{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,3]],"date-time":"2026-11-03T10:22:17Z","timestamp":1793701337192,"version":"4.5.1"},"reference-count":90,"publisher":"American Society of Hematology","issue":"18","license":[{"start":{"date-parts":[[2025,7,15]],"date-time":"2025-07-15T00:00:00Z","timestamp":1752537600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":["ashpublications.org"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2025,10,30]]},"abstract":"<jats:sec>\n                    <jats:title>Abstract<\/jats:title>\n                    <jats:p>Deficiency of factor VIII (FVIII) causes hemophilia A (HA), and excess FVIII function increases venous thromboembolic risk. The phenotypic consequences of aberrant FVIII function underscore the importance of understanding mechanisms that downregulate activated FVIII (FVIIIa) to inform disease pathology and therapeutic drug design. Spontaneous A2-domain dissociation and activated protein C (APC) proteolysis are established mechanisms of FVIIIa inactivation. However, we know very little about how FVIIIa binding interactions with FIXa and FX affect FVIIIa inactivation in vivo. Here, we investigate this using recombinant FVIIIa variants to probe A2-domain dissociation (FVIIIa-D519V,E665V) and APC cleavage (FVIIIa-R336Q,R562Q), or both (FVIIIa-R336Q,R562Q\/D519V,E665V), in biochemical assays and in HA mouse injury models. We found that FIXa binding to FVIIIa stabilized the A2 domain and increased the contribution of APC to FVIIIa inactivation. Additional studies using individual APC cleavage site variants (FVIIIa-R336Q and FVIIIa-R562Q) demonstrated that FIXa and FX can protect FVIIIa from APC cleavage at Arg562 and Arg336, respectively, in a manner that is incomplete in vivo. Data also demonstrate that APC inactivation of FVIIIa exceeds FVIII, suggesting differential APC recognition of FVIIIa relative to FVIII. Hemostatic studies of FVIII variants with altered inactivation demonstrated that both A2-domain dissociation and APC cleavage contribute to in vivo FVIIIa regulation. Specifically, stabilizing the A2 domain, inhibiting APC cleavage, or both, improved potency 2.4-, 4.8-, and &amp;gt;10-fold, respectively, over wild-type FVIII in a mouse hemostatic assay. Data support that both mechanisms of FVIIIa inactivation and FIXa interactions could be leveraged to enhance FVIII function for therapeutic benefit.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1182\/blood.2024027705","type":"journal-article","created":{"date-parts":[[2025,7,15]],"date-time":"2025-07-15T20:42:44Z","timestamp":1752612164000},"page":"2259-2271","update-policy":"https:\/\/doi.org\/10.1182\/blood.2019cm0000","source":"Crossref","is-referenced-by-count":3,"title":["Factor IXa and factor X influence factor VIIIa stability and inactivation mechanisms in vitro and in vivo"],"prefix":"10.1182","volume":"146","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1235-8950","authenticated-orcid":false,"given":"Johnathan J.","family":"Morris","sequence":"first","affiliation":[{"name":"1Perelman Center for Cellular and Molecular Therapeutics and Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA"},{"name":"2Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-7696-1434","authenticated-orcid":false,"given":"Nicole A.","family":"Parsons","sequence":"additional","affiliation":[{"name":"1Perelman Center for Cellular and Molecular Therapeutics and Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA"},{"name":"2Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9365-0042","authenticated-orcid":false,"given":"Amelia R.","family":"Wilhelm","sequence":"additional","affiliation":[{"name":"1Perelman Center for Cellular and Molecular Therapeutics and Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert J.","family":"Davidson","sequence":"additional","affiliation":[{"name":"1Perelman Center for Cellular and Molecular Therapeutics and Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA"},{"name":"2Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-5617-786X","authenticated-orcid":false,"given":"Lauren K.","family":"Olenick","sequence":"additional","affiliation":[{"name":"3Cell and Molecular Biology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7766-7880","authenticated-orcid":false,"given":"Connor T.","family":"Watson","sequence":"additional","affiliation":[{"name":"1Perelman Center for Cellular and Molecular Therapeutics and Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew","family":"Vanden Heuvel","sequence":"additional","affiliation":[{"name":"1Perelman Center for Cellular and Molecular Therapeutics and Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lindsey A.","family":"George","sequence":"additional","affiliation":[{"name":"1Perelman Center for Cellular and Molecular Therapeutics and Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA"},{"name":"2Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA"},{"name":"3Cell and Molecular Biology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"234","reference":[{"issue":"23","key":"2025103011212120100_bib1","doi-asserted-by":"crossref","first-page":"1773","DOI":"10.1056\/NEJM200106073442307","article-title":"The hemophilias \u2014 from royal genes to gene therapy","volume":"344","author":"Mannucci","year":"2001","journal-title":"N Engl J Med"},{"issue":"1","key":"2025103011212120100_bib2","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1111\/jth.14343","article-title":"High levels of coagulation factors and venous thrombosis risk: strongest association for factor VIII and von Willebrand factor","volume":"17","author":"Rietveld","year":"2019","journal-title":"J Thromb Haemost"},{"issue":"6","key":"2025103011212120100_bib3","doi-asserted-by":"crossref","first-page":"653","DOI":"10.1111\/j.1365-2141.2012.09134.x","article-title":"Elevated factor VIII levels and risk of venous thrombosis","volume":"157","author":"Jenkins","year":"2012","journal-title":"Br J Haematol"},{"issue":"1","key":"2025103011212120100_bib4","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1055\/s-0037-1613747","article-title":"High plasma concentration of factor VIIIc is a major risk factor for venous thromboembolism","volume":"83","author":"Kraaijenhagen","year":"2000","journal-title":"Thromb Haemost"},{"issue":"suppl 1","key":"2025103011212120100_bib5","doi-asserted-by":"crossref","first-page":"1053","DOI":"10.1182\/blood-2024-208486","article-title":"Efficacy and safety of giroctocogene fitelparvovec in adults with moderately severe to severe hemophilia \u0391: primary analysis results from the phase 3 \u0391FFINE gene therapy trial","volume":"144","author":"Leavitt","year":"2024","journal-title":"Blood"},{"issue":"21","key":"2025103011212120100_bib6","doi-asserted-by":"crossref","first-page":"2185","DOI":"10.1182\/blood.2023023275","article-title":"Coagulation factor VIII: biological basis of emerging hemophilia A therapies","volume":"144","author":"Samelson-Jones","year":"2024","journal-title":"Blood"},{"issue":"3","key":"2025103011212120100_bib7","doi-asserted-by":"crossref","first-page":"599","DOI":"10.1042\/bj2230599","article-title":"Binding of human blood-coagulation Factors IXa and X to phospholipid membranes","volume":"223","author":"Mertens","year":"1984","journal-title":"Biochem J"},{"issue":"7","key":"2025103011212120100_bib8","doi-asserted-by":"crossref","first-page":"3433","DOI":"10.1016\/S0021-9258(19)69627-4","article-title":"The role of phospholipid and factor VIIIa in the activation of bovine factor X","volume":"256","author":"van Dieijen","year":"1981","journal-title":"J Biol Chem"},{"issue":"11","key":"2025103011212120100_bib9","doi-asserted-by":"crossref","first-page":"6027","DOI":"10.1074\/jbc.271.11.6027","article-title":"Model for the factor VIIIa-dependent decay of the intrinsic factor Xase: role of subunit dissociation and factor IXa-catalyzed proteolysis (\u2217)","volume":"271","author":"Fay","year":"1996","journal-title":"J Biol Chem"},{"issue":"23","key":"2025103011212120100_bib10","doi-asserted-by":"crossref","first-page":"8883","DOI":"10.1016\/S0021-9258(19)40667-4","article-title":"Inhibition of human factor IXa by human antithrombin","volume":"250","author":"Rosenberg","year":"1975","journal-title":"J Biol Chem"},{"issue":"2","key":"2025103011212120100_bib11","doi-asserted-by":"crossref","first-page":"645","DOI":"10.1073\/pnas.0910144107","article-title":"Molecular basis of factor IXa recognition by heparin-activated antithrombin revealed by a 1.7-\u00c5 structure of the ternary complex","volume":"107","author":"Johnson","year":"2010","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"10","key":"2025103011212120100_bib12","doi-asserted-by":"crossref","first-page":"2387","DOI":"10.1161\/ATVBAHA.112.250928","article-title":"Inhibition of intrinsic Xase by protein