{"gene":"F2","run_date":"2026-06-14T21:06:12+00:00","timeline":{"discoveries":[{"year":2022,"finding":"Cryo-EM structure of the prothrombin-prothrombinase complex revealed that the Gla domains of fXa and prothrombin align on a membrane plane with the C1 and C2 domains of fVa; the 672ESTVMATRKMHDRLEPEDEE691 segment of the fVa A2 domain closes on the fXa protease domain like a lid to fix orientation of the active site; and the 696YDYQNRL702 segment binds prothrombin, sequestering R271 against D697 and directing R320 toward the fXa active site to establish the meizothrombin activation pathway.","method":"Cryo-EM structure determination (4.1-Å resolution) of fVa-fXa-prothrombin complex on nanodiscs","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1 / Strong — near-atomic cryo-EM structure with two independent complexes (free and substrate-bound), providing direct structural evidence for the activation mechanism","pmids":["35427420"],"is_preprint":false},{"year":2013,"finding":"Enzymic specificity of prothrombinase for prothrombin is dominated by exosite binding interactions between substrate and enzyme rather than specific recognition of sequences flanking the scissile bond; additionally, thrombin can reversibly interconvert between zymogen-like and proteinase-like forms depending on the complement of ligands bound, establishing ligand-dependent allostery of the proteinase.","method":"Biochemical and biophysical analysis of prothrombinase-substrate interactions including exosite mutagenesis and kinetic studies reviewed from primary experiments","journal":"Journal of thrombosis and haemostasis : JTH","confidence":"High","confidence_rationale":"Tier 1 / Strong — synthesis of multiple independent biochemical/biophysical reconstitution and mutagenesis studies, replicated across labs","pmids":["23809130"],"is_preprint":false},{"year":2019,"finding":"Limited proteolysis with chymotrypsin attacking W468 in the flexible autolysis loop of the protease domain revealed that prothrombin exists in two alternative conformations (open and closed): the closed form promotes cleavage at R320 (meizothrombin pathway) and the open form promotes cleavage at R271 (prethrombin-2 pathway); R296 in the A chain of the protease domain was identified as a critical link between the allosteric open-closed equilibrium and exposure of the R271 and R320 cleavage sites; removal of the Gla domain, kringles, or linkers stabilizes the open form and switches the activation pathway.","method":"Limited proteolysis with chymotrypsin combined with domain deletion experiments on recombinant prothrombin variants","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct biochemical assay with defined domain-deletion variants and site-specific readout, single lab with multiple orthogonal approaches","pmids":["30992526"],"is_preprint":false},{"year":2024,"finding":"Heterozygous prothrombin mutations Phe382Ser and Phe382Leu severely impaired thrombomodulin-binding ability of the resulting thrombin, leading to markedly reduced protein C activation; Asp597Tyr mutation mildly reduced both antithrombin inactivation and protein C activation; Arg596Gln showed the highest thrombin generation potential and Arg541Trp the second highest among thrombosis-associated mutations; all five mutations potentiate coagulation by either conferring antithrombin resistance and/or impairing the protein C pathway.","method":"Recombinant mutant prothrombin expression in HEK293T cells, thrombin generation test, functional assays for antithrombin inactivation and protein C activation","journal":"Thrombosis and haemostasis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — recombinant expression with functional assays in single lab; multiple mutations tested with consistent mechanistic readouts","pmids":["38914130"],"is_preprint":false},{"year":1988,"finding":"The bovine prothrombin gene comprises 14 exons interrupted by 13 introns spanning ~15.4 kb; exons encoding the prepro-leader peptide and Gla domain are similar in organization to factor IX and protein C genes (likely arising from gene duplication and exon shuffling), while exons encoding the kringles and serine protease domain differ in organization from homologous regions in other coagulation factor genes, indicating introns were inserted after initial gene duplication events.","method":"Partial DNA sequence analysis of cloned bovine prothrombin gene including 5' and 3' flanking sequences and all intron-exon junctions","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct sequencing of the cloned gene with comparative structural analysis; single study but comprehensive coverage","pmids":["3379642"],"is_preprint":false},{"year":2001,"finding":"Homozygous deletion of prothrombin in mice results in partial embryonic lethality (~50% die during mid-gestation at E9.5–11.5 due to loss of yolk sac vascular integrity from failed coagulation); surviving null mice die soon after birth from excessive bleeding, establishing that prothrombin is essential for both developmental hemostasis and postnatal survival.","method":"Gene targeting (knockout) in mice with phenotypic analysis of embryonic and neonatal lethality","journal":"Frontiers in bioscience : a journal and virtual library","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic knockout with defined developmental and hemostatic phenotypes, corroborated by independent reports in the corpus (PMID 9714898)","pmids":["11171556","9714898"],"is_preprint":false},{"year":1997,"finding":"A novel membrane-associated prothrombin activator (MAPA) was found in normal murine liver, kidney, lung, and heart; MAPA activity increased ~100-fold in CCl4-injured liver and ~5-fold in HgCl2-injured kidney within 48 and 12 hours respectively; phospholipids are required for MAPA-mediated activation of prothrombin on cell surfaces, suggesting MAPA activates prothrombin locally during tissue injury and participates in inflammation and regeneration.","method":"Enzymatic activity assays on tissue fractions from normal and chemically injured mouse tissues; comparison with fibroblast-derived MAPA; phospholipid requirement assay","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct enzymatic assay in tissue fractions with defined substrate and cofactor requirement; single lab, multiple tissue conditions","pmids":["9276460"],"is_preprint":false},{"year":2023,"finding":"A FRET-based plasma assay monitoring cleavage at R271 of prothrombin demonstrated that FV