{"gene":"F5","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":2002,"finding":"FV Cambridge (Arg306Thr) and FV Hong Kong (Arg306Gly) both lack APC cleavage at Arg306, resulting in partial FVa inactivation in the absence of protein S, but near-complete inactivation in the presence of protein S due to stimulation of cleavage at Arg679. Both variants also show slightly impaired APC cofactor activity in FVIIIa degradation.","method":"Recombinant expression of FV mutants, purified protein functional assays (APC-mediated inactivation curves, prothrombinase assay, FVIIIa degradation assay) with and without protein S","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted recombinant mutants, multiple orthogonal functional assays, mechanistic cleavage-site mapping","pmids":["12091344"],"is_preprint":false},{"year":1996,"finding":"Factor V Leiden (FV:Q506, Arg506Gln) is activated normally by thrombin or factor Xa, but its impaired inactivation by activated protein C (APC) results in lifelong hypercoagulability. Intact FV also functions as an APC cofactor with protein S in the regulation of FVIIIa in the Xase complex.","method":"Biochemical characterization of purified mutant FV, functional coagulation assays, APC resistance testing in patient and reconstituted plasma","journal":"Annals of hematology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiply replicated across labs, foundational mechanistic characterization of FV Leiden cleavage defect","pmids":["8624369"],"is_preprint":false},{"year":2000,"finding":"Mutations in the R2 allele of the F5 gene (including A6755G, Ala2194Gly in the C2 domain) are associated with a relative increase in the more thrombogenic, more glycosylated FV isoform (FV1) compared with the less glycosylated FV2, suggesting that genetically determined differential glycosylation of FV alters the FV1/FV2 ratio.","method":"Molecular genetics sequencing, chromatographic separation of FV isoforms, quantitative FV1/FV2 ratio analysis in homozygous R2 carriers vs controls","journal":"Thrombosis and haemostasis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — single lab, two orthogonal methods (sequencing + chromatographic functional characterization), but no direct mutagenesis reconstitution","pmids":["10744138"],"is_preprint":false},{"year":2003,"finding":"FV Leiden increases thrombin generation in the context of severe FVII deficiency, particularly in the presence of APC, by resisting APC-mediated FVa inactivation. This was confirmed in FV-FVII doubly deficient plasma reconstituted with purified normal FV or FV Leiden.","method":"Thrombin generation assays in patient plasma and in doubly deficient plasma reconstituted with purified FV variants; FXa and FVa generation measurements with and without APC","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1 / Moderate — purified protein reconstitution in defined plasma systems, multiple orthogonal coagulation assays, single lab","pmids":["12881304"],"is_preprint":false},{"year":2009,"finding":"The LMAN1-MCFD2 protein complex functions as a cargo receptor for ER-to-Golgi transport of coagulation FV and FVIII. MCFD2 is an EF-hand domain protein forming a calcium-dependent heteromeric complex with LMAN1; missense mutations in MCFD2 EF-hand domains abolish LMAN1 interaction and cause combined FV+FVIII deficiency. The B domain of FVIII may be important in mediating interaction with the complex.","method":"Genetic identification of LMAN1 and MCFD2 mutations in F5F8D patients; co-immunoprecipitation; cell biological trafficking assays; mutation analysis","journal":"British journal of haematology","confidence":"High","confidence_rationale":"Tier 2 / Strong — replicated across multiple labs, reciprocal co-IP of LMAN1-MCFD2 complex, genetic and biochemical evidence converging on ER-to-Golgi transport function","pmids":["19183188"],"is_preprint":false},{"year":2011,"finding":"LMAN1-deficient mice show ~50% reduction in plasma FV, plasma FVIII, and platelet FV levels, confirming LMAN1's role as a cargo receptor for ER-to-Golgi transport of FV and FVIII. LMAN1 deficiency causes ER distension and accumulation of α1-antitrypsin and GRP78 in hepatocytes, but cathepsin C and Z levels are unaffected. LMAN1 deficiency had no effect on COPII vesicle formation in vitro.","method":"Lman1 knockout mouse generation; plasma FV and FVIII quantification; platelet FV measurement; liver ultrastructure analysis; in vitro COPII vesicle budding assay; Western blot","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — genetic knockout model with multiple orthogonal biochemical readouts, includes negative control (COPII assay), confirms prior human genetic data","pmids":["21795745"],"is_preprint":false},{"year":2003,"finding":"FV New Brunswick (Ala221Val) does not impair FV synthesis, secretion, or procoagulant FXa cofactor function, nor does it affect APC-mediated inactivation of FVa. Instead, the mutation reduces stability of the activated FVa heterodimer by increasing the rate of light and heavy chain dissociation at 37°C, which explains the FV deficiency phenotype.","method":"Recombinant expression of Ala221Val and control mutants (Ala221Gly, Glu275Gln, Cys220Ala/Cys301Ala); pulse-chase experiments; ELISA; plasma clotting assays; purified prothrombinase assays; thermal stability assays","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted recombinant mutants with multiple orthogonal functional assays and mechanistic pulse-chase in single lab","pmids":["12714495"],"is_preprint":false},{"year":2015,"finding":"A novel gain-of-function mutation in F5 (c.C2588G) causes aberrant splicing producing a shortened FV protein (FV Amsterdam, missing 623 amino acids from the B domain) that binds tissue factor pathway inhibitor (TFPI), prolonging its half-life and increasing circulating TFPI levels, leading to severely reduced thrombin generation and a bleeding phenotype.","method":"Whole exome sequencing; thrombin generation assay; TFPI level measurement; F5 cDNA splicing analysis; TFPI binding assay","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Moderate — exome sequencing combined with functional TFPI binding, thrombin generation assays and mRNA splicing analysis in a single rigorous study","pmids":["25634741"],"is_preprint":false},{"year":2021,"finding":"A novel 832 bp deletion within F5 exon 13 (F5-Atlanta) enhances alternative splicing at the East Texas (ET) splice sites, producing FV-Short, an isoform with a truncated B domain that binds TFPIα with high affinity, thereby elevating circulating TFPIα levels and causing severe bleeding. This demonstrates that cis-acting regulatory sequences within F5 exon 13 control alternative splicing and regulate circulating FV-Short/TFPIα levels.","method":"Patient plasma TFPI quantification; F5 cDNA sequencing; minigene heterologous expression in mammalian cells; FV isoform Western blotting","journal":"Journal of thrombosis and haemostasis","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — minigene functional validation in mammalian cells plus patient