S","volume":"32","author":"Chattopadhyay","year":"2012","journal-title":"Arterioscler Thromb Vasc Biol"},{"issue":"40","key":"2025103011212120100_bib13","doi-asserted-by":"crossref","first-page":"33819","DOI":"10.1074\/jbc.M506502200","article-title":"Down-regulation of factor IXa in the factor Xase complex by protein Z-dependent protease inhibitor","volume":"280","author":"Heeb","year":"2005","journal-title":"J Biol Chem"},{"issue":"5","key":"2025103011212120100_bib14","doi-asserted-by":"crossref","first-page":"2540","DOI":"10.1172\/JCI116863","article-title":"Protease nexin-2\/amyloid beta protein precursor. A tight-binding inhibitor of coagulation factor IXa","volume":"92","author":"Schmaier","year":"1993","journal-title":"J Clin Invest"},{"issue":"17","key":"2025103011212120100_bib15","doi-asserted-by":"crossref","first-page":"17241","DOI":"10.1074\/jbc.M312827200","article-title":"Formation of factors IXa and Xa by the extrinsic pathway: differential regulation by tissue factor pathway inhibitor and antithrombin III","volume":"279","author":"Lu","year":"2004","journal-title":"J Biol Chem"},{"issue":"2","key":"2025103011212120100_bib16","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1160\/TH09-08-0585","article-title":"Contribution of exosite occupancy by heparin to the regulation of coagulation proteases by antithrombin","volume":"103","author":"Yang","year":"2010","journal-title":"Thromb Haemost"},{"issue":"19","key":"2025103011212120100_bib17","doi-asserted-by":"crossref","first-page":"12481","DOI":"10.1016\/S0021-9258(18)98924-6","article-title":"Structural basis for the decreased procoagulant activity of human factor VIII compared to the porcine homolog","volume":"266","author":"Lollar","year":"1991","journal-title":"J Biol Chem"},{"issue":"30","key":"2025103011212120100_bib18","doi-asserted-by":"crossref","first-page":"20139","DOI":"10.1016\/S0021-9258(18)54901-2","article-title":"Activated protein C-catalyzed inactivation of human factor VIII and factor VIIIa. Identification of cleavage sites and correlation of proteolysis with cofactor activity","volume":"266","author":"Fay","year":"1991","journal-title":"J Biol Chem"},{"issue":"1","key":"2025103011212120100_bib19","doi-asserted-by":"crossref","first-page":"322","DOI":"10.1016\/0003-9861(87)90037-3","article-title":"Inactivation of factor VIII by activated protein C and protein S","volume":"252","author":"Walker","year":"1987","journal-title":"Arch Biochem Biophys"},{"issue":"16","key":"2025103011212120100_bib20","doi-asserted-by":"crossref","first-page":"2007","DOI":"10.1182\/blood-2016-04-713289","article-title":"Life in the shadow of a dominant partner: the FVIII-VWF association and its clinical implications for hemophilia A","volume":"128","author":"Pipe","year":"2016","journal-title":"Blood"},{"issue":"3","key":"2025103011212120100_bib21","doi-asserted-by":"crossref","first-page":"476","DOI":"10.1182\/blood.V57.3.476.476","article-title":"The activation and inactivation of human factor VIII by thrombin: effect of inhibitors of thrombin","volume":"57","author":"Hultin","year":"1981","journal-title":"Blood"},{"issue":"4","key":"2025103011212120100_bib22","doi-asserted-by":"crossref","first-page":"846","DOI":"10.1111\/j.1365-2141.1994.tb06749.x","article-title":"Familial discrepancy between the one-stage and two-stage factor VIII methods in a subgroup of patients with haemophilia A","volume":"87","author":"Duncan","year":"1994","journal-title":"Br J Haematol"},{"issue":"5","key":"2025103011212120100_bib23","doi-asserted-by":"crossref","first-page":"781","DOI":"10.1055\/s-0037-1613302","article-title":"Hemophilia A mutations within the factor VIII A2-A3 subunit interface destabilize factor VIIIa and cause one-stage\/two-stage activity discrepancy","volume":"88","author":"Hakeos","year":"2002","journal-title":"Thromb Haemost"},{"issue":"2","key":"2025103011212120100_bib24","doi-asserted-by":"crossref","first-page":"392","DOI":"10.1006\/geno.1993.1073","article-title":"Spectrum of mutations in CRM-positive and CRM-reduced hemophilia A","volume":"15","author":"McGinniss","year":"1993","journal-title":"Genomics"},{"issue":"1","key":"2025103011212120100_bib25","doi-asserted-by":"crossref","first-page":"176","DOI":"10.1182\/blood.V93.1.176","article-title":"Mild hemophilia A caused by increased rate of factor VIII A2 subunit dissociation: evidence for nonproteolytic inactivation of factor VIIIa in