonal regeneration, and angiogenesis [PMID:2166055, PMID:10402467, PMID:27986809, PMID:16456079]. uPA also cleaves pro-HGF/SF to its active heterodimer, degrades extracellular matrix in 3D tumour models, and its kringle domain independently engages αVβ3 integrin to promote migration [PMID:1334458, PMID:38020586, PMID:16525582]. Tandem duplication of the PLAU locus causes Quebec platelet disorder, a dominant bleeding disorder driven by megakaryocyte-specific >100-fold overexpression of uPA [PMID:20007542, PMID:28301587].\",\n  \"teleology\": [\n    {\n      \"year\": 1982,\n      \"claim\": \"Determination of the complete primary structure of the uPA A-chain revealed a modular architecture — growth factor, kringle, and connecting peptide domains — establishing the domain logic that underlies all subsequent functional mapping.\",\n      \"evidence\": \"Edman degradation and multiple fragmentation strategies on purified high-MW urokinase\",\n      \"pmids\": [\"6754569\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure–function assignments for individual domains yet\", \"Receptor identity unknown\"]\n    },\n    {\n      \"year\": 1984,\n      \"claim\": \"Isolation and sequencing of the PLAU gene (6.4 kb, 11 exons) showed that intron–exon boundaries separate functional domains, providing the genomic framework for understanding splicing, transcriptional regulation, and evolutionary relationships.\",\n      \"evidence\": \"Gene cloning, nucleotide sequencing, S1 mapping, primer extension, cDNA analysis\",\n      \"pmids\": [\"2987867\", \"6589620\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Promoter regulatory elements beyond CAAT/TATA not yet defined\", \"No information on tissue-specific regulation\"]\n    },\n    {\n      \"year\": 1987,\n      \"claim\": \"Mapping of the receptor-binding determinant to the growth factor module (residues 12–32) answered how uPA engages the cell surface and showed the catalytic domain is dispensable for receptor binding.\",\n      \"evidence\": \"Synthetic peptide competition binding assays with ¹²⁵I-ATF on intact cells\",\n      \"pmids\": [\"3031025\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor identity still uncloned\", \"Three-dimensional basis of binding unknown\"]\n    },\n    {\n      \"year\": 1990,\n      \"claim\": \"Cloning of uPAR as a 313-aa GPI-anchored protein and demonstration that receptor-bound uPA localizes to the leading edge of migrating cells established the paradigm of receptor-focused pericellular proteolysis.\",\n      \"evidence\": \"cDNA cloning with heterologous reconstitution; immunofluorescence on chemotactically migrating monocytes; kinetic inhibition studies with PAI-1/PAI-2\",\n      \"pmids\": [\"1689240\", \"2166055\", \"2161846\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of uPA–uPAR interaction unknown\", \"Intracellular signaling consequences not addressed\"]\n    },\n    {\n      \"year\": 1991,\n      \"claim\": \"Quantitative enzymology revealed that uPAR binding drops the Km for plasminogen activation 40-fold (to 0.67 µM, below physiological [plasminogen]) and protects product plasmin from α2-antiplasmin, explaining why cell-surface localization matters for in vivo fibrinolysis and invasion.\",\n      \"evidence\": \"Kinetic analysis on U937 cells and purified isolated uPAR\",\n      \"pmids\": [\"1829461\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural explanation for kinetic enhancement unknown\", \"Relative contribution of plasminogen co-receptors not resolved\"]\n    },\n    {\n      \"year\": 1991,\n      \"claim\": \"Identification of cathepsin B as a pro-uPA activator cleaving the same Lys158–Ile159 bond as plasmin revealed that tumor-derived cysteine proteases can initiate the uPA activation cascade independently of plasmin feedback.\",\n      \"evidence\": \"In vitro cleavage with purified cathepsin B, N-terminal sequencing, E-64 inhibitor control\",\n      \"pmids\": [\"1900515\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of cathepsin B activation vs. plasmin autoactivation not determined\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Discovery that uPA converts pro-HGF/SF to active HGF/SF expanded the substrate repertoire beyond plasminogen, linking uPA to receptor tyrosine kinase signaling (c-Met) and cell scattering.\",\n      \"evidence\": \"In vitro cleavage of purified pro-HGF/SF with functional scatter and Met phosphorylation readouts\",\n      \"pmids\": [\"1334458\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative in vivo contribution of uPA vs. other HGF activases (e.g., HGFA) unresolved\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Demonstration that PAI-1 blocks migration by competing with αVβ3 integrin for vitronectin — independent of its protease-inhibitory activity — revealed a non-canonical regulatory mechanism where serpin–protease complex formation restores migration by lowering PAI-1 affinity for vitronectin.\",\n      \"evidence\": \"SMC migration assays with PAI-1 mutants, integrin blocking antibodies\",\n      \"pmids\": [\"8837777\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of PAI-1–vitronectin–integrin competition not resolved at atomic level\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Elucidation of composite PEA3/AP-1 enhancer elements at −2.4 kb and −6.9 kb, combined with later JNK/AP-1 pathway mapping and Fra-1 studies, defined the transcriptional logic driving PLAU induction in response to growth factors and genotoxic stress.\",\n      \"evidence\": \"DNase I hypersensitivity, deletion reporters, EMSA, dominant-negative kinases; ChIP and RNAi of Fra-1\",\n      \"pmids\": [\"9409785\", \"10942386\", \"25200076\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Chromatin remodeling steps at the PLAU locus incompletely mapped\", \"Megakaryocyte-specific regulatory elements not identified\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Discovery that uPAR is recycled back to the cell surface after LRP-mediated endocytosis of uPA:serpin complexes explained how cells maintain receptor availability for successive rounds of pericellular proteolysis.\",\n      \"evidence\": \"Surface biotinylation recycling assay, PI-PLC sensitivity, immunoelectron microscopy\",\n      \"pmids\": [\"9184208\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Sorting signals directing uPAR recycling vs. LRP degradation not identified\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Genetic epistasis with dominant-negative Ras/MEK and MLCK inhibitors established the first complete intracellular signaling cascade (Ras→MEK→ERK→MLCK→myosin II) downstream of uPA–uPAR ligation driving cell migration, demonstrating that uPA is a bona fide signaling ligand beyond its protease role.