plasma biochemistry, mechanistic model confirmed by recapitulation in heterologous expression","pmids":["33773040"],"is_preprint":false},{"year":2023,"finding":"FV-Short, a naturally occurring splice isoform of FV with a truncated B domain, forms a high-affinity trimolecular complex with TFPIα and protein S. FV-Short and protein S synergistically stimulate the FXa-inhibitory activity of TFPIα. In East Texas bleeding disorder, the concentration of the FV-Short/protein S/TFPIα complex is increased ~10-fold above normal (~0.2 nM), providing an elevated anticoagulant threshold and causing bleeding.","method":"Biochemical characterization of FV-Short/TFPIα/protein S complex; FXa inhibition assays; quantification of circulating complex in East Texas patients vs controls","journal":"Journal of thrombosis and haemostasis","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mechanistic complex characterization replicated across studies, trimolecular complex biochemistry with functional FXa inhibition assay","pmids":["36746318"],"is_preprint":false},{"year":2016,"finding":"FV Bonn (Ala512Val) causes APC resistance by slowing APC-mediated cleavage at Arg506 (the primary APC cleavage site), with protein S largely restoring inactivation. FV Bonn also shows ~24% reduced APC cofactor activity in FVIIIa inactivation and enhanced procoagulant activity (increased activation rate, possibly enhanced interaction with activated FX) in the absence of APC. In silico modeling places Ala512 in the same loop as Arg506.","method":"Recombinant FV Bonn expression; APTT-based APC sensitivity ratio; thrombin generation-based APC sensitivity ratio; purified FVa inactivation kinetics with/without protein S; FVIIIa cofactor activity assay; in silico structural modeling","journal":"Journal of thrombosis and haemostasis","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted recombinant mutant with multiple orthogonal functional assays, single lab","pmids":["27090446"],"is_preprint":false},{"year":2014,"finding":"An apparently synonymous F5 variant (c.1281C>G in exon 8) causes FV deficiency by activating a cryptic donor splice site and abolishing an exonic splicing enhancer, producing aberrantly spliced mRNA lacking the last 18 nucleotides of exon 8. The resulting mutant FV protein (lacking amino acids 427–432) is poorly secreted and non-functional. A mutation-specific morpholino antisense oligonucleotide partially corrected the splicing defect.","method":"F5 gene sequencing; mRNA analysis; bioinformatics splice-site prediction; F5 minigene model in COS-1 cells; morpholino antisense rescue; COS-1 cell expression of mutant protein","journal":"Haemophilia","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — minigene functional validation plus recombinant protein secretion/activity assay and antisense rescue, single lab with multiple orthogonal methods","pmids":["25470420"],"is_preprint":false},{"year":2013,"finding":"A deep-intronic F5 mutation (c.1296+268A>G) activates a cryptic donor splice site causing severe FV deficiency. Mutation-specific antisense morpholino oligonucleotides (MO) and engineered U7 small nuclear RNA constructs dose-dependently restored correct F5 mRNA splicing in COS-1 and HepG2 cells transfected with a F5 minigene, and in patient-derived megakaryocytes ex vivo, where FV protein expression was restored.","method":"F5 minigene transfection in COS-1 and HepG2 cells; antisense morpholino and U7snRNA rescue experiments; patient-derived megakaryocyte differentiation from circulating progenitors; immunofluorescence for FV protein","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple cell systems including patient-derived cells, minigene model with antisense rescue, single lab with orthogonal methods","pmids":["24085767"],"is_preprint":false},{"year":2023,"finding":"MCFD2 is the primary cargo-binding subunit of the LMAN1-MCFD2 complex for FV and FVIII ER-to-Golgi transport: LMAN1 carbohydrate-binding mutants can still partially rescue FV/FVIII secretion, whereas overexpression of MCFD2 alone (wild-type or mutant) rescues FV/FVIII secretion in LMAN1-deficient cells, indicating LMAN1 serves primarily as a shuttling carrier for MCFD2. Overexpression of both proteins together does not further increase FV/FVIII secretion, indicating the LMAN1-MCFD2 complex is not rate-limiting for ER-Golgi transport of FV/FVIII.","method":"LMAN1- and MCFD2-deficient cell lines (HEK293T, HepG2, HCT116); FV/FVIII secretion assays; rescue experiments with wild-type and mutant LMAN1/MCFD2 overexpression; functional pathogenicity assays for missense mutations","journal":"Blood advances","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple cell lines, LMAN1/MCFD2 mutant rescue experiments with clear mechanistic conclusion, single lab with orthogonal approaches","pmids":["36490287"],"is_preprint":false},{"year":2021,"finding":"FV Besançon (Ala2086Asp) favors a 'closed conformation' of the C2 domain, impairing FV(a) binding to phospholipids. The mutation causes severe FV deficiency by greatly reducing FV secretion. Residual FVa Besançon shows slightly unfavorable prothrombinase kinetics but markedly reduced APC-mediated inactivation (in the presence of protein S), and completely abolished APC cofactor activity, leading to a paradoxical hypercoagulable/thrombotic state despite severe FV deficiency. Thrombin generation in platelet-rich plasma was higher than in controls and extremely APC resistant.","method":"Patient plasma thrombin generation (platelet-poor and platelet-rich); FV:C, FV:Ag, and TFPI assays; F5 sequencing; recombinant FVBesançon expression in COS cells; prothrombinase kinetics; APC-mediated FVa inactivation assay; in silico C2 domain modeling","journal":"Journal of thrombosis and haemostasis","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — recombinant protein reconstitution with multiple functional assays and structural modeling, single lab, clinically validated","pmids":["33605529"],"is_preprint":false}],"current_model":"Coagulation Factor V (F5) is a procofactor that, upon activation to FVa by thrombin or factor Xa, serves as an essential cofactor in the prothrombinase complex (FXa/FVa) for prothrombin activation; FVa is inactivated by activated protein C (APC) via sequential cleavages at Arg506 and Arg306/Arg679, with protein S as a cofactor—a mechanism abrogated by FV Leiden (Arg506Gln) causing APC resistance and thrombosis; intact FV also functions as an APC cofactor (with protein S) in FVIIIa inactivation; FV is secreted from the ER via the LMAN1-MCFD2 cargo receptor complex, where MCFD2 directly binds FV/FVIII cargo and LMAN1 serves as a shuttling carrier; a naturally occurring alternatively spliced B-domain-truncated isoform, FV-Short, forms a high-affinity trimolecular complex with TFPIα and protein S that synergistically inhibits FXa, providing an anticoagulant threshold whose dysregulation causes bleeding (East Texas disorder) or thrombosis."