vivo","volume":"93","author":"Pipe","year":"1999","journal-title":"Blood"},{"issue":"3","key":"2025103011212120100_bib26","doi-asserted-by":"crossref","first-page":"685","DOI":"10.1182\/blood.V97.3.685","article-title":"Hemophilia A mutations associated with 1-stage\/2-stage activity discrepancy disrupt protein-protein interactions within the triplicated A domains of thrombin-activated factor VIIIa","volume":"97","author":"Pipe","year":"2001","journal-title":"Blood"},{"issue":"2","key":"2025103011212120100_bib27","doi-asserted-by":"crossref","first-page":"400","DOI":"10.1046\/j.1365-2141.1996.d01-1792.x","article-title":"Mutations in a subgroup of patients with mild haemophilia A and a familial discrepancy between the one-stage and two-stage factor VIII: C methods","volume":"94","author":"Rudzki","year":"1996","journal-title":"Br J Haematol"},{"issue":"2","key":"2025103011212120100_bib28","doi-asserted-by":"crossref","first-page":"392","DOI":"10.1182\/blood-2014-02-555656","article-title":"Noncovalent stabilization of the factor VIII A2 domain enhances efficacy in hemophilia A mouse vascular injury models","volume":"125","author":"Leong","year":"2015","journal-title":"Blood"},{"issue":"11","key":"2025103011212120100_bib29","doi-asserted-by":"crossref","first-page":"4708","DOI":"10.1182\/blood.V87.11.4708.bloodjournal87114708","article-title":"Comparison of activated protein C\/protein S-mediated inactivation of human factor VIII and factor V","volume":"87","author":"Lu","year":"1996","journal-title":"Blood"},{"issue":"18","key":"2025103011212120100_bib30","doi-asserted-by":"crossref","first-page":"2532","DOI":"10.1182\/blood.2020007562","article-title":"Activated protein C has a regulatory role in factor VIII function","volume":"137","author":"Wilhelm","year":"2021","journal-title":"Blood"},{"issue":"1","key":"2025103011212120100_bib31","doi-asserted-by":"crossref","first-page":"7193","DOI":"10.1038\/s41467-024-51296-8","article-title":"Pre-clinical evaluation of an enhanced-function factor VIII variant for durable hemophilia A gene therapy in male mice","volume":"15","author":"Sternberg","year":"2024","journal-title":"Nat Commun"},{"issue":"6","key":"2025103011212120100_bib32","doi-asserted-by":"crossref","first-page":"1303","DOI":"10.1182\/blood.V63.6.1303.1303","article-title":"Stabilization of thrombin-activated porcine factor VIII:C by factor IXa phospholipid","volume":"63","author":"Lollar","year":"1984","journal-title":"Blood"},{"issue":"6","key":"2025103011212120100_bib33","doi-asserted-by":"crossref","first-page":"3725","DOI":"10.1016\/S0021-9258(19)50585-3","article-title":"Factor IXa enhances reconstitution of factor VIIIa from isolated A2 subunit and A1\/A3-C1-C2 dimer","volume":"267","author":"Lamphear","year":"1992","journal-title":"J Biol Chem"},{"issue":"13","key":"2025103011212120100_bib34","doi-asserted-by":"crossref","first-page":"9445","DOI":"10.1016\/S0021-9258(17)36901-6","article-title":"Factor IXa protects factor VIIIa from activated protein C. Factor IXa inhibits activated protein C-catalyzed cleavage of factor VIIIa at Arg562","volume":"269","author":"Regan","year":"1994","journal-title":"J Biol Chem"},{"issue":"4","key":"2025103011212120100_bib35","doi-asserted-by":"crossref","first-page":"2082","DOI":"10.1074\/jbc.272.4.2082","article-title":"Localization of a factor X interactive site in the A1 subunit of factor VIIIa","volume":"272","author":"Lapan","year":"1997","journal-title":"J Biol Chem"},{"issue":"8","key":"2025103011212120100_bib36","doi-asserted-by":"crossref","first-page":"3982","DOI":"10.1074\/jbc.271.8.3982","article-title":"Activated protein C-catalyzed proteolysis of factor VIIIa alters its interactions within factor Xase","volume":"271","author":"Regan","year":"1996","journal-title":"J Biol Chem"},{"issue":"5","key":"2025103011212120100_bib37","doi-asserted-by":"crossref","first-page":"1714","DOI":"10.1182\/blood.V95.5.1714.005k40_1714_1720","article-title":"Regulation of factor VIIIa by human activated protein C and protein S: inactivation of cofactor in the intrinsic factor Xase","volume":"95","author":"O'Brien","year":"2000","journal-title":"Blood"},{"issue":"3","key":"2025103011212120100_bib38","doi-asserted-by":"crossref","first-page":"558","DOI":"10.1016\/0167-4838(83)90273-X","article-title":"Solution composition dependent variation in extinction coefficients for p-nitroaniline","volume":"742","author":"Lottenberg","year":"1983","journal-title":"Biochim