{"gene":"THBD","run_date":"2026-04-28T21:42:59","timeline":{"discoveries":[{"year":1987,"finding":"Human thrombomodulin cDNA was cloned and the complete 575-amino acid sequence deduced, revealing a domain structure comprising a signal peptide, N-terminal domain, six EGF-like domains, an O-glycosylation-rich region, a 24-residue transmembrane domain, and a cytoplasmic tail. Transfection of COS cells with the cDNA produced immunoreactive, functionally active thrombomodulin.","method":"cDNA cloning, expression in COS cells, functional assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — original structural characterization by cDNA sequencing and heterologous expression with functional validation","pmids":["2820710"],"is_preprint":false},{"year":1987,"finding":"Complete cDNA sequence of human thrombomodulin confirmed a 575-amino acid protein with six EGF-type repeats, no introns in the coding region, and putative regulatory elements in the promoter. The gene was localized to chromosome 20.","method":"cDNA and genomic cloning, Northern blot, chromosomal mapping","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — independent full-length sequence determination with genomic characterization","pmids":["2822087"],"is_preprint":false},{"year":1987,"finding":"The thrombomodulin gene contains no introns within its coding region, contains six EGF-type B repeats, has homology to the LDL receptor, and the promoter region contains potential regulatory elements. The intronless coding region was established by comparison of cDNA and genomic sequences.","method":"Genomic and cDNA cloning, nucleotide sequencing","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — direct genomic sequence comparison establishing gene structure","pmids":["2819876"],"is_preprint":false},{"year":1985,"finding":"Thrombomodulin is present as a soluble form in human plasma and urine. Soluble thrombomodulin retains intrinsic protein C-activating cofactor activity with the same apparent Km for protein C as the cellular form, but requires 3–5-fold higher thrombin concentration for half-maximal activity, suggesting loss of the membrane-anchoring domain alters thrombin affinity.","method":"Radioimmunoassay, immunoaffinity chromatography, immunoblot, protein C activation assay","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1 — purified protein with in vitro functional kinetic characterization","pmids":["3001144"],"is_preprint":false},{"year":1989,"finding":"Thrombomodulin acts as a thrombin receptor on endothelium that functions as a cofactor for protein C activation, converting thrombin from a procoagulant to an anticoagulant enzyme. The thrombin–thrombomodulin complex loses ability to clot fibrinogen and activate platelets while gaining ability to rapidly activate protein C zymogen.","method":"Biochemical reconstitution, enzyme kinetics, functional assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro biochemical mechanism, foundational study replicated extensively","pmids":["2538457"],"is_preprint":false},{"year":1995,"finding":"Thrombomodulin's chondroitin sulfate moiety binds to a basic surface on thrombin also involved in heparin interaction, enhancing thrombin affinity for thrombomodulin 10–20-fold, altering thrombin conformation and specificity, and accelerating thrombin inhibition by antithrombin. The EGF-like domains of thrombomodulin block thrombin's procoagulant substrate binding sites (fibrinogen, platelet receptor), while conformational changes in the thrombin active site facilitate protein C docking.","method":"Structural analysis, kinetic studies, competition assays, mutagenesis","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 1 — multiple orthogonal biochemical methods establishing mechanism","pmids":["7615164"],"is_preprint":false},{"year":1995,"finding":"The first thrombomodulin gene mutation associated with thromboembolism was identified: a heterozygous G1456T substitution predicting Asp468Tyr, located between the transmembrane domain and the sixth EGF-like domain, in a patient with pulmonary embolism.","method":"PCR-SSCP, DNA sequencing","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 — genetic identification of disease-causing mutation with structural implication, single case","pmids":["7811989"],"is_preprint":false},{"year":1996,"finding":"The thrombin–thrombomodulin complex, rather than free thrombin, is the physiologic activator of TAFI (thrombin-activatable fibrinolysis inhibitor/procarboxypeptidase B). Thrombomodulin increases the catalytic efficiency of TAFI activation 1250-fold, primarily by increasing kcat. The reaction follows a ternary complex model: thrombin binds thrombomodulin (Kd = 8.6 nM) or TAFI (Km = 1.0 µM), and the binary complex recruits the third component to form an active ternary complex. Activated TAFI inhibits fibrinolysis half-maximally at ~1 nM.","method":"In vitro reconstitution, enzyme kinetics, purified components","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — rigorous in vitro reconstitution with full kinetic characterization","pmids":["8663147"],"is_preprint":false},{"year":2000,"finding":"Crystal structure of human α-thrombin bound to TME456 (EGF-like domains 4–5–6 of thrombomodulin, the smallest fragment sufficient for full protein C cofactor activity) at 2.3 Å resolution. Thrombomodulin binds thrombin's anion-binding exosite-I in a Y-shaped configuration, sterically blocking procoagulant substrate binding without inducing marked allosteric rearrangements at the thrombin active site. Modeling indicates TME45 may orient protein C's cleavage site optimally toward the unaltered thrombin active site.","method":"X-ray crystallography (2.3 Å), molecular docking","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — high-resolution crystal structure with functional validation","pmids":["10761923"],"is_preprint":false},{"year":2003,"finding":"Thrombomodulin mediates Ca²⁺-dependent cell-to-cell adhesion via its lectin-like domain. In TM-negative melanoma cells transfected with GFP-tagged TM, cells formed closely clustered colonies with TM localized at intercellular boundaries, reduced monolayer permeability in vitro, and decreased tumor growth in vivo. Deletion of the lectin-like domain abolished these effects. Adhesion was inhibited by anti-lectin-domain antibody and by mannose, chondroitin sulfate A, or chondroitin sulfate C.","method":"Transfection of TM-negative cells, domain-deletion mutants, confocal microscopy, permeability assay, in vivo tumor model, antibody inhibition","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — domain-deletion mutagenesis with multiple orthogonal functional readouts","pmids":["12951323"],"is_preprint":false},{"year":2004,"finding":"Thrombomodulin–protein C–EPCR system is integrated to regulate both coagulation and inflammation. Thrombomodulin serves as a cofactor for thrombin-mediated protein C activation (further amplified by EPCR), and activated protein C exerts anticoagulant, anti-inflammatory, profibrinolytic, and cytoprotective effects. The covalently associated chondroitin sulfate moiety of TM additionally enhances thrombin binding and modulates TAFI activation and complement regulation.","method":"Review of biochemical, cell biology, and in vivo experimental data","journal":"Arteriosclerosis, thrombosis, and vascular biology","confidence":"High","confidence_rationale":"Tier 1–2 — synthesis of multiply replicated mechanistic studies across labs","pmids":["15178554"],"is_preprint":false},{"year":2005,"finding":"Inflammatory cytokines (TNF-α) down-regulate endothelial thrombomodulin expression through NF-κB activation. NF-κB does not bind the TM promoter directly but competes for limited pools of the coactivator p300, which is required by Ets transcription factors for TM promoter transactivation. Blocking NF-κB activation prevented cytokine-induced TM down-regulation in vitro and in a mouse TNF-α lung injury model.","method":"EMSA, chromatin immunoprecipitation, promoter deletion/mutation analysis, NF-κB inhibition, p300 overexpression, mouse model","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal molecular methods plus in vivo validation","pmids":["15677570"],"is_preprint":false},{"year":2005,"finding":"KLF2 (Krüppel-like factor 2) is required for statin-induced upregulation of thrombomodulin in endothelial cells. Statin treatment induces KLF2 mRNA via Rho pathway inhibition and de novo transcription through a myocyte enhancer factor binding site in the KLF2 promoter. siRNA-mediated KLF2 knockdown strongly attenuated mevastatin-induced thrombomodulin protein accumulation.","method":"siRNA knockdown, promoter deletion/mutation analysis, pharmacological inhibition, Western blot","journal":"Circulation","confidence":"High","confidence_rationale":"Tier 2 — siRNA epistasis plus promoter analysis, replicated with multiple statins","pmids":["16043642"],"is_preprint":false},{"year":2007,"finding":"Thrombomodulin expression in vascular endothelial cells is clock-controlled: TM mRNA and protein show circadian oscillation in mouse lung and heart. The CLOCK/BMAL2 heterodimer directly binds an E-box element in the TM promoter to drive transcription, as shown by reporter assays, EMSA, and ChIP. Oscillation was abolished in Clock mutant mice and phase-shifted by temporal feeding restriction.","method":"Microarray, reporter assay, EMSA, chromatin immunoprecipitation, Clock mutant mice, temporal feeding restriction","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — direct binding of CLOCK/BMAL2 to TM promoter confirmed by ChIP and EMSA, validated in vivo in mutant mice","pmids":["17848551"],"is_preprint":false},{"year":2007,"finding":"Endothelial thrombomodulin-dependent activated protein C (APC) formation is reduced in diabetic mice and causally linked to diabetic nephropathy. APC prevents glucose-induced apoptosis in endothelial cells and podocytes via protease-activated receptor PAR-1 and endothelial protein C receptor EPCR, modulating the mitochondrial apoptosis pathway. Loss of thrombomodulin-dependent APC disrupts endothelial–podocyte crosstalk, causing glomerular apoptosis.","method":"Diabetic mouse model, endothelial cell and podocyte in vitro assays, PAR-1/EPCR pathway analysis, apoptosis assays","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 — in vivo genetic model combined with in vitro mechanistic pathway dissection","pmids":["17982464"],"is_preprint":false},{"year":2009,"finding":"THBD mutations cause ~5% of atypical hemolytic-uremic syndrome (aHUS) cases. Mechanistically, thrombomodulin binds C3b and factor H and negatively regulates complement by accelerating factor I-mediated C3b inactivation (requiring cofactors CFH or C4b-binding protein). TM also accelerates inactivation of anaphylatoxins C3a and C5a via procarboxypeptidase B activation. aHUS-associated missense mutations in THBD reduce C3b inactivation and procarboxypeptidase B activation in cell-expression systems.","method":"THBD gene sequencing, purified protein binding assays, cell-expression systems, complement activation assays","journal":"The New England journal of medicine","confidence":"High","confidence_rationale":"Tier 1–2 — direct binding assays with purified proteins plus functional cell-expression mutagenesis, large patient cohort","pmids":["19625716"],"is_preprint":false},{"year":2011,"finding":"Thrombomodulin exhibits anti-inflammatory properties through multiple mechanisms: (1) cofactor for thrombin-mediated protein C activation; (2) TAFI activation to inhibit fibrinolysis; (3) complement regulation via C3b inactivation; (4) binding and sequestration of the pro-inflammatory cytokine HMGB1 via its lectin-like domain; (5) interaction with Lewis Y antigen. TM expression is tightly regulated by cytokines (down-regulated by TNF-α, IL-1), shear stress, and thrombin.","method":"Review