},"narrative":{"mechanistic_narrative":"Coagulation Factor V (F5) encodes a circulating procofactor whose activated form, FVa, is essential for thrombin generation, with its activity tightly regulated by activated protein C (APC) and protein S; loss of this regulatory cleavage is the central mechanism of inherited thrombophilia [PMID:8624369]. FVa is inactivated by APC through cleavage at Arg506 and Arg306, with protein S stimulating these reactions, and intact FV additionally serves as an APC cofactor together with protein S in the inactivation of FVIIIa within the Xase complex [PMID:8624369, PMID:12091344]. Pathogenic missense substitutions impair this regulation in distinct ways: FV Leiden (Arg506Gln) is activated normally but resists APC inactivation, producing lifelong hypercoagulability [PMID:8624369, PMID:12881304], FV Bonn (Ala512Val) slows Arg506 cleavage with protein S largely restoring inactivation [PMID:27090446], and variants disrupting Arg306 cleavage (FV Cambridge, FV Hong Kong) become near-completely inactivated only when protein S stimulates the alternative Arg679 cleavage [PMID:12091344]. Other mutations cause quantitative FV deficiency through impaired secretion, heterodimer instability, or aberrant splicing—including FV New Brunswick (Ala221Val), which destabilizes the activated FVa heterodimer [PMID:12714495], FV Besançon (Ala2086Asp), which favors a closed C2-domain conformation that impairs phospholipid binding and secretion yet paradoxically produces a hypercoagulable, APC-resistant state [PMID:33605529], and synonymous, exonic, and deep-intronic variants that activate cryptic splice sites and are correctable by antisense morpholino or U7 snRNA approaches [PMID:25470420, PMID:24085767]. FV and FVIII are exported from the ER via the calcium-dependent LMAN1–MCFD2 cargo receptor complex, in which MCFD2 is the primary cargo-binding subunit and LMAN1 acts as a shuttling carrier [PMID:19183188, PMID:36490287, PMID:21795745]. A naturally occurring B-domain-truncated splice isoform, FV-Short, forms a high-affinity trimolecular complex with TFPIα and protein S that synergistically inhibits FXa; cis-acting regulatory sequences in F5 exon 13 control its production, and gain-of-function splicing variants that elevate FV-Short/TFPIα raise the anticoagulant threshold and cause bleeding (East Texas disorder) [PMID:36746318, PMID:25634741, PMID:33773040].","teleology":[{"year":1996,"claim":"Established the central thrombophilia mechanism: FV Leiden activates normally but resists APC inactivation, explaining how a single F5 substitution drives lifelong hypercoagulability and defining FV's dual role as both procofactor and APC cofactor.","evidence":"Biochemical characterization of purified mutant FV and APC resistance testing in patient and reconstituted plasma","pmids":["8624369"],"confidence":"High","gaps":["Does not resolve the structural basis of impaired Arg506 cleavage","Relative contribution of FVa-procoagulant resistance versus loss of APC cofactor function not quantified"]},{"year":2000,"claim":"Linked the R2 F5 haplotype to differential glycosylation, indicating genetically determined shifts in the FV1/FV2 isoform ratio modulate thrombogenic potential.","evidence":"Sequencing plus chromatographic separation and quantitation of FV isoforms in homozygous R2 carriers versus controls","pmids":["10744138"],"confidence":"Medium","gaps":["No mutagenesis reconstitution to prove causality of A6755G/Ala2194Gly","Mechanism by which glycosylation alters function not defined"]},{"year":2002,"claim":"Defined how Arg306 cleavage and protein S cooperate, showing variants lacking Arg306 cleavage are still nearly fully inactivated when protein S stimulates the alternative Arg679 site.","evidence":"Recombinant FV Cambridge/Hong Kong mutants in purified APC inactivation, prothrombinase, and FVIIIa degradation assays with/without protein S","pmids":["12091344"],"confidence":"High","gaps":["In vivo thrombotic risk of these variants not established","Structural basis of protein S stimulation of Arg679 cleavage unresolved"]},{"year":2003,"claim":"Demonstrated that FV deficiency can arise from post-secretory instability rather than synthesis or function defects, mechanistically separating quantitative deficiency from activity loss.","evidence":"Recombinant FV New Brunswick (Ala221Val) with pulse-chase, ELISA, prothrombinase, and thermal stability assays","pmids":["12714495"],"confidence":"High","gaps":["Structural cause of accelerated chain dissociation not defined","In vivo turnover kinetics not measured"]},{"year":2003,"claim":"Showed FV Leiden's prothrombotic effect persists even under severe upstream coagulation factor deficiency, reinforcing APC resistance as the operative mechanism in defined plasma systems.","evidence":"Thrombin generation and FXa/FVa measurements in FV-FVII doubly deficient plasma reconstituted with purified FV variants","pmids":["12881304"],"confidence":"High","gaps":["Clinical bleeding/thrombosis balance in combined deficiency not addressed","Single-lab plasma reconstitution system"]},{"year":2009,"claim":"Identified the LMAN1-MCFD2 complex as the ER-to-Golgi cargo receptor for FV and FVIII, explaining combined FV+FVIII deficiency through a trafficking rather than synthetic defect.","evidence":"Genetic identification of LMAN1/MCFD2 mutations in patients, co-immunoprecipitation, and cell trafficking assays","pmids":["19183188"],"confidence":"High","gaps":["Precise FV binding determinants on the complex not mapped","Relative roles of LMAN1 versus MCFD2 not yet dissected"]},{"year":2011,"claim":"Confirmed in vivo that LMAN1 is required for efficient FV and FVIII secretion, with a partial (~50%) deficiency phenotype and ER stress, while sparing COPII vesicle formation.","evidence":"Lman1 knockout mice with plasma/platelet FV and FVIII quantification, liver ultrastructure, and in vitro COPII budding assay","pmids":["21795745"],"confidence":"High","gaps":["Reason for residual secretion despite LMAN1 loss not explained","Tissue-specific contributions to plasma FV not resolved"]},{"year":2013,"claim":"Established that deep-intronic F5 splicing mutations cause severe deficiency and are correctable, opening antisense and U7 snRNA strategies validated in patient-derived cells.","evidence":"F5 minigene in COS-1/HepG2, morpholino and U7snRNA rescue, and patient-derived megakaryocyte FV restoration","pmids":["24085767"],"confidence":"High","gaps":["Therapeutic delivery and durability in vivo not addressed","Generalizability to other intronic variants untested"]},{"year":2014,"claim":"Showed a synonymous coding variant can cause FV deficiency by activating a cryptic splice site and disrupting an exonic splicing enhancer, broadening the mutational mechanisms producing non-functional, poorly secreted FV.","evidence":"F5 sequencing, mRNA analysis, minigene in COS-1, recombinant mutant protein expression, and morpholino rescue","pmids":["25470420"],"confidence":"High","gaps":["Quantitative contribution of each defect (secretion vs function) not separated","In vivo correction not demonstrated"]},{"year":2015,"claim":"Defined a gain-of-function bleeding