availability strongly influences the rate of prothrombin activation; thrombin-catalyzed feedback reactions amplifying coagulation play an important role in generating sufficient FVa for prothrombinase assembly; congenital deficiencies of FVIII and FIX significantly slow cleavage at R271; FXI deficiency perturbs R271 cleavage only when coagulation is triggered along the intrinsic pathway.","method":"Förster resonance energy transfer (FRET) assay in plasma depleted of specific coagulation factors (FV, FIX, FVIII, FXI)","journal":"Journal of thrombosis and haemostasis : JTH","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct functional assay with defined factor-depleted plasmas, single lab but multiple orthogonal depletion conditions","pmids":["36931601"],"is_preprint":false},{"year":2025,"finding":"Prothrombin knockdown via antisense oligonucleotide in a rat puromycin aminonucleoside glomerular disease model significantly reduced prothrombin colocalization to podocytes, podocyte foot process effacement, podocytopathy, podocytopenia, and proteinuria with improved plasma albumin; conversely, intravenous prothrombin infusions (hyperprothrombinemia) significantly increased podocytopathy and proteinuria, establishing that circulating prothrombin/thrombin drives podocyte injury in glomerular disease.","method":"Antisense oligonucleotide knockdown and protein infusion in rat PAN-induced glomerular disease model; histology, electron microscopy, co-localization imaging, proteinuria measurement","journal":"Journal of the American Society of Nephrology : JASN","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo loss- and gain-of-function with multiple orthogonal readouts; single lab","pmids":["40152945"],"is_preprint":false},{"year":2023,"finding":"Recombinant prothrombin carrying the Belgrade mutation (c.1787G>A, located in the antithrombin and sodium-binding site) showed no significant difference from wild-type in overall hemostasis potential, fibrinolysis, or fibrin network density, but did show significant differences in slope and slope time parameters of clot formation kinetics and in fibrin fiber thickness, indicating the mutation affects clot architecture but not net fibrinolytic capacity.","method":"Recombinant mutant prothrombin expressed in HEK293T cells; reconstituted plasma assays (OHP, clot turbidity), confocal and electron microscopy of fibrin clots","journal":"International journal of laboratory hematology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — recombinant protein with multiple functional assays and imaging; single lab","pmids":["37918971"],"is_preprint":false},{"year":2025,"finding":"ApoM overexpression in HepG2 hepatocytes and in mice decreased secreted/plasma prothrombin levels and increased intracellular/hepatic prothrombin; ApoM knockout had the opposite effect; Arid5B knockdown increased prothrombin secretion into culture medium and decreased cellular levels, antagonizing ApoM's inhibitory effect; this ApoM-mediated suppression of prothrombin secretion is independent of S1P receptors and operates through upregulation of Arid5B.","method":"ApoM overexpression and knockout in mice and HepG2 cells; Arid5B knockdown; RNA sequencing; S1P receptor knockdown; prothrombin measurement in plasma/medium and cells","journal":"Thrombosis and haemostasis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function for both ApoM and Arid5B with mechanistic linkage; single lab with multiple orthogonal approaches","pmids":["40719152"],"is_preprint":false},{"year":1992,"finding":"Direct sequencing of exons I and II of the prothrombin gene in PIVKA-II-secreting hepatocellular carcinoma cell lines (PLC/PRF/5 and huH-2) and fresh HCC tumor samples found no mutations in the Gla domain, leader sequence carboxylase recognition site, or splice sites; a single synonymous nucleotide change (nt.554 A→G) was detected, establishing that PIVKA-II production in HCC is not caused by mutation in these regions of the prothrombin gene.","method":"PCR amplification and direct DNA sequencing of prothrombin gene exons I and II, plus cDNA sequencing from HCC cell lines and fresh frozen tumor samples","journal":"Cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct sequencing across multiple samples establishing a negative result; single study","pmids":["1309675"],"is_preprint":false},{"year":2026,"finding":"Chemical denaturation studies using single-molecule and ensemble spectroscopic techniques showed that under strongly denaturing conditions the four domains of prothrombin (Gla, kringle 1, kringle 2, protease) separate and the protein becomes elongated; refolding proceeds through acquisition of secondary and tertiary structure and hydrophobic core organization, followed by formation of interdomain contacts that stabilize the closed conformation; proper domain folding is required for formation of the interdomain contacts defining the physiologically predominant closed form.","method":"Chemical denaturation with single-molecule and ensemble spectroscopic techniques (FRET/fluorescence) on recombinant prothrombin","journal":"Journal of thrombosis and haemostasis : JTH","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — rigorous biophysical methods (single-molecule + ensemble spectroscopy) but single study, not yet replicated","pmids":["42173288"],"is_preprint":false}],"current_model":"Prothrombin (F2) is a multidomain zymogen activated to thrombin by the prothrombinase complex (fXa/fVa/Ca2+/phospholipids) via cleavage at R320 (meizothrombin pathway, favored by the closed conformation) or R271 (prethrombin-2 pathway, favored by the open conformation); the cryo-EM structure shows that fVa's A2 domain orients the fXa active site and directs R320 for cleavage while sequestering R271, enzymic specificity is dominated by exosite interactions rather than scissile-bond recognition, thrombin itself can reversibly interconvert between zymogen-like and proteinase-like states depending on bound ligands, thrombosis-associated mutations impair antithrombin inactivation or protein C activation, and prothrombin is essential for developmental and postnatal hemostasis in vivo as demonstrated by complete knockout lethality in mice."