integrating published biochemical and in vivo experimental data","journal":"Seminars in immunopathology","confidence":"High","confidence_rationale":"Tier 1–2 — synthesis of replicated mechanistic studies, multiple independent labs","pmids":["21805323"],"is_preprint":false},{"year":2013,"finding":"Loss of endothelial thrombomodulin (TM) and EPCR at sites of malaria-infected erythrocyte cytoadherence in cerebral malaria is associated with cerebral fibrin clot formation. Using ex vivo subcutaneous microvessels, loss of TM and EPCR was detectable at sites of infected erythrocyte binding in non-fatal cerebral malaria, linking parasite sequestration to disruption of endothelial anticoagulant/anti-inflammatory protection.","method":"Autopsy immunostaining, novel ex vivo microvascular assay, coagulation activation assays","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 — direct localization with functional correlation, but primarily observational/ex vivo","pmids":["23741007"],"is_preprint":false},{"year":2014,"finding":"Regulatory sequences from the porcine THBD gene (7.6-kb upstream fragment) drive endothelial-specific expression of human thrombomodulin (hTM) in transgenic pigs. Endothelial cells from hTM transgenic pigs showed 3–4-fold increased clotting time in human whole blood assays compared to wild-type, demonstrating that hTM retains anticoagulant function in a xenograft context and can overcome human–pig coagulation incompatibilities.","method":"Transgenic pig generation (somatic cell nuclear transfer), endothelial cell coagulation assay with human whole blood","journal":"Transplantation","confidence":"Medium","confidence_rationale":"Tier 2 — functional demonstration in transgenic animal model with human blood assay","pmids":["24150517"],"is_preprint":false},{"year":2017,"finding":"Tissue-selective Thbd gene ablation in adult mice reveals that complete Thbd deficiency leads to low-grade consumptive coagulopathy and sudden-onset mortality. Supplementation with activated protein C (via a partially Thbd-independent PC transgene) prevented thrombotic pathologies but Thbd-deficient females showed near-complete pregnancy-induced morbidity. This demonstrates that Thbd function in nonendothelial embryonic tissues (placenta, yolk sac) influences the penetrance and severity of postnatal thrombosis through unknown mechanisms.","method":"Conditional Cre/loxP Thbd gene ablation, transgenic protein C supplementation, histopathology, coagulation assays","journal":"Blood advances","confidence":"High","confidence_rationale":"Tier 2 — rigorous conditional knockout with genetic rescue experiment in vivo","pmids":["28920104"],"is_preprint":false},{"year":2017,"finding":"A THBD c.1611C>A nonsense mutation (p.Cys537X) leads to a truncated thrombomodulin that has lost the last three amino acids of the transmembrane domain and the cytoplasmic tail. This truncated protein is partially membrane-anchored but is shed at ~6× higher levels than wild-type TM. Shedding is ~75% dependent on metalloprotease activity and is not affected by cathepsin G, elastase, reducing agents, or high shear stress. Other truncation mutants with shortened transmembrane domains also show increased shedding, indicating the transmembrane domain length is critical for membrane retention.","method":"Site-directed mutagenesis, transient transfection in COS-1 cells, Western blot, immunofluorescence, metalloprotease inhibitor assay, ELISA","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis series with multiple inhibitor conditions, mechanistic dissection of shedding","pmids":["29145514"],"is_preprint":false},{"year":2020,"finding":"Endogenous THBD promotes angiogenesis in the ischemic brain. Following middle cerebral artery occlusion in mice, Thbd expression increased in the peri-infarct area. Inducible, brain endothelial cell-specific Thbd deletion worsened stroke outcome without increasing coagulation, thrombosis, or inflammation, but instead decreased vessel diameters and impaired angiogenesis in the peri-infarct area, reducing overall vessel length 1 week post-stroke.","method":"Mouse MCA occlusion model, inducible Cre/loxP brain endothelial-specific Thbd knockout, histology, vessel morphometry","journal":"Arteriosclerosis, thrombosis, and vascular biology","confidence":"High","confidence_rationale":"Tier 2 — cell-type-specific conditional knockout with specific angiogenic phenotype, dissociated from coagulation effects","pmids":["33028093"],"is_preprint":false},{"year":2021,"finding":"miR-18a-5p negatively regulates THBD expression in endometrial cancer cells. Dual-luciferase assay confirmed direct targeting of THBD 3'UTR by miR-18a-5p. Overexpression of miR-18a-5p enhanced proliferation, migration, and invasion of EC cells, while THBD overexpression inhibited these abilities. Rescue experiments showed THBD overexpression reversed the oncogenic effects of miR-18a-5p, placing THBD downstream of miR-18a-5p in a regulatory axis suppressing EC cell aggressiveness.","method":"Dual-luciferase reporter assay, qRT-PCR, Western blot, CCK-8 assay, Transwell assay, rescue experiments","journal":"Critical reviews in eukaryotic gene expression","confidence":"Medium","confidence_rationale":"Tier 2–3 — direct miRNA-target validation by luciferase assay plus functional rescue, single lab","pmids":["34347980"],"is_preprint":false},{"year":2022,"finding":"BRG1 (a chromatin remodeling ATPase) epigenetically represses THBD transcription in endothelial cells, contributing to deep vein thrombosis. TNF-α stimulation decreases THBD expression; BRG1 knockdown or inhibition recovers THBD expression. Mechanistically, BRG1 directly upregulates DNMT1 expression after TNF-α treatment, increasing CpG methylation of the THBD promoter. Endothelial-specific BRG1 conditional knockout mice showed substantially decreased DVT susceptibility with decreased thrombus weight/size.","method":"Conditional endothelial BRG1 knockout mice (Cdh5-ERT-Cre × Brg1-loxP), siRNA/inhibitor experiments, CpG methylation analysis, DVT ligation model, small-molecule BRG1 inhibitor (PFI-3)","journal":"Thrombosis research","confidence":"High","confidence_rationale":"Tier 2 — conditional KO in vivo model plus mechanistic epigenetic pathway (DNMT1-mediated CpG methylation) with pharmacological validation","pmids":["36162255"],"is_preprint":false},{"year":2021,"finding":"The THBD c.1418C>T (p.Ala473Val) variant reduces protein C activation and impairs endothelial progenitor cell (EPC) migration and angiogenesis. Expression of p.Ala473Val in EPCs decreased VEGFRs, MMP1/2/3, VE-cadherin, Flk-1, eNOS, and TIE-2 expression, weakened wound healing, Transwell migration, and tube formation, mechanistically linking the variant to reduced angiogenic capacity.","method":"THBD cDNA transfection in EPCs, ELISA, wound healing, Transwell migration, tube formation, Western blot","journal":"International angiology","confidence":"Medium","confidence_rationale":"Tier 2–3 — cell-based functional assays with variant vs. wild-type, single lab","pmids":["34236150"],"is_preprint":false},{"year":2025,"finding":"Adrenomedullin (ADM) signaling through its receptor RAMP2 synergizes with shear stress to increase THBD expression in human pulmonary endothelial cells, enhancing anti-coagulant phenotype (increased THBD, decreased tissue factor F3). This effect was completely abolished by RAMP2 blockade, identifying ADM/RAMP2 as an upstream paracrine regulator of endothelial THBD expression.","method":"ECIS, XperT barrier assay, qPCR, pharmacological receptor blockade, shear stress apparatus","journal":"bioRxiv (preprint)","confidence":"Low","confidence_rationale":"Tier 3 — preprint, single lab, expression-level readout without direct promoter mechanism","pmids":[],"is_preprint":true},{"year":2024,"finding":"In pristane-induced lupus, MEK1/2-ERK1/2 pathway activation in B6 mice upregulates Thbd (thrombomodulin) gene expression and increases circulating soluble Thbd protein in the lung, contributing to endothelial anticoagulant dysregulation and diffuse alveolar hemorrhage. MEK1/2 inhibition (trametinib) normalized Thbd expression and circulating Thbd levels, and abolished DAH, linking the ERK pathway to Thbd-mediated hemostatic imbalance.","method":"MEK/ERK inhibitor treatment in mouse lupus model, qPCR, Western blot, ELISA, tail bleeding assay","journal":"bioRxiv (preprint)","confidence":"Low","confidence_rationale":"Tier 3 — preprint, pharmacological inhibition with expression-level THBD readout, no direct mechanistic dissection of THBD","pmids":[],"is_preprint":true}],"current_model":"THBD (thrombomodulin) is an endothelial transmembrane glycoprotein whose EGF-like domains 4–6 bind thrombin at anion-binding exosite-I (crystal structure resolved at 2.3 Å), sterically blocking procoagulant substrate access while optimally presenting protein C for activation; the associated chondroitin sulfate moiety enhances thrombin affinity ~10–20-fold; the thrombin–TM complex also activates TAFI (coupling coagulation to fibrinolysis inhibition, 1250-fold kcat enhancement) and accelerates complement C3b inactivation; TM's lectin-like domain mediates Ca²⁺-dependent cell adhesion and sequesters HMGB1; gene expression is regulated by CLOCK/BMAL2 (circadian), NF-κB/p300 competition (cytokine repression), KLF2 (statin induction), and BRG1/DNMT1-mediated CpG methylation; endothelial TM additionally promotes post-ischemic angiogenesis independently of its anticoagulant function; loss-of-function mutations in THBD cause ~5% of atypical HUS cases and are associated with venous thromboembolism."},"narrative":{"teleology":[{"year":1985,"claim":"The discovery of soluble thrombomodulin in plasma and urine, retaining protein C cofactor activity but with reduced thrombin affinity, established that thrombomodulin functions as a circulating anticoagulant cofactor and that membrane anchorage modulates thrombin interaction.","evidence":"Radioimmunoassay and immunoaffinity purification from human plasma/urine with kinetic protein C activation assays","pmids":["3001144"],"confidence":"High","gaps":["Structural basis for reduced thrombin affinity of soluble TM was unknown","Physiological relevance of soluble vs. membrane-bound TM not quantified in vivo"]},{"year":1987,"claim":"Cloning the full-length cDNA revealed thrombomodulin's modular architecture—signal peptide, N-terminal (lectin-like) domain, six EGF-like repeats, Ser/Thr-rich region, transmembrane segment, and cytoplasmic tail—encoded by an intronless gene on chromosome 20, providing the structural framework for all subsequent domain-function mapping.","evidence":"Independent cDNA/genomic cloning and sequencing with COS cell expression and functional validation","pmids":["2820710","2822087","2819876"],"confidence":"High","gaps":["Individual domain contributions to thrombin binding and protein C activation not yet dissected","Post-translational modifications (chondroitin sulfate, glycosylation) not structurally resolved"]},{"year":1989,"claim":"Biochemical reconstitution demonstrated that thrombomodulin converts thrombin from a procoagulant enzyme into an anticoagulant one by abolishing fibrinogen clotting and platelet activation while enabling rapid protein C activation, defining the core switch mechanism.","evidence":"In vitro reconstitution with purified components and enzyme kinetics","pmids":["2538457"],"confidence":"High","gaps":["Atomic-level binding interface unresolved","Relative contributions of steric blockade vs. allosteric change debated"]},{"year":1995,"claim":"Detailed kinetic and competition studies resolved the dual mechanism: chondroitin sulfate enhances thrombin affinity 10–20-fold by engaging a heparin-binding surface, while EGF-like domains sterically occlude procoagulant substrates; together these explain how thrombomodulin simultaneously inhibits procoagulant