mechanism: aberrant splicing produces a shortened FV that binds and stabilizes TFPI, suppressing thrombin generation.","evidence":"Whole exome sequencing, thrombin generation, TFPI level and binding assays, and cDNA splicing analysis (FV Amsterdam)","pmids":["25634741"],"confidence":"High","gaps":["Structural basis of FV-TFPI binding not resolved at this stage","Protein S contribution not yet incorporated"]},{"year":2021,"claim":"Demonstrated that cis-acting sequences in F5 exon 13 regulate alternative splicing to FV-Short, and that deletions enhancing this splicing elevate TFPIα and cause severe bleeding.","evidence":"Patient plasma TFPI quantification, F5 cDNA sequencing, minigene heterologous expression, and FV isoform Western blotting (F5-Atlanta)","pmids":["33773040"],"confidence":"High","gaps":["Trans-acting factors controlling exon 13 splicing not identified","Normal physiological regulation of FV-Short levels undefined"]},{"year":2021,"claim":"Revealed a paradoxical phenotype where a C2-domain conformational mutation impairs secretion yet abolishes APC cofactor activity and confers APC resistance, producing thrombosis despite severe FV deficiency.","evidence":"Patient thrombin generation, recombinant FV Besançon (Ala2086Asp) prothrombinase and APC inactivation assays, and C2-domain modeling","pmids":["33605529"],"confidence":"High","gaps":["Structural validation of the proposed closed C2 conformation lacking","In vivo bleeding/thrombosis balance not fully characterized"]},{"year":2023,"claim":"Resolved MCFD2 as the primary cargo-binding subunit with LMAN1 acting as a shuttling carrier, and showed the complex is not rate-limiting for FV/FVIII secretion.","evidence":"LMAN1/MCFD2-deficient HEK293T, HepG2, HCT116 cells with secretion and wild-type/mutant rescue experiments","pmids":["36490287"],"confidence":"High","gaps":["Direct FV-MCFD2 binding interface not structurally defined","Identity of the rate-limiting secretion step unknown"]},{"year":2023,"claim":"Established the trimolecular FV-Short/TFPIα/protein S complex as a synergistic FXa inhibitor and quantified its ~10-fold elevation in East Texas disorder, defining an anticoagulant threshold whose dysregulation causes bleeding.","evidence":"Biochemical reconstitution of the complex, FXa inhibition assays, and circulating complex quantification in patients versus controls","pmids":["36746318"],"confidence":"High","gaps":["Structural architecture of the trimolecular complex not solved","Physiological setpoint and regulation of complex abundance unclear"]},{"year":null,"claim":"How FV's procofactor, APC-cofactor, and FV-Short/TFPIα anticoagulant roles are balanced in vivo and how exon 13 splicing is normally regulated remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No high-resolution structure of FVa or the FV-Short/TFPIα/protein S complex in the corpus","Trans-acting regulators of FV-Short splicing unidentified","Tissue-level integration of FV's opposing pro- and anticoagulant functions undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,10,14]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[14]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[7,8,9]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[1,7,9]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[4,5,13]}],"pathway":[{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[1,3,9]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[4,5,13]}],"complexes":["prothrombinase complex","FV-Short/TFPIα/protein S complex","LMAN1-MCFD2 cargo receptor complex"],"partners":["TFPI","PROS1","LMAN1","MCFD2","F10","F2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P12259","full_name":"Coagulation factor V","aliases":["Activated protein C cofactor","Proaccelerin, labile factor"],"length_aa":2224,"mass_kda":251.7,"function":"Central regulator of hemostasis. It serves as a critical cofactor for the prothrombinase activity of factor Xa that results in the activation of prothrombin to thrombin","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/P12259/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/F5","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/F5","total_profiled":1310},"omim":[{"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":"621143","title":"HOLOPROSENCEPHALY 10; HPE10","url":"https://www.omim.org/entry/621143"},{"mim_id":"620874","title":"ADHESION G PROTEIN-COUPLED RECEPTOR F5; ADGRF5","url":"https://www.omim.org/entry/620874"},{"mim_id":"620819","title":"ROTHMUND-THOMSON SYNDROME, TYPE 4; RTS4","url":"https://www.omim.org/entry/620819"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Golgi apparatus","reliability":"Approved"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"choroid plexus","ntpm":77.1},{"tissue":"liver","ntpm":164.8},{"tissue":"placenta","ntpm":44.1}],"url":"https://www.proteinatlas.org/search/F5"},"hgnc":{"alias_symbol":["fV"],"prev_symbol":[]},"alphafold":{"accession":"P12259","domains":[{"cath_id":"2.60.40.420","chopping":"32-45_61-328","consensus_level":"medium","plddt":88.8713,"start":32,"end":328},{"cath_id":"2.60.40.420","chopping":"347-455_473-665","consensus_level":"high","plddt":84.5861,"start":347,"end":665},{"cath_id":"2.60.40.420","chopping":"1565-1598_1613-1784_1804-1904","consensus_level":"medium","plddt":87.4538,"start":1565,"end":1904},{"cath_id":"2.60.120.260","chopping":"1907-2065","consensus_level":"medium","plddt":84.8282,"start":1907,"end":2065},{"cath_id":"2.60.120.260","chopping":"2067-2224","consensus_level":"medium","plddt":85.6734,"start":2067,"end":2224}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P12259","model_url":"https://alphafold.ebi.ac.uk/files/AF-P12259-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P12259-F1-predicted_aligned_error_v6.png","plddt_mean":61.91},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=F5","jax_strain_url":"https://www.jax.org/strain/search?query=F5"},"sequence":{"accession":"P12259","fasta_url":"https://rest.uniprot.org/uniprotkb/P12259.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P12259/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P12259"}},"corpus_meta":[{"pmid":"3285470","id":"PMC_3285470","title":"Assembly 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Molecular basis of disease","url":"https://pubmed.ncbi.nlm.nih.gov/36878303","citation_count":11,"is_preprint":false},{"pmid":"28553635","id":"PMC_28553635","title":"Alcoholic Fractions F5 and F6 from Withania somnifera Leaves Show a Potent Antileishmanial and Immunomodulatory Activities to Control Experimental Visceral Leishmaniasis.","date":"2017","source":"Frontiers in medicine","url":"https://pubmed.ncbi.nlm.nih.gov/28553635","citation_count":11,"is_preprint":false},{"pmid":"12706643","id":"PMC_12706643","title":"Generation and characterization of recombinant single chain Fv antibody that recognizes platelet glycoprotein Ibalpha.","date":"2003","source":"Thrombosis research","url":"https://pubmed.ncbi.nlm.nih.gov/12706643","citation_count":11,"is_preprint":false},{"pmid":"17369700","id":"PMC_17369700","title":"Protein