},"narrative":{"mechanistic_narrative":"Prothrombin (F2) is the zymogen precursor of thrombin and is essential for both developmental and postnatal hemostasis, as homozygous knockout in mice causes mid-gestation lethality from failed yolk-sac vascular integrity and neonatal death from hemorrhage [PMID:11171556, PMID:9714898]. It is activated by the prothrombinase complex (fXa/fVa on a phospholipid membrane), and a cryo-EM structure of the prothrombin-prothrombinase complex shows that fVa's A2 domain closes like a lid on the fXa protease to fix active-site orientation while a YDYQNRL segment sequesters R271 against D697 and directs R320 toward the fXa active site, establishing the meizothrombin activation pathway [PMID:35427420]. Activation specificity is governed predominantly by exosite binding between substrate and enzyme rather than recognition of the scissile bond, and thrombin reversibly interconverts between zymogen-like and proteinase-like states depending on bound ligands [PMID:23809130]. Prothrombin itself populates two conformations: the closed form favors R320 cleavage (meizothrombin) and the open form favors R271 cleavage (prethrombin-2), with R296 linking the open-closed equilibrium to cleavage-site exposure and removal of the Gla domain, kringles, or linkers stabilizing the open form [PMID:30992526]; proper interdomain folding is required to stabilize the physiologically predominant closed conformation [PMID:42173288]. Thrombosis-associated mutations potentiate coagulation by conferring antithrombin resistance and/or impairing the protein C pathway, including Phe382 substitutions that disrupt thrombomodulin binding and reduce protein C activation [PMID:38914130]. Beyond its hemostatic role, circulating prothrombin/thrombin drives podocyte injury in glomerular disease [PMID:40152945], and hepatic prothrombin secretion is suppressed by ApoM acting through Arid5B [PMID:40719152].","teleology":[{"year":1988,"claim":"Resolving how the prothrombin gene is organized established its evolutionary relationship to other vitamin-K-dependent coagulation factors and the modular origin of its domains.","evidence":"Partial DNA sequencing of the cloned bovine prothrombin gene with intron-exon junction analysis","pmids":["3379642"],"confidence":"Medium","gaps":["Does not address protein function or activation mechanism","Bovine gene; human regulatory architecture not directly characterized here"]},{"year":1992,"claim":"Tested whether abnormal PIVKA-II prothrombin in hepatocellular carcinoma arises from coding mutations, ruling out Gla-domain and carboxylase-recognition-site lesions as the cause.","evidence":"PCR and direct sequencing of prothrombin exons I/II and cDNA from HCC cell lines and tumors","pmids":["1309675"],"confidence":"Medium","gaps":["Negative result; mechanism of PIVKA-II production left unresolved","Limited to two exons and a small sample set"]},{"year":1997,"claim":"Identified a membrane-associated prothrombin activator induced by tissue injury, indicating prothrombin can be activated locally outside classical plasma coagulation during inflammation and regeneration.","evidence":"Enzymatic activity assays on fractions from normal and chemically injured mouse tissues with phospholipid-requirement testing","pmids":["9276460"],"confidence":"Medium","gaps":["Molecular identity of MAPA not defined","Physiological role in inflammation/regeneration inferred, not demonstrated"]},{"year":2001,"claim":"Established that prothrombin is non-redundantly required in vivo by showing its loss causes embryonic and neonatal lethality, linking it to both developmental vascular integrity and postnatal hemostasis.","evidence":"Gene targeting (knockout) in mice with embryonic and neonatal phenotypic analysis","pmids":["11171556","9714898"],"confidence":"High","gaps":["Does not separate developmental coagulation from possible non-hemostatic roles","Molecular cause of yolk-sac defect not dissected at the cellular level"]},{"year":2013,"claim":"Reframed prothrombin activation specificity as exosite-driven rather than scissile-bond-driven and established that thrombin is an allosteric, ligand-responsive proteinase.","evidence":"Synthesis of biochemical/biophysical reconstitution and exosite mutagenesis studies","pmids":["23809130"],"confidence":"High","gaps":["Structural basis of exosite engagement not yet visualized at this stage","Does not define how cofactors direct cleavage-site choice"]},{"year":2019,"claim":"Demonstrated that prothrombin populates open and closed conformations that dictate which scissile bond (R320 vs R271) is cleaved, identifying R296 as the allosteric link.","evidence":"Limited chymotryptic proteolysis with domain-deletion variants of recombinant prothrombin","pmids":["30992526"],"confidence":"Medium","gaps":["Single lab; equilibrium populations under physiological conditions not quantified","Coupling to cofactor binding inferred indirectly"]},{"year":2022,"claim":"Provided the structural mechanism by which fVa orients fXa and positions prothrombin to enforce the meizothrombin pathway, explaining cofactor-directed cleavage-site selection.","evidence":"4.1-Å cryo-EM of the fVa-fXa-prothrombin complex on nanodiscs","pmids":["35427420"],"confidence":"High","gaps":["Captures one assembly state; dynamics of pathway switching not resolved","Does not address how mutations remodel the interface"]},{"year":2023,"claim":"Connected upstream coagulation factor availability to prothrombin activation kinetics, showing FV supply and thrombin feedback control R271 cleavage in plasma.","evidence":"FRET assay monitoring R271 cleavage in factor-depleted (FV, FVIII, FIX, FXI) plasmas","pmids":["36931601"],"confidence":"Medium","gaps":["Focuses on R271; meizothrombin (R320) pathway contribution not tracked","Single-lab assay system"]},{"year":2023,"claim":"Assessed how a sodium/antithrombin-binding-site mutation (Belgrade) alters clot properties, showing effects on clot architecture without changing net fibrinolytic capacity.","evidence":"Recombinant mutant prothrombin in reconstituted plasma assays with clot turbidity and microscopy","pmids":["37918971"],"confidence":"Medium","gaps":["Clinical thrombotic phenotype not established","Single-lab functional characterization"]},{"year":2024,"claim":"Defined the molecular routes by which thrombosis-associated prothrombin mutations potentiate coagulation, separating antithrombin resistance from protein C pathway impairment.","evidence":"Recombinant mutant expression in HEK293T with thrombin generation and antithrombin/protein C functional assays","pmids":["38914130"],"confidence":"Medium","gaps":["Heterozygous physiological context approximated in vitro","Structural basis of thrombomodulin-binding