{"gene":"VTN","run_date":"2026-04-28T23:00:23","timeline":{"discoveries":[{"year":1985,"finding":"The complete amino acid sequence of human vitronectin was deduced from cDNA clones isolated from a human liver library. The sequence revealed that vitronectin contains the entire 44-amino acid somatomedin B (SMB) peptide at its N-terminus, three potential glycosylation sites, a C-terminal glycosaminoglycan-binding domain rich in basic residues, and an Arg-Gly-Asp (RGD) sequence immediately after the SMB domain that constitutes the cell attachment site, showing functional similarity to fibronectin's cell attachment sequence.","method":"cDNA cloning and nucleotide sequencing, oligonucleotide probe screening of lambda gt11 library, cell attachment inhibition assays with synthetic RGD peptides","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — primary sequence determination with functional validation of RGD cell attachment site; foundational study replicated across labs","pmids":["2414098"],"is_preprint":false},{"year":1985,"finding":"S-protein (a complement regulatory protein) and vitronectin (serum spreading factor) were shown to be identical proteins by molecular cloning, sequence analysis, and immunological criteria. The single polypeptide chain of 459 amino acids (plus 19-residue leader peptide) encodes the SMB domain at its N-terminus, linking complement regulation, coagulation, and cell-substrate adhesion functions in one molecule.","method":"cDNA cloning from pEX expression library screened with monoclonal antibodies, sequence analysis, immunological cross-reactivity assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — direct molecular identification of protein identity; foundational cloning paper","pmids":["3004934"],"is_preprint":false},{"year":1990,"finding":"Integrin αvβ5 was purified from human placenta and identified as a vitronectin receptor that binds preferentially to vitronectin (over fibronectin, fibrinogen, or von Willebrand factor). The β5 subunit pairs with the αv subunit and is immunologically and structurally distinct from β3, with the ligand-binding site architecture differing from αvβ3.","method":"Immunodepletion of αvβ3 followed by monoclonal antibody affinity chromatography, wheat germ lectin chromatography, Western blot, vitronectin-binding assays, peptide mapping, N-terminal amino acid sequencing","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — purification to homogeneity with direct ligand-binding characterization and structural distinction from paralog","pmids":["1694173"],"is_preprint":false},{"year":1993,"finding":"The vitronectin-binding integrin αvβ5 was shown to bind to the basic heparin-binding domain of vitronectin (sequence KKQRFRHRNRKG) through a divalent cation-independent mechanism, distinct from RGD-mediated integrin binding. This defines an auxiliary integrin-binding specificity for basic amino acid sequences within vitronectin.","method":"Affinity chromatography with Tat-derived peptides, immunoprecipitation with anti-integrin antibodies, cell attachment inhibition assays, divalent cation chelation experiments","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 — affinity chromatography plus immunoprecipitation with functional cell attachment assays; defines a mechanistically distinct binding mode","pmids":["7682219"],"is_preprint":false},{"year":1996,"finding":"Active PAI-1 blocks smooth muscle cell (SMC) migration by competing with integrin αVβ3 for an overlapping binding site on vitronectin, preventing αVβ3-dependent cell motility on the vitronectin matrix. This inhibitory effect is independent of PAI-1's protease inhibitor activity and requires high-affinity PAI-1 binding to vitronectin; formation of a PAI-1/plasminogen activator complex reduces PAI-1 affinity for vitronectin and restores migration.","method":"SMC migration assays on vitronectin substrates, blocking antibodies against αVβ3, PAI-1 mutants deficient in protease inhibition but retaining vitronectin binding, PAI-1/uPA complex formation assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods including function-blocking mutants and antibodies; highly cited foundational paper replicated across labs","pmids":["8837777"],"is_preprint":false},{"year":1996,"finding":"uPAR and PAI-1 compete for binding to the same site within the N-terminal somatomedin B (SMB) domain of vitronectin. PAI-1 dissociates VN-bound uPAR and detaches cells from vitronectin substratum in a protease-inhibitor-independent manner, while uPA can rapidly reverse this PAI-1-mediated cell detachment. The uPAR-binding sequence was localized within the central region of the SMB domain.","method":"Domain swapping, site-directed mutagenesis of SMB domain, competitive binding assays, cell detachment assays with U937 cells on VN substrates","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 — mutagenesis plus competitive binding with cell-based functional readout; replicated finding consistent with complementary PAI-1/αVβ3 paper","pmids":["8830783"],"is_preprint":false},{"year":2002,"finding":"The PAI-1/vitronectin interaction maps to two binding regions: the primary high-affinity PAI-1 binding site resides within the N-terminal somatomedin B (SMB) domain of vitronectin, while at least one secondary low-affinity binding site exists in the C-terminal region of vitronectin involved in forming larger PAI-1/Vn complexes. On PAI-1, the region around α-helix E and α-helix F is important for vitronectin binding.","method":"Peptide competition assays, domain deletion mapping, mutagenesis studies reviewed across multiple experimental approaches","journal":"Biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 — systematic domain mapping with multiple approaches but review/summary format; consistent with structural data","pmids":["12437099"],"is_preprint":false},{"year":2003,"finding":"The crystal structure (2.3 Å) of the somatomedin B (SMB) domain of vitronectin in complex with PAI-1 revealed the molecular basis of their interaction: vitronectin binding stabilizes the active conformation of PAI-1 by engaging its reactive center loop region. The PAI-1 binding site on the SMB domain sterically overlaps with the binding surfaces for αVβ3/αVβ5 integrins and uPAR, explaining how PAI-1 competitively blocks integrin- and uPAR-mediated cell adhesion and motility.","method":"X-ray crystallography at 2.3 Å resolution of SMB domain–PAI-1 complex","journal":"Nature structural biology","confidence":"High","confidence_rationale":"Tier 1 — high-resolution crystal structure with mechanistic interpretation explaining multiple functional observations; landmark structural paper","pmids":["12808446"],"is_preprint":false},{"year":2004,"finding":"Adhesion of human mesenchymal stem cells (hMSCs) to vitronectin (and collagen I) promotes osteogenic differentiation, with cells on vitronectin showing the greatest induction of mineralized matrix, osteopontin, osteocalcin, collagen I, and alkaline phosphatase expression. hMSCs adhere to vitronectin through distinct integrin receptors compared to other ECM proteins, and ECM contact alone can be sufficient to induce osteogenic differentiation.","method":"Cell adhesion assays on purified ECM proteins, integrin-blocking antibodies, osteogenic differentiation marker assays (alkaline phosphatase, mineralization, immunostaining for osteopontin and osteocalcin) over 16-day time course","journal":"Journal of biomedicine & biotechnology","confidence":"Medium","confidence_rationale":"Tier 2 — clean functional assay with multiple differentiation markers and integrin-blocking experiments; single lab study","pmids":["15123885"],"is_preprint":false},{"year":2005,"finding":"Human granzyme B (GrB) efficiently cleaves vitronectin at a site after the Arg-Gly-Asp (RGD) motif within the integrin-binding region, disrupting the integrin–ECM interface. This GrB-mediated cleavage of vitronectin (along with fibronectin and laminin) causes detachment of endothelial cells and other cell types, induces anoikis in endothelial cells, and inhibits tumor cell spreading, migration, and invasion in vitro.","method":"In vitro cleavage assays with native and recombinant GrB, cell detachment assays, cell spreading/migration/invasion assays, identification of cleavage site by sequence analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro biochemical cleavage assay with defined cleavage site plus multiple cell-based functional readouts","pmids":["15843372"],"is_preprint":false},{"year":2008,"finding":"MMP-2 secreted by ovarian cancer (OvCa) cells cleaves vitronectin (and fibronectin) into small fragments that enhance OvCa cell attachment to peritoneal surfaces. This cleavage exposes cryptic binding sites recognized by αVβ3 integrin on OvCa cells, promoting adhesion. MMP-2 inhibition in OvCa cells (but not in host cells) reduced peritoneal adhesion and tumor metastasis in vivo.","method":"siRNA knockdown and pharmacological inhibition of MMP-2, in vitro cleavage of ECM proteins, cell adhesion assays to ECM fragments, integrin-blocking antibodies, in vivo mouse peritoneal metastasis model with Mmp2-null mice","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods including genetic KO mouse model plus in vitro biochemical and cell-based assays; identifies specific VTN cleavage mechanism","pmids":["18340378"],"is_preprint":false},{"year":2017,"finding":"BPIFB1 interacts with