function and promotes protein C activation.","evidence":"Mutagenesis, competition assays, and structural analysis of thrombin–TM interactions","pmids":["7615164"],"confidence":"High","gaps":["Three-dimensional structure of the complex not yet determined","Role of individual EGF domains not fully assigned"]},{"year":1995,"claim":"Identification of the first THBD coding mutation (Asp468Tyr) in a patient with pulmonary embolism linked THBD genetic variation to venous thromboembolism, opening the question of whether loss-of-function alleles are common thrombophilia risk factors.","evidence":"PCR-SSCP and DNA sequencing in a thromboembolism patient","pmids":["7811989"],"confidence":"Medium","gaps":["Single case report without family cosegregation data","Functional impact of Asp468Tyr on protein C activation not directly measured in this study"]},{"year":1996,"claim":"The discovery that thrombomodulin enhances TAFI activation by thrombin 1250-fold expanded thrombomodulin's role beyond anticoagulation into fibrinolysis regulation, establishing the thrombin–TM–TAFI ternary complex model.","evidence":"In vitro reconstitution with purified thrombin, TM, and TAFI; full steady-state kinetics","pmids":["8663147"],"confidence":"High","gaps":["In vivo quantitative contribution of TM-dependent TAFI activation to fibrinolysis control unclear","Whether TAFI activation shares or competes with protein C activation for TM binding not resolved"]},{"year":2000,"claim":"The 2.3 Å crystal structure of thrombin bound to TM EGF-like domains 4–6 resolved the longstanding question of mechanism: TM binds exosite-I in a Y-shaped configuration, sterically blocking procoagulant substrates without major active-site allosteric changes, and positioning the substrate-presentation surface for protein C docking.","evidence":"X-ray crystallography of thrombin–TME456 complex with molecular modeling","pmids":["10761923"],"confidence":"High","gaps":["No ternary structure with protein C or TAFI","Role of chondroitin sulfate not captured in the crystal"]},{"year":2003,"claim":"Functional dissection of the lectin-like domain revealed a non-hemostatic role: Ca²⁺-dependent cell adhesion mediated by this domain suppresses monolayer permeability and tumor growth, establishing TM as a multifunctional protein beyond coagulation.","evidence":"Domain-deletion mutants in TM-negative melanoma cells, confocal imaging, permeability assays, in vivo tumor model","pmids":["12951323"],"confidence":"High","gaps":["Endogenous ligand for the lectin-like domain in cell adhesion not identified","Structural basis for Ca²⁺-dependent interaction unknown"]},{"year":2005,"claim":"Two key transcriptional regulatory mechanisms were defined: TNF-α represses THBD via NF-κB competing for the coactivator p300 away from Ets factors at the TM promoter, while statins induce THBD through KLF2 upregulation via Rho pathway inhibition, explaining how inflammation and pharmacology modulate endothelial anticoagulant phenotype.","evidence":"ChIP, EMSA, promoter analysis, NF-κB inhibition, p300 overexpression, mouse lung injury model; KLF2 siRNA epistasis with statin treatment","pmids":["15677570","16043642"],"confidence":"High","gaps":["Relative contributions of Ets family members to basal TM transcription not resolved","Whether KLF2 binds the TM promoter directly or acts through intermediaries unclear"]},{"year":2007,"claim":"CLOCK/BMAL2 heterodimer binding to an E-box in the THBD promoter was shown to drive circadian oscillation of TM mRNA and protein in mouse vasculature, abolished in Clock mutant mice, establishing THBD as a clock-controlled gene and linking circadian biology to hemostatic capacity.","evidence":"ChIP, EMSA, luciferase reporter, Clock mutant mice, temporal feeding restriction","pmids":["17848551"],"confidence":"High","gaps":["Whether circadian TM variation translates to measurable oscillation in protein C activation rate in vivo not tested","Contribution of BMAL1 vs. BMAL2 to TM regulation not clarified"]},{"year":2009,"claim":"THBD mutations were shown to cause ~5% of atypical HUS by impairing complement regulation: TM binds C3b and factor H, accelerates factor I–mediated C3b inactivation, and promotes TAFI-dependent anaphylatoxin degradation—aHUS-associated mutations reduce both activities, revealing TM as a complement regulator.","evidence":"Patient cohort sequencing, purified protein binding assays, cell-based complement activation assays with mutant TM constructs","pmids":["19625716"],"confidence":"High","gaps":["Structural basis for TM–C3b interaction unknown","Whether complement dysregulation alone or combined coagulation defect drives aHUS pathology in THBD mutation carriers unclear"]},{"year":2017,"claim":"Conditional Thbd ablation in adult mice demonstrated that complete TM deficiency causes consumptive coagulopathy and sudden death, with APC supplementation rescuing thrombosis but not pregnancy-associated morbidity, revealing essential non-endothelial (placental) TM functions; separately, transmembrane domain truncation mutants showed that domain length governs metalloprotease-dependent shedding, explaining how naturally occurring mutations increase soluble TM.","evidence":"Inducible Cre/loxP Thbd knockout with transgenic PC rescue; site-directed mutagenesis/shedding assays in COS-1 cells","pmids":["28920104","29145514"],"confidence":"High","gaps":["Identity of the metalloprotease(s) responsible for TM shedding not determined","Mechanism of TM function in trophoblast biology uncharacterized"]},{"year":2020,"claim":"Brain endothelial–specific Thbd deletion worsened stroke outcome not through coagulopathy or inflammation but by impairing peri-infarct angiogenesis, establishing a coagulation-independent pro-angiogenic function for endothelial thrombomodulin.","evidence":"Inducible Cre/loxP brain endothelial Thbd knockout, MCA occlusion model, vessel morphometry","pmids":["33028093"],"confidence":"High","gaps":["Molecular pathway through which TM promotes angiogenesis (receptor, signaling cascade) not identified","Whether the lectin-like domain or EGF-like domains mediate the angiogenic effect unknown"]},{"year":2022,"claim":"An epigenetic repression axis was defined: BRG1 upregulates DNMT1 under TNF-α stimulation, increasing CpG methylation of the THBD promoter and silencing expression; endothelial-specific BRG1 knockout restored THBD and reduced DVT in mice, providing a druggable epigenetic mechanism for thrombosis.","evidence":"Conditional endothelial BRG1 knockout mice, DVT ligation model, CpG methylation analysis, pharmacological BRG1 inhibitor PFI-3","pmids":["36162255"],"confidence":"High","gaps":["Whether DNMT1-mediated THBD methylation is reversible with DNMT inhibitors in established DVT not tested","Relative contribution of THBD silencing vs. other BRG1 targets to DVT phenotype not isolated"]},{"year":null,"claim":"Major open questions include the structural basis for TM's complement-regulatory (C3b binding) and angiogenic functions, the identity of the sheddase(s), the signaling mechanism linking TM to angiogenesis independently of APC, and whether circadian TM oscillation measurably affects in vivo hemostatic thresholds.","evidence":"","pmids":[],"confidence":"High","gaps":["No ternary crystal structure of thrombin–TM–protein C or TM–C3b","Sheddase identity unknown","Pro-angiogenic signaling pathway downstream of TM uncharacterized","In vivo functional consequence of circadian TM oscillation untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4,5,7,8,10]},{"term_id":"GO:0098631","term_label":"cell adhesion mediator activity","supporting_discovery_ids":[9]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[4,5,8]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[16]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,4,8,9,20]},{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[3,20]}],"pathway":[{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[4,5,7,8,10,15,19]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[15,16]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[14]},{"term_id":"R-HSA-9909396","term_label":"Circadian clock","supporting_discovery_ids":[13]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[6,15]}],"complexes":["Thrombin–thrombomodulin complex","Thrombin–thrombomodulin–protein C ternary complex","Thrombin–thrombomodulin–TAFI ternary complex"],"partners":["F2","PROC","CPB2","C3","CFH","HMGB1","PROCR"],"other_free_text":[]},"mechanistic_narrative":"Thrombomodulin is an endothelial transmembrane glycoprotein that redirects thrombin from procoagulant to anticoagulant and anti-inflammatory functions by binding thrombin's anion-binding exosite-I through its EGF-like domains 4–6, thereby blocking fibrinogen and platelet-receptor access while optimally presenting protein C for activation; its chondroitin sulfate moiety enhances thrombin affinity 10–20-fold [PMID:2820710, PMID:10761923, PMID:7615164]. The thrombin–thrombomodulin complex also activates TAFI with a 1250-fold increase in catalytic efficiency, coupling coagulation to fibrinolysis inhibition, and accelerates factor I–mediated C3b inactivation to negatively regulate complement—loss-of-function THBD mutations that impair these activities cause approximately 5% of atypical hemolytic-uremic syndrome cases [PMID:8663147, PMID:19625716]. Beyond hemostasis, thrombomodulin's N-terminal lectin-like domain mediates Ca²⁺-dependent cell adhesion and sequesters HMGB1, and endothelial thrombomodulin promotes post-ischemic angiogenesis independently of its anticoagulant role [PMID:12951323, PMID:21805323, PMID:33028093]. THBD transcription is regulated by CLOCK/BMAL2 via an E-box element (conferring circadian oscillation), repressed by TNF-α through NF-κB/p300 coactivator competition and BRG1/DNMT1-mediated CpG methylation, and induced by statins via KLF2 [PMID:17848551, PMID:15677570, PMID:36162255, PMID:16043642]."},"prefetch_data":{"uniprot":{"accession":"P07204","full_name":"Thrombomodulin","aliases":["Fetomodulin"],"length_aa":575,"mass_kda":60.3,"function":"Endothelial cell receptor that plays a critical role in regulating several physiological processes including hemostasis, coagulation, fibrinolysis, inflammation, and angiogenesis (PubMed:10761923). Acts as a cofactor for thrombin activation of protein C/PROC on the surface of vascular endothelial cells leading to initiation of the activated protein C anticoagulant pathway (PubMed:29323190, PubMed:33836597, PubMed:9395524). Also accelerates the activation of the plasma carboxypeptidase B2/CPB2, which catalyzes removal of C-terminal basic amino acids from its substrates including kinins or anaphylatoxins leading to fibrinolysis inhibition (PubMed:26663133). Plays critical protective roles in changing the cleavage specificity of protease-activated receptor 1/PAR1, inhibiting endothelial cell permeability and inflammation (By similarity). 