loss not shown"]},{"year":2025,"claim":"Extended prothrombin biology beyond hemostasis by showing circulating prothrombin/thrombin drives podocyte injury, and identified ApoM-Arid5B as a hepatic regulator of prothrombin secretion.","evidence":"ASO knockdown and infusion in a rat glomerular disease model; ApoM/Arid5B gain- and loss-of-function in HepG2 and mice","pmids":["40152945","40719152"],"confidence":"Medium","gaps":["Direct podocyte receptor/effector mechanism not defined","ApoM-Arid5B regulatory link characterized in single labs"]},{"year":2026,"claim":"Showed that the physiologically predominant closed conformation depends on hierarchical domain folding and the formation of stabilizing interdomain contacts.","evidence":"Chemical denaturation with single-molecule and ensemble spectroscopy on recombinant prothrombin","pmids":["42173288"],"confidence":"Medium","gaps":["Single study, not yet replicated","Folding pathway in cellular context not validated"]},{"year":null,"claim":"How conformational equilibrium, cofactor engagement, and disease mutations are integrated to control the choice between meizothrombin and prethrombin-2 pathways in vivo remains incompletely defined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No in vivo measurement linking conformational state to pathway choice","Identity and regulation of tissue prothrombin activators undefined","Mechanism of non-hemostatic prothrombin signaling in target tissues unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[0,6]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[5,8,10]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,6]}],"pathway":[{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[0,5,7]}],"complexes":["prothrombinase complex"],"partners":["F5","F10"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P00734","full_name":"Prothrombin","aliases":["Coagulation factor II"],"length_aa":622,"mass_kda":70.0,"function":"Thrombin, which cleaves bonds after Arg and Lys, converts fibrinogen to fibrin and activates factors V, VII, VIII, XIII, and, in complex with thrombomodulin, protein C. Functions in blood homeostasis, inflammation and wound healing. Activates coagulation factor XI (F11); activation is promoted by the contact with negatively charged surfaces (PubMed:2019570, PubMed:21976677). Triggers the production of pro-inflammatory cytokines, such as MCP-1/CCL2 and IL8/CXCL8, in endothelial cells (PubMed:30568593, PubMed:9780208)","subcellular_location":"Secreted, extracellular space","url":"https://www.uniprot.org/uniprotkb/P00734/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/F2","classification":"Not Classified","n_dependent_lines":23,"n_total_lines":1208,"dependency_fraction":0.01903973509933775},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/F2","total_profiled":1310},"omim":[{"mim_id":"621543","title":"UBIQUITIN-ASSOCIATED PROTEIN 1-LIKE PROTEIN; UBAP1L","url":"https://www.omim.org/entry/621543"},{"mim_id":"621535","title":"SPINOCEREBELLAR ATAXIA 52; SCA52","url":"https://www.omim.org/entry/621535"},{"mim_id":"621424","title":"LEUKOENCEPHALOPATHY WITHOUT LACUNAE, ADULT-ONSET; LUCAO","url":"https://www.omim.org/entry/621424"},{"mim_id":"621349","title":"PEROXIREDOXIN-LIKE 2B; PRXL2B","url":"https://www.omim.org/entry/621349"},{"mim_id":"621125","title":"CILIARY DYSKINESIA, PRIMARY, 54; CILD54","url":"https://www.omim.org/entry/621125"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in single","driving_tissues":[{"tissue":"liver","ntpm":1138.4}],"url":"https://www.proteinatlas.org/search/F2"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P00734","domains":[{"cath_id":"2.40.20.10","chopping":"52-197","consensus_level":"medium","plddt":89.3003,"start":52,"end":197},{"cath_id":"2.40.20.10","chopping":"214-292","consensus_level":"medium","plddt":91.6213,"start":214,"end":292},{"cath_id":"2.40.10.10","chopping":"334-620","consensus_level":"medium","plddt":90.2657,"start":334,"end":620}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P00734","model_url":"https://alphafold.ebi.ac.uk/files/AF-P00734-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P00734-F1-predicted_aligned_error_v6.png","plddt_mean":83.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=F2","jax_strain_url":"https://www.jax.org/strain/search?query=F2"},"sequence":{"accession":"P00734","fasta_url":"https://rest.uniprot.org/uniprotkb/P00734.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P00734/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P00734"}},"corpus_meta":[{"pmid":"23809130","id":"PMC_23809130","title":"The 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and injured murine tissues.","date":"1997","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/9276460","citation_count":5,"is_preprint":false},{"pmid":"3530900","id":"PMC_3530900","title":"The prothrombin gene.","date":"1986","source":"Haemostasis","url":"https://pubmed.ncbi.nlm.nih.gov/3530900","citation_count":4,"is_preprint":false},{"pmid":"38309434","id":"PMC_38309434","title":"Prothrombin conversion and thrombin decay in patients with cirrhosis-role of prothrombin and antithrombin deficiencies.","date":"2024","source":"Journal of thrombosis and haemostasis : JTH","url":"https://pubmed.ncbi.nlm.nih.gov/38309434","citation_count":3,"is_preprint":false},{"pmid":"11171556","id":"PMC_11171556","title":"Gene targeting in hemostasis. Prothrombin.","date":"2001","source":"Frontiers in bioscience : a journal and virtual library","url":"https://pubmed.ncbi.nlm.nih.gov/11171556","citation_count":3,"is_preprint":false},{"pmid":"40152945","id":"PMC_40152945","title":"Effects of Prothrombin on Podocytopathy and Proteinuria in Glomerular Disease.","date":"2025","source":"Journal of the American Society of Nephrology : JASN","url":"https://pubmed.ncbi.nlm.nih.gov/40152945","citation_count":2,"is_preprint":false},{"pmid":"37918971","id":"PMC_37918971","title":"Effect of prothrombin Belgrade mutation, causing antithrombin resistance, on fibrin clot properties.","date":"2023","source":"International journal of laboratory hematology","url":"https://pubmed.ncbi.nlm.nih.gov/37918971","citation_count":2,"is_preprint":false},{"pmid":"41049578","id":"PMC_41049578","title":"Anti-Phosphatidylserine/Prothrombin Antibodies Identify a Distinct Form of ITP.","date":"2025","source":"EJHaem","url":"https://pubmed.ncbi.nlm.nih.gov/41049578","citation_count":2,"is_preprint":false},{"pmid":"36931601","id":"PMC_36931601","title":"Monitoring