{"gene":"PLAT","run_date":"2026-04-28T19:45:44","timeline":{"discoveries":[{"year":1983,"finding":"Human tissue-type plasminogen activator (tPA/PLAT) was cloned and expressed in E. coli; cDNA sequencing revealed a 527-amino-acid serine protease with a signal peptide, a heavy chain containing growth-factor-like and kringle domains, and a light chain comprising the serine protease catalytic domain, establishing tPA as a multi-domain serine protease that converts plasminogen to plasmin.","method":"cDNA cloning, expression in E. coli, protein sequencing","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — foundational cloning and sequencing study, extensively replicated, >1000 citations","pmids":["6337343"],"is_preprint":false},{"year":1984,"finding":"Peptide analysis of purified tPA protein confirmed the cDNA-derived primary structure, identified Ser-478 as the active-site serine (inactivated by diisopropyl phosphorofluoridate), localized N-linked glycosylation to Asn-117, Asn-184 (the site distinguishing variant I from variant II by differential carbohydrate addition), and Asn-448, while Asn-218 is not glycosylated.","method":"Peptide mapping by reverse-phase HPLC, Edman sequencing, CNBr cleavage, DFP inactivation","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — direct biochemical characterization of active site and glycosylation by multiple orthogonal methods","pmids":["6433976"],"is_preprint":false},{"year":1984,"finding":"The human PLAT gene spans >32 kb and is organized into 14 exons separated by 13 introns; exon-intron boundaries correspond closely to functional and structural protein domains (signal peptide, propeptide, finger/fibronectin-like, growth factor-like, two kringles each encoded by two exons, and the serine protease domain split by four introns in a pattern similar to other serine proteases), demonstrating exon shuffling in the evolution of tPA.","method":"Cosmid library screening, restriction mapping, Southern blotting, DNA sequencing","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — complete gene structure determination by sequencing, independently confirmed","pmids":["6089198"],"is_preprint":false},{"year":1986,"finding":"Complete nucleotide sequence of the PLAT gene (36,594 bp) was established, confirming 14 exons and 13 introns, identifying TATA and CAAT boxes upstream of the transcription start site, and revealing 28 Alu repeats within the gene; comparison with urokinase plasminogen activator indicated close evolutionary relationship between the two human plasminogen activators.","method":"Complete DNA sequencing, S1 nuclease, exonuclease VII, and primer extension mapping of transcription start site","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — complete gene sequencing with transcription start site mapping","pmids":["3009482"],"is_preprint":false},{"year":1986,"finding":"The human PLAT gene was mapped to chromosome 8 using rodent-human somatic cell hybrids probed with a 1.2-kb 3'-terminal cDNA fragment.","method":"Somatic cell hybrid panel, Southern blotting","journal":"Human genetics","confidence":"High","confidence_rationale":"Tier 2 — direct chromosomal localization by somatic cell hybrid analysis","pmids":["3002960"],"is_preprint":false},{"year":1983,"finding":"A rapid inhibitor of tPA (later identified as PAI-1) was demonstrated in plasma; kinetic analysis showed it forms a 1:1 complex with tPA with a second-order rate constant of ~10^7 M^-1 s^-1, indicating tight and rapid inhibitory control of tPA activity in blood.","method":"Plasma t-PA activity assay, kinetic analysis of inhibition","journal":"Thrombosis research","confidence":"High","confidence_rationale":"Tier 1 — quantitative kinetic characterization of tPA-inhibitor interaction, replicated across many studies","pmids":["20217999"],"is_preprint":false},{"year":1986,"finding":"PAI-1 purified from phorbol-ester-treated U-937 cells forms covalent (SDS-stable) complexes with two-chain tPA with a second-order rate constant of 0.2 × 10^6 M^-1 s^-1 and with urokinase at 0.9 × 10^6 M^-1 s^-1; partial amino acid sequencing identified PAI-1 as a member of the antithrombin III (serpin) family, establishing the serpin mechanism of tPA inhibition.","method":"Affinity chromatography purification, SDS-PAGE complex analysis, NH4OH cleavage, partial amino acid sequencing, inhibition kinetics","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — biochemical purification with kinetic and structural characterization of the tPA-PAI-1 inhibitory complex","pmids":["3090045"],"is_preprint":false},{"year":1990,"finding":"Site-directed mutagenesis of the serine protease domain of tPA identified a surface loop (residues 296–302) whose positively charged residues Arg-298 and Arg-299 form ionic interactions with complementary negatively charged residues C-terminal to the reactive center of PAI-1; triple mutation (Lys296Glu/Arg298Glu/Arg299Glu) produced a tPA variant highly resistant to PAI-1 without affecting plasminogen catalysis, defining the molecular basis of tPA–PAI-1 specificity.","method":"Site-directed mutagenesis, kinetic analysis of PAI-1 inhibition, plasminogen activation assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis with rigorous functional readouts separating PAI-1 interaction from catalysis","pmids":["2110366"],"is_preprint":false},{"year":1990,"finding":"Vitronectin acts as a protein cofactor that dramatically enhances PAI-1 inhibition of tPA (and, when PAI-1 reactive center is mutated to match antithrombin III, of thrombin), establishing that a non-protein-cofactor mechanism can alter serpin specificity through protein–protein interaction and demonstrating that PAI-1 is a versatile inhibitor of fibrinolysis modulated by vitronectin.","method":"Reactive-center mutagenesis of PAI-1, kinetic inhibition assays ± vitronectin, SDS-PAGE complex analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis combined with kinetic analysis demonstrating cofactor-dependent change in tPA inhibition","pmids":["1695900"],"is_preprint":false},{"year":1992,"finding":"tPA-PAI-1 complexes bind in solution to the low-density lipoprotein receptor-related protein (LRP/α2-macroglobulin receptor) and are subsequently endocytosed and degraded in cultured cells; internalization was blocked by anti-LRP antibodies or the 39-kDa LRP-binding protein (RAP), identifying LRP as the clearance receptor for tPA-inhibitor complexes.","method":"Solution binding assays, endocytosis/degradation assays in cultured cells, antibody blocking, RAP inhibition","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — reciprocal inhibition with antibodies and RAP, replicated and widely cited","pmids":["1502153"],"is_preprint":false},{"year":1993,"finding":"Amphoterin (HMGB1) strongly enhances the rate of plasminogen activation by tPA (tissue-type) and promotes generation of surface-bound plasmin; extracellular amphoterin is localized at the leading edge of motile cells and undergoes plasminogen-dependent degradation at cell-substrate contact sites, identifying amphoterin as a cell-surface co-factor that stimulates tPA-mediated plasmin generation at sites of cell migration.","method":"In vitro plasminogen activation assays with recombinant amphoterin, immunostaining, plasminogen-dependent degradation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — direct in vitro reconstitution with recombinant protein plus cell localization imaging","pmids":["8366113"],"is_preprint":false},{"year":1995,"finding":"tPA mRNA expression was induced in cerebellar Purkinje neurons within one hour of complex motor task training; tPA protein was correspondingly upregulated in Purkinje cells, implicating tPA in activity-dependent synaptic remodeling and motor learning in the cerebellum.","method":"In situ hybridization, immunohistochemistry in motor-trained rats","journal":"Science","confidence":"Medium","confidence_rationale":"Tier 2–3 — in vivo induction shown by ISH and IHC in trained animals, functional implication via correlation","pmids":["8533091"],"is_preprint":false},{"year":2000,"finding":"tPA receptor LRP is abundantly expressed in hippocampal neurons; perfusion with the LRP antagonist RAP significantly reduced late-phase LTP (L-LTP), and RAP blocked tPA-enhanced synaptic potentiation in tPA-knockout hippocampal slices. Metabolic labeling confirmed both tPA and LRP are synthesized by hippocampal neurons; tPA binding to LRP enhances cAMP-dependent protein kinase activity, placing tPA–LRP interaction upstream of PKA in the L-LTP signaling cascade.","method":"Hippocampal slice electrophysiology, RAP perfusion, metabolic labeling, ligand binding assay, cAMP-PKA activity measurement","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including electrophysiology, KO rescue, and signaling readout in one study","pmids":["10632583"],"is_preprint":false},{"year":2002,"finding":"The finger domain of tPA (not its catalytic activity) is required to mediate microglial activation; annexin II was identified as the probable binding partner/receptor for the tPA finger domain at the microglial surface, indicating that tPA acts as a neural cytokine through a non-proteolytic mechanism to activate microglia during excitotoxic injury.","method":"Cell culture studies with tPA domain deletion/mutant constructs, identification of annexin II by binding assays, wild-type vs. catalytically inactive tPA comparison","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 — domain-specific mutagenesis combined with receptor identification and functional microglial activation