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American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/7615164","citation_count":328,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"17982464","id":"PMC_17982464","title":"Activated protein C protects against diabetic nephropathy by inhibiting endothelial and podocyte apoptosis.","date":"2007","source":"Nature medicine","url":"https://pubmed.ncbi.nlm.nih.gov/17982464","citation_count":326,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"3001144","id":"PMC_3001144","title":"Thrombomodulin is present in human plasma and urine.","date":"1985","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/3001144","citation_count":311,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15178554","id":"PMC_15178554","title":"Thrombomodulin-protein C-EPCR system: integrated to regulate coagulation and inflammation.","date":"2004","source":"Arteriosclerosis, thrombosis, and vascular biology","url":"https://pubmed.ncbi.nlm.nih.gov/15178554","citation_count":298,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"16043642","id":"PMC_16043642","title":"Kruppel-like factor 2 as a novel mediator of statin effects in endothelial cells.","date":"2005","source":"Circulation","url":"https://pubmed.ncbi.nlm.nih.gov/16043642","citation_count":290,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"10761923","id":"PMC_10761923","title":"Structural basis for the anticoagulant activity of the thrombin-thrombomodulin complex.","date":"2000","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/10761923","citation_count":270,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"20513133","id":"PMC_20513133","title":"Mutations in alternative pathway complement proteins in American patients with atypical hemolytic uremic syndrome.","date":"2010","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/20513133","citation_count":258,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21805323","id":"PMC_21805323","title":"Thrombomodulin and its role in inflammation.","date":"2011","source":"Seminars in immunopathology","url":"https://pubmed.ncbi.nlm.nih.gov/21805323","citation_count":253,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21988832","id":"PMC_21988832","title":"Toward an understanding of the protein interaction network of the human liver.","date":"2011","source":"Molecular systems biology","url":"https://pubmed.ncbi.nlm.nih.gov/21988832","citation_count":207,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"2822087","id":"PMC_2822087","title":"Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene.","date":"1987","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/2822087","citation_count":203,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"23741007","id":"PMC_23741007","title":"Loss of endothelial protein C receptors links coagulation and inflammation to parasite sequestration in cerebral malaria in African children.","date":"2013","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/23741007","citation_count":176,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"11780052","id":"PMC_11780052","title":"The DNA sequence and comparative analysis of human chromosome 20.","date":"2001","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/11780052","citation_count":168,"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":"2819876","id":"PMC_2819876","title":"Human thrombomodulin gene is intron depleted: nucleic acid sequences of the cDNA and gene predict protein structure and suggest sites of regulatory control.","date":"1987","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/2819876","citation_count":142,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12951323","id":"PMC_12951323","title":"Thrombomodulin-mediated cell adhesion: involvement of its lectin-like domain.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12951323","citation_count":132,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"30639242","id":"PMC_30639242","title":"The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2.","date":"2019","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/30639242","citation_count":124,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"20379614","id":"PMC_20379614","title":"Personalized smoking cessation: interactions between nicotine dose, dependence and quit-success genotype score.","date":"2010","source":"Molecular medicine (Cambridge, Mass.)","url":"https://pubmed.ncbi.nlm.nih.gov/20379614","citation_count":108,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"17848551","id":"PMC_17848551","title":"Thrombomodulin is a clock-controlled gene in vascular endothelial cells.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17848551","citation_count":106,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"7811989","id":"PMC_7811989","title":"The first mutation identified in the thrombomodulin gene in a 45-year-old man presenting with thromboembolic disease.","date":"1995","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/7811989","citation_count":98,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15677570","id":"PMC_15677570","title":"Regulation of endothelial thrombomodulin expression by inflammatory cytokines is mediated by activation of nuclear factor-kappa B.","date":"2005","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/15677570","citation_count":96,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":41664,"output_tokens":2753,"usd":0.083143},"stage2":{"model":"claude-opus-4-6","input_tokens":5602,"output_tokens":2312,"usd":0.128715},"total_usd":0.582106,"stage1_batch_id":"msgbatch_01RPbVc9PDH9zQmYK5Hs6srm","stage2_batch_id":"msgbatch_01JVbVLzeKN2yVrsGp3zC8aM","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":54797,"output_tokens":7630,"usd":0.139421},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":10947,"output_tokens":3966,"usd":0.230827}},"stage1_raw":"Looking at the corpus, I need to identify papers about the human gene THBD (thrombomodulin), which encodes a transmembrane glycoprotein expressed on endothelial cells that acts as a cofactor for thrombin in activating protein C and thrombin-activatable fibrinolysis inhibitor (TAFI). \n\nThe majority of papers in this corpus are about CD141 as a dendritic cell marker (BDCA-3), not about THBD's mechanistic function as thrombomodulin. I need to extract only mechanistic discoveries about THBD protein function.\n\nKey mechanistic papers about THBD:\n- Paper 17 (PMID:24150517): hTM anticoagulant function on porcine endothelial cells\n- Paper 34 (PMID:33028093): Endogenous THBD in angiogenesis/ischemic brain\n- Paper 39 (PMID:28920104): Thbd-/- mice, protein C pathway, placental function\n- Paper 45 (PMID:29145514): TM shedding mechanism via metalloproteases, transmembrane domain truncation\n- Paper 52 (PMID:36162255): BRG1 epigenetic repression of THBD via DNMT1/CpG methylation\n- Paper 59 (PMID:34236150): THBD p.Ala473Val variant effects on protein C activation, EPC migration/angiogenesis\n- Paper 64 (PMID:30401399): TM administration prolongs cardiac allograft survival\n- Paper 73 (preprint): ADM/RAMP2 signaling increases THBD expression with shear stress\n- Paper 74 (preprint): MEK1/2-ERK1/2 pathway regulates Thbd expression in lupus DAH\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2017,\n      \"finding\": \"A THBD nonsense mutation (c.1611C>A, p.Cys537X) truncates the transmembrane domain, causing elevated soluble thrombomodulin shedding that is ~75% dependent on metalloprotease activity; serial truncation mutants showed that reducing the transmembrane domain length progressively increased shedding, establishing that transmembrane domain integrity controls metalloprotease-mediated ectodomain release.\",\n      \"method\": \"Site-directed mutagenesis of TM transmembrane domain truncations transfected into COS-1 cells; metalloprotease inhibitor treatment; Western blot and immunofluorescence; conditioned medium ELISA for soluble TM\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution with mutagenesis series and pharmacological inhibition in the same study, multiple orthogonal methods\",\n      \"pmids\": [\"29145514\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Complete endothelial deletion of Thbd in adult mice causes consumptive coagulopathy and sudden mortality; supplementing activated protein C by transgenic expression of a partially Thbd-independent protein C zymogen rescued the thrombotic phenotype, establishing that Thbd's primary anticoagulant function in adults operates through the thrombin-mediated protein C activation pathway.\",\n      \"method\": \"Conditional Cre/loxP Thbd knockout mice (Meox2Cre for embryonic rescue; inducible adult deletion); transgenic protein C supplementation; coagulation assays; histopathology\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic epistasis with rescue experiment in vivo, multiple mouse lines\",\n      \"pmids\": [\"28920104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Human thrombomodulin (hTM) expressed on porcine endothelial cells (driven by the porcine THBD endothelial regulatory region) increases human whole-blood clotting time 3–4 fold, demonstrating that hTM exerts anticoagulant activity at the endothelial surface by facilitating thrombin-mediated protein C activation in a xenogeneic context.\",\n      \"method\": \"Transgenic pig generation by somatic cell nuclear transfer; human whole-blood coagulation assay with transgenic porcine endothelial cells; histological and molecular characterization of transgene expression\",\n      \"journal\": \"Transplantation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — functional coagulation assay in primary endothelial cells from genetically defined animals\",\n      \"pmids\": [\"24150517\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Endogenous THBD in brain endothelial cells promotes angiogenesis in the peri-infarct area after ischemic stroke; inducible brain-endothelial-specific Thbd deletion worsened stroke outcome with reduced vessel diameter and impaired vessel proliferation without signs of increased coagulation or inflammation, revealing a coagulation-independent pro-angiogenic role for endothelial THBD.\",\n      \"method\": \"Inducible endothelial-specific Cre/loxP Thbd knockout mice (Cdh5-ERT-Cre); middle cerebral artery occlusion model; histological and vascular morphometry; coagulation and inflammatory marker analysis\",\n      \"journal\": \"Arteriosclerosis, thrombosis, and vascular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific conditional KO with defined vascular phenotype and exclusion of coagulation mechanism\",\n      \"pmids\": [\"33028093\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"BRG1 (a chromatin remodeling ATPase) epigenetically represses THBD transcription in endothelial cells by upregulating DNMT1 after TNF-α stimulation, increasing CpG methylation of the THBD promoter; endothelial-specific BRG1 knockout reduced DVT susceptibility and restored THBD expression, and pharmacological BRG1 inhibition (PFI-3) had preventive and therapeutic effects in a mouse DVT model.\",\n      \"method\": \"Endothelial-specific conditional BRG1 knockout mice (Cdh5-ERT-Cre × Brg1loxp/loxp); inferior vena cava ligation DVT model; siRNA knockdown and pharmacological inhibition in endothelial cells; bisulfite sequencing for CpG methylation; ChIP; Western blot\",\n      \"journal\": \"Thrombosis research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistatic genetic and pharmacological experiments in vivo and in vitro with epigenetic mechanism defined\",\n      \"pmids\": [\"36162255\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The THBD p.Ala473Val variant (from c.1418C>T polymorphism) expressed in endothelial progenitor cells (EPCs) significantly decreased protein C activation, impaired EPC migration (wound healing and Transwell assays), reduced angiogenic tube formation, and downregulated VEGFRs, MMP1/2/3, VE-cadherin, eNOS, and TIE-2, establishing that this missense variant disrupts both anticoagulant and angiogenic functions of THBD.\",\n      \"method\": \"Transfection of WT and mutant THBD cDNA into bone-marrow-derived EPCs; protein C activation assay; wound healing assay; Transwell migration; tube formation assay; Western blot\",\n      \"journal\": \"International angiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal functional assays in primary cells, single lab\",\n      \"pmids\": [\"34236150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"miR-18a-5p directly targets the 3'UTR of THBD mRNA and negatively regulates its expression, promoting proliferation, migration, and invasion of endometrial cancer cells; THBD overexpression rescued the oncogenic effects of miR-18a-5p, identifying THBD as a tumor-suppressive target of miR-18a-5p in endometrial cancer.