prothrombin activation in plasma through loss of Förster resonance energy transfer.","date":"2023","source":"Journal of thrombosis and haemostasis : JTH","url":"https://pubmed.ncbi.nlm.nih.gov/36931601","citation_count":2,"is_preprint":false},{"pmid":"12373814","id":"PMC_12373814","title":"[Prothrombin time and its standardization].","date":"2002","source":"Rinsho byori. The Japanese journal of clinical pathology","url":"https://pubmed.ncbi.nlm.nih.gov/12373814","citation_count":1,"is_preprint":false},{"pmid":"11075405","id":"PMC_11075405","title":"[G20210A transition in the prothrombin gene and venous thromboembolic disease].","date":"2000","source":"La Revue de medecine interne","url":"https://pubmed.ncbi.nlm.nih.gov/11075405","citation_count":1,"is_preprint":false},{"pmid":"34669612","id":"PMC_34669612","title":"Prothrombin complex concentrates in cardiac surgery: where are we?","date":"2022","source":"Current opinion in anaesthesiology","url":"https://pubmed.ncbi.nlm.nih.gov/34669612","citation_count":1,"is_preprint":false},{"pmid":"42173288","id":"PMC_42173288","title":"The folding pathway of prothrombin.","date":"2026","source":"Journal of thrombosis and haemostasis : JTH","url":"https://pubmed.ncbi.nlm.nih.gov/42173288","citation_count":0,"is_preprint":false},{"pmid":"31704834","id":"PMC_31704834","title":"Prothrombin 3'end Gene Variants in Patients With Sporadic Colon Adenocarcinoma.","date":"2019","source":"Anticancer research","url":"https://pubmed.ncbi.nlm.nih.gov/31704834","citation_count":0,"is_preprint":false},{"pmid":"38464017","id":"PMC_38464017","title":"Prothrombin Knockdown Protects Podocytes and Reduces Proteinuria in Glomerular Disease.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/38464017","citation_count":0,"is_preprint":false},{"pmid":"30970085","id":"PMC_30970085","title":"Genetic correlation between Prothrombin G20210A polymorphism and retinal vein occlusion risk.","date":"2019","source":"Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas","url":"https://pubmed.ncbi.nlm.nih.gov/30970085","citation_count":0,"is_preprint":false},{"pmid":"39011238","id":"PMC_39011238","title":"Prothrombin G20210A Mutation is Rare but not Absent Among North Indian Patients with Thromboembolic Events.","date":"2024","source":"Indian journal of hematology & blood transfusion : an official journal of Indian Society of Hematology and Blood Transfusion","url":"https://pubmed.ncbi.nlm.nih.gov/39011238","citation_count":0,"is_preprint":false},{"pmid":"36820119","id":"PMC_36820119","title":"Prothrombin Gene Mutation as a Teaching Tool: An Autobiographical Case Report.","date":"2023","source":"Cureus","url":"https://pubmed.ncbi.nlm.nih.gov/36820119","citation_count":0,"is_preprint":false},{"pmid":"40719152","id":"PMC_40719152","title":"Hepatic Apolipoprotein M Suppresses Hepatocyte Secretion of Prothrombin by Upregulating Arid5B.","date":"2025","source":"Thrombosis and haemostasis","url":"https://pubmed.ncbi.nlm.nih.gov/40719152","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"sonnet_model":"claude-sonnet-4-6","sonnet_input_tokens":19585,"sonnet_output_tokens":3782,"sonnet_usd":0.1155,"sonnet_elapsed_seconds":76.2,"stage1_stop_reason":"end_turn"},"stage2":{"opus_model":"claude-opus-4-8","opus_input_tokens":11513,"opus_output_tokens":3682,"opus_usd":0.1496,"opus_elapsed_seconds":40.9,"stage2_stop_reason":"end_turn"},"total_usd":0.2651},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2022,\n      \"finding\": \"Cryo-EM structure of the prothrombin-prothrombinase complex revealed that the Gla domains of fXa and prothrombin align on a membrane plane with the C1 and C2 domains of fVa; the 672ESTVMATRKMHDRLEPEDEE691 segment of the fVa A2 domain closes on the fXa protease domain like a lid to fix orientation of the active site; and the 696YDYQNRL702 segment binds prothrombin, sequestering R271 against D697 and directing R320 toward the fXa active site to establish the meizothrombin activation pathway.\",\n      \"method\": \"Cryo-EM structure determination (4.1-Å resolution) of fVa-fXa-prothrombin complex on nanodiscs\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — near-atomic cryo-EM structure with two independent complexes (free and substrate-bound), providing direct structural evidence for the activation mechanism\",\n      \"pmids\": [\"35427420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Enzymic specificity of prothrombinase for prothrombin is dominated by exosite binding interactions between substrate and enzyme rather than specific recognition of sequences flanking the scissile bond; additionally, thrombin can reversibly interconvert between zymogen-like and proteinase-like forms depending on the complement of ligands bound, establishing ligand-dependent allostery of the proteinase.\",\n      \"method\": \"Biochemical and biophysical analysis of prothrombinase-substrate interactions including exosite mutagenesis and kinetic studies reviewed from primary experiments\",\n      \"journal\": \"Journal of thrombosis and haemostasis : JTH\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — synthesis of multiple independent biochemical/biophysical reconstitution and mutagenesis studies, replicated across labs\",\n      \"pmids\": [\"23809130\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Limited proteolysis with chymotrypsin attacking W468 in the flexible autolysis loop of the protease domain revealed that prothrombin exists in two alternative conformations (open and closed): the closed form promotes cleavage at R320 (meizothrombin pathway) and the open form promotes cleavage at R271 (prethrombin-2 pathway); R296 in the A chain of the protease domain was identified as a critical link between the allosteric open-closed equilibrium and exposure of the R271 and R320 cleavage sites; removal of the Gla domain, kringles, or linkers stabilizes the open form and switches the activation pathway.\",\n      \"method\": \"Limited proteolysis with chymotrypsin combined with domain deletion experiments on recombinant prothrombin variants\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct biochemical assay with defined domain-deletion variants and site-specific readout, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"30992526\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Heterozygous prothrombin mutations Phe382Ser and Phe382Leu severely impaired thrombomodulin-binding ability of the resulting thrombin, leading to markedly reduced protein C activation; Asp597Tyr mutation mildly reduced both antithrombin inactivation and protein C activation; Arg596Gln showed the highest thrombin generation potential and Arg541Trp the second highest among thrombosis-associated mutations; all five mutations potentiate coagulation by either conferring antithrombin resistance and/or impairing the protein C pathway.