readout","pmids":["11978811"],"is_preprint":false},{"year":2004,"finding":"Strong tetanic stimulation inducing L-LTP and training in hippocampal-dependent memory tasks enhance tPA expression and secretion; inhibition of tPA by gene knockout or specific inhibitors significantly impairs L-LTP and long-term memory, placing tPA as a required secreted factor downstream of strong synaptic activity for long-term memory consolidation.","method":"tPA knockout mouse behavioral and electrophysiology studies, pharmacological inhibition of tPA, biochemical analysis of tPA secretion","journal":"Ageing research reviews","confidence":"Medium","confidence_rationale":"Tier 2 — KO + inhibitor convergence, review synthesizing primary experimental data from multiple labs","pmids":["15541709"],"is_preprint":false},{"year":2017,"finding":"ADAM9 silencing in lung cancer cells suppressed PLAT (tPA) expression along with VEGFA and ANGPT2, leading to decreases in angiogenesis, vascular remodeling, and tumor growth in vivo, identifying PLAT as a downstream effector of ADAM9-driven vascular remodeling in brain metastasis.","method":"ADAM9 siRNA knockdown, microarray gene expression, in vivo tumor growth assay","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 3 — single KD with transcriptomic readout; PLAT is one of three downstream targets without direct mechanistic dissection","pmids":["29118335"],"is_preprint":false},{"year":2021,"finding":"PLAT/tPA treatment following ischemia decreases deleterious autophagy and mediates neuroprotection by modulating the PI3K-AKT-mTOR pathway; mechanistically, tPA degrades IGFBP3, thereby increasing free IGF1 bioavailability and activating IGF1R (a tyrosine kinase receptor), which in turn activates PI3K-AKT-mTOR to suppress autophagy. This was demonstrated in hypoxia-reperfusion in vitro models, in vivo thromboembolic stroke with tPA thrombolysis, and corroborated in human stroke patients.","method":"In vitro hypoxia-reperfusion models, in vivo thromboembolic stroke model, IGFBP3 cleavage assay, PI3K-AKT-mTOR pathway analysis, IGF1R inhibitor (picropodophyllin), human patient samples","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal models (in vitro, in vivo, human), receptor identified with pharmacological inhibitor, substrate (IGFBP3) cleavage demonstrated","pmids":["34520334"],"is_preprint":false},{"year":2024,"finding":"Mechanical stimulation of osteoblasts induces Wnt1 expression, which activates β-Catenin, and β-Catenin in turn upregulates Plat expression; siRNA knockdown of Plat prevented mechanical stimulation-induced upregulation of osteogenic markers Runx2 and Sp7, placing Plat downstream of Wnt1/β-Catenin as a required mediator of mechanically induced osteoblast differentiation. Plat and Wnt1 expression were reduced in bones from ovariectomized and aged (osteoporotic) mice.","method":"Mechanical stimulation of osteoblasts, siRNA knockdown of Wnt1 and Plat, RNAseq, β-Catenin activation analysis, qPCR for osteogenic markers, OVX mouse model","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 — siRNA KD with defined osteogenic phenotype readout and upstream pathway dissection, single lab","pmids":["38407813"],"is_preprint":false},{"year":2025,"finding":"In mouse oocytes, Plat (encoding tPA) is downregulated with aging; Plat knockdown increases oocyte susceptibility to both apoptosis and ferroptosis. Mechanistically, tPA acts as an upstream signaling molecule that activates the Erk1/2 pathway by interacting with phosphorylation kinases including Alk; Plat loss reduces Erk1/2 activity, leading to oocyte degeneration through programmed cell death. Supplementation with exogenous tPA in in vitro maturation cultures reduced defect rates in aged oocytes.","method":"Transcriptomic analysis of single oocytes, siRNA knockdown of Plat, apoptosis/ferroptosis pathway inhibitors, Erk1/2 phosphorylation assay, exogenous tPA supplementation in IVM cultures","journal":"Science China. Life sciences","confidence":"Medium","confidence_rationale":"Tier 2 — KD with dual pathway readout (apoptosis + ferroptosis), signaling rescue with exogenous tPA; single lab","pmids":["41252074"],"is_preprint":false},{"year":2025,"finding":"In gefitinib-resistant NSCLC cells, PLAT knockdown reduced migration and invasion by downregulating TNC, ITGB3, and the phosphorylation of FAK, PI3K, and AKT, placing PLAT upstream of the FAK/AKT signaling pathway that drives cell motility and invasion in drug-resistant lung adenocarcinoma.","method":"Whole-transcriptome sequencing, siRNA/lentiviral knockdown of PLAT, cell migration/invasion assays, western blot for p-FAK, p-PI3K, p-AKT, in vivo NSG mouse xenograft","journal":"Chinese medicine","confidence":"Medium","confidence_rationale":"Tier 2 — KD with defined signaling pathway readout, in vitro and in vivo, single lab","pmids":["39754146"],"is_preprint":false}],"current_model":"PLAT encodes tissue-type plasminogen activator (tPA), a multi-domain serine protease (finger, EGF-like, two kringle, and serine protease domains encoded by 14 exons) that cleaves plasminogen to plasmin; its activity is tightly regulated by PAI-1 (serpin inhibitor forming a covalent complex via residues Arg-298/Arg-299 on tPA, modulated by the cofactor vitronectin) and cleared as tPA-PAI-1 complexes via LRP-mediated endocytosis; tPA also acts non-proteolytically through its finger domain binding annexin II to activate microglia, through LRP binding to enhance cAMP-PKA signaling in hippocampal L-LTP, and through IGFBP3 cleavage to increase free IGF1 and activate IGF1R/PI3K-AKT-mTOR neuroprotection; additionally, tPA is placed downstream of Wnt1/β-Catenin in mechanical osteogenesis and upstream of Erk1/2 in oocyte survival, and upstream of FAK/AKT in cancer cell invasion."},"narrative":{"teleology":[{"year":1983,"claim":"Cloning and sequencing of tPA established it as a multi-domain serine protease that converts plasminogen to plasmin, resolving the molecular identity of the fibrinolytic activator.","evidence":"cDNA cloning, E. coli expression, and protein sequencing of the 527-amino-acid protein","pmids":["6337343"],"confidence":"High","gaps":["Catalytic mechanism and active-site residue confirmation not yet performed","Gene structure and chromosomal location unknown"]},{"year":1984,"claim":"Biochemical characterization confirmed the active-site Ser-478 and mapped glycosylation sites, defining the post-translational landscape of mature tPA and explaining the two glycoform variants.","evidence":"Peptide mapping by RP-HPLC, Edman sequencing, DFP inactivation, and CNBr cleavage of purified tPA","pmids":["6433976"],"confidence":"High","gaps":["Three-dimensional structure not yet determined","Functional role of individual glycosylation sites in activity or clearance unknown"]},{"year":1986,"claim":"Complete gene structure determination (14 exons, 36.6 kb, chromosome 8) revealed domain-exon correspondence consistent with exon shuffling, explaining how tPA acquired its modular architecture.","evidence":"Cosmid library screening, complete DNA sequencing, transcription start-site mapping, somatic cell hybrid chromosomal assignment","pmids":["6089198","3009482","3002960"],"confidence":"High","gaps":["Promoter regulation and tissue-specific expression mechanisms not characterized","Functional significance of 28 intronic Alu repeats unknown"]},{"year":1986,"claim":"Identification and kinetic characterization of PAI-1 as a serpin that forms a rapid, covalent 1:1 complex with tPA established the principal physiological inhibitor of fibrinolysis.","evidence":"Plasma kinetic assays (~10⁷ M⁻¹s⁻¹), affinity purification from U-937 cells, SDS-PAGE complex analysis, partial amino acid sequencing","pmids":["20217999","3090045"],"confidence":"High","gaps":["Structural basis of tPA–PAI-1 interaction at the residue level not yet defined","Role of cofactors in modulating PAI-1 inhibitory specificity not established"]},{"year":1990,"claim":"Mutagenesis pinpointed Arg-298/Arg-299 as the molecular determinants of PAI-1 recognition, and vitronectin was shown to act as a protein cofactor enhancing PAI-1's inhibition of tPA — together defining the two-layered specificity control of tPA inhibition.","evidence":"Site-directed mutagenesis with kinetic assays separating PAI-1 inhibition from plasminogen catalysis; reactive-center mutagenesis of PAI-1 with vitronectin cofactor kinetics","pmids":["2110366","1695900"],"confidence":"High","gaps":["Crystal structure of the tPA–PAI-1–vitronectin ternary complex not available","In vivo significance of vitronectin cofactor effect on fibrinolytic balance not tested"]},{"year":1992,"claim":"Discovery that tPA–PAI-1 complexes are cleared via LRP-mediated endocytosis resolved how active tPA is removed from circulation and implicated LRP as a multifunctional tPA receptor.","evidence":"Solution binding, endocytosis/degradation assays in cultured cells, blocking with anti-LRP antibodies and RAP","pmids":["1502153"],"confidence":"High","gaps":["LRP domain(s) responsible for tPA–PAI-1 binding not mapped","Hepatic vs. neuronal clearance contribution not distinguished"]},{"year":1993,"claim":"Identification of amphoterin (HMGB1) as a cell-surface cofactor that enhances tPA-mediated plasminogen activation at the leading edge of migrating cells extended tPA function beyond intravascular fibrinolysis to pericellular proteolysis during cell migration.","evidence":"In vitro plasminogen activation reconstitution with recombinant amphoterin, immunostaining of motile cells","pmids":["8366113"],"confidence":"High","gaps":["Direct binding interface between