vitronectin (VTN) and reduces VTN expression and VTN–integrin αV complex formation in NPC cells, leading to inhibition of the FAK/Src/ERK signalling pathway downstream of VTN. BPIFB1 also attenuates VTN-induced epithelial-mesenchymal transition, thereby inhibiting NPC cell migration, invasion, and lung metastasis.","method":"Co-immunoprecipitation coupled with mass spectrometry to identify BPIFB1-binding proteins, western blotting, immunofluorescence, immunohistochemistry, in vitro migration/invasion assays, in vivo lung metastasis mouse model","journal":"British journal of cancer","confidence":"High","confidence_rationale":"Tier 2 — Co-IP/MS identification of interaction confirmed by western blot and functional assays in vitro and in vivo with multiple orthogonal methods","pmids":["29123267"],"is_preprint":false},{"year":2018,"finding":"VTN promotes NPC cell radioresistance by inducing cell proliferation and survival, G2/M phase arrest, DNA repair, and activation of the ATM-Chk2 and ATR-Chk1 DNA damage response pathways, as well as anti-apoptotic effects after ionizing radiation. BPIFB1 (which binds VTN) inhibits this VTN-mediated radioresistance, improving NPC radiosensitivity.","method":"Colony formation and cell survival assays, cell cycle analysis, western blotting for ATM-Chk2 and ATR-Chk1 pathway components, apoptosis assays, overexpression and knockdown of VTN and BPIFB1","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2-3 — defined pathway placement (ATM-Chk2, ATR-Chk1) with VTN overexpression/knockdown and functional readouts; single lab","pmids":["29568064"],"is_preprint":false},{"year":2019,"finding":"miR-30c regulates vitronectin (VN) protein levels in smooth muscle cells via targeting PAI-1: miR-30c reduces PAI-1 expression (validated by luciferase assay demonstrating direct 3'UTR targeting), and reduced PAI-1 leads to decreased VN levels, revealing a miR-30c → PAI-1 → VN regulatory axis relevant to coronary disease.","method":"miRNA transfection, luciferase reporter assays for PAI-1 3'UTR targeting, qRT-PCR, western blotting, ELISA for VN and PAI-1 in plasma, in vitro SMC assays","journal":"Life sciences","confidence":"Medium","confidence_rationale":"Tier 2-3 — luciferase assay validates direct miR-30c/PAI-1 interaction; VN regulation inferred from downstream PAI-1 effects; single lab","pmids":["31760103"],"is_preprint":false},{"year":2021,"finding":"A vitronectin fragment (VTN amino acids 381–397) competes with TGF-β1 for binding to αVβ6 integrin on human fibroblast-like synoviocytes (FLSs). By interacting with αVβ6 on FLS surfaces, this VTN fragment prevents αVβ6-mediated TGF-β1 activation and increases α-SMA expression, revealing that VTN can modulate TGF-β1 bioavailability through integrin competition.","method":"Competition binding assay between VTN fragment and αVβ6, flow cytometry and western blot for αVβ6 on primary FLSs, immunohistochemistry of synovial tissue, TGF-β bioassay, α-SMA immunostaining","journal":"Experimental & molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 — direct competition assay plus cell-based TGF-β bioassay; multiple orthogonal methods; single lab","pmids":["33526813"],"is_preprint":false},{"year":2022,"finding":"FBLN2 (fibulin-2) binds directly to VTN (vitronectin) and negatively regulates its expression in lung fibroblasts. FBLN2 knockdown increases VTN expression, which activates FAK signaling and promotes TGF-β1-induced cell proliferation, migration, MMP2/MMP9 upregulation, and fibrotic marker expression (α-SMA, collagen I, fibronectin); VTN overexpression partially rescues the anti-fibrotic effect of FBLN2 knockdown.","method":"Protein co-immunoprecipitation (confirmed FBLN2-VTN interaction), STRING database prediction, western blotting, siRNA knockdown and overexpression, wound-healing assay, CCK-8, immunofluorescence for α-SMA","journal":"Tissue & cell","confidence":"Medium","confidence_rationale":"Tier 2-3 — co-IP validates FBLN2-VTN interaction; rescue experiment with VTN overexpression demonstrates pathway position; single lab","pmids":["36608640"],"is_preprint":false},{"year":2024,"finding":"Tumor cell-secreted vitronectin (Vtn) binds to complement C1Q binding protein (C1qbp) on the surface of tumor-associated macrophages. This Vtn–C1qbp interaction inhibits macrophage phagocytosis of tumor cells and shifts macrophages toward an M2-like subtype by facilitating FcγRIIIA/CD16-induced Shp1 recruitment, which reduces Syk phosphorylation, thereby suppressing pro-phagocytic signaling. Vtn knockdown combined with anti-CD47 antibody synergistically enhanced macrophage phagocytosis and reduced tumor growth in vivo.","method":"Genome-wide CRISPR screen for anti-phagocytic genes, cell-to-cell interaction database analysis, siRNA knockdown, flow cytometry for phagocytosis and macrophage polarization, RNA sequencing, co-immunoprecipitation, mass spectrometry, immunofluorescence, syngeneic mouse tumor models","journal":"Theranostics","confidence":"High","confidence_rationale":"Tier 1-2 — CRISPR screen discovery validated by Co-IP/MS, mechanistic pathway (Shp1/Syk) defined, confirmed in vivo; multiple orthogonal methods","pmids":["38773982"],"is_preprint":false},{"year":2025,"finding":"VTN overexpression in pancreatic cancer cells suppresses proliferation, invasion, and migration in vitro, and inhibits tumor growth in a syngeneic mouse model, acting as a tumor suppressor. VTN expression is linked to immune regulatory pathways, and VTN overexpression synergizes with anti-PD1 therapy to enhance antitumor efficacy in vivo.","method":"VTN knockdown and overexpression in pancreatic cancer cell lines, proliferation/invasion/migration assays, syngeneic mouse subcutaneous tumor model with anti-PD1 treatment, single-cell RNA sequencing analysis, immune pathway analysis","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2-3 — defined cell-autonomous tumor suppressor role with in vivo validation; mechanistic pathway not fully resolved; single lab","pmids":["40433359"],"is_preprint":false},{"year":2025,"finding":"Insufficient VTN expression in trophoblast cells impairs migration, invasion, and endothelial-like tube formation via the HEY1/autophagy pathway. VTN overexpression upregulates HEY1 (a Notch signaling downstream target), while VTN knockdown increases LC3II expression indicating enhanced autophagy; HEY1 overexpression alleviates autophagy induced by VTN knockdown, and autophagy inhibition with 3-MA partially restores trophoblast function suppressed by VTN knockdown.","method":"Functional assays in HTR8/SVneo cells, HUVECs, and primary EVTs; VTN overexpression/knockdown; transcriptome sequencing; LC3II western blotting for autophagy; 3-MA autophagy inhibitor; HEY1 overexpression rescue experiments","journal":"Placenta","confidence":"Medium","confidence_rationale":"Tier 2-3 — defined pathway (VTN → HEY1 → autophagy suppression) with rescue experiments; multiple cell types tested; single lab","pmids":["40516241"],"is_preprint":false}],"current_model":"Vitronectin (VTN) is a multifunctional extracellular matrix and plasma glycoprotein whose N-terminal somatomedin B (SMB) domain serves as a high-affinity docking site for PAI-1 (stabilizing its active conformation) and competitively overlaps with binding sites for αVβ3/αVβ5 integrins and uPAR, thereby controlling cell adhesion and migration; its central RGD sequence mediates integrin-dependent cell attachment and can be cleaved by MMP-2 and granzyme B to generate pro-adhesive fragments or induce anoikis; its heparin-binding domain engages αvβ5 through a divalent cation-independent basic-sequence mechanism; and beyond matrix roles, VTN acts as a 'don't eat me' signal by binding C1qbp on macrophages to suppress phagocytosis via Shp1-mediated Syk dephosphorylation, while also regulating trophoblast function through a HEY1/autophagy pathway and modulating TGF-β1 bioavailability through competition with αVβ6 integrin."},"narrative":{"teleology":[{"year":1985,"claim":"Molecular cloning resolved vitronectin's primary structure and established its domain architecture — the N-terminal somatomedin B domain, the RGD cell-attachment sequence, and the C-terminal heparin-binding domain — while simultaneously proving that S-protein (complement regulator) and serum spreading factor are the same polypeptide.","evidence":"cDNA cloning from human liver libraries, nucleotide sequencing, and immunological cross-reactivity assays","pmids":["2414098","3004934"],"confidence":"High","gaps":["No receptor or binding-partner specificity defined at this stage","Three-dimensional structure of VTN domains not yet determined"]},{"year":1993,"claim":"Identification of αVβ5 as a second vitronectin receptor, and discovery that αVβ5 engages the heparin-binding domain through a divalent-cation-independent basic-sequence mechanism, established that VTN uses multiple integrin-binding modes beyond the RGD motif.","evidence":"Purification of αVβ5 from human placenta with ligand-binding assays; affinity chromatography with Tat-derived peptides and cation-chelation experiments","pmids":["1694173","7682219"],"confidence":"High","gaps":["Structural basis of the αVβ5–heparin-binding domain interaction not resolved","Relative contributions of RGD versus heparin-binding domain engagement in vivo unknown"]},{"year":1996,"claim":"Functional studies revealed that PAI-1 and uPAR compete for an overlapping