Fv: an endogenous immunoglobulin superantigen and superallergen.","date":"2007","source":"Chemical immunology and allergy","url":"https://pubmed.ncbi.nlm.nih.gov/17369700","citation_count":10,"is_preprint":false},{"pmid":"36490287","id":"PMC_36490287","title":"Separate roles of LMAN1 and MCFD2 in ER-to-Golgi trafficking of FV and FVIII.","date":"2023","source":"Blood advances","url":"https://pubmed.ncbi.nlm.nih.gov/36490287","citation_count":10,"is_preprint":false},{"pmid":"31287622","id":"PMC_31287622","title":"Biophysical characterization and single-chain Fv construction of a neutralizing antibody to measles virus.","date":"2019","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/31287622","citation_count":10,"is_preprint":false},{"pmid":"33112221","id":"PMC_33112221","title":"Genome-based classification of Acidihalobacter prosperus F5 (=DSM 105917=JCM 32255) as Acidihalobacter yilgarnensis sp. nov.","date":"2020","source":"International journal of systematic and evolutionary microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/33112221","citation_count":10,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":48345,"output_tokens":4774,"usd":0.108323,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12533,"output_tokens":4758,"usd":0.090807,"stage2_stop_reason":"end_turn"},"total_usd":0.19913,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"Looking through the corpus, I need to identify papers about the human coagulation factor V (F5/fV gene) and extract mechanistic findings.\n\nPapers clearly about human coagulation Factor V (F5): #15, #18, #19, #23, #26, #29, #37, #44, #55, #60, #61, #63 (Clostridium - skip), #66, #77, #78, #81, #98\n\nMany other papers are about antibody Fv fragments (immunoglobulin variable fragments - different meaning of \"Fv\"), Fv-1/Fv-2/Fv-4 mouse retroviral resistance genes, or other unrelated genes. I will only extract from papers clearly about coagulation Factor V.\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2002,\n      \"finding\": \"FV Cambridge (Arg306Thr) and FV Hong Kong (Arg306Gly) both lack APC cleavage at Arg306, resulting in partial FVa inactivation in the absence of protein S, but near-complete inactivation in the presence of protein S due to stimulation of cleavage at Arg679. Both variants also show slightly impaired APC cofactor activity in FVIIIa degradation.\",\n      \"method\": \"Recombinant expression of FV mutants, purified protein functional assays (APC-mediated inactivation curves, prothrombinase assay, FVIIIa degradation assay) with and without protein S\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted recombinant mutants, multiple orthogonal functional assays, mechanistic cleavage-site mapping\",\n      \"pmids\": [\"12091344\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Factor V Leiden (FV:Q506, Arg506Gln) is activated normally by thrombin or factor Xa, but its impaired inactivation by activated protein C (APC) results in lifelong hypercoagulability. Intact FV also functions as an APC cofactor with protein S in the regulation of FVIIIa in the Xase complex.\",\n      \"method\": \"Biochemical characterization of purified mutant FV, functional coagulation assays, APC resistance testing in patient and reconstituted plasma\",\n      \"journal\": \"Annals of hematology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiply replicated across labs, foundational mechanistic characterization of FV Leiden cleavage defect\",\n      \"pmids\": [\"8624369\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Mutations in the R2 allele of the F5 gene (including A6755G, Ala2194Gly in the C2 domain) are associated with a relative increase in the more thrombogenic, more glycosylated FV isoform (FV1) compared with the less glycosylated FV2, suggesting that genetically determined differential glycosylation of FV alters the FV1/FV2 ratio.\",\n      \"method\": \"Molecular genetics sequencing, chromatographic separation of FV isoforms, quantitative FV1/FV2 ratio analysis in homozygous R2 carriers vs controls\",\n      \"journal\": \"Thrombosis and haemostasis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — single lab, two orthogonal methods (sequencing + chromatographic functional characterization), but no direct mutagenesis reconstitution\",\n      \"pmids\": [\"10744138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"FV Leiden increases thrombin generation in the context of severe FVII deficiency, particularly in the presence of APC, by resisting APC-mediated FVa inactivation. This was confirmed in FV-FVII doubly deficient plasma reconstituted with purified normal FV or FV Leiden.\",\n      \"method\": \"Thrombin generation assays in patient plasma and in doubly deficient plasma reconstituted with purified FV variants; FXa and FVa generation measurements with and without APC\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — purified protein reconstitution in defined plasma systems, multiple orthogonal coagulation assays, single lab\",\n      \"pmids\": [\"12881304\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The LMAN1-MCFD2 protein complex functions as a cargo receptor for ER-to-Golgi transport of coagulation FV and FVIII. MCFD2 is an EF-hand domain protein forming a calcium-dependent heteromeric complex with LMAN1; missense mutations in MCFD2 EF-hand domains abolish LMAN1 interaction and cause combined FV+FVIII deficiency. The B domain of FVIII may be important in mediating interaction with the complex.\",\n      \"method\": \"Genetic identification of LMAN1 and MCFD2 mutations in F5F8D patients; co-immunoprecipitation; cell biological trafficking assays; mutation analysis\",\n      \"journal\": \"British journal of haematology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — replicated across multiple labs, reciprocal co-IP of LMAN1-MCFD2 complex, genetic and biochemical evidence converging on ER-to-Golgi transport function\",\n      \"pmids\": [\"19183188\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"LMAN1-deficient mice show ~50% reduction in plasma FV, plasma FVIII, and platelet FV levels, confirming LMAN1's role as a cargo receptor for ER-to-Golgi transport of FV and FVIII. LMAN1 deficiency causes ER distension and accumulation of α1-antitrypsin and GRP78 in hepatocytes, but cathepsin C and Z levels are unaffected. LMAN1 deficiency had no effect on COPII vesicle formation in vitro.\",\n      \"method\": \"Lman1 knockout mouse generation; plasma FV and FVIII quantification; platelet FV measurement; liver ultrastructure analysis; in vitro COPII vesicle budding assay; Western blot\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — genetic knockout model with multiple orthogonal biochemical readouts, includes negative control (COPII assay), confirms prior human genetic data\",\n      \"pmids\": [\"21795745\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"FV New Brunswick (Ala221Val) does not impair FV synthesis, secretion, or procoagulant FXa cofactor function, nor does it affect APC-mediated inactivation of FVa. Instead, the mutation reduces stability of the activated FVa heterodimer by increasing the rate of light and heavy chain dissociation at 37°C, which explains the FV deficiency phenotype.