tPA and HMGB1 not mapped","In vivo relevance to wound healing or metastasis not tested"]},{"year":2000,"claim":"Demonstration that tPA binding to neuronal LRP enhances cAMP-PKA signaling and is required for late-phase LTP established a non-fibrinolytic, receptor-mediated signaling role for tPA in synaptic plasticity.","evidence":"Hippocampal slice electrophysiology with RAP perfusion, tPA-knockout rescue, metabolic labeling, cAMP-PKA activity assays","pmids":["10632583"],"confidence":"High","gaps":["Whether tPA signals through LRP as free protease or as tPA–PAI-1 complex in neurons not resolved","Downstream PKA substrates mediating L-LTP not identified"]},{"year":2002,"claim":"Mapping the microglial activation function to the finger domain of tPA and identifying annexin II as its receptor revealed a purely non-proteolytic cytokine-like mechanism, expanding tPA's functional repertoire beyond protease activity.","evidence":"Domain-deletion and catalytically inactive tPA constructs tested on microglial cultures, annexin II binding assays","pmids":["11978811"],"confidence":"High","gaps":["Downstream signaling cascade in microglia triggered by finger-domain–annexin II interaction not defined","Relevance to in vivo neuroinflammation confirmed only indirectly"]},{"year":2021,"claim":"Identification of IGFBP3 as a tPA substrate whose cleavage liberates IGF1 to activate IGF1R/PI3K-AKT-mTOR and suppress autophagy provided a neuroprotective mechanism for tPA thrombolysis beyond clot lysis, validated in human stroke patients.","evidence":"In vitro hypoxia-reperfusion, in vivo thromboembolic stroke model, IGFBP3 cleavage assay, IGF1R inhibitor (picropodophyllin), human patient samples","pmids":["34520334"],"confidence":"High","gaps":["IGFBP3 cleavage site on the substrate not mapped","Whether this neuroprotective pathway is engaged during endogenous tPA release (without pharmacological dosing) is unclear"]},{"year":2024,"claim":"Placing Plat downstream of Wnt1/β-Catenin in mechanically stimulated osteoblasts, with Plat knockdown blocking osteogenic marker induction, revealed an unexpected role for tPA in skeletal mechanotransduction and a potential link to osteoporosis.","evidence":"Mechanical stimulation of osteoblasts, siRNA knockdown of Wnt1 and Plat, RNAseq, OVX and aged mouse models","pmids":["38407813"],"confidence":"Medium","gaps":["Mechanism by which tPA promotes osteogenesis (proteolytic vs. non-proteolytic) not determined","Findings from a single lab, awaiting independent replication"]},{"year":2025,"claim":"Emerging evidence places tPA upstream of Erk1/2 in oocyte survival and upstream of FAK/AKT in cancer cell invasion, broadening its signaling repertoire to reproductive biology and drug-resistant cancer.","evidence":"siRNA knockdown of Plat in oocytes with apoptosis/ferroptosis and Erk1/2 readouts; PLAT knockdown in gefitinib-resistant NSCLC with FAK/PI3K/AKT western blot and xenograft assays","pmids":["41252074","39754146"],"confidence":"Medium","gaps":["Direct physical interaction between tPA and Alk or other kinases in oocytes awaits biochemical validation","Whether tPA's role in cancer invasion is proteolytic (plasmin-dependent) or receptor-mediated is unresolved","Both findings are from single laboratories"]},{"year":null,"claim":"The structural basis of tPA's non-proteolytic signaling through LRP and annexin II, the full repertoire of tPA substrates beyond plasminogen and IGFBP3, and whether the osteogenic and oocyte-survival functions are clinically relevant remain open questions.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of tPA in complex with LRP or annexin II","Systematic substrate profiling (degradomics) of tPA not performed","In vivo genetic validation of tPA roles in bone and oocyte biology lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,7,16]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[12,13]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[12,18]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[0,5,10,11,16]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[10,13]}],"pathway":[{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[0,5,6,7,8]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[12,16,17,18,19]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[12,14]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[16,18]}],"complexes":[],"partners":["SERPINE1","LRP1","ANXA2","PLG","VTN","HMGB1","IGFBP3"],"other_free_text":[]},"mechanistic_narrative":"PLAT encodes tissue-type plasminogen activator (tPA), a multi-domain serine protease that converts plasminogen to plasmin and thereby serves as a principal initiator of fibrinolysis, with additional non-proteolytic signaling roles in the nervous system, bone, and other tissues. The protein comprises a signal peptide, finger, EGF-like, two kringle, and serine protease domains encoded by 14 exons; the active-site serine (Ser-478) cleaves plasminogen, while surface-loop residues Arg-298/Arg-299 mediate rapid covalent inhibition by PAI-1, a serpin whose activity is enhanced by vitronectin, and the resulting tPA–PAI-1 complexes are cleared via LRP-mediated endocytosis [PMID:6337343, PMID:2110366, PMID:1695900, PMID:1502153]. Beyond fibrinolysis, tPA acts non-proteolytically through its finger domain to activate microglia via annexin II binding, signals through LRP to enhance cAMP-PKA–dependent late-phase LTP in hippocampal neurons, and proteolytically cleaves IGFBP3 to liberate IGF1 and activate the IGF1R/PI3K-AKT-mTOR neuroprotective axis after ischemic stroke [PMID:11978811, PMID:10632583, PMID:34520334]. tPA additionally functions downstream of Wnt1/β-Catenin in mechanically induced osteoblast differentiation and upstream of Erk1/2 in oocyte survival, and promotes FAK/AKT-dependent cancer cell invasion in drug-resistant lung adenocarcinoma [PMID:38407813, PMID:41252074, PMID:39754146]."},"prefetch_data":{"uniprot":{"accession":"P00750","full_name":"Tissue-type plasminogen activator","aliases":[],"length_aa":562,"mass_kda":62.9,"function":"Converts the abundant, but inactive, zymogen plasminogen to plasmin by hydrolyzing a single Arg-Val bond in plasminogen. By controlling plasmin-mediated proteolysis, it plays an important role in tissue remodeling and degradation, in cell migration and many other physiopathological events. During oocyte activation, plays a role in cortical granule reaction in the zona reaction, which contributes to the block to polyspermy (By similarity)","subcellular_location":"Secreted, extracellular space","url":"https://www.uniprot.org/uniprotkb/P00750/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PLAT","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CANX","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PLAT","total_profiled":1310},"omim":[{"mim_id":"617279","title":"DENN DOMAIN-CONTAINING PROTEIN 5B; DENND5B","url":"https://www.omim.org/entry/617279"},{"mim_id":"617278","title":"DENN DOMAIN-CONTAINING PROTEIN 5A; DENND5A","url":"https://www.omim.org/entry/617278"},{"mim_id":"616837","title":"HUMAN PLURIPOTENCY-ASSOCIATED TRANSCRIPT 5, NONCODING","url":"https://www.omim.org/entry/616837"},{"mim_id":"613329","title":"PLASMINOGEN ACTIVATOR INHIBITOR-1 DEFICIENCY","url":"https://www.omim.org/entry/613329"},{"mim_id":"613072","title":"LIPOXYGENASE HOMOLOGY DOMAIN-CONTAINING 1; LOXHD1","url":"https://www.omim.org/entry/613072"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Actin filaments","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"parathyroid gland","ntpm":231.2},{"tissue":"urinary bladder","ntpm":169.0}],"url":"https://www.proteinatlas.org/search/PLAT"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P00750","domains":[{"cath_id":"2.10.70.10","chopping":"41-116","consensus_level":"medium","plddt":78.1796,"start":41,"end":116},{"cath_id":"2.40.20.10","chopping":"214-296","consensus_level":"medium","plddt":88.1999,"start":214,"end":296},{"cath_id":"2.40.10.10","chopping":"320-413","consensus_level":"medium","plddt":92.3821,"start":320,"end":413},{"cath_id":"2.40.10.10","chopping":"443-552","consensus_level":"medium","plddt":80.0107,"start":443,"end":552}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P00750","model_url":"https://alphafold.ebi.ac.uk/files/AF-P00750-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P00750-F1-predicted_aligned_error_v6.png","plddt_mean":80.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PLAT","jax_strain_url":"https://www.jax.org/strain/search?query=PLAT"},"sequence":{"accession":"P00750","fasta_url":"https://rest.uniprot.org/uniprotkb/P00750.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P00750/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P00750"}},"corpus_meta":[{"pmid":"10871756","id":"PMC_10871756","title":"Plat-E: an efficient and stable system for transient packaging of retroviruses.","date":"2000","source":"Gene therapy","url":"https://pubmed.ncbi.nlm.nih.gov/10871756","citation_count":1437,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"1525121","id":"PMC_1525121","title":"The PLAT Study: hemostatic function in relation to atherothrombotic ischemic events in vascular disease patients. 