\",\n      \"method\": \"Dual-luciferase reporter assay confirming miR-18a-5p binding to THBD 3'UTR; qRT-PCR and Western blot for expression; CCK-8 proliferation assay; Transwell migration/invasion assay; rescue experiment with THBD overexpression\",\n      \"journal\": \"Critical reviews in eukaryotic gene expression\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct 3'UTR binding validated by luciferase, functional rescue, single lab\",\n      \"pmids\": [\"34347980\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Administration of soluble thrombomodulin (TM) protein dose-dependently prolonged fully MHC-mismatched cardiac allograft survival in mice and suppressed perivascular inflammatory cell infiltration, demonstrating an immunomodulatory/anti-inflammatory role for soluble THBD in vivo independent of its direct endothelial localization.\",\n      \"method\": \"Murine heterotopic cardiac transplantation model (CBA→C57BL/6); intraperitoneal TM administration at 0.2, 2.0, 20.0 μg/day; allograft survival monitoring; histological assessment of inflammation and infiltration\",\n      \"journal\": \"Transplantation proceedings\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo dose-response experiment with defined cellular phenotype, single lab\",\n      \"pmids\": [\"30401399\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"THBD (thrombomodulin) is an endothelial transmembrane glycoprotein whose primary anticoagulant function is to act as a cofactor for thrombin-mediated activation of protein C (established by conditional knockout rescue experiments); its ectodomain is shed by metalloproteases in a manner controlled by transmembrane domain integrity; BRG1 epigenetically represses THBD transcription via DNMT1-mediated promoter CpG methylation downstream of TNF-α; endogenous THBD additionally promotes post-ischemic angiogenesis in brain endothelium through a coagulation-independent mechanism; and miR-18a-5p directly targets the THBD 3'UTR to suppress its tumor-suppressive functions in endometrial cancer cells.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll systematically classify each paper before extracting discoveries.\n\n**Classification:**\n\nPapers [1]-[11], [13]-[15], [18]-[24], [26]-[30], [32]-[33], [35]-[41], [43], [47], [49], [51]-[53], [55], [57]-[58], [62], [65], [67]-[68], [70]-[71] from the retrieved corpus: These are about CD141 as a dendritic cell marker (BDCA-3), NOT about thrombomodulin protein function. CD141 is indeed the same protein (thrombomodulin/THBD), but these papers use it purely as a DC surface marker without describing THBD's mechanistic function. I need to carefully distinguish: papers that describe thrombomodulin's molecular mechanism vs. papers using CD141 as a DC marker.\n\nLet me re-examine carefully for THBD mechanistic findings:\n\n- Papers about DC biology using CD141 as a surface marker (no THBD mechanism): EXCLUDE for mechanistic extraction purposes (they don't describe HOW THBD works mechanistically).\n- Papers describing THBD protein function, structure, regulation: KEEP.\n\n**KEEP papers with mechanistic findings:**\n\nRetrieved corpus: [16] (TM expression on DCs, TH2), [17] (transgenic pigs with hTM, anticoagulant function), [25] (SNPs/GVHD - no mechanism), [31] (variants/VTE - no mechanism), [34] (angiogenesis role), [39] (Thbd gene ablation, protein C pathway), [42] (miR-18a-5p/THBD), [44] (epistasis - no mechanism), [45] (TM shedding mechanism), [50] (THBD/signaling in HF), [52] (BRG1/DNMT1 epigenetic regulation), [59] (c.1418C>T, EPC migration), [64] (TM administration, cardiac allograft), [72] (ThBD = thalidomide-binding domain - EXCLUDE: alias collision), [73] (THBD expression under shear stress/ADM), [74] (Thbd in lupus DAH model), [75] (THBD in liver organoids - marker use)\n\nGene2pubmed papers: [2], [6], [8], [10], [11], [12], [13], [14], [15], [16], [18], [20], [21], [24], [25], [28], [29], [30] — all about canonical THBD protein.\n\nPaper [72] from retrieved: \"ThBD\" = thalidomide-binding domain of CRBN — EXCLUDE (alias collision).\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1987,\n      \"finding\": \"Human thrombomodulin cDNA was cloned and the complete 575-amino acid sequence deduced, revealing a domain structure comprising a signal peptide, N-terminal domain, six EGF-like domains, an O-glycosylation-rich region, a 24-residue transmembrane domain, and a cytoplasmic tail. Transfection of COS cells with the cDNA produced immunoreactive, functionally active thrombomodulin.\",\n      \"method\": \"cDNA cloning, expression in COS cells, functional assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — original structural characterization by cDNA sequencing and heterologous expression with functional validation\",\n      \"pmids\": [\"2820710\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1987,\n      \"finding\": \"Complete cDNA sequence of human thrombomodulin confirmed a 575-amino acid protein with six EGF-type repeats, no introns in the coding region, and putative regulatory elements in the promoter. The gene was localized to chromosome 20.\",\n      \"method\": \"cDNA and genomic cloning, Northern blot, chromosomal mapping\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — independent full-length sequence determination with genomic characterization\",\n      \"pmids\": [\"2822087\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1987,\n      \"finding\": \"The thrombomodulin gene contains no introns within its coding region, contains six EGF-type B repeats, has homology to the LDL receptor, and the promoter region contains potential regulatory elements. The intronless coding region was established by comparison of cDNA and genomic sequences.\",\n      \"method\": \"Genomic and cDNA cloning, nucleotide sequencing\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct genomic sequence comparison establishing gene structure\",\n      \"pmids\": [\"2819876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1985,\n      \"finding\": \"Thrombomodulin is present as a soluble form in human plasma and urine. Soluble thrombomodulin retains intrinsic protein C-activating cofactor activity with the same apparent Km for protein C as the cellular form, but requires 3–5-fold higher thrombin concentration for half-maximal activity, suggesting loss of the membrane-anchoring domain alters thrombin affinity.\",\n      \"method\": \"Radioimmunoassay, immunoaffinity chromatography, immunoblot, protein C activation assay\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — purified protein with in vitro functional kinetic characterization\",\n      \"pmids\": [\"3001144\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1989,\n      \"finding\": \"Thrombomodulin acts as a thrombin receptor on endothelium that functions as a cofactor for protein C activation, converting thrombin from a procoagulant to an anticoagulant enzyme. The thrombin–thrombomodulin complex loses ability to clot fibrinogen and activate platelets while gaining ability to rapidly activate protein C zymogen.\",\n      \"method\": \"Biochemical reconstitution, enzyme kinetics, functional assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro biochemical mechanism, foundational study replicated extensively\",\n      \"pmids\": [\"2538457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Thrombomodulin's chondroitin sulfate moiety binds to a basic surface on thrombin also involved in heparin interaction, enhancing thrombin affinity for thrombomodulin 10–20-fold, altering thrombin conformation and specificity, and accelerating thrombin inhibition by antithrombin. The EGF-like domains of thrombomodulin block thrombin's procoagulant substrate binding sites (fibrinogen, platelet receptor), while conformational changes in the thrombin active site facilitate protein C docking.\",\n      \"method\": \"Structural analysis, kinetic studies, competition assays, mutagenesis\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple orthogonal biochemical methods establishing mechanism\",\n      \"pmids\": [\"7615164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"The first thrombomodulin gene mutation associated with thromboembolism was identified: a heterozygous G1456T substitution predicting Asp468Tyr, located between the transmembrane domain and the sixth EGF-like domain, in a patient with pulmonary embolism.\",\n      \"method\": \"PCR-SSCP, DNA sequencing\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic identification of disease-causing mutation with structural implication, single case\",\n      \"pmids\": [\"7811989\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"The thrombin–thrombomodulin complex, rather than free thrombin, is the physiologic activator of TAFI (thrombin-activatable fibrinolysis inhibitor/procarboxypeptidase B). Thrombomodulin increases the catalytic efficiency of TAFI activation 1250-fold, primarily by increasing kcat. The reaction follows a ternary complex model: thrombin binds thrombomodulin (Kd = 8.6 nM) or TAFI (Km = 1.0 µM), and the binary complex recruits the third component to form an active ternary complex. Activated TAFI inhibits fibrinolysis half-maximally at ~1 nM.\",\n      \"method\": \"In vitro reconstitution, enzyme kinetics, purified components\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — rigorous in vitro reconstitution with full kinetic characterization\",\n      \"pmids\": [\"8663147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Crystal structure of human α-thrombin bound to TME456 (EGF-like domains 4–5–6 of thrombomodulin, the smallest fragment sufficient for full protein C cofactor activity) at 2.3 Å resolution. Thrombomodulin binds thrombin's anion-binding exosite-I in a Y-shaped configuration, sterically blocking procoagulant substrate binding without inducing marked allosteric rearrangements at the thrombin active site. Modeling indicates TME45 may orient protein C's cleavage site optimally toward the unaltered thrombin active site.\",\n      \"method\": \"X-ray crystallography (2.3 Å), molecular docking\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution crystal structure with functional validation\",\n      \"pmids\": [\"10761923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Thrombomodulin mediates Ca²⁺-dependent cell-to-cell adhesion via its lectin-like domain. In TM-negative melanoma cells transfected with GFP-tagged TM, cells formed closely clustered colonies with TM localized at intercellular boundaries, reduced monolayer permeability in vitro, and decreased tumor growth in vivo. Deletion of the lectin-like domain abolished these effects. Adhesion was inhibited by anti-lectin-domain antibody and by mannose, chondroitin sulfate A, or chondroitin sulfate C.\",\n      \"method\": \"Transfection of TM-negative cells, domain-deletion mutants, confocal microscopy, permeability assay, in vivo tumor model, antibody inhibition\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — domain-deletion mutagenesis with multiple orthogonal functional readouts\",\n      \"pmids\": [\"12951323\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Thrombomodulin–protein C–EPCR system is integrated to regulate both coagulation and inflammation. Thrombomodulin serves as a cofactor for thrombin-mediated protein C activation (further amplified by EPCR), and activated protein C exerts anticoagulant, anti-inflammatory, profibrinolytic, and cytoprotective effects. The covalently associated chondroitin sulfate moiety of TM additionally enhances thrombin binding and modulates TAFI activation and complement regulation.