binding site on the SMB domain, and that PAI-1 binding — independent of its protease-inhibitory activity — blocks αVβ3-dependent cell migration and detaches uPAR-anchored cells, establishing VTN as a switchable adhesion platform.","evidence":"SMC migration assays with PAI-1 mutants; domain-swap mutagenesis and competitive binding with U937 cell detachment readout","pmids":["8837777","8830783"],"confidence":"High","gaps":["Atomic-resolution mapping of the competitive binding interface not yet available","In vivo relevance of PAI-1/uPAR competition on VTN in wound healing or thrombosis not demonstrated"]},{"year":2003,"claim":"The 2.3 Å crystal structure of the SMB domain–PAI-1 complex provided the atomic explanation for how VTN stabilizes PAI-1's active conformation and why PAI-1 binding sterically excludes integrins and uPAR, unifying a decade of competitive-binding observations.","evidence":"X-ray crystallography of the SMB–PAI-1 complex at 2.3 Å resolution","pmids":["12808446"],"confidence":"High","gaps":["Full-length VTN structure remains unsolved","Conformational changes in intact VTN upon PAI-1 engagement not resolved"]},{"year":2008,"claim":"Proteolytic processing of VTN by granzyme B and MMP-2 was shown to remodel the adhesion landscape: GrB cleaves after the RGD motif to induce anoikis, while MMP-2 generates fragments that expose cryptic αVβ3-binding sites promoting metastatic adhesion, demonstrating that VTN fragmentation is a biologically active event.","evidence":"In vitro cleavage assays with defined sites, cell detachment and anoikis assays (GrB); MMP-2 siRNA/KO with peritoneal metastasis mouse model and integrin-blocking antibodies","pmids":["15843372","18340378"],"confidence":"High","gaps":["Precise MMP-2 cleavage site(s) on VTN not mapped at single-residue resolution","Whether GrB cleavage of VTN occurs in immune-mediated tissue surveillance in vivo is unconfirmed"]},{"year":2021,"claim":"A VTN fragment (aa 381–397) was found to compete with TGF-β1 for αVβ6 integrin binding on fibroblast-like synoviocytes, revealing a mechanism by which VTN modulates TGF-β1 bioavailability beyond its classical adhesion roles.","evidence":"Competition binding assay between VTN peptide and αVβ6; TGF-β bioassay and α-SMA immunostaining in primary FLSs","pmids":["33526813"],"confidence":"Medium","gaps":["Whether endogenous VTN fragments of this size are generated in vivo is not established","No structural data for VTN fragment–αVβ6 interaction","Relevance to arthritis or fibrosis pathology not validated in animal models"]},{"year":2024,"claim":"A genome-wide CRISPR screen identified VTN as a tumor-secreted anti-phagocytic signal: VTN binds C1qbp on macrophages, recruits Shp1 via FcγRIIIA/CD16, dephosphorylates Syk, and suppresses phagocytosis — defining a 'don't eat me' pathway parallel to the CD47–SIRPα axis.","evidence":"CRISPR screen, Co-IP/MS for VTN–C1qbp interaction, Shp1/Syk phosphorylation assays, syngeneic tumor models with combined VTN KD and anti-CD47","pmids":["38773982"],"confidence":"High","gaps":["Whether C1qbp is the sole macrophage receptor for VTN's anti-phagocytic function is not resolved","Clinical relevance of VTN–C1qbp axis in human tumors not yet validated","Structural basis of VTN–C1qbp binding unknown"]},{"year":2025,"claim":"VTN was linked to trophoblast function through a HEY1/autophagy pathway and to pancreatic cancer suppression with immunotherapy synergy, expanding its roles to placental biology and tumor-immune regulation beyond adhesion.","evidence":"VTN overexpression/knockdown in trophoblast cells with HEY1 rescue and autophagy inhibitor experiments; syngeneic pancreatic cancer model with anti-PD1 treatment","pmids":["40516241","40433359"],"confidence":"Medium","gaps":["Mechanism connecting extracellular VTN to intracellular HEY1/Notch signaling not defined","Tumor-suppressive versus pro-tumorigenic roles of VTN across cancer types remain contradictory and unresolved","Whether VTN's immunotherapy synergy operates through the C1qbp–Shp1 axis or a distinct mechanism is unknown"]},{"year":null,"claim":"The full-length three-dimensional structure of VTN, the conformational switch between its monomeric plasma form and its multimeric ECM-deposited form, and the integration of its anti-phagocytic (C1qbp) and adhesion (integrin) functions in the tumor microenvironment remain major unresolved questions.","evidence":"","pmids":[],"confidence":"Low","gaps":["No full-length VTN crystal or cryo-EM structure exists","Monomer-to-multimer conversion mechanism not structurally characterized","Relative in vivo contributions of VTN's anti-phagocytic versus adhesion functions in tumor immune evasion undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098631","term_label":"cell adhesion mediator activity","supporting_discovery_ids":[0,2,3,8,9]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[4,5,7,16]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4,5,14,16]}],"localization":[{"term_id":"GO:0031012","term_label":"extracellular matrix","supporting_discovery_ids":[0,8,9,10]},{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[0,1,16,17]}],"pathway":[{"term_id":"R-HSA-1474244","term_label":"Extracellular matrix organization","supporting_discovery_ids":[0,8,9,10]},{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[4,5,7]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[16,17]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[11,15,16]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[0,2,3,8]}],"complexes":[],"partners":["SERPINE1","PLAUR","ITGAV","ITGB3","ITGB5","C1QBP","FBLN2","BPIFB1"],"other_free_text":[]},"mechanistic_narrative":"Vitronectin (VTN) is a multifunctional plasma and extracellular matrix glycoprotein that integrates cell adhesion, pericellular proteolysis, complement regulation, and immune modulation through distinct structural domains. Its N-terminal somatomedin B (SMB) domain serves as a high-affinity binding site for PAI-1, stabilizing PAI-1's active conformation; this site sterically overlaps with binding surfaces for αVβ3/αVβ5 integrins and uPAR, enabling PAI-1 to competitively block integrin- and uPAR-dependent cell adhesion and migration [PMID:12808446, PMID:8837777, PMID:8830783]. The central RGD motif mediates canonical integrin-dependent cell attachment and is a substrate for proteolytic cleavage by granzyme B and MMP-2, which can induce anoikis or expose cryptic pro-adhesive fragments that promote metastatic adhesion [PMID:15843372, PMID:18340378]. Beyond matrix functions, tumor-secreted VTN binds C1qbp on macrophages and suppresses phagocytosis through Shp1-mediated Syk dephosphorylation, acting as an anti-phagocytic signal that synergizes with CD47 blockade when depleted [PMID:38773982]."},"prefetch_data":{"uniprot":{"accession":"P04004","full_name":"Vitronectin","aliases":["S-protein","Serum-spreading factor","V75"],"length_aa":478,"mass_kda":54.3,"function":"Vitronectin is a cell adhesion and spreading factor found in serum and tissues. Vitronectin interact with glycosaminoglycans and proteoglycans. Is recognized by certain members of the integrin family and serves as a cell-to-substrate adhesion molecule. Inhibitor of the membrane-damaging effect of the terminal cytolytic complement pathway Somatomedin-B is a growth hormone-dependent serum factor with protease-inhibiting activity","subcellular_location":"Parasitophorous vacuole","url":"https://www.uniprot.org/uniprotkb/P04004/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/VTN","classification":"Not Classified","n_dependent_lines":18,"n_total_lines":1208,"dependency_fraction":0.014900662251655629},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/VTN","total_profiled":1310},"omim":[{"mim_id":"615825","title":"SUSHI DOMAIN-CONTAINING PROTEIN 2; SUSD2","url":"https://www.omim.org/entry/615825"},{"mim_id":"613504","title":"ZINC FINGER FYVE DOMAIN-CONTAINING PROTEIN 21; ZFYVE21","url":"https://www.omim.org/entry/613504"},{"mim_id":"607732","title":"STERILE ALPHA AND TIR MOTIFS-CONTAINING PROTEIN 1; SARM1","url":"https://www.omim.org/entry/607732"},{"mim_id":"606018","title":"EGF-LIKE REPEATS- AND DISCOIDIN I-LIKE DOMAINS-CONTAINING PROTEIN 3; EDIL3","url":"https://www.omim.org/entry/606018"},{"mim_id":"602182","title":"ECTONUCLEOTIDE PYROPHOSPHATASE/PHOSPHODIESTERASE 3; ENPP3","url":"https://www.omim.org/entry/602182"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Endoplasmic reticulum","reliability":"Supported"},{"location":"Vesicles","reliability":"Supported"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"liver","ntpm":5644.8}],"url":"https://www.proteinatlas.org/search/VTN"},"hgnc":{"alias_symbol":["VN"],"prev_symbol":[]},"alphafold":{"accession":"P04004","domains":[{"cath_id":"4.10.410","chopping":"23-59","consensus_level":"medium","plddt":82.1714,"start":23,"end":59},{"cath_id":"2.110.10.10","chopping":"162-356_430-469","consensus_level":"high","plddt":86.9616,"start":162,"end":469}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P04004","model_url":"https://alphafold.ebi.ac.uk/files/AF-P04004-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P04004-F1-predicted_aligned_error_v6.png","plddt_mean":67.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=VTN","jax_strain_url":"https://www.jax.org/strain/search?query=VTN"},"sequence":{"accession":"P04004","fasta_url":"https://rest.uniprot.org/uniprotkb/P04004.