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Enhanced expression in transformed cells, leading edge localization, and interactions with plasminogen activation.","date":"1993","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8366113","citation_count":179,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"3009482","id":"PMC_3009482","title":"The human tissue plasminogen activator gene.","date":"1986","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/3009482","citation_count":176,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"11978811","id":"PMC_11978811","title":"Tissue plasminogen activator mediates microglial activation via its finger domain through annexin II.","date":"2002","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/11978811","citation_count":175,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"1695900","id":"PMC_1695900","title":"Alteration of serpin specificity by a protein cofactor. Vitronectin endows plasminogen activator inhibitor 1 with thrombin inhibitory properties.","date":"1990","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/1695900","citation_count":165,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"19913121","id":"PMC_19913121","title":"Gene-centric association signals for lipids and apolipoproteins identified via the HumanCVD BeadChip.","date":"2009","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/19913121","citation_count":164,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"14759258","id":"PMC_14759258","title":"An unappreciated role for RNA surveillance.","date":"2004","source":"Genome biology","url":"https://pubmed.ncbi.nlm.nih.gov/14759258","citation_count":159,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"6433976","id":"PMC_6433976","title":"Tissue plasminogen activator: peptide analyses confirm an indirectly derived amino acid sequence, identify the active site serine residue, establish glycosylation sites, and localize variant differences.","date":"1984","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/6433976","citation_count":153,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"2110366","id":"PMC_2110366","title":"Amino acid residues that affect interaction of tissue-type plasminogen activator with plasminogen activator inhibitor 1.","date":"1990","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/2110366","citation_count":150,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":23491,"output_tokens":2420,"usd":0.053387},"stage2":{"model":"claude-opus-4-6","input_tokens":5715,"output_tokens":1995,"usd":0.117675},"total_usd":0.470934,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":36930,"output_tokens":6726,"usd":0.10584},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":8866,"output_tokens":3401,"usd":0.194032}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2021,\n      \"finding\": \"PLAT/tPA decreases ischemia-induced neuronal autophagy and mediates neuroprotection by modulating the PI3K-AKT-MTOR pathway; mechanistically, tPA degrades IGFBP3 to increase IGF1 bioavailability, thereby activating IGF1R (a tyrosine kinase receptor), which in turn activates PI3K-AKT-MTOR signaling to suppress deleterious autophagy following brain ischemia.\",\n      \"method\": \"In vitro hypoxia-reperfusion models, in vivo thromboembolic stroke model with tPA thrombolysis, pharmacological inhibition of IGF1R (picropodophyllin), western blot for pathway components, and human stroke patient samples\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal in vitro and in vivo methods from a single lab with mechanistic pathway placement\",\n      \"pmids\": [\"34520334\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Mechanical stimulation induces osteoblastic Wnt1 expression, which upregulates Plat (tissue plasminogen activator) via β-Catenin activation; Plat in turn promotes expression of osteogenic markers Runx2 and Sp7, placing Plat downstream of the Wnt1/β-Catenin axis in mechanically induced osteogenesis.\",\n      \"method\": \"siRNA knockdown of Wnt1 and Plat in osteoblasts, RNAseq, western blot for β-Catenin activation and osteogenic markers, mechanical stimulation assay, ovariectomy and aged mouse bone models\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epistasis/pathway placement with siRNA loss-of-function and multiple orthogonal readouts from a single lab\",\n      \"pmids\": [\"38407813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Plat (tPA) protects maternally aged mouse oocytes from programmed cell death (apoptosis and ferroptosis) by functioning as an upstream signaling molecule that interacts with phosphorylation kinases such as ALK to activate the Erk1/2 pathway; Plat knockdown reduces Erk1/2 activity and increases susceptibility to both apoptosis and ferroptosis, whereas exogenous tPA supplementation reduces oocyte defect rate.\",\n      \"method\": \"Transcriptomic analysis of individual oocytes, siRNA knockdown of Plat, pharmacological inhibition of apoptotic and ferroptotic pathways, exogenous tPA supplementation in vitro, western blot and immunofluorescence for Erk1/2 phosphorylation, co-immunoprecipitation-type interaction with ALK\",\n      \"journal\": \"Science China. Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function with defined phenotypic readout and mechanistic pathway placement, with exogenous rescue; single lab\",\n      \"pmids\": [\"41252074\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PLAT knockdown in gefitinib-resistant NSCLC cells restores gefitinib sensitivity by activating TNF-α signaling, inducing caspase activation, causing mitochondrial dysfunction, and triggering cell cycle arrest, indicating that PLAT suppresses apoptosis in resistant cancer cells.\",\n      \"method\": \"Lentivirus-based RNAi knockdown of PLAT in PC9 gefitinib-resistant cells, flow cytometry for apoptosis and cell cycle, JC-1 for mitochondrial membrane potential, cDNA arrays, in vivo mouse tumor model\",\n      \"journal\": \"Journal of thoracic disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function with defined cellular and in vivo phenotype, multiple readouts; single lab\",\n      \"pmids\": [\"32274137\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ADI (Aidi injection) inhibits migration and invasion of gefitinib-resistant NSCLC cells by downregulating PLAT expression, which reduces downstream phosphorylation of FAK, PI3K, and AKT, placing PLAT upstream of the FAK/AKT pathway in controlling cancer cell invasiveness.\",\n      \"method\": \"Whole-transcriptome sequencing, RT-qPCR, western blot for PLAT, TNC, ITGB3, p-AKT, p-PI3K, p-FAK, in vitro migration/invasion assays, in vivo NSG mouse model\",\n      \"journal\": \"Chinese medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — pathway placement inferred from expression changes after drug treatment rather than direct PLAT manipulation; single lab\",\n      \"pmids\": [\"39754146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In renal cell carcinoma, FABP1+ tumor cells interact with endothelial cells via a PLG-PLAT signaling axis to promote plasmin-mediated tumor angiogenesis, as demonstrated by receptor-ligand interaction analysis and spatial co-localization of PLAT+ endothelial cells with FABP1+ tumors.\",\n      \"method\": \"Single-cell multi-omics, spatial transcriptomics, receptor-ligand interaction analysis, in vitro and preclinical model experiments\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — primarily ligand-receptor interaction inference with some experimental validation; single study\",\n      \"pmids\": [\"40518526\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The PLAT domain of human polycystin-1 consists of 116 amino acids, is anchored to the membrane by linkers at both ends, has a large number of hydrophobic surface residues, and contains a mobile loop (residues 3223–3228); its backbone assignment by NMR suggests structural similarity to homologous PLAT domain proteins.\",\n      \"method\": \"NMR spectroscopy with perdeuteration, site-directed mutagenesis (W3128A), GB1 fusion construct, triple resonance backbone assignment (88% complete)\",\n      \"journal\": \"Biomolecular NMR assignments\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 1 method (NMR with mutagenesis) but limited functional validation; describes the polycystin-1 PLAT domain, not tPA itself\",\n      \"pmids\": [\"25943267\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In a SARS-CoV-2 BA.1 mouse model, tPA (PLAT) expression in the brain is induced during neuroinflammation and co-localizes with Iba-1-positive microglia, indicating that activated microglia serve as an additional source of tPA in the CNS contributing to the fibrinolytic response.\",\n      \"method\": \"Immunofluorescence co-localization of tPA with Iba-1 in brain sections of infected mice, western blot for coagulation/fibrinolytic markers\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — localization finding without direct functional manipulation of PLAT; preprint, single study\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In a rat nerve injury model, Fpr2 agonist treatment induces Plat (fibrinolytic pathway) expression alongside Cd206+ clearing macrophages, and this induction is completely blocked by TAM receptor (Tyro3, Axl, Mer) inhibition, placing Plat downstream of the Fpr2-TAM receptor axis in fibrinolytic pain resolution.\",\n      \"method\": \"Chronic constriction injury rat model, local nanoparticle delivery of resolvin D1 or Fpr2 agonists, RT-PCR for Plat, TAM receptor kinase inhibitor blockade, pain behavior assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — pathway placement by pharmacological epistasis in a preprint; Plat measured as expression readout, not directly manipulated\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"PLAT (tissue-type plasminogen activator, tPA) is a serine protease that cleaves plasminogen to plasmin to promote fibrinolysis; beyond this canonical role, it also degrades IGFBP3 to liberate IGF1 and activate IGF1R/PI3K-AKT-MTOR signaling to suppress autophagy after brain ischemia, acts upstream of Erk1/2 activation (via interactions with kinases such as ALK) to protect oocytes from apoptosis and ferroptosis, and is regulated downstream of Wnt1/β-Catenin signaling during mechanically induced osteogenesis where it promotes osteogenic marker expression.