Biophys Acta"},{"issue":"1","key":"2025103011212120100_bib39","doi-asserted-by":"crossref","first-page":"290","DOI":"10.1182\/blood-2010-08-300756","article-title":"Zymogen-like factor Xa variants restore thrombin generation and effectively bypass the intrinsic pathway in vitro","volume":"117","author":"Bunce","year":"2011","journal-title":"Blood"},{"issue":"45","key":"2025103011212120100_bib40","doi-asserted-by":"crossref","first-page":"15198","DOI":"10.1074\/jbc.RA120.015325","article-title":"Exosite binding drives substrate affinity for the activation of coagulation factor X by the intrinsic Xase complex","volume":"295","author":"Basavaraj","year":"2020","journal-title":"J Biol Chem"},{"issue":"27","key":"2025103011212120100_bib41","doi-asserted-by":"crossref","first-page":"16126","DOI":"10.1074\/jbc.271.27.16126","article-title":"Role of the activation peptide domain in human factor X activation by the extrinsic Xase complex","volume":"271","author":"Baugh","year":"1996","journal-title":"J Biol Chem"},{"issue":"29","key":"2025103011212120100_bib42","doi-asserted-by":"crossref","first-page":"26689","DOI":"10.1074\/jbc.M202507200","article-title":"Nematode anticoagulant protein c2 reveals a site on factor Xa that is important for macromolecular substrate binding to human prothrombinase","volume":"277","author":"Buddai","year":"2002","journal-title":"J Biol Chem"},{"issue":"4","key":"2025103011212120100_bib43","doi-asserted-by":"crossref","first-page":"698","DOI":"10.1021\/bi00623a022","article-title":"A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S","volume":"16","author":"Di Scipio","year":"1977","journal-title":"Biochemistry"},{"issue":"22","key":"2025103011212120100_bib44","doi-asserted-by":"crossref","first-page":"4508","DOI":"10.1021\/bi00719a006","article-title":"The mechanism of activation of bovine factor IX (Christmas factor) by bovine factor XIa (activated plasma thromboplastin antecedent)","volume":"13","author":"Fujikawa","year":"1974","journal-title":"Biochemistry"},{"key":"2025103011212120100_bib45","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1016\/S0076-6879(76)45017-6","article-title":"Thrombin","volume":"45","author":"Lundblad","year":"1976","journal-title":"Methods Enzymol"},{"issue":"2","key":"2025103011212120100_bib46","doi-asserted-by":"crossref","first-page":"438","DOI":"10.1016\/0003-9861(84)90465-X","article-title":"Inhibition of activated porcine Factor IX by dansyl-glutamyl-glycyl-arginyl-chloromethylketone","volume":"233","author":"Lollar","year":"1984","journal-title":"Arch Biochem Biophys"},{"issue":"11","key":"2025103011212120100_bib47","doi-asserted-by":"crossref","first-page":"2925","DOI":"10.1182\/blood.V81.11.2925.2925","article-title":"Biochemical, immunological, and in vivo functional characterization of B-domain-deleted FVIII","volume":"81","author":"Pittman","year":"1993","journal-title":"Blood"},{"issue":"3","key":"2025103011212120100_bib48","doi-asserted-by":"crossref","first-page":"438","DOI":"10.1111\/j.1538-7836.2008.03256.x","article-title":"Combining mutations of charged residues at the A2 domain interface enhances factor VIII stability over single point mutations","volume":"7","author":"Wakabayashi","year":"2009","journal-title":"J Thromb Haemost"},{"issue":"20","key":"2025103011212120100_bib49","doi-asserted-by":"crossref","first-page":"4562","DOI":"10.1182\/blood-2009-05-220327","article-title":"Recombinant canine B-domain-deleted FVIII exhibits high specific activity and is safe in the canine hemophilia A model","volume":"114","author":"Sabatino","year":"2009","journal-title":"Blood"},{"issue":"8","key":"2025103011212120100_bib50","doi-asserted-by":"crossref","first-page":"6246","DOI":"10.1016\/S0021-9258(17)37594-4","article-title":"Isolation and characterization of thrombin-activated human factor VIII","volume":"269","author":"Curtis","year":"1994","journal-title":"J Biol Chem"},{"issue":"2","key":"2025103011212120100_bib51","doi-asserted-by":"crossref","first-page":"666","DOI":"10.1021\/bi00428a038","article-title":"Subunit structure of thrombin-activated porcine factor VIII","volume":"28","author":"Lollar","year":"1989","journal-title":"Biochemistry"},{"issue":"11","key":"2025103011212120100_bib52","doi-asserted-by":"crossref","first-page":"2041","DOI":"10.1111\/jth.13134","article-title":"Thrombin generation assay using factor XIa to measure factors VIII and