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P04004/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P04004"}},"corpus_meta":[{"pmid":"25591066","id":"PMC_25591066","title":"Discovery and development of Galeterone (TOK-001 or VN/124-1) for the treatment of all stages of prostate cancer.","date":"2015","source":"Journal of medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25591066","citation_count":148,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"31830338","id":"PMC_31830338","title":"A Dual-Functional Conductive Framework Embedded with TiN-VN Heterostructures for Highly Efficient Polysulfide and Lithium Regulation toward Stable Li-S Full Batteries.","date":"2019","source":"Advanced materials (Deerfield Beach, Fla.)","url":"https://pubmed.ncbi.nlm.nih.gov/31830338","citation_count":148,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"2999288","id":"PMC_2999288","title":"Diversity in the germline antibody repertoire. 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BPIFB1 reduces VTN expression and disrupts this complex, leading to inhibition of the FAK/Src/ERK signalling pathway and suppression of epithelial-mesenchymal transition, thereby inhibiting cancer cell migration and invasion.\",\n      \"method\": \"Co-immunoprecipitation coupled with mass spectrometry, western blotting, immunofluorescence, in vitro migration/invasion assays, in vivo lung metastasis model\",\n      \"journal\": \"British journal of cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP/MS identifying binding partners, multiple orthogonal functional assays, in vivo validation\",\n      \"pmids\": [\"29123267\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"VTN promotes radioresistance in nasopharyngeal carcinoma cells by inducing G2/M phase arrest, DNA repair, and activation of the ATM-Chk2 and ATR-Chk1 pathways, as well as exerting anti-apoptotic effects after ionising radiation; BPIFB1 (a previously identified VTN-binding partner) inhibits this VTN-mediated radioresistance.\",\n      \"method\": \"Colony formation assay, cell survival assay, cell cycle analysis, western blotting (ATM-Chk2/ATR-Chk1 pathway), apoptosis assay, VTN overexpression/knockdown\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple functional readouts in one lab; mechanism placed downstream of VTN via defined signalling pathway\",\n      \"pmids\": [\"29568064\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"PAI-1 binds vitronectin (VTN) via a region around alpha-helices E and F of PAI-1, while the primary high-affinity PAI-1-binding site on VTN is localised within the N-terminal somatomedin B (SMB) domain; a secondary low-affinity site exists in the C-terminal region of VTN and is involved in formation of larger PAI-1/VTN complexes.\",\n      \"method\": \"Binding site mapping studies (mutagenesis, domain deletion, biochemical binding assays as reviewed)\",\n      \"journal\": \"Biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — domain-mapping review summarising multiple biochemical studies; replicated across multiple approaches but presented as a review/minireview\",\n      \"pmids\": [\"12437099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A VTN fragment (amino acids 381–397) interacts with αVβ6 integrin on fibroblast-like synoviocytes and competes with TGF-β1 activation; αVβ6 integrin mediates TGF-β1 bioavailability, and VTN(381-397) prevents TGF-β1 activation by blocking this integrin interaction, thereby modulating fibrosis-related signalling (increased α-SMA) in osteoarthritis synovial cells.\",\n      \"method\": \"Competition binding assay, western blot, flow cytometry, immunohistochemistry, TGF-β bioassay, nano-LC/Chip MS-MS\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (competition assay, bioassay, proteomics) from a single lab; mechanistic link between VTN fragment, integrin, and TGF-β1 activation established\",\n      \"pmids\": [\"33526813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FBLN2 (fibulin-2) binds VTN and negatively regulates its expression; VTN in turn promotes TGF-β1-induced pulmonary fibroblast proliferation, migration, and fibrosis through FAK signalling; FBLN2 knockdown suppresses VTN expression and attenuates fibrosis, an effect rescued by VTN overexpression.\",\n      \"method\": \"Protein co-immunoprecipitation, western blot, qPCR, wound healing assay, immunofluorescence, gene knockdown/overexpression, STRING database interaction prediction\",\n      \"journal\": \"Tissue & cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — Co-IP confirms FBLN2-VTN binding; epistasis via rescue experiment places VTN downstream of FBLN2 in FAK signalling; single lab\",\n      \"pmids\": [\"36608640\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Tumour cell-secreted VTN interacts with complement C1Q binding protein (C1qbp) on the surface of tumour-associated macrophages, inhibiting macrophage phagocytosis of tumour cells and shifting macrophages toward an M2-like phenotype; mechanistically, the VTN-C1qbp axis facilitates FcγRIIIA/CD16-induced Shp1 recruitment, reducing Syk phosphorylation and thereby dampening pro-phagocytic signalling.\",\n      \"method\": \"Genome-wide CRISPR screen, immunoprecipitation, mass spectrometry, RNA sequencing, immunofluorescence, flow cytometry, in vivo mouse tumour models, knockdown functional assays\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — unbiased CRISPR screen plus reciprocal IP/MS identifying VTN-C1qbp interaction, mechanistic pathway delineated (Shp1/Syk), multiple orthogonal methods including in vivo validation\",\n      \"pmids\": [\"38773982\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"miR-30c regulates VTN protein levels in smooth muscle cells by targeting PAI-1; reduction of PAI-1 by miR-30c leads to decreased VTN expression, establishing a miR-30c → PAI-1 → VTN regulatory axis relevant to vascular disease.\",\n      \"method\": \"Luciferase reporter assay, miRNA transfection, qRT-PCR, western blot, ELISA in SMC and ex vivo plasma\",\n      \"journal\": \"Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — luciferase assay establishes PAI-1 as miR-30c target; VTN regulation downstream confirmed by multiple methods; single lab\",\n      \"pmids\": [\"31760103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"VTN overexpression suppresses pancreatic cancer cell proliferation, invasion, and migration in vitro; VTN expression is linked to immune regulatory pathways and its overexpression in a syngeneic mouse model inhibits tumour growth and synergises with anti-PD1 therapy, suggesting VTN acts as a tumour suppressor and modulator of the immunotherapy response in pancreatic cancer.\",\n      \"method\": \"VTN knockdown/overexpression functional assays (proliferation, invasion, migration), syngeneic mouse subcutaneous tumour model, single-cell RNA sequencing analysis\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — loss-of-function and gain-of-function with defined cellular phenotypes plus in vivo model; mechanism linked to immune regulatory pathways but molecular mechanism only partially defined\",\n      \"pmids\": [\"40433359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Insufficient VTN expression impairs trophoblast cell migration, invasion, and endothelial-like tube formation; mechanistically, VTN overexpression upregulates HEY1 (a Notch signalling downstream target) and reduces LC3II-marked autophagy; HEY1 overexpression alleviates VTN-knockdown-induced autophagy, and autophagy inhibition with 3-MA partially restores function suppressed by VTN knockdown, placing VTN upstream of a HEY1/autophagy pathway in trophoblast biology.\",\n      \"method\": \"VTN knockdown/overexpression in HTR8/SVneo cells and primary EVTs, transcriptome sequencing, LC3II western blot, 3-MA autophagy inhibition rescue experiment, HEY1 overexpression epistasis\",\n      \"journal\": \"Placenta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pathway placement via epistasis (HEY1 overexpression rescues, 3-MA rescues), transcriptome sequencing plus functional assays; single lab\",\n      \"pmids\": [\"40516241\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Vitronectin (VTN) is a multifunctional extracellular matrix glycoprotein that acts through integrin αV-containing complexes (αVβ3, αVβ5, αVβ6) and soluble binding partners (PAI-1 via the SMB domain; C1qbp on macrophages) to regulate cell adhesion, migration, and survival signalling (FAK/Src/ERK); secreted VTN suppresses macrophage phagocytosis via a C1qbp–Shp1–Syk axis, promotes cancer cell radioresistance through ATM-Chk2/ATR-Chk1 DNA-damage pathways, supports trophoblast invasion via a HEY1/autophagy axis, and is itself regulated transcriptionally by a miR-30c/PAI-1 circuit and by binding partners BPIFB1 and FBLN2 that modulate its expression and complex formation.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries from KEEP papers only.