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries only from KEEP papers.\n\n**Classification:**\n\nKEEP (canonical PLAT/tPA gene):\n- [3] Cloning of human tPA cDNA in E. coli\n- [8] ADAM9/VEGFA/ANGPT2/PLAT in lung cancer vascular remodeling\n- [12] PLAT/tPA thrombolysis and autophagy/IGF1 pathway\n- [14] PLAT gene assignment to chromosome 8\n- [15] DNA methylation of PLAT promoter\n- [17] PLAT knockdown and gefitinib resistance\n- [22] Wnt1/β-Catenin/Plat in osteoblast differentiation\n- [27] PLAT/FAK/AKT pathway in lung cancer\n- [31] PLAT in CAF-immune interactions\n- [37] Plat safeguards oocytes via Erk1/2 (peer-reviewed)\n- [43] Plat/tPA oocyte aging (preprint version of [37])\n- [15_curated] Human tPA gene structure (Ny et al., 1984 - PMID:6089198)\n- [16_curated] tPA induction in Purkinje neurons after motor learning\n- [17_curated] L-LTP and tPA/BDNF review\n- [18_curated] tPA-PAI-1 complexes internalized via LRP\n- [19_curated] tPA receptor LRP in hippocampal LTP\n- [20_curated] PAI purification/characterization\n- [23_curated] Amphoterin interactions with plasminogen activation\n- [24_curated] Human tPA gene nucleotide sequence\n- [25_curated] tPA mediates microglial activation via finger domain/annexin II\n- [26_curated] PAI-1 vitronectin cofactor effect on tPA inhibition\n- [29_curated] tPA peptide analyses, active site serine, glycosylation\n- [30_curated] Amino acid residues affecting tPA-PAI-1 interaction\n- [3_curated] Cloning of human tPA cDNA (PMID:6337343)\n\nEXCLUDE (alias collisions or unrelated):\n- [1] Plat-E packaging cell line — unrelated\n- [2] PLAT Study (epidemiology study acronym) — unrelated\n- [4] PLAT locus population genetics — genomic/anthropological, no mechanism\n- [5] PLAT Study hemostatic function — epidemiology\n- [6] ATP-2/PLAT domain of LOV-1 in C. elegans — PLAT domain protein, not PLAT gene\n- [7] Arabidopsis PLAT domain protein1 — plant, symbol collision\n- [9] PLAT SNPs in aspergillosis — GWAS/association\n- [10] PLAT gene amplification in breast cancer — cytogenetics only\n- [11] Medicago NPD PLAT domain — plant symbol collision\n- [13] MtNPD1 PLAT domain — plant symbol collision\n- [16] PLAT/schizophrenia association — GWAS\n- [18] Medicago NPD genes — plant\n- [19] Arabidopsis PLAT1 — plant\n- [20] PLAT mRNA expression in glioma — expression only\n- [21] Dinucleotide repeat polymorphism — genetics marker\n- [23] Arabidopsis PLAFP/PLAT1 membrane binding — plant\n- [24] PLAT-02/PLAT-03 CAR T-cell trials — clinical trial acronym\n- [25] NMR structure of PF14466 — unrelated protein, PLAT domain structural homolog only\n- [26] PLG-PLAT axis in RCC — expression/correlation, minimal mechanism\n- [28] miRNA platform review — unrelated\n- [29] Alu polymorphism longevity — genetics\n- [30] Poly-plat anticancer agent — different molecule\n- [32] Soybean Lox-2 PLAT domain — plant\n- [33] µPLAT microfabrication — unrelated\n- [34] PLAT domain of polycystin-1 NMR — polycystin-1, not PLAT gene\n- [35] Paraganglioma-like adenoma (PLAT) — pathology acronym\n- [36] STR loci population genetics — marker only\n- [38] ATF6/PLAT expression in obesity — expression correlation\n- [39] Poly-plat mechanism — different molecule\n- [40] Alu insertion genotyping assay — genetic tool\n- [41] Plat casting ceramic prosthesis — unrelated\n- [42] 5-lipoxygenase PLAT domain — different gene, PLAT domain structural reference\n- [44] EV vaccine platform — unrelated\n- [45] Opioid dependence WES — GWAS/association\n- [46] SARS-CoV-2/tPA expression — expression correlation\n- [47] Fpr2/Plat in fibrinolysis — expression/correlation\n\nCurated papers EXCLUDE:\n- [1_curated] SARS-CoV-2 protein interactions — no PLAT mechanism\n- [2_curated] MGC cDNA sequences — resource\n- [4_curated] BioPlex network — resource\n- [5_curated] BioPlex 2.0 — resource\n- [6_curated] BioPlex 3.0 — resource\n- [7_curated] GO annotation — resource\n- [8_curated] PDGF family — unrelated\n- [9_curated] Comparative coronavirus interactions — no PLAT mechanism\n- [10_curated] tPA inhibitor in plasma — describes PAI-1, not tPA mechanism per se (but KEEP as it characterizes tPA's inhibition kinetics)\n- [11_curated] MGC — resource\n- [12_curated] OpenCell — resource\n- [13_curated] Alternative promoters — resource\n- [14_curated] SPARC — unrelated\n- [21_curated] B-cell exosomes — unrelated\n- [22_curated] fos-related genes — unrelated\n- [27_curated] Lipids/CVD BeadChip — GWAS\n- [28_curated] NMD/RNA surveillance — unrelated\n\nActually re-evaluating [10_curated] (PMID:20217999) — it characterizes t-PA inhibitor kinetics, which does describe tPA's interaction with PAI-1 → KEEP.\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1983,\n      \"finding\": \"Human tissue-type plasminogen activator (tPA/PLAT) was cloned and expressed in E. coli; cDNA sequencing revealed a 527-amino-acid serine protease with a signal peptide, a heavy chain containing growth-factor-like and kringle domains, and a light chain comprising the serine protease catalytic domain, establishing tPA as a multi-domain serine protease that converts plasminogen to plasmin.\",\n      \"method\": \"cDNA cloning, expression in E. coli, protein sequencing\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — foundational cloning and sequencing study, extensively replicated, >1000 citations\",\n      \"pmids\": [\"6337343\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1984,\n      \"finding\": \"Peptide analysis of purified tPA protein confirmed the cDNA-derived primary structure, identified Ser-478 as the active-site serine (inactivated by diisopropyl phosphorofluoridate), localized N-linked glycosylation to Asn-117, Asn-184 (the site distinguishing variant I from variant II by differential carbohydrate addition), and Asn-448, while Asn-218 is not glycosylated.\",\n      \"method\": \"Peptide mapping by reverse-phase HPLC, Edman sequencing, CNBr cleavage, DFP inactivation\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct biochemical characterization of active site and glycosylation by multiple orthogonal methods\",\n      \"pmids\": [\"6433976\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1984,\n      \"finding\": \"The human PLAT gene spans >32 kb and is organized into 14 exons separated by 13 introns; exon-intron boundaries correspond closely to functional and structural protein domains (signal peptide, propeptide, finger/fibronectin-like, growth factor-like, two kringles each encoded by two exons, and the serine protease domain split by four introns in a pattern similar to other serine proteases), demonstrating exon shuffling in the evolution of tPA.\",\n      \"method\": \"Cosmid library screening, restriction mapping, Southern blotting, DNA sequencing\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — complete gene structure determination by sequencing, independently confirmed\",\n      \"pmids\": [\"6089198\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1986,\n      \"finding\": \"Complete nucleotide sequence of the PLAT gene (36,594 bp) was established, confirming 14 exons and 13 introns, identifying TATA and CAAT boxes upstream of the transcription start site, and revealing 28 Alu repeats within the gene; comparison with urokinase plasminogen activator indicated close evolutionary relationship between the two human plasminogen activators.\",\n      \"method\": \"Complete DNA sequencing, S1 nuclease, exonuclease VII, and primer extension mapping of transcription start site\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — complete gene sequencing with transcription start site mapping\",\n      \"pmids\": [\"3009482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1986,\n      \"finding\": \"The human PLAT gene was mapped to chromosome 8 using rodent-human somatic cell hybrids probed with a 1.2-kb 3'-terminal cDNA fragment.\",\n      \"method\": \"Somatic cell hybrid panel, Southern blotting\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct chromosomal localization by somatic cell hybrid analysis\",\n      \"pmids\": [\"3002960\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1983,\n      \"finding\": \"A rapid inhibitor of tPA (later identified as PAI-1) was demonstrated in plasma; kinetic analysis showed it forms a 1:1 complex with tPA with a second-order rate constant of ~10^7 M^-1 s^-1, indicating tight and rapid inhibitory control of tPA activity in blood.\",\n      \"method\": \"Plasma t-PA activity assay, kinetic analysis of inhibition\",\n      \"journal\": \"Thrombosis research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — quantitative kinetic characterization of tPA-inhibitor interaction, replicated across many studies\",\n      \"pmids\": [\"20217999\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1986,\n      \"finding\": \"PAI-1 purified from phorbol-ester-treated U-937 cells forms covalent (SDS-stable) complexes with two-chain tPA with a second-order rate constant of 0.2 × 10^6 M^-1 s^-1 and with urokinase at 0.9 × 10^6 M^-1 s^-1; partial amino acid sequencing identified PAI-1 as a member of the antithrombin III (serpin) family, establishing the serpin mechanism of tPA inhibition.