IX and their glycoPEGylated derivatives is robust and sensitive","volume":"13","author":"Waters","year":"2015","journal-title":"J Thromb Haemost"},{"issue":"17","key":"2025103011212120100_bib53","doi-asserted-by":"crossref","first-page":"1484","DOI":"10.1182\/blood.2019001292","article-title":"BIVV001, a new class of factor VIII replacement for hemophilia A that is independent of von Willebrand factor in primates and mice","volume":"135","author":"Seth Chhabra","year":"2020","journal-title":"Blood"},{"issue":"1","key":"2025103011212120100_bib54","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1038\/ng0595-119","article-title":"Targeted disruption of the mouse factor VIII gene produces a model of haemophilia A","volume":"10","author":"Bi","year":"1995","journal-title":"Nat Genet"},{"issue":"11","key":"2025103011212120100_bib55","doi-asserted-by":"crossref","first-page":"1028","DOI":"10.1038\/nbt.1995","article-title":"A zymogen-like factor Xa variant corrects the coagulation defect in hemophilia","volume":"29","author":"Ivanciu","year":"2011","journal-title":"Nat Biotechnol"},{"issue":"24","key":"2025103011212120100_bib56","doi-asserted-by":"crossref","first-page":"16355","DOI":"10.1074\/jbc.M708985200","article-title":"Detailed mechanisms of the inactivation of factor VIIIa by activated protein C in the presence of its cofactors, protein S and factor V","volume":"283","author":"Gale","year":"2008","journal-title":"J Biol Chem"},{"issue":"24","key":"2025103011212120100_bib57","doi-asserted-by":"crossref","first-page":"17006","DOI":"10.1016\/S0021-9258(18)41885-6","article-title":"Binding of factor VIIIa and factor VIII to factor IXa on phospholipid vesicles","volume":"267","author":"Duffy","year":"1992","journal-title":"J Biol Chem"},{"issue":"6","key":"2025103011212120100_bib58","doi-asserted-by":"crossref","first-page":"2497","DOI":"10.1172\/JCI117259","article-title":"Inhibition of human factor VIIIa by anti-A2 subunit antibodies","volume":"93","author":"Lollar","year":"1994","journal-title":"J Clin Invest"},{"issue":"7","key":"2025103011212120100_bib59","doi-asserted-by":"crossref","first-page":"2761","DOI":"10.1182\/blood-2008-02-142158","article-title":"Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface","volume":"112","author":"Wakabayashi","year":"2008","journal-title":"Blood"},{"issue":"6","key":"2025103011212120100_bib60","doi-asserted-by":"crossref","first-page":"2292","DOI":"10.1182\/blood.V87.6.2292.bloodjournal8762292","article-title":"Requirements of von Willebrand factor to protect factor VIII from inactivation by activated protein C","volume":"87","author":"Koppelman","year":"1996","journal-title":"Blood"},{"issue":"32","key":"2025103011212120100_bib61","doi-asserted-by":"crossref","first-page":"20522","DOI":"10.1016\/S0021-9258(17)32024-0","article-title":"Factor VIIIa A2 subunit residues 558-565 represent a factor IXa interactive site","volume":"269","author":"Fay","year":"1994","journal-title":"J Biol Chem"},{"issue":"12","key":"2025103011212120100_bib62","doi-asserted-by":"crossref","first-page":"3120","DOI":"10.1182\/blood.V80.12.3120.3120","article-title":"Proteolytic interactions of factor IXa with human factor VIII and factor VIIIa","volume":"80","author":"Lamphear","year":"1992","journal-title":"Blood"},{"key":"2025103011212120100_bib63","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1016\/j.omtm.2018.12.007","article-title":"Protein-engineered coagulation factors for hemophilia gene therapy","volume":"12","author":"Samelson-Jones","year":"2019","journal-title":"Mol Ther Methods Clin Dev"},{"issue":"10","key":"2025103011212120100_bib64","doi-asserted-by":"crossref","first-page":"7150","DOI":"10.1016\/S0021-9258(17)37260-5","article-title":"Identification of a binding site for blood coagulation factor IXa on the light chain of human factor VIII","volume":"269","author":"Lenting","year":"1994","journal-title":"J Biol Chem"},{"issue":"4","key":"2025103011212120100_bib65","doi-asserted-by":"crossref","first-page":"1935","DOI":"10.1074\/jbc.271.4.1935","article-title":"The sequence Glu1811-Lys1818 of human blood coagulation factor VIII comprises a binding site for activated factor IX","volume":"271","author":"Lenting","year":"1996","journal-title":"J Biol Chem"},{"issue":"2","key":"2025103011212120100_bib66","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1016\/0005-2744(79)90103-7","article-title":"The inhibition