\n\n**Classification:**\n\nKEEP (canonical VTN/vitronectin protein):\n- [4] BPIFB1 inhibits NPC via VTN/VIM interactions\n- [5] BPIFB1 inhibits radioresistance via VTN\n- [10] VTN(381-397) fragment interacts with αVβ6 integrin, TGF-β1 activation\n- [11] Candida albicans VN receptors (αvβ3/αvβ5-like) mediating adhesion to VTN\n- [12] PAI-1/vitronectin binding site mapping\n- [15] miR-30c regulates PAI-1/VN interactions\n- [17] FBLN2 binds VTN, regulates FAK signaling in fibrosis\n- [26] Vtn-C1qbp interaction inhibits macrophage phagocytosis\n- [36] VTN role in pancreatic cancer progression\n- [42] VTN/HEY1/autophagy in preeclampsia\n- gene2pubmed [10] PAI-1 blocks αVβ3 binding to vitronectin → SMC migration\n- gene2pubmed [13] uPAR/PAI-1 compete for VN somatomedin B domain\n- gene2pubmed [14] Complete amino acid sequence of vitronectin, RGD cell attachment site\n- gene2pubmed [17] Vitronectin promotes osteogenic differentiation of hMSC\n- gene2pubmed [18] MMP-2 cleaves vitronectin to promote OvCa metastasis\n- gene2pubmed [20] αvβ5 binds basic domain of Tat and vitronectin heparin-binding domain\n- gene2pubmed [24] Purification of integrin αvβ5 as vitronectin receptor\n- gene2pubmed [25] S-protein = vitronectin; molecular cloning\n- gene2pubmed [26] Granzyme B cleaves vitronectin after RGD motif\n- gene2pubmed [27] Crystal structure of somatomedin B domain of vitronectin with PAI-1\n\nEXCLUDE (alias collision, material science VN=vanadium nitride, vestibular neuritis, antibody VN gene, other drugs named VN/xxx, etc.):\n- [1],[6],[7],[8],[13],[22],[30],[34] — VN/124-1, VN/14-1, VN/66-1, VN/87-1 drug compounds\n- [2],[19],[24],[25],[27],[28],[31],[41],[43],[45] — vanadium nitride materials\n- [3] — T15 VN antibody gene family\n- [9] — Brevibacterium VN-15 strain\n- [16] — GaN/VN synthesis chemistry\n- [18] — vestibular neuritis (VN)\n- [20] — hollow Mo/MoSVn nanoreactors\n- [21] — RAMBA VN/12-1 breast cancer drug\n- [23] — equine arteritis virus neutralization (VN) test\n- [29] — Vitex Negundo nanocatalyst\n- [33] — MTHFR VN polymorphism (different gene)\n- [35] — type V-N CRISPR-Cas12n (not VTN)\n- [37] — VN-EGNN graph neural networks\n- [38] — contryphan Vn cone snail peptide\n- [39] — VN/14-1 drug\n- [40] — Hericium VN compound from mushroom\n- [44] — DMN-VN brain network connectivity\n- [46] — VN-NsLAAO fusion protein (liver cancer gene therapy, not vitronectin)\n- [47] — preprint: VTN as biomarker (expression/ML, no mechanistic finding)\n- gene2pubmed [1] — global phosphoproteomics (VTN incidental)\n- gene2pubmed [2],[12] — MGC cDNA sequencing projects\n- gene2pubmed [3],[4],[5],[6],[7] — large interactome/proteome screens (VTN incidental)\n- gene2pubmed [8],[15],[21],[22] — plasma/proteomics surveys\n- gene2pubmed [9] — GO annotation methods\n- gene2pubmed [11] — αvβ3/VEGFR2 cooperation (vitronectin as substrate context only, no VTN mechanism)\n- gene2pubmed [16] — organelle proteomics\n- gene2pubmed [19] — PCK1/INSIG phosphorylation (unrelated)\n- gene2pubmed [23] — integrin αvβ6/fibronectin (VTN incidental)\n- gene2pubmed [28],[29],[30] — cilia proteomics, CFTR interactome, VEGFR2/integrin cooperation\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1985,\n      \"finding\": \"The complete amino acid sequence of human vitronectin was deduced from cDNA clones isolated from a human liver library. The sequence revealed that vitronectin contains the entire 44-amino acid somatomedin B (SMB) peptide at its N-terminus, three potential glycosylation sites, a C-terminal glycosaminoglycan-binding domain rich in basic residues, and an Arg-Gly-Asp (RGD) sequence immediately after the SMB domain that constitutes the cell attachment site, showing functional similarity to fibronectin's cell attachment sequence.\",\n      \"method\": \"cDNA cloning and nucleotide sequencing, oligonucleotide probe screening of lambda gt11 library, cell attachment inhibition assays with synthetic RGD peptides\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — primary sequence determination with functional validation of RGD cell attachment site; foundational study replicated across labs\",\n      \"pmids\": [\"2414098\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1985,\n      \"finding\": \"S-protein (a complement regulatory protein) and vitronectin (serum spreading factor) were shown to be identical proteins by molecular cloning, sequence analysis, and immunological criteria. The single polypeptide chain of 459 amino acids (plus 19-residue leader peptide) encodes the SMB domain at its N-terminus, linking complement regulation, coagulation, and cell-substrate adhesion functions in one molecule.\",\n      \"method\": \"cDNA cloning from pEX expression library screened with monoclonal antibodies, sequence analysis, immunological cross-reactivity assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct molecular identification of protein identity; foundational cloning paper\",\n      \"pmids\": [\"3004934\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"Integrin αvβ5 was purified from human placenta and identified as a vitronectin receptor that binds preferentially to vitronectin (over fibronectin, fibrinogen, or von Willebrand factor). The β5 subunit pairs with the αv subunit and is immunologically and structurally distinct from β3, with the ligand-binding site architecture differing from αvβ3.\",\n      \"method\": \"Immunodepletion of αvβ3 followed by monoclonal antibody affinity chromatography, wheat germ lectin chromatography, Western blot, vitronectin-binding assays, peptide mapping, N-terminal amino acid sequencing\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — purification to homogeneity with direct ligand-binding characterization and structural distinction from paralog\",\n      \"pmids\": [\"1694173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"The vitronectin-binding integrin αvβ5 was shown to bind to the basic heparin-binding domain of vitronectin (sequence KKQRFRHRNRKG) through a divalent cation-independent mechanism, distinct from RGD-mediated integrin binding. This defines an auxiliary integrin-binding specificity for basic amino acid sequences within vitronectin.\",\n      \"method\": \"Affinity chromatography with Tat-derived peptides, immunoprecipitation with anti-integrin antibodies, cell attachment inhibition assays, divalent cation chelation experiments\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — affinity chromatography plus immunoprecipitation with functional cell attachment assays; defines a mechanistically distinct binding mode\",\n      \"pmids\": [\"7682219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Active PAI-1 blocks smooth muscle cell (SMC) migration by competing with integrin αVβ3 for an overlapping binding site on vitronectin, preventing αVβ3-dependent cell motility on the vitronectin matrix. This inhibitory effect is independent of PAI-1's protease inhibitor activity and requires high-affinity PAI-1 binding to vitronectin; formation of a PAI-1/plasminogen activator complex reduces PAI-1 affinity for vitronectin and restores migration.\",\n      \"method\": \"SMC migration assays on vitronectin substrates, blocking antibodies against αVβ3, PAI-1 mutants deficient in protease inhibition but retaining vitronectin binding, PAI-1/uPA complex formation assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods including function-blocking mutants and antibodies; highly cited foundational paper replicated across labs\",\n      \"pmids\": [\"8837777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"uPAR and PAI-1 compete for binding to the same site within the N-terminal somatomedin B (SMB) domain of vitronectin. PAI-1 dissociates VN-bound uPAR and detaches cells from vitronectin substratum in a protease-inhibitor-independent manner, while uPA can rapidly reverse this PAI-1-mediated cell detachment. The uPAR-binding sequence was localized within the central region of the SMB domain.\",\n      \"method\": \"Domain swapping, site-directed mutagenesis of SMB domain, competitive binding assays, cell detachment assays with U937 cells on VN substrates\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis plus competitive binding with cell-based functional readout; replicated finding consistent with complementary PAI-1/αVβ3 paper\",\n      \"pmids\": [\"8830783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The PAI-1/vitronectin interaction maps to two binding regions: the primary high-affinity PAI-1 binding site resides within the N-terminal somatomedin B (SMB) domain of vitronectin, while at least one secondary low-affinity binding site exists in the C-terminal region of vitronectin involved in forming larger PAI-1/Vn complexes. On PAI-1, the region around α-helix E and α-helix F is important for vitronectin binding.\",\n      \"method\": \"Peptide competition assays, domain deletion mapping, mutagenesis studies reviewed across multiple experimental approaches\",\n      \"journal\": \"Biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — systematic domain mapping with multiple approaches but review/summary format; consistent with structural data\",\n      \"pmids\": [\"12437099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The crystal structure (2.3 Å) of the somatomedin B (SMB) domain of vitronectin in complex with PAI-1 revealed the molecular basis of their interaction: vitronectin binding stabilizes the active conformation of PAI-1 by engaging its reactive center loop region. The PAI-1 binding site on the SMB domain sterically overlaps with the binding surfaces for αVβ3/αVβ5 integrins and uPAR, explaining how PAI-1 competitively blocks integrin- and uPAR-mediated cell adhesion and motility.