\",\n      \"method\": \"Affinity chromatography purification, SDS-PAGE complex analysis, NH4OH cleavage, partial amino acid sequencing, inhibition kinetics\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — biochemical purification with kinetic and structural characterization of the tPA-PAI-1 inhibitory complex\",\n      \"pmids\": [\"3090045\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"Site-directed mutagenesis of the serine protease domain of tPA identified a surface loop (residues 296–302) whose positively charged residues Arg-298 and Arg-299 form ionic interactions with complementary negatively charged residues C-terminal to the reactive center of PAI-1; triple mutation (Lys296Glu/Arg298Glu/Arg299Glu) produced a tPA variant highly resistant to PAI-1 without affecting plasminogen catalysis, defining the molecular basis of tPA–PAI-1 specificity.\",\n      \"method\": \"Site-directed mutagenesis, kinetic analysis of PAI-1 inhibition, plasminogen activation assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis with rigorous functional readouts separating PAI-1 interaction from catalysis\",\n      \"pmids\": [\"2110366\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"Vitronectin acts as a protein cofactor that dramatically enhances PAI-1 inhibition of tPA (and, when PAI-1 reactive center is mutated to match antithrombin III, of thrombin), establishing that a non-protein-cofactor mechanism can alter serpin specificity through protein–protein interaction and demonstrating that PAI-1 is a versatile inhibitor of fibrinolysis modulated by vitronectin.\",\n      \"method\": \"Reactive-center mutagenesis of PAI-1, kinetic inhibition assays ± vitronectin, SDS-PAGE complex analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis combined with kinetic analysis demonstrating cofactor-dependent change in tPA inhibition\",\n      \"pmids\": [\"1695900\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"tPA-PAI-1 complexes bind in solution to the low-density lipoprotein receptor-related protein (LRP/α2-macroglobulin receptor) and are subsequently endocytosed and degraded in cultured cells; internalization was blocked by anti-LRP antibodies or the 39-kDa LRP-binding protein (RAP), identifying LRP as the clearance receptor for tPA-inhibitor complexes.\",\n      \"method\": \"Solution binding assays, endocytosis/degradation assays in cultured cells, antibody blocking, RAP inhibition\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal inhibition with antibodies and RAP, replicated and widely cited\",\n      \"pmids\": [\"1502153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Amphoterin (HMGB1) strongly enhances the rate of plasminogen activation by tPA (tissue-type) and promotes generation of surface-bound plasmin; extracellular amphoterin is localized at the leading edge of motile cells and undergoes plasminogen-dependent degradation at cell-substrate contact sites, identifying amphoterin as a cell-surface co-factor that stimulates tPA-mediated plasmin generation at sites of cell migration.\",\n      \"method\": \"In vitro plasminogen activation assays with recombinant amphoterin, immunostaining, plasminogen-dependent degradation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct in vitro reconstitution with recombinant protein plus cell localization imaging\",\n      \"pmids\": [\"8366113\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"tPA mRNA expression was induced in cerebellar Purkinje neurons within one hour of complex motor task training; tPA protein was correspondingly upregulated in Purkinje cells, implicating tPA in activity-dependent synaptic remodeling and motor learning in the cerebellum.\",\n      \"method\": \"In situ hybridization, immunohistochemistry in motor-trained rats\",\n      \"journal\": \"Science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — in vivo induction shown by ISH and IHC in trained animals, functional implication via correlation\",\n      \"pmids\": [\"8533091\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"tPA receptor LRP is abundantly expressed in hippocampal neurons; perfusion with the LRP antagonist RAP significantly reduced late-phase LTP (L-LTP), and RAP blocked tPA-enhanced synaptic potentiation in tPA-knockout hippocampal slices. Metabolic labeling confirmed both tPA and LRP are synthesized by hippocampal neurons; tPA binding to LRP enhances cAMP-dependent protein kinase activity, placing tPA–LRP interaction upstream of PKA in the L-LTP signaling cascade.\",\n      \"method\": \"Hippocampal slice electrophysiology, RAP perfusion, metabolic labeling, ligand binding assay, cAMP-PKA activity measurement\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including electrophysiology, KO rescue, and signaling readout in one study\",\n      \"pmids\": [\"10632583\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The finger domain of tPA (not its catalytic activity) is required to mediate microglial activation; annexin II was identified as the probable binding partner/receptor for the tPA finger domain at the microglial surface, indicating that tPA acts as a neural cytokine through a non-proteolytic mechanism to activate microglia during excitotoxic injury.\",\n      \"method\": \"Cell culture studies with tPA domain deletion/mutant constructs, identification of annexin II by binding assays, wild-type vs. catalytically inactive tPA comparison\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — domain-specific mutagenesis combined with receptor identification and functional microglial activation readout\",\n      \"pmids\": [\"11978811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Strong tetanic stimulation inducing L-LTP and training in hippocampal-dependent memory tasks enhance tPA expression and secretion; inhibition of tPA by gene knockout or specific inhibitors significantly impairs L-LTP and long-term memory, placing tPA as a required secreted factor downstream of strong synaptic activity for long-term memory consolidation.\",\n      \"method\": \"tPA knockout mouse behavioral and electrophysiology studies, pharmacological inhibition of tPA, biochemical analysis of tPA secretion\",\n      \"journal\": \"Ageing research reviews\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO + inhibitor convergence, review synthesizing primary experimental data from multiple labs\",\n      \"pmids\": [\"15541709\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ADAM9 silencing in lung cancer cells suppressed PLAT (tPA) expression along with VEGFA and ANGPT2, leading to decreases in angiogenesis, vascular remodeling, and tumor growth in vivo, identifying PLAT as a downstream effector of ADAM9-driven vascular remodeling in brain metastasis.\",\n      \"method\": \"ADAM9 siRNA knockdown, microarray gene expression, in vivo tumor growth assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single KD with transcriptomic readout; PLAT is one of three downstream targets without direct mechanistic dissection\",\n      \"pmids\": [\"29118335\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PLAT/tPA treatment following ischemia decreases deleterious autophagy and mediates neuroprotection by modulating the PI3K-AKT-mTOR pathway; mechanistically, tPA degrades IGFBP3, thereby increasing free IGF1 bioavailability and activating IGF1R (a tyrosine kinase receptor), which in turn activates PI3K-AKT-mTOR to suppress autophagy. This was demonstrated in hypoxia-reperfusion in vitro models, in vivo thromboembolic stroke with tPA thrombolysis, and corroborated in human stroke patients.\",\n      \"method\": \"In vitro hypoxia-reperfusion models, in vivo thromboembolic stroke model, IGFBP3 cleavage assay, PI3K-AKT-mTOR pathway analysis, IGF1R inhibitor (picropodophyllin), human patient samples\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal models (in vitro, in vivo, human), receptor identified with pharmacological inhibitor, substrate (IGFBP3) cleavage demonstrated\",\n      \"pmids\": [\"34520334\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Mechanical stimulation of osteoblasts induces Wnt1 expression, which activates β-Catenin, and β-Catenin in turn upregulates Plat expression; siRNA knockdown of Plat prevented mechanical stimulation-induced upregulation of osteogenic markers Runx2 and Sp7, placing Plat downstream of Wnt1/β-Catenin as a required mediator of mechanically induced osteoblast differentiation. Plat and Wnt1 expression were reduced in bones from ovariectomized and aged (osteoporotic) mice.\",\n      \"method\": \"Mechanical stimulation of osteoblasts, siRNA knockdown of Wnt1 and Plat, RNAseq, β-Catenin activation analysis, qPCR for osteogenic markers, OVX mouse model\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — siRNA KD with defined osteogenic phenotype readout and upstream pathway dissection, single lab\",\n      \"pmids\": [\"38407813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In mouse oocytes, Plat (encoding tPA) is downregulated with aging; Plat knockdown increases oocyte susceptibility to both apoptosis and ferroptosis. Mechanistically, tPA acts as an upstream signaling molecule that activates the Erk1/2 pathway by interacting with phosphorylation kinases including Alk; Plat loss reduces Erk1/2 activity, leading to oocyte degeneration through programmed cell death. Supplementation with exogenous tPA in in vitro maturation cultures reduced defect rates in aged oocytes.\",\n      \"method\": \"Transcriptomic analysis of single oocytes, siRNA knockdown of Plat, apoptosis/ferroptosis pathway inhibitors, Erk1/2 phosphorylation assay, exogenous tPA supplementation in IVM cultures\",\n      \"journal\": \"Science China. Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KD with dual pathway readout (apoptosis + ferroptosis), signaling rescue with exogenous tPA; single lab\",\n      \"pmids\": [\"41252074\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In gefitinib-resistant NSCLC cells, PLAT knockdown reduced migration and invasion by downregulating TNC, ITGB3, and the phosphorylation of FAK, PI3K, and AKT, placing PLAT upstream of the FAK/AKT signaling pathway that drives cell motility and invasion in drug-resistant lung adenocarcinoma.\",\n      \"method\": \"Whole-transcriptome sequencing, siRNA/lentiviral knockdown of PLAT, cell migration/invasion assays, western blot for p-FAK, p-PI3K, p-AKT, in vivo NSG mouse xenograft\",\n      \"journal\": \"Chinese medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KD with defined signaling pathway readout, in vitro and in vivo, single lab\",\n      \"pmids\": [\"39754146\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PLAT encodes tissue-type plasminogen activator (tPA), a multi-domain serine protease (finger, EGF-like, two kringle, and serine protease domains encoded by 14 exons) that cleaves plasminogen to plasmin; its activity is tightly regulated by PAI-1 (serpin inhibitor forming a covalent complex via residues Arg-298/Arg-299 on tPA, modulated by the cofactor vitronectin) and cleared as tPA-PAI-1 complexes via LRP-mediated endocytosis; tPA also acts non-proteolytically through its finger domain binding annexin II to activate microglia, through LRP binding to enhance cAMP-PKA signaling in hippocampal L-LTP, and through IGFBP3 cleavage to increase free IGF1 and activate IGF1R/PI3K-AKT-mTOR neuroprotection; additionally, tPA is placed downstream of Wnt1/β-Catenin in mechanical osteogenesis and upstream of Erk1/2 in oocyte survival, and upstream of FAK/AKT in cancer cell invasion.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PLAT (tissue-type plasminogen activator, tPA) is a serine protease whose canonical function is cleavage of plasminogen to plasmin for fibrinolysis, but it also operates as a signaling molecule in multiple non-fibrinolytic contexts. In brain ischemia, tPA degrades IGFBP3 to liberate IGF1, thereby activating IGF1R/PI3K-AKT-MTOR signaling and suppressing deleterious neuronal autophagy [PMID:34520334]. In oocytes, tPA interacts with ALK to activate Erk1/2 signaling and protect against apoptosis and ferroptosis [PMID:41252074], and in osteoblasts it functions downstream of Wnt1/β-Catenin to promote expression of osteogenic transcription factors Runx2 and Sp7 [PMID:38407813]. In gefitinib-resistant non-small-cell lung cancer cells, PLAT knockdown restores drug sensitivity by activating TNF-α signaling, caspase-mediated apoptosis, and mitochondrial dysfunction, indicating a pro-survival role in resistant tumors [PMID:32274137].\",\n  \"teleology\": [\n    {\n      \"year\": 2015,\n      \"claim\": \"NMR structural characterization of a PLAT-domain protein (polycystin-1) provided the first atomic-level description of PLAT-domain architecture, revealing a membrane-anchored fold with extensive hydrophobic surface and a mobile loop, though this addressed polycystin-1 rather than tPA itself.\",\n      \"evidence\": \"NMR backbone assignment of human polycystin-1 PLAT domain with perdeuteration and mutagenesis\",\n      \"pmids\": [\"25943267\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Describes polycystin-1 PLAT domain, not tPA; functional relevance to PLAT/tPA unclear\",\n        \"No functional assay linking structural features to enzymatic or signaling activity\",\n        \"Single structural study without independent validation\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Establishing that PLAT sustains a pro-survival state in drug-resistant cancer cells answered whether tPA has non-fibrinolytic roles in tumor biology; PLAT knockdown in gefitinib-resistant NSCLC cells restored apoptotic sensitivity via TNF-α signaling, caspase activation, and mitochondrial dysfunction.\",\n      \"evidence\": \"Lentiviral RNAi knockdown in PC9 gefitinib-resistant NSCLC cells with flow cytometry, JC-1 mitochondrial assay, and in vivo xenograft model\",\n      \"pmids\": [\"32274137\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct molecular target of PLAT in cancer cells not identified—unclear whether proteolytic activity or receptor-mediated signaling is responsible\",\n        \"Single cancer cell line; generalizability to other resistant tumors untested\",\n        \"Upstream regulation of PLAT overexpression in resistant cells not defined\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrating that tPA suppresses post-ischemic neuronal autophagy via IGFBP3 degradation and IGF1R/PI3K-AKT-MTOR activation established a non-fibrinolytic neuroprotective signaling pathway for tPA in stroke.\",\n      \"evidence\": \"In vitro hypoxia-reperfusion, in vivo thromboembolic stroke model, IGF1R inhibitor (picropodophyllin), western blot for pathway components, and human stroke patient samples\",\n      \"pmids\": [\"34520334\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether tPA's proteolytic cleavage of IGFBP3 is the sole mechanism or additional substrates contribute is unresolved\",\n        \"Therapeutic window and dose-dependency of autophagy suppression not established\",\n        \"Single-lab finding; independent replication in additional stroke models needed\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Placing Plat downstream of Wnt1/β-Catenin in mechanically stimulated osteoblasts revealed an unexpected role for tPA in bone formation, where it promotes Runx2 and Sp7 expression during osteogenesis.\",\n      \"evidence\": \"siRNA knockdown of Wnt1 and Plat in osteoblasts, RNAseq, western blot, mechanical stimulation assay, ovariectomy and aged mouse bone models\",\n      \"pmids\": [\"38407813\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether tPA acts via proteolytic liberation of a growth factor or via receptor-mediated signaling in osteoblasts is unknown\",\n        \"Direct transcriptional targets of tPA in osteogenesis not defined\",\n        \"In vivo bone phenotype of Plat-specific knockout under mechanical loading not tested\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identifying tPA as a protector of maternally aged oocytes from apoptosis and ferroptosis via ALK-Erk1/2 signaling expanded its non-fibrinolytic repertoire to reproductive biology and ferroptosis regulation.\",\n      \"evidence\": \"Single-oocyte transcriptomics, siRNA knockdown, exogenous tPA rescue, western blot and immunofluorescence for p-Erk1/2, co-immunoprecipitation with ALK\",\n      \"pmids\": [\"41252074\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"ALK interaction demonstrated by co-IP without reciprocal validation or structural mapping of the binding interface\",\n        \"Whether tPA's proteolytic activity or a non-catalytic interaction with ALK drives Erk1/2 activation is unresolved\",\n        \"Ferroptosis protection mechanism downstream of Erk1/2 not delineated\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A unifying question remains: whether tPA's diverse non-fibrinolytic signaling roles (autophagy suppression, osteogenesis, oocyte protection, cancer survival) depend on its serine protease catalytic activity or on non-enzymatic receptor interactions, and whether these pathways converge on common downstream effectors.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Catalytic-dead tPA mutants have not been tested systematically across the non-fibrinolytic contexts described\",\n        \"No structural model of tPA-ALK or tPA-IGF1R complex exists\",\n        \"Cross-tissue comparison of tPA signaling outputs has not been performed\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3, 5]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 2, 1]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"IGFBP3\",\n      \"IGF1R\",\n      \"ALK\",\n      \"PLG\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"PLAT encodes tissue-type plasminogen activator (tPA), a multi-domain serine protease that converts plasminogen to plasmin and thereby serves as a principal initiator of fibrinolysis, with additional non-proteolytic signaling roles in the nervous system, bone, and other tissues. The protein comprises a signal peptide, finger, EGF-like, two kringle, and serine protease domains encoded by 14 exons; the active-site serine (Ser-478) cleaves plasminogen, while surface-loop residues Arg-298/Arg-299 mediate rapid covalent inhibition by PAI-1, a serpin whose activity is enhanced by vitronectin, and the resulting tPA–PAI-1 complexes are cleared via LRP-mediated endocytosis [PMID:6337343, PMID:2110366, PMID:1695900, PMID:1502153]. Beyond fibrinolysis, tPA acts non-proteolytically through its finger domain to activate microglia via annexin II binding, signals through LRP to enhance cAMP-PKA–dependent late-phase LTP in hippocampal neurons, and proteolytically cleaves IGFBP3 to liberate IGF1 and activate the IGF1R/PI3K-AKT-mTOR neuroprotective axis after ischemic stroke [PMID:11978811, PMID:10632583, PMID:34520334]. tPA additionally functions downstream of Wnt1/β-Catenin in mechanically induced osteoblast differentiation and upstream of Erk1/2 in oocyte survival, and promotes FAK/AKT-dependent cancer cell invasion in drug-resistant lung adenocarcinoma [PMID:38407813, PMID:41252074, PMID:39754146].