\",\n      \"method\": \"Review of biochemical, cell biology, and in vivo experimental data\",\n      \"journal\": \"Arteriosclerosis, thrombosis, and vascular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — synthesis of multiply replicated mechanistic studies across labs\",\n      \"pmids\": [\"15178554\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Inflammatory cytokines (TNF-α) down-regulate endothelial thrombomodulin expression through NF-κB activation. NF-κB does not bind the TM promoter directly but competes for limited pools of the coactivator p300, which is required by Ets transcription factors for TM promoter transactivation. Blocking NF-κB activation prevented cytokine-induced TM down-regulation in vitro and in a mouse TNF-α lung injury model.\",\n      \"method\": \"EMSA, chromatin immunoprecipitation, promoter deletion/mutation analysis, NF-κB inhibition, p300 overexpression, mouse model\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal molecular methods plus in vivo validation\",\n      \"pmids\": [\"15677570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"KLF2 (Krüppel-like factor 2) is required for statin-induced upregulation of thrombomodulin in endothelial cells. Statin treatment induces KLF2 mRNA via Rho pathway inhibition and de novo transcription through a myocyte enhancer factor binding site in the KLF2 promoter. siRNA-mediated KLF2 knockdown strongly attenuated mevastatin-induced thrombomodulin protein accumulation.\",\n      \"method\": \"siRNA knockdown, promoter deletion/mutation analysis, pharmacological inhibition, Western blot\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — siRNA epistasis plus promoter analysis, replicated with multiple statins\",\n      \"pmids\": [\"16043642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Thrombomodulin expression in vascular endothelial cells is clock-controlled: TM mRNA and protein show circadian oscillation in mouse lung and heart. The CLOCK/BMAL2 heterodimer directly binds an E-box element in the TM promoter to drive transcription, as shown by reporter assays, EMSA, and ChIP. Oscillation was abolished in Clock mutant mice and phase-shifted by temporal feeding restriction.\",\n      \"method\": \"Microarray, reporter assay, EMSA, chromatin immunoprecipitation, Clock mutant mice, temporal feeding restriction\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct binding of CLOCK/BMAL2 to TM promoter confirmed by ChIP and EMSA, validated in vivo in mutant mice\",\n      \"pmids\": [\"17848551\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Endothelial thrombomodulin-dependent activated protein C (APC) formation is reduced in diabetic mice and causally linked to diabetic nephropathy. APC prevents glucose-induced apoptosis in endothelial cells and podocytes via protease-activated receptor PAR-1 and endothelial protein C receptor EPCR, modulating the mitochondrial apoptosis pathway. Loss of thrombomodulin-dependent APC disrupts endothelial–podocyte crosstalk, causing glomerular apoptosis.\",\n      \"method\": \"Diabetic mouse model, endothelial cell and podocyte in vitro assays, PAR-1/EPCR pathway analysis, apoptosis assays\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic model combined with in vitro mechanistic pathway dissection\",\n      \"pmids\": [\"17982464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"THBD mutations cause ~5% of atypical hemolytic-uremic syndrome (aHUS) cases. Mechanistically, thrombomodulin binds C3b and factor H and negatively regulates complement by accelerating factor I-mediated C3b inactivation (requiring cofactors CFH or C4b-binding protein). TM also accelerates inactivation of anaphylatoxins C3a and C5a via procarboxypeptidase B activation. aHUS-associated missense mutations in THBD reduce C3b inactivation and procarboxypeptidase B activation in cell-expression systems.\",\n      \"method\": \"THBD gene sequencing, purified protein binding assays, cell-expression systems, complement activation assays\",\n      \"journal\": \"The New England journal of medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct binding assays with purified proteins plus functional cell-expression mutagenesis, large patient cohort\",\n      \"pmids\": [\"19625716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Thrombomodulin exhibits anti-inflammatory properties through multiple mechanisms: (1) cofactor for thrombin-mediated protein C activation; (2) TAFI activation to inhibit fibrinolysis; (3) complement regulation via C3b inactivation; (4) binding and sequestration of the pro-inflammatory cytokine HMGB1 via its lectin-like domain; (5) interaction with Lewis Y antigen. TM expression is tightly regulated by cytokines (down-regulated by TNF-α, IL-1), shear stress, and thrombin.\",\n      \"method\": \"Review integrating published biochemical and in vivo experimental data\",\n      \"journal\": \"Seminars in immunopathology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — synthesis of replicated mechanistic studies, multiple independent labs\",\n      \"pmids\": [\"21805323\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Loss of endothelial thrombomodulin (TM) and EPCR at sites of malaria-infected erythrocyte cytoadherence in cerebral malaria is associated with cerebral fibrin clot formation. Using ex vivo subcutaneous microvessels, loss of TM and EPCR was detectable at sites of infected erythrocyte binding in non-fatal cerebral malaria, linking parasite sequestration to disruption of endothelial anticoagulant/anti-inflammatory protection.\",\n      \"method\": \"Autopsy immunostaining, novel ex vivo microvascular assay, coagulation activation assays\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization with functional correlation, but primarily observational/ex vivo\",\n      \"pmids\": [\"23741007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Regulatory sequences from the porcine THBD gene (7.6-kb upstream fragment) drive endothelial-specific expression of human thrombomodulin (hTM) in transgenic pigs. Endothelial cells from hTM transgenic pigs showed 3–4-fold increased clotting time in human whole blood assays compared to wild-type, demonstrating that hTM retains anticoagulant function in a xenograft context and can overcome human–pig coagulation incompatibilities.\",\n      \"method\": \"Transgenic pig generation (somatic cell nuclear transfer), endothelial cell coagulation assay with human whole blood\",\n      \"journal\": \"Transplantation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional demonstration in transgenic animal model with human blood assay\",\n      \"pmids\": [\"24150517\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Tissue-selective Thbd gene ablation in adult mice reveals that complete Thbd deficiency leads to low-grade consumptive coagulopathy and sudden-onset mortality. Supplementation with activated protein C (via a partially Thbd-independent PC transgene) prevented thrombotic pathologies but Thbd-deficient females showed near-complete pregnancy-induced morbidity. This demonstrates that Thbd function in nonendothelial embryonic tissues (placenta, yolk sac) influences the penetrance and severity of postnatal thrombosis through unknown mechanisms.\",\n      \"method\": \"Conditional Cre/loxP Thbd gene ablation, transgenic protein C supplementation, histopathology, coagulation assays\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — rigorous conditional knockout with genetic rescue experiment in vivo\",\n      \"pmids\": [\"28920104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"A THBD c.1611C>A nonsense mutation (p.Cys537X) leads to a truncated thrombomodulin that has lost the last three amino acids of the transmembrane domain and the cytoplasmic tail. This truncated protein is partially membrane-anchored but is shed at ~6× higher levels than wild-type TM. Shedding is ~75% dependent on metalloprotease activity and is not affected by cathepsin G, elastase, reducing agents, or high shear stress. Other truncation mutants with shortened transmembrane domains also show increased shedding, indicating the transmembrane domain length is critical for membrane retention.\",\n      \"method\": \"Site-directed mutagenesis, transient transfection in COS-1 cells, Western blot, immunofluorescence, metalloprotease inhibitor assay, ELISA\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis series with multiple inhibitor conditions, mechanistic dissection of shedding\",\n      \"pmids\": [\"29145514\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Endogenous THBD promotes angiogenesis in the ischemic brain. Following middle cerebral artery occlusion in mice, Thbd expression increased in the peri-infarct area. Inducible, brain endothelial cell-specific Thbd deletion worsened stroke outcome without increasing coagulation, thrombosis, or inflammation, but instead decreased vessel diameters and impaired angiogenesis in the peri-infarct area, reducing overall vessel length 1 week post-stroke.\",\n      \"method\": \"Mouse MCA occlusion model, inducible Cre/loxP brain endothelial-specific Thbd knockout, histology, vessel morphometry\",\n      \"journal\": \"Arteriosclerosis, thrombosis, and vascular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific conditional knockout with specific angiogenic phenotype, dissociated from coagulation effects\",\n      \"pmids\": [\"33028093\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"miR-18a-5p negatively regulates THBD expression in endometrial cancer cells. Dual-luciferase assay confirmed direct targeting of THBD 3'UTR by miR-18a-5p. Overexpression of miR-18a-5p enhanced proliferation, migration, and invasion of EC cells, while THBD overexpression inhibited these abilities. Rescue experiments showed THBD overexpression reversed the oncogenic effects of miR-18a-5p, placing THBD downstream of miR-18a-5p in a regulatory axis suppressing EC cell aggressiveness.\",\n      \"method\": \"Dual-luciferase reporter assay, qRT-PCR, Western blot, CCK-8 assay, Transwell assay, rescue experiments\",\n      \"journal\": \"Critical reviews in eukaryotic gene expression\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — direct miRNA-target validation by luciferase assay plus functional rescue, single lab\",\n      \"pmids\": [\"34347980\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"BRG1 (a chromatin remodeling ATPase) epigenetically represses THBD transcription in endothelial cells, contributing to deep vein thrombosis. TNF-α stimulation decreases THBD expression; BRG1 knockdown or inhibition recovers THBD expression. Mechanistically, BRG1 directly upregulates DNMT1 expression after TNF-α treatment, increasing CpG methylation of the THBD promoter. Endothelial-specific BRG1 conditional knockout mice showed substantially decreased DVT susceptibility with decreased thrombus weight/size.\",\n      \"method\": \"Conditional endothelial BRG1 knockout mice (Cdh5-ERT-Cre × Brg1-loxP), siRNA/inhibitor experiments, CpG methylation analysis, DVT ligation model, small-molecule BRG1 inhibitor (PFI-3)\",\n      \"journal\": \"Thrombosis research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO in vivo model plus mechanistic epigenetic pathway (DNMT1-mediated CpG methylation) with pharmacological validation\",\n      \"pmids\": [\"36162255\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The THBD c.1418C>T (p.Ala473Val) variant reduces protein C activation and impairs endothelial progenitor cell (EPC) migration and angiogenesis. Expression of p.Ala473Val in EPCs decreased VEGFRs, MMP1/2/3, VE-cadherin, Flk-1, eNOS, and TIE-2 expression, weakened wound healing, Transwell migration, and tube formation, mechanistically linking the variant to reduced angiogenic capacity.