\",\n      \"method\": \"Recombinant mutant prothrombin expression in HEK293T cells, thrombin generation test, functional assays for antithrombin inactivation and protein C activation\",\n      \"journal\": \"Thrombosis and haemostasis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — recombinant expression with functional assays in single lab; multiple mutations tested with consistent mechanistic readouts\",\n      \"pmids\": [\"38914130\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1988,\n      \"finding\": \"The bovine prothrombin gene comprises 14 exons interrupted by 13 introns spanning ~15.4 kb; exons encoding the prepro-leader peptide and Gla domain are similar in organization to factor IX and protein C genes (likely arising from gene duplication and exon shuffling), while exons encoding the kringles and serine protease domain differ in organization from homologous regions in other coagulation factor genes, indicating introns were inserted after initial gene duplication events.\",\n      \"method\": \"Partial DNA sequence analysis of cloned bovine prothrombin gene including 5' and 3' flanking sequences and all intron-exon junctions\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct sequencing of the cloned gene with comparative structural analysis; single study but comprehensive coverage\",\n      \"pmids\": [\"3379642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Homozygous deletion of prothrombin in mice results in partial embryonic lethality (~50% die during mid-gestation at E9.5–11.5 due to loss of yolk sac vascular integrity from failed coagulation); surviving null mice die soon after birth from excessive bleeding, establishing that prothrombin is essential for both developmental hemostasis and postnatal survival.\",\n      \"method\": \"Gene targeting (knockout) in mice with phenotypic analysis of embryonic and neonatal lethality\",\n      \"journal\": \"Frontiers in bioscience : a journal and virtual library\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic knockout with defined developmental and hemostatic phenotypes, corroborated by independent reports in the corpus (PMID 9714898)\",\n      \"pmids\": [\"11171556\", \"9714898\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"A novel membrane-associated prothrombin activator (MAPA) was found in normal murine liver, kidney, lung, and heart; MAPA activity increased ~100-fold in CCl4-injured liver and ~5-fold in HgCl2-injured kidney within 48 and 12 hours respectively; phospholipids are required for MAPA-mediated activation of prothrombin on cell surfaces, suggesting MAPA activates prothrombin locally during tissue injury and participates in inflammation and regeneration.\",\n      \"method\": \"Enzymatic activity assays on tissue fractions from normal and chemically injured mouse tissues; comparison with fibroblast-derived MAPA; phospholipid requirement assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct enzymatic assay in tissue fractions with defined substrate and cofactor requirement; single lab, multiple tissue conditions\",\n      \"pmids\": [\"9276460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"A FRET-based plasma assay monitoring cleavage at R271 of prothrombin demonstrated that FV availability strongly influences the rate of prothrombin activation; thrombin-catalyzed feedback reactions amplifying coagulation play an important role in generating sufficient FVa for prothrombinase assembly; congenital deficiencies of FVIII and FIX significantly slow cleavage at R271; FXI deficiency perturbs R271 cleavage only when coagulation is triggered along the intrinsic pathway.\",\n      \"method\": \"Förster resonance energy transfer (FRET) assay in plasma depleted of specific coagulation factors (FV, FIX, FVIII, FXI)\",\n      \"journal\": \"Journal of thrombosis and haemostasis : JTH\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct functional assay with defined factor-depleted plasmas, single lab but multiple orthogonal depletion conditions\",\n      \"pmids\": [\"36931601\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Prothrombin knockdown via antisense oligonucleotide in a rat puromycin aminonucleoside glomerular disease model significantly reduced prothrombin colocalization to podocytes, podocyte foot process effacement, podocytopathy, podocytopenia, and proteinuria with improved plasma albumin; conversely, intravenous prothrombin infusions (hyperprothrombinemia) significantly increased podocytopathy and proteinuria, establishing that circulating prothrombin/thrombin drives podocyte injury in glomerular disease.\",\n      \"method\": \"Antisense oligonucleotide knockdown and protein infusion in rat PAN-induced glomerular disease model; histology, electron microscopy, co-localization imaging, proteinuria measurement\",\n      \"journal\": \"Journal of the American Society of Nephrology : JASN\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo loss- and gain-of-function with multiple orthogonal readouts; single lab\",\n      \"pmids\": [\"40152945\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Recombinant prothrombin carrying the Belgrade mutation (c.1787G>A, located in the antithrombin and sodium-binding site) showed no significant difference from wild-type in overall hemostasis potential, fibrinolysis, or fibrin network density, but did show significant differences in slope and slope time parameters of clot formation kinetics and in fibrin fiber thickness, indicating the mutation affects clot architecture but not net fibrinolytic capacity.\",\n      \"method\": \"Recombinant mutant prothrombin expressed in HEK293T cells; reconstituted plasma assays (OHP, clot turbidity), confocal and electron microscopy of fibrin clots\",\n      \"journal\": \"International journal of laboratory hematology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — recombinant protein with multiple functional assays and imaging; single lab\",\n      \"pmids\": [\"37918971\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ApoM overexpression in HepG2 hepatocytes and in mice decreased secreted/plasma prothrombin levels and increased intracellular/hepatic prothrombin; ApoM knockout had the opposite effect; Arid5B knockdown increased prothrombin secretion into culture medium and decreased cellular levels, antagonizing ApoM's inhibitory effect; this ApoM-mediated suppression of prothrombin secretion is independent of S1P receptors and operates through upregulation of Arid5B.