\",\n      \"method\": \"X-ray crystallography at 2.3 Å resolution of SMB domain–PAI-1 complex\",\n      \"journal\": \"Nature structural biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution crystal structure with mechanistic interpretation explaining multiple functional observations; landmark structural paper\",\n      \"pmids\": [\"12808446\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Adhesion of human mesenchymal stem cells (hMSCs) to vitronectin (and collagen I) promotes osteogenic differentiation, with cells on vitronectin showing the greatest induction of mineralized matrix, osteopontin, osteocalcin, collagen I, and alkaline phosphatase expression. hMSCs adhere to vitronectin through distinct integrin receptors compared to other ECM proteins, and ECM contact alone can be sufficient to induce osteogenic differentiation.\",\n      \"method\": \"Cell adhesion assays on purified ECM proteins, integrin-blocking antibodies, osteogenic differentiation marker assays (alkaline phosphatase, mineralization, immunostaining for osteopontin and osteocalcin) over 16-day time course\",\n      \"journal\": \"Journal of biomedicine & biotechnology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean functional assay with multiple differentiation markers and integrin-blocking experiments; single lab study\",\n      \"pmids\": [\"15123885\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Human granzyme B (GrB) efficiently cleaves vitronectin at a site after the Arg-Gly-Asp (RGD) motif within the integrin-binding region, disrupting the integrin–ECM interface. This GrB-mediated cleavage of vitronectin (along with fibronectin and laminin) causes detachment of endothelial cells and other cell types, induces anoikis in endothelial cells, and inhibits tumor cell spreading, migration, and invasion in vitro.\",\n      \"method\": \"In vitro cleavage assays with native and recombinant GrB, cell detachment assays, cell spreading/migration/invasion assays, identification of cleavage site by sequence analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro biochemical cleavage assay with defined cleavage site plus multiple cell-based functional readouts\",\n      \"pmids\": [\"15843372\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"MMP-2 secreted by ovarian cancer (OvCa) cells cleaves vitronectin (and fibronectin) into small fragments that enhance OvCa cell attachment to peritoneal surfaces. This cleavage exposes cryptic binding sites recognized by αVβ3 integrin on OvCa cells, promoting adhesion. MMP-2 inhibition in OvCa cells (but not in host cells) reduced peritoneal adhesion and tumor metastasis in vivo.\",\n      \"method\": \"siRNA knockdown and pharmacological inhibition of MMP-2, in vitro cleavage of ECM proteins, cell adhesion assays to ECM fragments, integrin-blocking antibodies, in vivo mouse peritoneal metastasis model with Mmp2-null mice\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods including genetic KO mouse model plus in vitro biochemical and cell-based assays; identifies specific VTN cleavage mechanism\",\n      \"pmids\": [\"18340378\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"BPIFB1 interacts with vitronectin (VTN) and reduces VTN expression and VTN–integrin αV complex formation in NPC cells, leading to inhibition of the FAK/Src/ERK signalling pathway downstream of VTN. BPIFB1 also attenuates VTN-induced epithelial-mesenchymal transition, thereby inhibiting NPC cell migration, invasion, and lung metastasis.\",\n      \"method\": \"Co-immunoprecipitation coupled with mass spectrometry to identify BPIFB1-binding proteins, western blotting, immunofluorescence, immunohistochemistry, in vitro migration/invasion assays, in vivo lung metastasis mouse model\",\n      \"journal\": \"British journal of cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP/MS identification of interaction confirmed by western blot and functional assays in vitro and in vivo with multiple orthogonal methods\",\n      \"pmids\": [\"29123267\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"VTN promotes NPC cell radioresistance by inducing cell proliferation and survival, G2/M phase arrest, DNA repair, and activation of the ATM-Chk2 and ATR-Chk1 DNA damage response pathways, as well as anti-apoptotic effects after ionizing radiation. BPIFB1 (which binds VTN) inhibits this VTN-mediated radioresistance, improving NPC radiosensitivity.\",\n      \"method\": \"Colony formation and cell survival assays, cell cycle analysis, western blotting for ATM-Chk2 and ATR-Chk1 pathway components, apoptosis assays, overexpression and knockdown of VTN and BPIFB1\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — defined pathway placement (ATM-Chk2, ATR-Chk1) with VTN overexpression/knockdown and functional readouts; single lab\",\n      \"pmids\": [\"29568064\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"miR-30c regulates vitronectin (VN) protein levels in smooth muscle cells via targeting PAI-1: miR-30c reduces PAI-1 expression (validated by luciferase assay demonstrating direct 3'UTR targeting), and reduced PAI-1 leads to decreased VN levels, revealing a miR-30c → PAI-1 → VN regulatory axis relevant to coronary disease.\",\n      \"method\": \"miRNA transfection, luciferase reporter assays for PAI-1 3'UTR targeting, qRT-PCR, western blotting, ELISA for VN and PAI-1 in plasma, in vitro SMC assays\",\n      \"journal\": \"Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — luciferase assay validates direct miR-30c/PAI-1 interaction; VN regulation inferred from downstream PAI-1 effects; single lab\",\n      \"pmids\": [\"31760103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A vitronectin fragment (VTN amino acids 381–397) competes with TGF-β1 for binding to αVβ6 integrin on human fibroblast-like synoviocytes (FLSs). By interacting with αVβ6 on FLS surfaces, this VTN fragment prevents αVβ6-mediated TGF-β1 activation and increases α-SMA expression, revealing that VTN can modulate TGF-β1 bioavailability through integrin competition.\",\n      \"method\": \"Competition binding assay between VTN fragment and αVβ6, flow cytometry and western blot for αVβ6 on primary FLSs, immunohistochemistry of synovial tissue, TGF-β bioassay, α-SMA immunostaining\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct competition assay plus cell-based TGF-β bioassay; multiple orthogonal methods; single lab\",\n      \"pmids\": [\"33526813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FBLN2 (fibulin-2) binds directly to VTN (vitronectin) and negatively regulates its expression in lung fibroblasts. FBLN2 knockdown increases VTN expression, which activates FAK signaling and promotes TGF-β1-induced cell proliferation, migration, MMP2/MMP9 upregulation, and fibrotic marker expression (α-SMA, collagen I, fibronectin); VTN overexpression partially rescues the anti-fibrotic effect of FBLN2 knockdown.\",\n      \"method\": \"Protein co-immunoprecipitation (confirmed FBLN2-VTN interaction), STRING database prediction, western blotting, siRNA knockdown and overexpression, wound-healing assay, CCK-8, immunofluorescence for α-SMA\",\n      \"journal\": \"Tissue & cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — co-IP validates FBLN2-VTN interaction; rescue experiment with VTN overexpression demonstrates pathway position; single lab\",\n      \"pmids\": [\"36608640\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Tumor cell-secreted vitronectin (Vtn) binds to complement C1Q binding protein (C1qbp) on the surface of tumor-associated macrophages. This Vtn–C1qbp interaction inhibits macrophage phagocytosis of tumor cells and shifts macrophages toward an M2-like subtype by facilitating FcγRIIIA/CD16-induced Shp1 recruitment, which reduces Syk phosphorylation, thereby suppressing pro-phagocytic signaling. Vtn knockdown combined with anti-CD47 antibody synergistically enhanced macrophage phagocytosis and reduced tumor growth in vivo.\",\n      \"method\": \"Genome-wide CRISPR screen for anti-phagocytic genes, cell-to-cell interaction database analysis, siRNA knockdown, flow cytometry for phagocytosis and macrophage polarization, RNA sequencing, co-immunoprecipitation, mass spectrometry, immunofluorescence, syngeneic mouse tumor models\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — CRISPR screen discovery validated by Co-IP/MS, mechanistic pathway (Shp1/Syk) defined, confirmed in vivo; multiple orthogonal methods\",\n      \"pmids\": [\"38773982\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"VTN overexpression in pancreatic cancer cells suppresses proliferation, invasion, and migration in vitro, and inhibits tumor growth in a syngeneic mouse model, acting as a tumor suppressor. VTN expression is linked to immune regulatory pathways, and VTN overexpression synergizes with anti-PD1 therapy to enhance antitumor efficacy in vivo.\",\n      \"method\": \"VTN knockdown and overexpression in pancreatic cancer cell lines, proliferation/invasion/migration assays, syngeneic mouse subcutaneous tumor model with anti-PD1 treatment, single-cell RNA sequencing analysis, immune pathway analysis\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — defined cell-autonomous tumor suppressor role with in vivo validation; mechanistic pathway not fully resolved; single lab\",\n      \"pmids\": [\"40433359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Insufficient VTN expression in trophoblast cells impairs migration, invasion, and endothelial-like tube formation via the HEY1/autophagy pathway. VTN overexpression upregulates HEY1 (a Notch signaling downstream target), while VTN knockdown increases LC3II expression indicating enhanced autophagy; HEY1 overexpression alleviates autophagy induced by VTN knockdown, and autophagy inhibition with 3-MA partially restores trophoblast function suppressed by VTN knockdown.