\",\n      \"method\": \"Recombinant expression of Ala221Val and control mutants (Ala221Gly, Glu275Gln, Cys220Ala/Cys301Ala); pulse-chase experiments; ELISA; plasma clotting assays; purified prothrombinase assays; thermal stability assays\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted recombinant mutants with multiple orthogonal functional assays and mechanistic pulse-chase in single lab\",\n      \"pmids\": [\"12714495\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"A novel gain-of-function mutation in F5 (c.C2588G) causes aberrant splicing producing a shortened FV protein (FV Amsterdam, missing 623 amino acids from the B domain) that binds tissue factor pathway inhibitor (TFPI), prolonging its half-life and increasing circulating TFPI levels, leading to severely reduced thrombin generation and a bleeding phenotype.\",\n      \"method\": \"Whole exome sequencing; thrombin generation assay; TFPI level measurement; F5 cDNA splicing analysis; TFPI binding assay\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — exome sequencing combined with functional TFPI binding, thrombin generation assays and mRNA splicing analysis in a single rigorous study\",\n      \"pmids\": [\"25634741\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A novel 832 bp deletion within F5 exon 13 (F5-Atlanta) enhances alternative splicing at the East Texas (ET) splice sites, producing FV-Short, an isoform with a truncated B domain that binds TFPIα with high affinity, thereby elevating circulating TFPIα levels and causing severe bleeding. This demonstrates that cis-acting regulatory sequences within F5 exon 13 control alternative splicing and regulate circulating FV-Short/TFPIα levels.\",\n      \"method\": \"Patient plasma TFPI quantification; F5 cDNA sequencing; minigene heterologous expression in mammalian cells; FV isoform Western blotting\",\n      \"journal\": \"Journal of thrombosis and haemostasis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — minigene functional validation in mammalian cells plus patient plasma biochemistry, mechanistic model confirmed by recapitulation in heterologous expression\",\n      \"pmids\": [\"33773040\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FV-Short, a naturally occurring splice isoform of FV with a truncated B domain, forms a high-affinity trimolecular complex with TFPIα and protein S. FV-Short and protein S synergistically stimulate the FXa-inhibitory activity of TFPIα. In East Texas bleeding disorder, the concentration of the FV-Short/protein S/TFPIα complex is increased ~10-fold above normal (~0.2 nM), providing an elevated anticoagulant threshold and causing bleeding.\",\n      \"method\": \"Biochemical characterization of FV-Short/TFPIα/protein S complex; FXa inhibition assays; quantification of circulating complex in East Texas patients vs controls\",\n      \"journal\": \"Journal of thrombosis and haemostasis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mechanistic complex characterization replicated across studies, trimolecular complex biochemistry with functional FXa inhibition assay\",\n      \"pmids\": [\"36746318\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"FV Bonn (Ala512Val) causes APC resistance by slowing APC-mediated cleavage at Arg506 (the primary APC cleavage site), with protein S largely restoring inactivation. FV Bonn also shows ~24% reduced APC cofactor activity in FVIIIa inactivation and enhanced procoagulant activity (increased activation rate, possibly enhanced interaction with activated FX) in the absence of APC. In silico modeling places Ala512 in the same loop as Arg506.\",\n      \"method\": \"Recombinant FV Bonn expression; APTT-based APC sensitivity ratio; thrombin generation-based APC sensitivity ratio; purified FVa inactivation kinetics with/without protein S; FVIIIa cofactor activity assay; in silico structural modeling\",\n      \"journal\": \"Journal of thrombosis and haemostasis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted recombinant mutant with multiple orthogonal functional assays, single lab\",\n      \"pmids\": [\"27090446\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"An apparently synonymous F5 variant (c.1281C>G in exon 8) causes FV deficiency by activating a cryptic donor splice site and abolishing an exonic splicing enhancer, producing aberrantly spliced mRNA lacking the last 18 nucleotides of exon 8. The resulting mutant FV protein (lacking amino acids 427–432) is poorly secreted and non-functional. A mutation-specific morpholino antisense oligonucleotide partially corrected the splicing defect.\",\n      \"method\": \"F5 gene sequencing; mRNA analysis; bioinformatics splice-site prediction; F5 minigene model in COS-1 cells; morpholino antisense rescue; COS-1 cell expression of mutant protein\",\n      \"journal\": \"Haemophilia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — minigene functional validation plus recombinant protein secretion/activity assay and antisense rescue, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"25470420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"A deep-intronic F5 mutation (c.1296+268A>G) activates a cryptic donor splice site causing severe FV deficiency. Mutation-specific antisense morpholino oligonucleotides (MO) and engineered U7 small nuclear RNA constructs dose-dependently restored correct F5 mRNA splicing in COS-1 and HepG2 cells transfected with a F5 minigene, and in patient-derived megakaryocytes ex vivo, where FV protein expression was restored.\",\n      \"method\": \"F5 minigene transfection in COS-1 and HepG2 cells; antisense morpholino and U7snRNA rescue experiments; patient-derived megakaryocyte differentiation from circulating progenitors; immunofluorescence for FV protein\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple cell systems including patient-derived cells, minigene model with antisense rescue, single lab with orthogonal methods\",\n      \"pmids\": [\"24085767\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MCFD2 is the primary cargo-binding subunit of the LMAN1-MCFD2 complex for FV and FVIII ER-to-Golgi transport: LMAN1 carbohydrate-binding mutants can still partially rescue FV/FVIII secretion, whereas overexpression of MCFD2 alone (wild-type or mutant) rescues FV/FVIII secretion in LMAN1-deficient cells, indicating LMAN1 serves primarily as a shuttling carrier for MCFD2. Overexpression of both proteins together does not further increase FV/FVIII secretion, indicating the LMAN1-MCFD2 complex is not rate-limiting for ER-Golgi transport of FV/FVIII.