\",\n      \"method\": \"ApoM overexpression and knockout in mice and HepG2 cells; Arid5B knockdown; RNA sequencing; S1P receptor knockdown; prothrombin measurement in plasma/medium and cells\",\n      \"journal\": \"Thrombosis and haemostasis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function for both ApoM and Arid5B with mechanistic linkage; single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"40719152\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"Direct sequencing of exons I and II of the prothrombin gene in PIVKA-II-secreting hepatocellular carcinoma cell lines (PLC/PRF/5 and huH-2) and fresh HCC tumor samples found no mutations in the Gla domain, leader sequence carboxylase recognition site, or splice sites; a single synonymous nucleotide change (nt.554 A→G) was detected, establishing that PIVKA-II production in HCC is not caused by mutation in these regions of the prothrombin gene.\",\n      \"method\": \"PCR amplification and direct DNA sequencing of prothrombin gene exons I and II, plus cDNA sequencing from HCC cell lines and fresh frozen tumor samples\",\n      \"journal\": \"Cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct sequencing across multiple samples establishing a negative result; single study\",\n      \"pmids\": [\"1309675\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Chemical denaturation studies using single-molecule and ensemble spectroscopic techniques showed that under strongly denaturing conditions the four domains of prothrombin (Gla, kringle 1, kringle 2, protease) separate and the protein becomes elongated; refolding proceeds through acquisition of secondary and tertiary structure and hydrophobic core organization, followed by formation of interdomain contacts that stabilize the closed conformation; proper domain folding is required for formation of the interdomain contacts defining the physiologically predominant closed form.\",\n      \"method\": \"Chemical denaturation with single-molecule and ensemble spectroscopic techniques (FRET/fluorescence) on recombinant prothrombin\",\n      \"journal\": \"Journal of thrombosis and haemostasis : JTH\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — rigorous biophysical methods (single-molecule + ensemble spectroscopy) but single study, not yet replicated\",\n      \"pmids\": [\"42173288\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Prothrombin (F2) is a multidomain zymogen activated to thrombin by the prothrombinase complex (fXa/fVa/Ca2+/phospholipids) via cleavage at R320 (meizothrombin pathway, favored by the closed conformation) or R271 (prethrombin-2 pathway, favored by the open conformation); the cryo-EM structure shows that fVa's A2 domain orients the fXa active site and directs R320 for cleavage while sequestering R271, enzymic specificity is dominated by exosite interactions rather than scissile-bond recognition, thrombin itself can reversibly interconvert between zymogen-like and proteinase-like states depending on bound ligands, thrombosis-associated mutations impair antithrombin inactivation or protein C activation, and prothrombin is essential for developmental and postnatal hemostasis in vivo as demonstrated by complete knockout lethality in mice.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"Prothrombin (F2) is the zymogen precursor of thrombin and is essential for both developmental and postnatal hemostasis, as homozygous knockout in mice causes mid-gestation lethality from failed yolk-sac vascular integrity and neonatal death from hemorrhage [#5]. It is activated by the prothrombinase complex (fXa/fVa on a phospholipid membrane), and a cryo-EM structure of the prothrombin-prothrombinase complex shows that fVa's A2 domain closes like a lid on the fXa protease to fix active-site orientation while a YDYQNRL segment sequesters R271 against D697 and directs R320 toward the fXa active site, establishing the meizothrombin activation pathway [#0]. Activation specificity is governed predominantly by exosite binding between substrate and enzyme rather than recognition of the scissile bond, and thrombin reversibly interconverts between zymogen-like and proteinase-like states depending on bound ligands [#1]. Prothrombin itself populates two conformations: the closed form favors R320 cleavage (meizothrombin) and the open form favors R271 cleavage (prethrombin-2), with R296 linking the open-closed equilibrium to cleavage-site exposure and removal of the Gla domain, kringles, or linkers stabilizing the open form [#2]; proper interdomain folding is required to stabilize the physiologically predominant closed conformation [#12]. Thrombosis-associated mutations potentiate coagulation by conferring antithrombin resistance and/or impairing the protein C pathway, including Phe382 substitutions that disrupt thrombomodulin binding and reduce protein C activation [#3]. Beyond its hemostatic role, circulating prothrombin/thrombin drives podocyte injury in glomerular disease [#8], and hepatic prothrombin secretion is suppressed by ApoM acting through Arid5B [#10].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 1988,\n      \"claim\": \"Resolving how the prothrombin gene is organized established its evolutionary relationship to other vitamin-K-dependent coagulation factors and the modular origin of its domains.\",\n      \"evidence\": \"Partial DNA sequencing of the cloned bovine prothrombin gene with intron-exon junction analysis\",\n      \"pmids\": [\"3379642\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not address protein function or activation mechanism\", \"Bovine gene; human regulatory architecture not directly characterized here\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Tested whether abnormal PIVKA-II prothrombin in hepatocellular carcinoma arises from coding mutations, ruling out Gla-domain and carboxylase-recognition-site lesions as the cause.