\",\n      \"method\": \"THBD cDNA transfection in EPCs, ELISA, wound healing, Transwell migration, tube formation, Western blot\",\n      \"journal\": \"International angiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — cell-based functional assays with variant vs. wild-type, single lab\",\n      \"pmids\": [\"34236150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Adrenomedullin (ADM) signaling through its receptor RAMP2 synergizes with shear stress to increase THBD expression in human pulmonary endothelial cells, enhancing anti-coagulant phenotype (increased THBD, decreased tissue factor F3). This effect was completely abolished by RAMP2 blockade, identifying ADM/RAMP2 as an upstream paracrine regulator of endothelial THBD expression.\",\n      \"method\": \"ECIS, XperT barrier assay, qPCR, pharmacological receptor blockade, shear stress apparatus\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — preprint, single lab, expression-level readout without direct promoter mechanism\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In pristane-induced lupus, MEK1/2-ERK1/2 pathway activation in B6 mice upregulates Thbd (thrombomodulin) gene expression and increases circulating soluble Thbd protein in the lung, contributing to endothelial anticoagulant dysregulation and diffuse alveolar hemorrhage. MEK1/2 inhibition (trametinib) normalized Thbd expression and circulating Thbd levels, and abolished DAH, linking the ERK pathway to Thbd-mediated hemostatic imbalance.\",\n      \"method\": \"MEK/ERK inhibitor treatment in mouse lupus model, qPCR, Western blot, ELISA, tail bleeding assay\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — preprint, pharmacological inhibition with expression-level THBD readout, no direct mechanistic dissection of THBD\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"THBD (thrombomodulin) is an endothelial transmembrane glycoprotein whose EGF-like domains 4–6 bind thrombin at anion-binding exosite-I (crystal structure resolved at 2.3 Å), sterically blocking procoagulant substrate access while optimally presenting protein C for activation; the associated chondroitin sulfate moiety enhances thrombin affinity ~10–20-fold; the thrombin–TM complex also activates TAFI (coupling coagulation to fibrinolysis inhibition, 1250-fold kcat enhancement) and accelerates complement C3b inactivation; TM's lectin-like domain mediates Ca²⁺-dependent cell adhesion and sequesters HMGB1; gene expression is regulated by CLOCK/BMAL2 (circadian), NF-κB/p300 competition (cytokine repression), KLF2 (statin induction), and BRG1/DNMT1-mediated CpG methylation; endothelial TM additionally promotes post-ischemic angiogenesis independently of its anticoagulant function; loss-of-function mutations in THBD cause ~5% of atypical HUS cases and are associated with venous thromboembolism.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"THBD (thrombomodulin) is an endothelial transmembrane glycoprotein that serves as the principal cofactor for thrombin-mediated activation of protein C, a function demonstrated to be essential for adult hemostasis since complete endothelial Thbd deletion causes lethal consumptive coagulopathy rescuable by activated protein C supplementation [PMID:28920104, PMID:24150517]. Beyond anticoagulation, endothelial THBD promotes post-ischemic angiogenesis in a coagulation-independent manner, as brain-endothelial-specific Thbd knockout impairs peri-infarct vessel proliferation without altering coagulation or inflammatory markers [PMID:33028093], and soluble thrombomodulin exerts immunomodulatory effects that prolong allograft survival in vivo [PMID:30401399]. Ectodomain shedding of THBD is controlled by transmembrane domain integrity, with progressive transmembrane truncation increasing metalloprotease-mediated release of soluble thrombomodulin [PMID:29145514]. THBD transcription is epigenetically repressed by BRG1-recruited DNMT1-mediated CpG methylation of its promoter downstream of TNF-α, and disruption of this axis restores THBD expression and reduces deep vein thrombosis susceptibility [PMID:36162255].\",\n  \"teleology\": [\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrating that human thrombomodulin expressed on endothelial surfaces is sufficient to activate protein C and prolong clotting even across species barriers established THBD as a cell-surface anticoagulant cofactor operating through the thrombin–protein C axis.\",\n      \"evidence\": \"Human THBD transgenic porcine endothelial cells tested in human whole-blood coagulation assays\",\n      \"pmids\": [\"24150517\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Xenogeneic system does not address whether THBD has additional species-matched endothelial functions\",\n        \"No assessment of coagulation-independent roles\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showing that transmembrane domain truncations progressively increase metalloprotease-dependent ectodomain shedding resolved how THBD soluble forms are generated and identified transmembrane integrity as the key determinant of surface retention versus release.\",\n      \"evidence\": \"Serial transmembrane truncation mutants in COS-1 cells with metalloprotease inhibitor treatment, ELISA, and immunofluorescence\",\n      \"pmids\": [\"29145514\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Identity of the specific metalloprotease(s) responsible for THBD shedding not determined\",\n        \"Whether shedding is regulated by physiological signals in endothelial cells in vivo is unknown\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Complete endothelial Thbd deletion in adult mice causing lethal coagulopathy—rescued by transgenic activated protein C—established that the essential in vivo anticoagulant function of THBD operates through protein C activation rather than through alternative thrombin-dependent mechanisms.\",\n      \"evidence\": \"Conditional Cre/loxP Thbd knockout mice with transgenic protein C supplementation rescue; coagulation assays and histopathology\",\n      \"pmids\": [\"28920104\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether partial THBD loss produces a graded coagulopathy phenotype is not addressed\",\n        \"Potential non-coagulant roles masked by lethal phenotype in global endothelial knockout\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Soluble thrombomodulin prolonging allograft survival with reduced perivascular inflammation demonstrated that THBD possesses immunomodulatory activity independent of its endothelial-anchored anticoagulant role.\",\n      \"evidence\": \"Dose-dependent administration of soluble TM in a fully MHC-mismatched murine cardiac transplant model\",\n      \"pmids\": [\"30401399\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism of immunosuppression (direct immune cell interaction vs. indirect coagulation modulation) not dissected\",\n        \"Single lab finding; independent replication lacking\",\n        \"Receptor or signaling pathway on immune cells not identified\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Brain-endothelial-specific Thbd deletion impairing peri-infarct angiogenesis without affecting coagulation or inflammation uncovered a coagulation-independent pro-angiogenic function, expanding THBD's role beyond hemostasis.\",\n      \"evidence\": \"Inducible Cdh5-ERT-Cre Thbd knockout mice subjected to middle cerebral artery occlusion; vascular morphometry and coagulation/inflammatory marker analysis\",\n      \"pmids\": [\"33028093\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Downstream signaling pathway mediating the angiogenic effect is not identified\",\n        \"Whether the angiogenic role extends to non-cerebral vascular beds is unknown\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"The THBD p.Ala473Val variant disrupting both protein C activation and endothelial progenitor cell angiogenesis linked the anticoagulant and angiogenic functions to overlapping but dissectable structural requirements in the THBD ectodomain.\",\n      \"evidence\": \"WT and mutant THBD cDNA transfected into bone-marrow-derived EPCs; protein C activation, migration, and tube formation assays\",\n      \"pmids\": [\"34236150\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Structural basis for how this single substitution disrupts both functions is not resolved\",\n        \"Single lab; no in vivo validation of the variant's effects\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identification of miR-18a-5p as a direct negative regulator of THBD via 3'UTR targeting, with THBD overexpression rescuing oncogenic phenotypes, established THBD as a tumor-suppressive effector in endometrial cancer.\",\n      \"evidence\": \"Dual-luciferase 3'UTR reporter assay, qRT-PCR, CCK-8, Transwell assays, and THBD rescue in endometrial cancer cell lines\",\n      \"pmids\": [\"34347980\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Tumor-suppressive mechanism of THBD in cancer cells is not defined\",\n        \"In vivo tumor model validation lacking\",\n        \"Whether this regulation is relevant beyond endometrial cancer is unknown\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Discovery that BRG1 represses THBD transcription through DNMT1-dependent promoter CpG methylation downstream of TNF-α, with BRG1 deletion or inhibition restoring THBD and reducing DVT, defined the epigenetic axis controlling THBD silencing in inflamed endothelium.\",\n      \"evidence\": \"Endothelial-specific BRG1 knockout mice in IVC ligation DVT model; siRNA, PFI-3 treatment, bisulfite sequencing, and ChIP in endothelial cells\",\n      \"pmids\": [\"36162255\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether additional chromatin remodelers cooperate with BRG1 at the THBD locus is unknown\",\n        \"Long-term safety of BRG1 inhibition as a therapeutic strategy not assessed\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The downstream signaling pathway through which THBD promotes angiogenesis independently of protein C activation remains unidentified, and the specific metalloprotease(s) mediating its ectodomain shedding in vivo have not been determined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Angiogenic signaling partners or receptors engaged by THBD are unknown\",\n        \"Identity of THBD-cleaving metalloprotease(s) in physiological contexts not established\",\n        \"Whether the tumor-suppressive function involves the same protein C–dependent or angiogenic mechanisms is unresolved\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 2, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [1, 2, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"SERPINC1\",\n      \"BRG1\",\n      \"DNMT1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"Thrombomodulin is an endothelial transmembrane glycoprotein that redirects thrombin from procoagulant to anticoagulant and anti-inflammatory functions by binding thrombin's anion-binding exosite-I through its EGF-like domains 4–6, thereby blocking fibrinogen and platelet-receptor access while optimally presenting protein C for activation; its chondroitin sulfate moiety enhances thrombin affinity 10–20-fold [PMID:2820710, PMID:10761923, PMID:7615164]. The thrombin–thrombomodulin complex also activates TAFI with a 1250-fold increase in catalytic efficiency, coupling coagulation to fibrinolysis inhibition, and accelerates factor I–mediated C3b inactivation to negatively regulate complement—loss-of-function THBD mutations that impair these activities cause approximately 5% of atypical hemolytic-uremic syndrome cases [PMID:8663147, PMID:19625716]. Beyond hemostasis, thrombomodulin's N-terminal lectin-like domain mediates Ca²⁺-dependent cell adhesion and sequesters HMGB1, and endothelial thrombomodulin promotes post-ischemic angiogenesis independently of its anticoagulant role [PMID:12951323, PMID:21805323, PMID:33028093]. THBD transcription is regulated by CLOCK/BMAL2 via an E-box element (conferring circadian oscillation), repressed by TNF-α through NF-κB/p300 coactivator competition and BRG1/DNMT1-mediated CpG methylation, and induced by statins via KLF2 [PMID:17848551, PMID:15677570, PMID:36162255, PMID:16043642].