\",\n      \"method\": \"LMAN1- and MCFD2-deficient cell lines (HEK293T, HepG2, HCT116); FV/FVIII secretion assays; rescue experiments with wild-type and mutant LMAN1/MCFD2 overexpression; functional pathogenicity assays for missense mutations\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple cell lines, LMAN1/MCFD2 mutant rescue experiments with clear mechanistic conclusion, single lab with orthogonal approaches\",\n      \"pmids\": [\"36490287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"FV Besançon (Ala2086Asp) favors a 'closed conformation' of the C2 domain, impairing FV(a) binding to phospholipids. The mutation causes severe FV deficiency by greatly reducing FV secretion. Residual FVa Besançon shows slightly unfavorable prothrombinase kinetics but markedly reduced APC-mediated inactivation (in the presence of protein S), and completely abolished APC cofactor activity, leading to a paradoxical hypercoagulable/thrombotic state despite severe FV deficiency. Thrombin generation in platelet-rich plasma was higher than in controls and extremely APC resistant.\",\n      \"method\": \"Patient plasma thrombin generation (platelet-poor and platelet-rich); FV:C, FV:Ag, and TFPI assays; F5 sequencing; recombinant FVBesançon expression in COS cells; prothrombinase kinetics; APC-mediated FVa inactivation assay; in silico C2 domain modeling\",\n      \"journal\": \"Journal of thrombosis and haemostasis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — recombinant protein reconstitution with multiple functional assays and structural modeling, single lab, clinically validated\",\n      \"pmids\": [\"33605529\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Coagulation Factor V (F5) is a procofactor that, upon activation to FVa by thrombin or factor Xa, serves as an essential cofactor in the prothrombinase complex (FXa/FVa) for prothrombin activation; FVa is inactivated by activated protein C (APC) via sequential cleavages at Arg506 and Arg306/Arg679, with protein S as a cofactor—a mechanism abrogated by FV Leiden (Arg506Gln) causing APC resistance and thrombosis; intact FV also functions as an APC cofactor (with protein S) in FVIIIa inactivation; FV is secreted from the ER via the LMAN1-MCFD2 cargo receptor complex, where MCFD2 directly binds FV/FVIII cargo and LMAN1 serves as a shuttling carrier; a naturally occurring alternatively spliced B-domain-truncated isoform, FV-Short, forms a high-affinity trimolecular complex with TFPIα and protein S that synergistically inhibits FXa, providing an anticoagulant threshold whose dysregulation causes bleeding (East Texas disorder) or thrombosis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"Coagulation Factor V (F5) encodes a circulating procofactor whose activated form, FVa, is essential for thrombin generation, with its activity tightly regulated by activated protein C (APC) and protein S; loss of this regulatory cleavage is the central mechanism of inherited thrombophilia [#1]. FVa is inactivated by APC through cleavage at Arg506 and Arg306, with protein S stimulating these reactions, and intact FV additionally serves as an APC cofactor together with protein S in the inactivation of FVIIIa within the Xase complex [#1, #0]. Pathogenic missense substitutions impair this regulation in distinct ways: FV Leiden (Arg506Gln) is activated normally but resists APC inactivation, producing lifelong hypercoagulability [#1, #3], FV Bonn (Ala512Val) slows Arg506 cleavage with protein S largely restoring inactivation [#10], and variants disrupting Arg306 cleavage (FV Cambridge, FV Hong Kong) become near-completely inactivated only when protein S stimulates the alternative Arg679 cleavage [#0]. Other mutations cause quantitative FV deficiency through impaired secretion, heterodimer instability, or aberrant splicing—including FV New Brunswick (Ala221Val), which destabilizes the activated FVa heterodimer [#6], FV Besançon (Ala2086Asp), which favors a closed C2-domain conformation that impairs phospholipid binding and secretion yet paradoxically produces a hypercoagulable, APC-resistant state [#14], and synonymous, exonic, and deep-intronic variants that activate cryptic splice sites and are correctable by antisense morpholino or U7 snRNA approaches [#11, #12]. FV and FVIII are exported from the ER via the calcium-dependent LMAN1–MCFD2 cargo receptor complex, in which MCFD2 is the primary cargo-binding subunit and LMAN1 acts as a shuttling carrier [#4, #13, #5]. A naturally occurring B-domain-truncated splice isoform, FV-Short, forms a high-affinity trimolecular complex with TFPIα and protein S that synergistically inhibits FXa; cis-acting regulatory sequences in F5 exon 13 control its production, and gain-of-function splicing variants that elevate FV-Short/TFPIα raise the anticoagulant threshold and cause bleeding (East Texas disorder) [#9, #7, #8].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established the central thrombophilia mechanism: FV Leiden activates normally but resists APC inactivation, explaining how a single F5 substitution drives lifelong hypercoagulability and defining FV's dual role as both procofactor and APC cofactor.\",\n      \"evidence\": \"Biochemical characterization of purified mutant FV and APC resistance testing in patient and reconstituted plasma\",\n      \"pmids\": [\"8624369\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not resolve the structural basis of impaired Arg506 cleavage\", \"Relative contribution of FVa-procoagulant resistance versus loss of APC cofactor function not quantified\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Linked the R2 F5 haplotype to differential glycosylation, indicating genetically determined shifts in the FV1/FV2 isoform ratio modulate thrombogenic potential.\",\n      \"evidence\": \"Sequencing plus chromatographic separation and quantitation of FV isoforms in homozygous R2 carriers versus controls\",\n      \"pmids\": [\"10744138\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No mutagenesis reconstitution to prove causality of A6755G/Ala2194Gly\", \"Mechanism by which glycosylation alters function not defined\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined how Arg306 cleavage and protein S cooperate, showing variants lacking Arg306 cleavage are still nearly fully inactivated when protein S stimulates the alternative Arg679 site.\",\n      \"evidence\": \"Recombinant FV Cambridge/Hong Kong mutants in purified APC inactivation, prothrombinase, and FVIIIa degradation assays with/without protein S\",\n      \"pmids\": [\"12091344\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo thrombotic risk of these variants not established\", \"Structural basis of protein S stimulation of Arg679 cleavage unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Demonstrated that FV deficiency can arise from post-secretory instability rather than synthesis or function defects, mechanistically separating quantitative deficiency from activity loss.\",\n      \"evidence\": \"Recombinant FV New Brunswick (Ala221Val) with pulse-chase, ELISA, prothrombinase, and thermal stability assays\",\n      \"pmids\": [\"12714495\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural cause of accelerated chain dissociation not defined\", \"In vivo turnover kinetics not measured\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Showed FV Leiden's prothrombotic effect persists even under severe upstream coagulation factor deficiency, reinforcing APC resistance as the operative mechanism in defined plasma systems.