\",\n      \"method\": \"Functional assays in HTR8/SVneo cells, HUVECs, and primary EVTs; VTN overexpression/knockdown; transcriptome sequencing; LC3II western blotting for autophagy; 3-MA autophagy inhibitor; HEY1 overexpression rescue experiments\",\n      \"journal\": \"Placenta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — defined pathway (VTN → HEY1 → autophagy suppression) with rescue experiments; multiple cell types tested; single lab\",\n      \"pmids\": [\"40516241\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Vitronectin (VTN) is a multifunctional extracellular matrix and plasma glycoprotein whose N-terminal somatomedin B (SMB) domain serves as a high-affinity docking site for PAI-1 (stabilizing its active conformation) and competitively overlaps with binding sites for αVβ3/αVβ5 integrins and uPAR, thereby controlling cell adhesion and migration; its central RGD sequence mediates integrin-dependent cell attachment and can be cleaved by MMP-2 and granzyme B to generate pro-adhesive fragments or induce anoikis; its heparin-binding domain engages αvβ5 through a divalent cation-independent basic-sequence mechanism; and beyond matrix roles, VTN acts as a 'don't eat me' signal by binding C1qbp on macrophages to suppress phagocytosis via Shp1-mediated Syk dephosphorylation, while also regulating trophoblast function through a HEY1/autophagy pathway and modulating TGF-β1 bioavailability through competition with αVβ6 integrin.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"Vitronectin (VTN) is a secreted extracellular matrix glycoprotein that signals through αV-containing integrins and soluble binding partners to regulate cell adhesion, migration, survival, and immune evasion. VTN forms complexes with integrin αV (via αVβ3/αVβ5/αVβ6) and activates FAK/Src/ERK signalling to promote epithelial-mesenchymal transition, cell migration, and fibroblast proliferation, while its N-terminal somatomedin B domain provides the high-affinity docking site for PAI-1, whose levels in turn control VTN abundance through a miR-30c→PAI-1→VTN regulatory axis [PMID:29123267, PMID:12437099, PMID:31760103, PMID:36608640]. Tumour cell-secreted VTN binds C1qbp on tumour-associated macrophages, recruiting Shp1 to FcγRIIIA/CD16 and suppressing Syk-dependent phagocytic signalling, thereby enabling immune evasion [PMID:38773982]. VTN also promotes radioresistance via ATM-Chk2/ATR-Chk1 DNA-damage checkpoint activation, modulates TGF-β1 bioavailability by competing for αVβ6 integrin binding, and supports trophoblast invasion through a HEY1/autophagy pathway [PMID:29568064, PMID:33526813, PMID:40516241].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Defining the molecular interface between VTN and PAI-1 established that the somatomedin B (SMB) domain is the primary high-affinity binding site, providing the structural basis for understanding how VTN–PAI-1 complexes assemble.\",\n      \"evidence\": \"Domain-mapping studies using mutagenesis, domain deletion, and biochemical binding assays (reviewed)\",\n      \"pmids\": [\"12437099\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Functional significance of the secondary low-affinity C-terminal binding site in vivo remains unclear\",\n        \"Crystal structure of the full-length VTN–PAI-1 complex not resolved\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identifying VTN as an integrin αV partner that activates FAK/Src/ERK signalling and drives EMT in cancer cells revealed the core pro-migratory signalling axis downstream of extracellular VTN.\",\n      \"evidence\": \"Co-IP/mass spectrometry, immunofluorescence, in vitro migration/invasion assays, and in vivo lung metastasis model in nasopharyngeal carcinoma cells\",\n      \"pmids\": [\"29123267\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Specific integrin αV heterodimer (αVβ3/β5/β6) mediating EMT signalling was not resolved\",\n        \"Contribution of BPIFB1-mediated VTN downregulation versus complex disruption was not separated\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrating that VTN activates ATM-Chk2 and ATR-Chk1 DNA-damage checkpoints after ionising radiation expanded VTN's role from adhesion/migration to DNA-damage response and radioresistance.\",\n      \"evidence\": \"Colony formation, cell cycle analysis, apoptosis assays, and western blotting of checkpoint kinases following VTN overexpression/knockdown in NPC cells\",\n      \"pmids\": [\"29568064\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct versus integrin-mediated activation of ATM/ATR by VTN not distinguished\",\n        \"In vivo radioresistance phenotype not tested\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identification of a miR-30c→PAI-1→VTN regulatory axis showed that VTN protein levels are indirectly controlled by a microRNA circuit through its binding partner PAI-1, linking vascular disease-relevant gene regulation to VTN abundance.\",\n      \"evidence\": \"Luciferase reporter assay confirming PAI-1 as miR-30c target, miRNA transfection, western blot, and ELISA in smooth muscle cells\",\n      \"pmids\": [\"31760103\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism by which PAI-1 reduction decreases VTN protein (transcriptional versus post-translational stabilisation) not resolved\",\n        \"In vivo vascular disease relevance not directly tested\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A VTN fragment (aa 381–397) was shown to compete with αVβ6 integrin for TGF-β1 activation, demonstrating that VTN can modulate fibrosis by controlling latent TGF-β1 bioavailability.\",\n      \"evidence\": \"Competition binding assay, TGF-β bioassay, flow cytometry, and immunohistochemistry in fibroblast-like synoviocytes from osteoarthritis patients\",\n      \"pmids\": [\"33526813\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether full-length VTN exerts the same competitive effect as the synthetic fragment is untested\",\n        \"Relevance to pulmonary or hepatic fibrosis not examined\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"FBLN2 was identified as a VTN-binding partner that negatively regulates VTN expression and thereby controls TGF-β1-induced FAK-dependent fibroblast proliferation and fibrosis, placing VTN as an effector downstream of FBLN2.\",\n      \"evidence\": \"Co-immunoprecipitation, knockdown/overexpression epistasis rescue experiments in pulmonary fibroblasts\",\n      \"pmids\": [\"36608640\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism by which FBLN2 binding reduces VTN expression (transcriptional or degradation) not defined\",\n        \"Single lab; independent replication lacking\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"A genome-wide CRISPR screen revealed that tumour-secreted VTN binds macrophage-surface C1qbp and inhibits phagocytosis through a Shp1–Syk axis, establishing VTN as an immune checkpoint-like molecule in the tumour microenvironment.\",\n      \"evidence\": \"CRISPR screen, reciprocal IP/MS, RNA-seq, flow cytometry, and in vivo mouse tumour models\",\n      \"pmids\": [\"38773982\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Therapeutic blockade of VTN–C1qbp interaction not tested\",\n        \"Whether VTN–C1qbp axis operates beyond the tumour microenvironment (e.g. infection) is unknown\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Two studies extended VTN's roles: VTN overexpression suppresses pancreatic cancer growth and synergises with anti-PD1 therapy, while insufficient VTN impairs trophoblast invasion through a HEY1/autophagy pathway, broadening VTN's functional repertoire to immune modulation and placental development.