\",\n  \"teleology\": [\n    {\n      \"year\": 1983,\n      \"claim\": \"Cloning and sequencing of tPA established it as a multi-domain serine protease that converts plasminogen to plasmin, resolving the molecular identity of the fibrinolytic activator.\",\n      \"evidence\": \"cDNA cloning, E. coli expression, and protein sequencing of the 527-amino-acid protein\",\n      \"pmids\": [\"6337343\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Catalytic mechanism and active-site residue confirmation not yet performed\",\n        \"Gene structure and chromosomal location unknown\"\n      ]\n    },\n    {\n      \"year\": 1984,\n      \"claim\": \"Biochemical characterization confirmed the active-site Ser-478 and mapped glycosylation sites, defining the post-translational landscape of mature tPA and explaining the two glycoform variants.\",\n      \"evidence\": \"Peptide mapping by RP-HPLC, Edman sequencing, DFP inactivation, and CNBr cleavage of purified tPA\",\n      \"pmids\": [\"6433976\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Three-dimensional structure not yet determined\",\n        \"Functional role of individual glycosylation sites in activity or clearance unknown\"\n      ]\n    },\n    {\n      \"year\": 1986,\n      \"claim\": \"Complete gene structure determination (14 exons, 36.6 kb, chromosome 8) revealed domain-exon correspondence consistent with exon shuffling, explaining how tPA acquired its modular architecture.\",\n      \"evidence\": \"Cosmid library screening, complete DNA sequencing, transcription start-site mapping, somatic cell hybrid chromosomal assignment\",\n      \"pmids\": [\"6089198\", \"3009482\", \"3002960\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Promoter regulation and tissue-specific expression mechanisms not characterized\",\n        \"Functional significance of 28 intronic Alu repeats unknown\"\n      ]\n    },\n    {\n      \"year\": 1986,\n      \"claim\": \"Identification and kinetic characterization of PAI-1 as a serpin that forms a rapid, covalent 1:1 complex with tPA established the principal physiological inhibitor of fibrinolysis.\",\n      \"evidence\": \"Plasma kinetic assays (~10⁷ M⁻¹s⁻¹), affinity purification from U-937 cells, SDS-PAGE complex analysis, partial amino acid sequencing\",\n      \"pmids\": [\"20217999\", \"3090045\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of tPA–PAI-1 interaction at the residue level not yet defined\",\n        \"Role of cofactors in modulating PAI-1 inhibitory specificity not established\"\n      ]\n    },\n    {\n      \"year\": 1990,\n      \"claim\": \"Mutagenesis pinpointed Arg-298/Arg-299 as the molecular determinants of PAI-1 recognition, and vitronectin was shown to act as a protein cofactor enhancing PAI-1's inhibition of tPA — together defining the two-layered specificity control of tPA inhibition.\",\n      \"evidence\": \"Site-directed mutagenesis with kinetic assays separating PAI-1 inhibition from plasminogen catalysis; reactive-center mutagenesis of PAI-1 with vitronectin cofactor kinetics\",\n      \"pmids\": [\"2110366\", \"1695900\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Crystal structure of the tPA–PAI-1–vitronectin ternary complex not available\",\n        \"In vivo significance of vitronectin cofactor effect on fibrinolytic balance not tested\"\n      ]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Discovery that tPA–PAI-1 complexes are cleared via LRP-mediated endocytosis resolved how active tPA is removed from circulation and implicated LRP as a multifunctional tPA receptor.\",\n      \"evidence\": \"Solution binding, endocytosis/degradation assays in cultured cells, blocking with anti-LRP antibodies and RAP\",\n      \"pmids\": [\"1502153\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"LRP domain(s) responsible for tPA–PAI-1 binding not mapped\",\n        \"Hepatic vs. neuronal clearance contribution not distinguished\"\n      ]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Identification of amphoterin (HMGB1) as a cell-surface cofactor that enhances tPA-mediated plasminogen activation at the leading edge of migrating cells extended tPA function beyond intravascular fibrinolysis to pericellular proteolysis during cell migration.\",\n      \"evidence\": \"In vitro plasminogen activation reconstitution with recombinant amphoterin, immunostaining of motile cells\",\n      \"pmids\": [\"8366113\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct binding interface between tPA and HMGB1 not mapped\",\n        \"In vivo relevance to wound healing or metastasis not tested\"\n      ]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Demonstration that tPA binding to neuronal LRP enhances cAMP-PKA signaling and is required for late-phase LTP established a non-fibrinolytic, receptor-mediated signaling role for tPA in synaptic plasticity.\",\n      \"evidence\": \"Hippocampal slice electrophysiology with RAP perfusion, tPA-knockout rescue, metabolic labeling, cAMP-PKA activity assays\",\n      \"pmids\": [\"10632583\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether tPA signals through LRP as free protease or as tPA–PAI-1 complex in neurons not resolved\",\n        \"Downstream PKA substrates mediating L-LTP not identified\"\n      ]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Mapping the microglial activation function to the finger domain of tPA and identifying annexin II as its receptor revealed a purely non-proteolytic cytokine-like mechanism, expanding tPA's functional repertoire beyond protease activity.\",\n      \"evidence\": \"Domain-deletion and catalytically inactive tPA constructs tested on microglial cultures, annexin II binding assays\",\n      \"pmids\": [\"11978811\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Downstream signaling cascade in microglia triggered by finger-domain–annexin II interaction not defined\",\n        \"Relevance to in vivo neuroinflammation confirmed only indirectly\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identification of IGFBP3 as a tPA substrate whose cleavage liberates IGF1 to activate IGF1R/PI3K-AKT-mTOR and suppress autophagy provided a neuroprotective mechanism for tPA thrombolysis beyond clot lysis, validated in human stroke patients.\",\n      \"evidence\": \"In vitro hypoxia-reperfusion, in vivo thromboembolic stroke model, IGFBP3 cleavage assay, IGF1R inhibitor (picropodophyllin), human patient samples\",\n      \"pmids\": [\"34520334\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"IGFBP3 cleavage site on the substrate not mapped\",\n        \"Whether this neuroprotective pathway is engaged during endogenous tPA release (without pharmacological dosing) is unclear\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Placing Plat downstream of Wnt1/β-Catenin in mechanically stimulated osteoblasts, with Plat knockdown blocking osteogenic marker induction, revealed an unexpected role for tPA in skeletal mechanotransduction and a potential link to osteoporosis.\",\n      \"evidence\": \"Mechanical stimulation of osteoblasts, siRNA knockdown of Wnt1 and Plat, RNAseq, OVX and aged mouse models\",\n      \"pmids\": [\"38407813\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism by which tPA promotes osteogenesis (proteolytic vs. non-proteolytic) not determined\",\n        \"Findings from a single lab, awaiting independent replication\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Emerging evidence places tPA upstream of Erk1/2 in oocyte survival and upstream of FAK/AKT in cancer cell invasion, broadening its signaling repertoire to reproductive biology and drug-resistant cancer.\",\n      \"evidence\": \"siRNA knockdown of Plat in oocytes with apoptosis/ferroptosis and Erk1/2 readouts; PLAT knockdown in gefitinib-resistant NSCLC with FAK/PI3K/AKT western blot and xenograft assays\",\n      \"pmids\": [\"41252074\", \"39754146\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct physical interaction between tPA and Alk or other kinases in oocytes awaits biochemical validation\",\n        \"Whether tPA's role in cancer invasion is proteolytic (plasmin-dependent) or receptor-mediated is unresolved\",\n        \"Both findings are from single laboratories\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The structural basis of tPA's non-proteolytic signaling through LRP and annexin II, the full repertoire of tPA substrates beyond plasminogen and IGFBP3, and whether the osteogenic and oocyte-survival functions are clinically relevant remain open questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No high-resolution structure of tPA in complex with LRP or annexin II\",\n        \"Systematic substrate profiling (degradomics) of tPA not performed\",\n        \"In vivo genetic validation of tPA roles in bone and oocyte biology lacking\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 7, 16]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [12, 13]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [12, 18]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0, 5, 10, 11, 16]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [10, 13]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [0, 5, 6, 7, 8]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [12, 16, 17, 18, 19]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [12, 14]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [16, 18]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"SERPINE1\",\n      \"LRP1\",\n      \"ANXA2\",\n      \"PLG\",\n      \"VTN\",\n      \"HMGB1\",\n      \"IGFBP3\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}