\",\n      \"evidence\": \"Dominant-negative constructs, pharmacological inhibitors, migration assays on vitronectin with integrin blocking antibodies\",\n      \"pmids\": [\"10402467\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Proximal signaling link between GPI-anchored uPAR and cytoplasmic Ras not identified\", \"Lipid raft requirement not yet established\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"The 1.9 Å crystal structure of the uPA–uPAR complex revealed uPAR's three-domain concave architecture with a central cone-shaped cavity accommodating the uPA amino-terminal fragment, providing the atomic framework for understanding receptor engagement and enabling structure-based drug design.\",\n      \"evidence\": \"X-ray crystallography of uPA:uPAR:antibody ternary complex\",\n      \"pmids\": [\"16456079\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length uPA structure in complex not determined\", \"Conformational dynamics upon uPAR engagement not characterized\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"The uPA kringle domain was shown to bind αVβ3 integrin directly and independently of uPAR, identifying a second cell-surface tethering mechanism that enhances plasminogen activation on uPAR-negative cells and promotes integrin-dependent migration.\",\n      \"evidence\": \"Binding assays on uPAR-depleted CHO cells and purified αVβ3, blocking antibodies\",\n      \"pmids\": [\"16525582\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of kringle–integrin interaction unknown\", \"Physiological context where this uPAR-independent mechanism dominates not defined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstration that uPA binding drives uPAR into lipid rafts — independently of catalytic activity — and that raft disruption abolishes ERK signaling provided a mechanistic link between GPI-anchored uPAR and cytoplasmic signaling.\",\n      \"evidence\": \"Sucrose gradient DRM fractionation, methyl-β-cyclodextrin treatment, ERK phosphorylation assays\",\n      \"pmids\": [\"17963689\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the raft-resident transmembrane partner transducing the signal not determined\", \"Single lab observation\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identification of a 78-kb tandem duplication encompassing PLAU as the cause of Quebec platelet disorder — with megakaryocyte-specific >100-fold overexpression — established uPA gene dosage as a Mendelian disease mechanism and revealed cell-type-specific regulatory vulnerability.\",\n      \"evidence\": \"Array CGH, FISH in 38 QPD subjects and 425 controls; RNA-seq and allele-specific transcript analysis in megakaryocytes\",\n      \"pmids\": [\"20007542\", \"28301587\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Regulatory element within the duplication conferring megakaryocyte specificity not mapped\", \"Therapeutic strategy to normalize uPA in megakaryocytes not developed\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"uPA–uPAR signaling was shown to promote CNS axonal regeneration through a plasminogen-independent, LRP1/β1-integrin/Rac1 pathway, expanding uPA function from proteolysis and migration to neural repair.\",\n      \"evidence\": \"In vitro axonal injury and in vivo CNS injury models with LRP1 inhibition, β1-integrin blocking, Rac1 activity assays\",\n      \"pmids\": [\"27986809\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this pathway operates in peripheral nerve injury not tested\", \"LRP1 binding determinants on uPA for this non-proteolytic function not mapped\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Discovery that METTL3 stabilizes PLAU mRNA through m6A modification introduced an epitranscriptomic layer of uPA regulation, linking RNA methylation to metastatic potential via MAPK/ERK signaling.\",\n      \"evidence\": \"m6A-seq, METTL3 overexpression/knockdown, RNA stability assays, in vivo tumour models\",\n      \"pmids\": [\"35567945\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific m6A site(s) on PLAU mRNA not mapped at single-nucleotide resolution\", \"m6A reader(s) responsible for stabilization not identified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"CRISPR knockout of PLAU completely abolished matrix remodeling and invasion in 3D tumouroid models, confirming with genetic precision that uPA enzymatic activity is the dominant driver of ECM degradation during cancer cell invasion.\",\n      \"evidence\": \"CRISPR-Cas9 KO, AFM stiffness measurements, pharmacological inhibitor UK-371,801, 3D tumouroid culture\",\n      \"pmids\": [\"38020586\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Redundancy with tPA or other MMPs in different tumour types not assessed\", \"In vivo metastasis suppression by PLAU KO not shown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the identity of the transmembrane co-receptor that couples GPI-anchored uPAR to intracellular kinase cascades, the structural basis for the kringle–αVβ3 interaction, the megakaryocyte-specific regulatory element disrupted in QPD, and whether therapeutic targeting of the uPA–uPAR interface can achieve anti-metastatic efficacy in vivo.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Transmembrane signaling partner of uPAR not conclusively identified\", \"No in vivo efficacy data for uPA–uPAR PPI inhibitors in metastasis models\", \"Full-length active uPA crystal structure still lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [7, 8, 9, 14, 24, 37]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [7, 8, 9, 37]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [13, 27, 30]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [2, 7, 9, 37]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [5, 7, 20]},\n      {\"term_id\": \"GO:0031012\", \"supporting_discovery_ids\": [37]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [7, 21, 28]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [13, 16, 19, 27, 33, 38]},\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [37]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [33, 36, 38]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"PLAUR\",\n      \"SERPINE1\",\n      \"LRP1\",\n      \"ITGAV\",\n      \"ITGB3\",\n      \"ST14\",\n      \"TM4SF1\",\n      \"HGF\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}