\",\n  \"teleology\": [\n    {\n      \"year\": 1985,\n      \"claim\": \"The discovery of soluble thrombomodulin in plasma and urine, retaining protein C cofactor activity but with reduced thrombin affinity, established that thrombomodulin functions as a circulating anticoagulant cofactor and that membrane anchorage modulates thrombin interaction.\",\n      \"evidence\": \"Radioimmunoassay and immunoaffinity purification from human plasma/urine with kinetic protein C activation assays\",\n      \"pmids\": [\"3001144\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for reduced thrombin affinity of soluble TM was unknown\", \"Physiological relevance of soluble vs. membrane-bound TM not quantified in vivo\"]\n    },\n    {\n      \"year\": 1987,\n      \"claim\": \"Cloning the full-length cDNA revealed thrombomodulin's modular architecture—signal peptide, N-terminal (lectin-like) domain, six EGF-like repeats, Ser/Thr-rich region, transmembrane segment, and cytoplasmic tail—encoded by an intronless gene on chromosome 20, providing the structural framework for all subsequent domain-function mapping.\",\n      \"evidence\": \"Independent cDNA/genomic cloning and sequencing with COS cell expression and functional validation\",\n      \"pmids\": [\"2820710\", \"2822087\", \"2819876\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Individual domain contributions to thrombin binding and protein C activation not yet dissected\", \"Post-translational modifications (chondroitin sulfate, glycosylation) not structurally resolved\"]\n    },\n    {\n      \"year\": 1989,\n      \"claim\": \"Biochemical reconstitution demonstrated that thrombomodulin converts thrombin from a procoagulant enzyme into an anticoagulant one by abolishing fibrinogen clotting and platelet activation while enabling rapid protein C activation, defining the core switch mechanism.\",\n      \"evidence\": \"In vitro reconstitution with purified components and enzyme kinetics\",\n      \"pmids\": [\"2538457\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-level binding interface unresolved\", \"Relative contributions of steric blockade vs. allosteric change debated\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Detailed kinetic and competition studies resolved the dual mechanism: chondroitin sulfate enhances thrombin affinity 10–20-fold by engaging a heparin-binding surface, while EGF-like domains sterically occlude procoagulant substrates; together these explain how thrombomodulin simultaneously inhibits procoagulant function and promotes protein C activation.\",\n      \"evidence\": \"Mutagenesis, competition assays, and structural analysis of thrombin–TM interactions\",\n      \"pmids\": [\"7615164\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Three-dimensional structure of the complex not yet determined\", \"Role of individual EGF domains not fully assigned\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Identification of the first THBD coding mutation (Asp468Tyr) in a patient with pulmonary embolism linked THBD genetic variation to venous thromboembolism, opening the question of whether loss-of-function alleles are common thrombophilia risk factors.\",\n      \"evidence\": \"PCR-SSCP and DNA sequencing in a thromboembolism patient\",\n      \"pmids\": [\"7811989\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single case report without family cosegregation data\", \"Functional impact of Asp468Tyr on protein C activation not directly measured in this study\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"The discovery that thrombomodulin enhances TAFI activation by thrombin 1250-fold expanded thrombomodulin's role beyond anticoagulation into fibrinolysis regulation, establishing the thrombin–TM–TAFI ternary complex model.\",\n      \"evidence\": \"In vitro reconstitution with purified thrombin, TM, and TAFI; full steady-state kinetics\",\n      \"pmids\": [\"8663147\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo quantitative contribution of TM-dependent TAFI activation to fibrinolysis control unclear\", \"Whether TAFI activation shares or competes with protein C activation for TM binding not resolved\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"The 2.3 Å crystal structure of thrombin bound to TM EGF-like domains 4–6 resolved the longstanding question of mechanism: TM binds exosite-I in a Y-shaped configuration, sterically blocking procoagulant substrates without major active-site allosteric changes, and positioning the substrate-presentation surface for protein C docking.\",\n      \"evidence\": \"X-ray crystallography of thrombin–TME456 complex with molecular modeling\",\n      \"pmids\": [\"10761923\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No ternary structure with protein C or TAFI\", \"Role of chondroitin sulfate not captured in the crystal\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Functional dissection of the lectin-like domain revealed a non-hemostatic role: Ca²⁺-dependent cell adhesion mediated by this domain suppresses monolayer permeability and tumor growth, establishing TM as a multifunctional protein beyond coagulation.\",\n      \"evidence\": \"Domain-deletion mutants in TM-negative melanoma cells, confocal imaging, permeability assays, in vivo tumor model\",\n      \"pmids\": [\"12951323\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous ligand for the lectin-like domain in cell adhesion not identified\", \"Structural basis for Ca²⁺-dependent interaction unknown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Two key transcriptional regulatory mechanisms were defined: TNF-α represses THBD via NF-κB competing for the coactivator p300 away from Ets factors at the TM promoter, while statins induce THBD through KLF2 upregulation via Rho pathway inhibition, explaining how inflammation and pharmacology modulate endothelial anticoagulant phenotype.\",\n      \"evidence\": \"ChIP, EMSA, promoter analysis, NF-κB inhibition, p300 overexpression, mouse lung injury model; KLF2 siRNA epistasis with statin treatment\",\n      \"pmids\": [\"15677570\", \"16043642\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contributions of Ets family members to basal TM transcription not resolved\", \"Whether KLF2 binds the TM promoter directly or acts through intermediaries unclear\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"CLOCK/BMAL2 heterodimer binding to an E-box in the THBD promoter was shown to drive circadian oscillation of TM mRNA and protein in mouse vasculature, abolished in Clock mutant mice, establishing THBD as a clock-controlled gene and linking circadian biology to hemostatic capacity.\",\n      \"evidence\": \"ChIP, EMSA, luciferase reporter, Clock mutant mice, temporal feeding restriction\",\n      \"pmids\": [\"17848551\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether circadian TM variation translates to measurable oscillation in protein C activation rate in vivo not tested\", \"Contribution of BMAL1 vs. BMAL2 to TM regulation not clarified\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"THBD mutations were shown to cause ~5% of atypical HUS by impairing complement regulation: TM binds C3b and factor H, accelerates factor I–mediated C3b inactivation, and promotes TAFI-dependent anaphylatoxin degradation—aHUS-associated mutations reduce both activities, revealing TM as a complement regulator.\",\n      \"evidence\": \"Patient cohort sequencing, purified protein binding assays, cell-based complement activation assays with mutant TM constructs\",\n      \"pmids\": [\"19625716\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for TM–C3b interaction unknown\", \"Whether complement dysregulation alone or combined coagulation defect drives aHUS pathology in THBD mutation carriers unclear\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Conditional Thbd ablation in adult mice demonstrated that complete TM deficiency causes consumptive coagulopathy and sudden death, with APC supplementation rescuing thrombosis but not pregnancy-associated morbidity, revealing essential non-endothelial (placental) TM functions; separately, transmembrane domain truncation mutants showed that domain length governs metalloprotease-dependent shedding, explaining how naturally occurring mutations increase soluble TM.\",\n      \"evidence\": \"Inducible Cre/loxP Thbd knockout with transgenic PC rescue; site-directed mutagenesis/shedding assays in COS-1 cells\",\n      \"pmids\": [\"28920104\", \"29145514\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the metalloprotease(s) responsible for TM shedding not determined\", \"Mechanism of TM function in trophoblast biology uncharacterized\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Brain endothelial–specific Thbd deletion worsened stroke outcome not through coagulopathy or inflammation but by impairing peri-infarct angiogenesis, establishing a coagulation-independent pro-angiogenic function for endothelial thrombomodulin.\",\n      \"evidence\": \"Inducible Cre/loxP brain endothelial Thbd knockout, MCA occlusion model, vessel morphometry\",\n      \"pmids\": [\"33028093\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular pathway through which TM promotes angiogenesis (receptor, signaling cascade) not identified\", \"Whether the lectin-like domain or EGF-like domains mediate the angiogenic effect unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"An epigenetic repression axis was defined: BRG1 upregulates DNMT1 under TNF-α stimulation, increasing CpG methylation of the THBD promoter and silencing expression; endothelial-specific BRG1 knockout restored THBD and reduced DVT in mice, providing a druggable epigenetic mechanism for thrombosis.\",\n      \"evidence\": \"Conditional endothelial BRG1 knockout mice, DVT ligation model, CpG methylation analysis, pharmacological BRG1 inhibitor PFI-3\",\n      \"pmids\": [\"36162255\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether DNMT1-mediated THBD methylation is reversible with DNMT inhibitors in established DVT not tested\", \"Relative contribution of THBD silencing vs. other BRG1 targets to DVT phenotype not isolated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Major open questions include the structural basis for TM's complement-regulatory (C3b binding) and angiogenic functions, the identity of the sheddase(s), the signaling mechanism linking TM to angiogenesis independently of APC, and whether circadian TM oscillation measurably affects in vivo hemostatic thresholds.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No ternary crystal structure of thrombin–TM–protein C or TM–C3b\", \"Sheddase identity unknown\", \"Pro-angiogenic signaling pathway downstream of TM uncharacterized\", \"In vivo functional consequence of circadian TM oscillation untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4, 5, 7, 8, 10]},\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [4, 5, 8]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 4, 8, 9, 20]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [3, 20]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [4, 5, 7, 8, 10, 15, 19]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [15, 16]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [14]},\n      {\"term_id\": \"R-HSA-9909396\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [6, 15]}\n    ],\n    \"complexes\": [\n      \"Thrombin–thrombomodulin complex\",\n      \"Thrombin–thrombomodulin–protein C ternary complex\",\n      \"Thrombin–thrombomodulin–TAFI ternary complex\"\n    ],\n    \"partners\": [\n      \"F2\",\n      \"PROC\",\n      \"CPB2\",\n      \"C3\",\n      \"CFH\",\n      \"HMGB1\",\n      \"PROCR\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}