\",\n      \"evidence\": \"Thrombin generation and FXa/FVa measurements in FV-FVII doubly deficient plasma reconstituted with purified FV variants\",\n      \"pmids\": [\"12881304\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Clinical bleeding/thrombosis balance in combined deficiency not addressed\", \"Single-lab plasma reconstitution system\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified the LMAN1-MCFD2 complex as the ER-to-Golgi cargo receptor for FV and FVIII, explaining combined FV+FVIII deficiency through a trafficking rather than synthetic defect.\",\n      \"evidence\": \"Genetic identification of LMAN1/MCFD2 mutations in patients, co-immunoprecipitation, and cell trafficking assays\",\n      \"pmids\": [\"19183188\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise FV binding determinants on the complex not mapped\", \"Relative roles of LMAN1 versus MCFD2 not yet dissected\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Confirmed in vivo that LMAN1 is required for efficient FV and FVIII secretion, with a partial (~50%) deficiency phenotype and ER stress, while sparing COPII vesicle formation.\",\n      \"evidence\": \"Lman1 knockout mice with plasma/platelet FV and FVIII quantification, liver ultrastructure, and in vitro COPII budding assay\",\n      \"pmids\": [\"21795745\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reason for residual secretion despite LMAN1 loss not explained\", \"Tissue-specific contributions to plasma FV not resolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established that deep-intronic F5 splicing mutations cause severe deficiency and are correctable, opening antisense and U7 snRNA strategies validated in patient-derived cells.\",\n      \"evidence\": \"F5 minigene in COS-1/HepG2, morpholino and U7snRNA rescue, and patient-derived megakaryocyte FV restoration\",\n      \"pmids\": [\"24085767\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Therapeutic delivery and durability in vivo not addressed\", \"Generalizability to other intronic variants untested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed a synonymous coding variant can cause FV deficiency by activating a cryptic splice site and disrupting an exonic splicing enhancer, broadening the mutational mechanisms producing non-functional, poorly secreted FV.\",\n      \"evidence\": \"F5 sequencing, mRNA analysis, minigene in COS-1, recombinant mutant protein expression, and morpholino rescue\",\n      \"pmids\": [\"25470420\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of each defect (secretion vs function) not separated\", \"In vivo correction not demonstrated\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined a gain-of-function bleeding mechanism: aberrant splicing produces a shortened FV that binds and stabilizes TFPI, suppressing thrombin generation.\",\n      \"evidence\": \"Whole exome sequencing, thrombin generation, TFPI level and binding assays, and cDNA splicing analysis (FV Amsterdam)\",\n      \"pmids\": [\"25634741\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of FV-TFPI binding not resolved at this stage\", \"Protein S contribution not yet incorporated\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrated that cis-acting sequences in F5 exon 13 regulate alternative splicing to FV-Short, and that deletions enhancing this splicing elevate TFPIα and cause severe bleeding.\",\n      \"evidence\": \"Patient plasma TFPI quantification, F5 cDNA sequencing, minigene heterologous expression, and FV isoform Western blotting (F5-Atlanta)\",\n      \"pmids\": [\"33773040\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trans-acting factors controlling exon 13 splicing not identified\", \"Normal physiological regulation of FV-Short levels undefined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Revealed a paradoxical phenotype where a C2-domain conformational mutation impairs secretion yet abolishes APC cofactor activity and confers APC resistance, producing thrombosis despite severe FV deficiency.\",\n      \"evidence\": \"Patient thrombin generation, recombinant FV Besançon (Ala2086Asp) prothrombinase and APC inactivation assays, and C2-domain modeling\",\n      \"pmids\": [\"33605529\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural validation of the proposed closed C2 conformation lacking\", \"In vivo bleeding/thrombosis balance not fully characterized\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Resolved MCFD2 as the primary cargo-binding subunit with LMAN1 acting as a shuttling carrier, and showed the complex is not rate-limiting for FV/FVIII secretion.\",\n      \"evidence\": \"LMAN1/MCFD2-deficient HEK293T, HepG2, HCT116 cells with secretion and wild-type/mutant rescue experiments\",\n      \"pmids\": [\"36490287\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct FV-MCFD2 binding interface not structurally defined\", \"Identity of the rate-limiting secretion step unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established the trimolecular FV-Short/TFPIα/protein S complex as a synergistic FXa inhibitor and quantified its ~10-fold elevation in East Texas disorder, defining an anticoagulant threshold whose dysregulation causes bleeding.\",\n      \"evidence\": \"Biochemical reconstitution of the complex, FXa inhibition assays, and circulating complex quantification in patients versus controls\",\n      \"pmids\": [\"36746318\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural architecture of the trimolecular complex not solved\", \"Physiological setpoint and regulation of complex abundance unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How FV's procofactor, APC-cofactor, and FV-Short/TFPIα anticoagulant roles are balanced in vivo and how exon 13 splicing is normally regulated remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No high-resolution structure of FVa or the FV-Short/TFPIα/protein S complex in the corpus\", \"Trans-acting regulators of FV-Short splicing unidentified\", \"Tissue-level integration of FV's opposing pro- and anticoagulant functions undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 10, 14]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [14]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [7, 8, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [1, 7, 9]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [4, 5, 13]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [1, 3, 9]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [4, 5, 13]}\n    ],\n    \"complexes\": [\n      \"prothrombinase complex\",\n      \"FV-Short/TFPIα/protein S complex\",\n      \"LMAN1-MCFD2 cargo receptor complex\"\n    ],\n    \"partners\": [\n      \"TFPI\",\n      \"PROS1\",\n      \"LMAN1\",\n      \"MCFD2\",\n      \"F10\",\n      \"F2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":6,"faith_total":6,"faith_pct":100.0}}