\",\n      \"evidence\": \"Knockdown/overexpression functional assays, syngeneic mouse models with anti-PD1, transcriptome sequencing, and autophagy epistasis experiments in trophoblast cells\",\n      \"pmids\": [\"40433359\", \"40516241\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Molecular link between VTN and HEY1 upregulation (direct transcriptional target or indirect) not defined\",\n        \"Opposing tumour-suppressive versus pro-tumour roles of VTN across cancer types not reconciled\",\n        \"Receptor mediating VTN's immune-modulatory effect in pancreatic cancer not identified\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How VTN signals context-dependently through different integrin heterodimers and non-integrin receptors (C1qbp) to produce opposing outcomes (pro-migratory/pro-survival versus tumour-suppressive/anti-phagocytic) in different tissue contexts remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No structural model of full-length VTN in complex with any receptor\",\n        \"Integrin-specific versus C1qbp-specific downstream signalling branches not dissected in a single system\",\n        \"In vivo genetic models (conditional knockouts) examining VTN in immunity, fibrosis, and placentation are lacking\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 3]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"GO:0031012\", \"supporting_discovery_ids\": [0, 3, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 4, 5]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [5, 7]},\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [0, 3, 4]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"ITGAV\",\n      \"SERPINE1\",\n      \"C1QBP\",\n      \"FBLN2\",\n      \"BPIFB1\",\n      \"HEY1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"Vitronectin (VTN) is a multifunctional plasma and extracellular matrix glycoprotein that integrates cell adhesion, pericellular proteolysis, complement regulation, and immune modulation through distinct structural domains. Its N-terminal somatomedin B (SMB) domain serves as a high-affinity binding site for PAI-1, stabilizing PAI-1's active conformation; this site sterically overlaps with binding surfaces for αVβ3/αVβ5 integrins and uPAR, enabling PAI-1 to competitively block integrin- and uPAR-dependent cell adhesion and migration [PMID:12808446, PMID:8837777, PMID:8830783]. The central RGD motif mediates canonical integrin-dependent cell attachment and is a substrate for proteolytic cleavage by granzyme B and MMP-2, which can induce anoikis or expose cryptic pro-adhesive fragments that promote metastatic adhesion [PMID:15843372, PMID:18340378]. Beyond matrix functions, tumor-secreted VTN binds C1qbp on macrophages and suppresses phagocytosis through Shp1-mediated Syk dephosphorylation, acting as an anti-phagocytic signal that synergizes with CD47 blockade when depleted [PMID:38773982].\",\n  \"teleology\": [\n    {\n      \"year\": 1985,\n      \"claim\": \"Molecular cloning resolved vitronectin's primary structure and established its domain architecture — the N-terminal somatomedin B domain, the RGD cell-attachment sequence, and the C-terminal heparin-binding domain — while simultaneously proving that S-protein (complement regulator) and serum spreading factor are the same polypeptide.\",\n      \"evidence\": \"cDNA cloning from human liver libraries, nucleotide sequencing, and immunological cross-reactivity assays\",\n      \"pmids\": [\"2414098\", \"3004934\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No receptor or binding-partner specificity defined at this stage\",\n        \"Three-dimensional structure of VTN domains not yet determined\"\n      ]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Identification of αVβ5 as a second vitronectin receptor, and discovery that αVβ5 engages the heparin-binding domain through a divalent-cation-independent basic-sequence mechanism, established that VTN uses multiple integrin-binding modes beyond the RGD motif.\",\n      \"evidence\": \"Purification of αVβ5 from human placenta with ligand-binding assays; affinity chromatography with Tat-derived peptides and cation-chelation experiments\",\n      \"pmids\": [\"1694173\", \"7682219\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of the αVβ5–heparin-binding domain interaction not resolved\",\n        \"Relative contributions of RGD versus heparin-binding domain engagement in vivo unknown\"\n      ]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Functional studies revealed that PAI-1 and uPAR compete for an overlapping binding site on the SMB domain, and that PAI-1 binding — independent of its protease-inhibitory activity — blocks αVβ3-dependent cell migration and detaches uPAR-anchored cells, establishing VTN as a switchable adhesion platform.\",\n      \"evidence\": \"SMC migration assays with PAI-1 mutants; domain-swap mutagenesis and competitive binding with U937 cell detachment readout\",\n      \"pmids\": [\"8837777\", \"8830783\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Atomic-resolution mapping of the competitive binding interface not yet available\",\n        \"In vivo relevance of PAI-1/uPAR competition on VTN in wound healing or thrombosis not demonstrated\"\n      ]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"The 2.3 Å crystal structure of the SMB domain–PAI-1 complex provided the atomic explanation for how VTN stabilizes PAI-1's active conformation and why PAI-1 binding sterically excludes integrins and uPAR, unifying a decade of competitive-binding observations.\",\n      \"evidence\": \"X-ray crystallography of the SMB–PAI-1 complex at 2.3 Å resolution\",\n      \"pmids\": [\"12808446\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Full-length VTN structure remains unsolved\",\n        \"Conformational changes in intact VTN upon PAI-1 engagement not resolved\"\n      ]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Proteolytic processing of VTN by granzyme B and MMP-2 was shown to remodel the adhesion landscape: GrB cleaves after the RGD motif to induce anoikis, while MMP-2 generates fragments that expose cryptic αVβ3-binding sites promoting metastatic adhesion, demonstrating that VTN fragmentation is a biologically active event.\",\n      \"evidence\": \"In vitro cleavage assays with defined sites, cell detachment and anoikis assays (GrB); MMP-2 siRNA/KO with peritoneal metastasis mouse model and integrin-blocking antibodies\",\n      \"pmids\": [\"15843372\", \"18340378\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Precise MMP-2 cleavage site(s) on VTN not mapped at single-residue resolution\",\n        \"Whether GrB cleavage of VTN occurs in immune-mediated tissue surveillance in vivo is unconfirmed\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A VTN fragment (aa 381–397) was found to compete with TGF-β1 for αVβ6 integrin binding on fibroblast-like synoviocytes, revealing a mechanism by which VTN modulates TGF-β1 bioavailability beyond its classical adhesion roles.\",\n      \"evidence\": \"Competition binding assay between VTN peptide and αVβ6; TGF-β bioassay and α-SMA immunostaining in primary FLSs\",\n      \"pmids\": [\"33526813\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether endogenous VTN fragments of this size are generated in vivo is not established\",\n        \"No structural data for VTN fragment–αVβ6 interaction\",\n        \"Relevance to arthritis or fibrosis pathology not validated in animal models\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"A genome-wide CRISPR screen identified VTN as a tumor-secreted anti-phagocytic signal: VTN binds C1qbp on macrophages, recruits Shp1 via FcγRIIIA/CD16, dephosphorylates Syk, and suppresses phagocytosis — defining a 'don't eat me' pathway parallel to the CD47–SIRPα axis.\",\n      \"evidence\": \"CRISPR screen, Co-IP/MS for VTN–C1qbp interaction, Shp1/Syk phosphorylation assays, syngeneic tumor models with combined VTN KD and anti-CD47\",\n      \"pmids\": [\"38773982\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether C1qbp is the sole macrophage receptor for VTN's anti-phagocytic function is not resolved\",\n        \"Clinical relevance of VTN–C1qbp axis in human tumors not yet validated\",\n        \"Structural basis of VTN–C1qbp binding unknown\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"VTN was linked to trophoblast function through a HEY1/autophagy pathway and to pancreatic cancer suppression with immunotherapy synergy, expanding its roles to placental biology and tumor-immune regulation beyond adhesion.\",\n      \"evidence\": \"VTN overexpression/knockdown in trophoblast cells with HEY1 rescue and autophagy inhibitor experiments; syngeneic pancreatic cancer model with anti-PD1 treatment\",\n      \"pmids\": [\"40516241\", \"40433359\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism connecting extracellular VTN to intracellular HEY1/Notch signaling not defined\",\n        \"Tumor-suppressive versus pro-tumorigenic roles of VTN across cancer types remain contradictory and unresolved\",\n        \"Whether VTN's immunotherapy synergy operates through the C1qbp–Shp1 axis or a distinct mechanism is unknown\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The full-length three-dimensional structure of VTN, the conformational switch between its monomeric plasma form and its multimeric ECM-deposited form, and the integration of its anti-phagocytic (C1qbp) and adhesion (integrin) functions in the tumor microenvironment remain major unresolved questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No full-length VTN crystal or cryo-EM structure exists\",\n        \"Monomer-to-multimer conversion mechanism not structurally characterized\",\n        \"Relative in vivo contributions of VTN's anti-phagocytic versus adhesion functions in tumor immune evasion undefined\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": [0, 2, 3, 8, 9]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [4, 5, 7, 16]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4, 5, 14, 16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031012\", \"supporting_discovery_ids\": [0, 8, 9, 10]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0, 1, 16, 17]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [0, 8, 9, 10]},\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [4, 5, 7]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [16, 17]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [11, 15, 16]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [0, 2, 3, 8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"SERPINE1\",\n      \"PLAUR\",\n      \"ITGAV\",\n      \"ITGB3\",\n      \"ITGB5\",\n      \"C1QBP\",\n      \"FBLN2\",\n      \"BPIFB1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}