\",\n      \"evidence\": \"PCR and direct sequencing of prothrombin exons I/II and cDNA from HCC cell lines and tumors\",\n      \"pmids\": [\"1309675\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Negative result; mechanism of PIVKA-II production left unresolved\", \"Limited to two exons and a small sample set\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Identified a membrane-associated prothrombin activator induced by tissue injury, indicating prothrombin can be activated locally outside classical plasma coagulation during inflammation and regeneration.\",\n      \"evidence\": \"Enzymatic activity assays on fractions from normal and chemically injured mouse tissues with phospholipid-requirement testing\",\n      \"pmids\": [\"9276460\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular identity of MAPA not defined\", \"Physiological role in inflammation/regeneration inferred, not demonstrated\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Established that prothrombin is non-redundantly required in vivo by showing its loss causes embryonic and neonatal lethality, linking it to both developmental vascular integrity and postnatal hemostasis.\",\n      \"evidence\": \"Gene targeting (knockout) in mice with embryonic and neonatal phenotypic analysis\",\n      \"pmids\": [\"11171556\", \"9714898\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not separate developmental coagulation from possible non-hemostatic roles\", \"Molecular cause of yolk-sac defect not dissected at the cellular level\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Reframed prothrombin activation specificity as exosite-driven rather than scissile-bond-driven and established that thrombin is an allosteric, ligand-responsive proteinase.\",\n      \"evidence\": \"Synthesis of biochemical/biophysical reconstitution and exosite mutagenesis studies\",\n      \"pmids\": [\"23809130\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of exosite engagement not yet visualized at this stage\", \"Does not define how cofactors direct cleavage-site choice\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrated that prothrombin populates open and closed conformations that dictate which scissile bond (R320 vs R271) is cleaved, identifying R296 as the allosteric link.\",\n      \"evidence\": \"Limited chymotryptic proteolysis with domain-deletion variants of recombinant prothrombin\",\n      \"pmids\": [\"30992526\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; equilibrium populations under physiological conditions not quantified\", \"Coupling to cofactor binding inferred indirectly\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Provided the structural mechanism by which fVa orients fXa and positions prothrombin to enforce the meizothrombin pathway, explaining cofactor-directed cleavage-site selection.\",\n      \"evidence\": \"4.1-Å cryo-EM of the fVa-fXa-prothrombin complex on nanodiscs\",\n      \"pmids\": [\"35427420\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Captures one assembly state; dynamics of pathway switching not resolved\", \"Does not address how mutations remodel the interface\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected upstream coagulation factor availability to prothrombin activation kinetics, showing FV supply and thrombin feedback control R271 cleavage in plasma.\",\n      \"evidence\": \"FRET assay monitoring R271 cleavage in factor-depleted (FV, FVIII, FIX, FXI) plasmas\",\n      \"pmids\": [\"36931601\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Focuses on R271; meizothrombin (R320) pathway contribution not tracked\", \"Single-lab assay system\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Assessed how a sodium/antithrombin-binding-site mutation (Belgrade) alters clot properties, showing effects on clot architecture without changing net fibrinolytic capacity.\",\n      \"evidence\": \"Recombinant mutant prothrombin in reconstituted plasma assays with clot turbidity and microscopy\",\n      \"pmids\": [\"37918971\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Clinical thrombotic phenotype not established\", \"Single-lab functional characterization\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined the molecular routes by which thrombosis-associated prothrombin mutations potentiate coagulation, separating antithrombin resistance from protein C pathway impairment.\",\n      \"evidence\": \"Recombinant mutant expression in HEK293T with thrombin generation and antithrombin/protein C functional assays\",\n      \"pmids\": [\"38914130\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Heterozygous physiological context approximated in vitro\", \"Structural basis of thrombomodulin-binding loss not shown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended prothrombin biology beyond hemostasis by showing circulating prothrombin/thrombin drives podocyte injury, and identified ApoM-Arid5B as a hepatic regulator of prothrombin secretion.\",\n      \"evidence\": \"ASO knockdown and infusion in a rat glomerular disease model; ApoM/Arid5B gain- and loss-of-function in HepG2 and mice\",\n      \"pmids\": [\"40152945\", \"40719152\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct podocyte receptor/effector mechanism not defined\", \"ApoM-Arid5B regulatory link characterized in single labs\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Showed that the physiologically predominant closed conformation depends on hierarchical domain folding and the formation of stabilizing interdomain contacts.\",\n      \"evidence\": \"Chemical denaturation with single-molecule and ensemble spectroscopy on recombinant prothrombin\",\n      \"pmids\": [\"42173288\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single study, not yet replicated\", \"Folding pathway in cellular context not validated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How conformational equilibrium, cofactor engagement, and disease mutations are integrated to control the choice between meizothrombin and prethrombin-2 pathways in vivo remains incompletely defined.\",\n      \"evidence\": null,\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vivo measurement linking conformational state to pathway choice\", \"Identity and regulation of tissue prothrombin activators undefined\", \"Mechanism of non-hemostatic prothrombin signaling in target tissues unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [5, 8, 10]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [0, 5, 7]}\n    ],\n    \"complexes\": [\"prothrombinase complex\"],\n    \"partners\": [\"F5\", \"F10\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win"}}