of blood coagulation by activated Protein C through the selective inactivation of activated Factor V","volume":"571","author":"Walker","year":"1979","journal-title":"Biochim Biophys Acta"},{"issue":"3","key":"2025103011212120100_bib67","doi-asserted-by":"crossref","first-page":"1443","DOI":"10.1016\/S0021-9258(19)68213-X","article-title":"Studies of the capacity of factor Xa to protect factor Va from inactivation by activated protein C","volume":"257","author":"Nesheim","year":"1982","journal-title":"J Biol Chem"},{"issue":"3","key":"2025103011212120100_bib68","doi-asserted-by":"crossref","first-page":"1914","DOI":"10.1016\/S0021-9258(18)33076-X","article-title":"Inactivation of human coagulation factor V by activated protein C","volume":"258","author":"Suzuki","year":"1983","journal-title":"J Biol Chem"},{"issue":"42","key":"2025103011212120100_bib69","doi-asserted-by":"crossref","first-page":"31486","DOI":"10.1016\/S0021-9258(19)84061-9","article-title":"Effects of factor Xa and protein S on the individual activated protein C-mediated cleavages of coagulation factor Va","volume":"281","author":"Norstr\u00f8m","year":"2006","journal-title":"J Biol Chem"},{"issue":"39","key":"2025103011212120100_bib70","doi-asserted-by":"crossref","first-page":"28353","DOI":"10.1074\/jbc.M704316200","article-title":"Factor Va Residues 311\u2013325 represent an activated protein C binding region","volume":"282","author":"Yegneswaran","year":"2007","journal-title":"J Biol Chem"},{"issue":"1","key":"2025103011212120100_bib71","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1160\/TH08-07-0491","article-title":"Prothrombin amino terminal region helps protect coagulation factor Va from proteolytic inactivation by activated protein C","volume":"101","author":"Yegneswaran","year":"2009","journal-title":"Thromb Haemost"},{"issue":"46","key":"2025103011212120100_bib72","doi-asserted-by":"crossref","first-page":"27852","DOI":"10.1074\/jbc.270.46.27852","article-title":"Effects of protein S and factor Xa on peptide bond cleavages during inactivation of factor Va and factor VaR506Q by activated protein C","volume":"270","author":"Rosing","year":"1995","journal-title":"J Biol Chem"},{"issue":"11","key":"2025103011212120100_bib73","doi-asserted-by":"crossref","first-page":"6648","DOI":"10.1074\/jbc.M708036200","article-title":"Effects of prothrombin on the individual activated protein C-mediated cleavages of coagulation factor Va","volume":"283","author":"Tran","year":"2008","journal-title":"J Biol Chem"},{"issue":"2","key":"2025103011212120100_bib74","doi-asserted-by":"crossref","first-page":"374","DOI":"10.1111\/j.1742-4658.2005.05070.x","article-title":"Factor VIIIa regulates substrate delivery to the intrinsic factor X-activating complex","volume":"273","author":"Panteleev","year":"2006","journal-title":"The FEBS J"},{"issue":"21","key":"2025103011212120100_bib75","doi-asserted-by":"crossref","first-page":"4396","DOI":"10.1182\/blood-2012-10-464164","article-title":"Minimal modification in the factor VIII B-domain sequence ameliorates the murine hemophilia A phenotype","volume":"121","author":"Siner","year":"2013","journal-title":"Blood"},{"issue":"6475","key":"2025103011212120100_bib76","doi-asserted-by":"crossref","first-page":"64","DOI":"10.1038\/369064a0","article-title":"Mutation in blood coagulation factor V associated with resistance to activated protein C","volume":"369","author":"Bertina","year":"1994","journal-title":"Nature"},{"issue":"35","key":"2025103011212120100_bib77","doi-asserted-by":"crossref","first-page":"20794","DOI":"10.1074\/jbc.270.35.20794","article-title":"The mechanism of inactivation of human platelet factor Va from normal and activated protein C-resistant individuals (\u2217)","volume":"270","author":"Camire","year":"1995","journal-title":"J Biol Chem"},{"issue":"8","key":"2025103011212120100_bib78","doi-asserted-by":"crossref","first-page":"4053","DOI":"10.1074\/jbc.270.8.4053","article-title":"Characterization of the molecular defect in factor VR506A(\u2217)","volume":"270","author":"Kalafatis","year":"1995","journal-title":"J Biol Chem"},{"issue":"3","key":"2025103011212120100_bib79","doi-asserted-by":"crossref","first-page":"1004","DOI":"10.1073\/pnas.90.3.1004","article-title":"Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C","volume":"90","author":"Dahlb\u00e4ck","year":"1993","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"05","key":"2025103011212120100_bib80","doi-asserted-by":"crossref","first-page":"739","DOI":"10.1055\/s-0038-1653860","article-title":"Resistance to activated protein C, the Arg506 to gin mutation in the factor V gene, and venous thrombosis","volume":"73","author":"Dahlb\u00e4ck","year":"1995","journal-title":"Thromb Haemost"},{"issue":"6","key":"2025103011212120100_bib81","doi-asserted-by":"crossref","first-page":"1504","DOI":"10.1182\/blood.V85.6.1504.bloodjournal8561504","article-title":"High risk of thrombosis in patients homozygous for factor V Leiden (activated protein C resistance)","volume":"85","author":"Rosendaal","year":"1995","journal-title":"Blood"},{"issue":"1","key":"2025103011212120100_bib82","doi-asserted-by":"crossref","first-page":"483","DOI":"10.1055\/s-0038-1657574","article-title":"Resistance to activated protein C caused by the factor VR506Q mutation is a common risk factor for venous thrombosis","volume":"78","author":"Dahlb\u00e4ck","year":"1997","journal-title":"Thromb Haemost"},{"issue":"8943","key":"2025103011212120100_bib83","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1016\/S0140-6736(95)90166-3","article-title":"Role of clotting factor VIII in effect of von Willebrand factor on occurrence of deep-vein thrombosis","volume":"345","author":"Koster","year":"1995","journal-title":"Lancet"},{"issue":"23","key":"2025103011212120100_bib84","doi-asserted-by":"crossref","first-page":"2215","DOI":"10.1056\/NEJMoa1708538","article-title":"Hemophilia B gene therapy with a high-specific-activity factor IX variant","volume":"377","author":"George","year":"2017","journal-title":"N Engl J Med"},{"issue":"8","key":"2025103011212120100_bib85","doi-asserted-by":"crossref","first-page":"706","DOI":"10.1056\/NEJMoa2211644","article-title":"Gene therapy with etranacogene dezaparvovec for Hemophilia B","volume":"388","author":"Pipe","year":"2023","journal-title":"N Engl J Med"},{"issue":"8","key":"2025103011212120100_bib86","doi-asserted-by":"crossref","first-page":"694","DOI":"10.1056\/NEJMoa2211075","article-title":"Two-year outcomes of valoctocogene roxaparvovec therapy for hemophilia A","volume":"388","author":"Mahlangu","year":"2023","journal-title":"N Engl J Med Overseas Ed"},{"issue":"21","key":"2025103011212120100_bib87","doi-asserted-by":"crossref","first-page":"1961","DOI":"10.1056\/NEJMoa2104205","article-title":"Multiyear factor VIII expression after AAV gene transfer for hemophilia A","volume":"385","author":"George","year":"2021","journal-title":"N Engl J Med"},{"issue":"9","key":"2025103011212120100_bib88","doi-asserted-by":"crossref","first-page":"796","DOI":"10.1182\/blood.2022018971","article-title":"Giroctocogene fitelparvovec gene therapy for severe hemophilia A: 104-week analysis of the phase 1\/2 Alta study","volume":"143","author":"Leavitt","year":"2024","journal-title":"Blood"},{"key":"2025103011212120100_bib89","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1146\/annurev-med-043021-033013","article-title":"Adeno-associated virus gene therapy for hemophilia","volume":"74","author":"Samelson-Jones","year":"2023","journal-title":"Annu Rev Med"},{"issue":"7","key":"2025103011212120100_bib90","doi-asserted-by":"crossref","first-page":"781","DOI":"10.1002\/ajh.23723","article-title":"Chromogenic factor VIII activity assay","volume":"89","author":"Moser","year":"2014","journal-title":"Am J Hematol"}],"container-title":["Blood"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/ashpublications.org\/blood\/article-pdf\/146\/18\/2259\/2418440\/blood_bld-2024-027705-main.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/ashpublications.org\/blood\/article-pdf\/146\/18\/2259\/2418440\/blood_bld-2024-027705-main.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,30]],"date-time":"2025-10-30T15:21:39Z","timestamp":1761837699000},"score":1,"resource":{"primary":{"URL":"https:\/\/ashpublications.org\/blood\/article\/146\/18\/2259\/546192\/Factor-IXa-and-factor-X-influence-factor-VIIIa"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,30]]},"references-count":90,"journal-issue":{"issue":"18","published-print":{"date-parts":[[2025,10,30]]}},"URL":"https:\/\/doi.org\/10.1182\/blood.2024027705","relation":{},"ISSN":["0006-4971","1528-0020"],"issn-type":[{"value":"0006-4971","type":"print"},{"value":"1528-0020","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2025,10,30]]},"published":{"date-parts":[[2025,10,30]]}}}