{"title":"FVIII 中 Asp519Val/Glu665Val 和 Lys1813Ala 突变的组合会显著增加凝血潜能。","authors":"Yuto Nakajima, Akihisa Oda, Nemekhbayar Baatartsogt, Yuji Kashiwakura, Tsukasa Ohmori, Keiji Nogami","doi":"10.1182/bloodadvances.2023012391","DOIUrl":null,"url":null,"abstract":"<p><strong>Abstract: </strong>A2 domain dissociation in activated factor VIII (FVIIIa) results in reduced activity. Previous studies demonstrated that some FVIII mutants (D519V/E665V and K1813A) with delayed A2 dissociation enhanced coagulation potential. We speculated, therefore, that FVIII encompassing a combination of these mutations might further enhance coagulant activity. The aim was to assess the D519V/E665V/K1813A-FVIII mutation as a gain of function. The FVIII mutants, D519V/E665V/K1813A, D519V/E665V, and K1813A were expressed in a baby hamster kidney cell system, and global coagulation potential of these mutants was compared with wild-type (WT) FVIII in vitro and in hemophilia A mice in vivo. Kinetic analyses indicated that the apparent Kd for FIXa on the tenase assembly with D519V/E665V and D519V/E665V/K1813A mutants were lower, and that the generated FXa for D519V/E665V/K1813A was significantly greater than WT-FVIII. WT-FVIII activity after thrombin activation increased by ∼12-fold within 5 minutes, and returned to initial levels within 30 minutes. In contrast, The FVIII-related activity of D519V/E665V/K1813A increased further with time after thrombin activation, and showed an ∼25-fold increase at 2 hours. The A2 dissociation rate of D519V/E665V/K1813A was ∼50-fold slower than the WT in a 1-stage clotting assay. Thrombin generation assays demonstrated that D519V/E665V/K1813A (0.125 nM) exhibited coagulation potential comparable with that of the WT (1 nM). In animal studies, rotational thromboelastometry and tail-clip assays showed that the coagulation potential of D519V/E665V/K1813A (0.25 μg/kg) was equal to that of the WT (2 μg/kg). FVIII-D519V/E665V/K1813A mutant could provide an approximately eightfold increase in hemostatic function of WT-FVIII because of increased FVIIIa stability and the association between FVIIIa and FIXa.</p>","PeriodicalId":9228,"journal":{"name":"Blood advances","volume":null,"pages":null},"PeriodicalIF":7.4000,"publicationDate":"2024-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11321387/pdf/","citationCount":"0","resultStr":"{\"title\":\"The combination of Asp519Val/Glu665Val and Lys1813Ala mutations in FVIII markedly increases coagulation potential.\",\"authors\":\"Yuto Nakajima, Akihisa Oda, Nemekhbayar Baatartsogt, Yuji Kashiwakura, Tsukasa Ohmori, Keiji Nogami\",\"doi\":\"10.1182/bloodadvances.2023012391\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Abstract: </strong>A2 domain dissociation in activated factor VIII (FVIIIa) results in reduced activity. Previous studies demonstrated that some FVIII mutants (D519V/E665V and K1813A) with delayed A2 dissociation enhanced coagulation potential. We speculated, therefore, that FVIII encompassing a combination of these mutations might further enhance coagulant activity. The aim was to assess the D519V/E665V/K1813A-FVIII mutation as a gain of function. The FVIII mutants, D519V/E665V/K1813A, D519V/E665V, and K1813A were expressed in a baby hamster kidney cell system, and global coagulation potential of these mutants was compared with wild-type (WT) FVIII in vitro and in hemophilia A mice in vivo. Kinetic analyses indicated that the apparent Kd for FIXa on the tenase assembly with D519V/E665V and D519V/E665V/K1813A mutants were lower, and that the generated FXa for D519V/E665V/K1813A was significantly greater than WT-FVIII. WT-FVIII activity after thrombin activation increased by ∼12-fold within 5 minutes, and returned to initial levels within 30 minutes. In contrast, The FVIII-related activity of D519V/E665V/K1813A increased further with time after thrombin activation, and showed an ∼25-fold increase at 2 hours. The A2 dissociation rate of D519V/E665V/K1813A was ∼50-fold slower than the WT in a 1-stage clotting assay. Thrombin generation assays demonstrated that D519V/E665V/K1813A (0.125 nM) exhibited coagulation potential comparable with that of the WT (1 nM). In animal studies, rotational thromboelastometry and tail-clip assays showed that the coagulation potential of D519V/E665V/K1813A (0.25 μg/kg) was equal to that of the WT (2 μg/kg). FVIII-D519V/E665V/K1813A mutant could provide an approximately eightfold increase in hemostatic function of WT-FVIII because of increased FVIIIa stability and the association between FVIIIa and FIXa.</p>\",\"PeriodicalId\":9228,\"journal\":{\"name\":\"Blood advances\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":7.4000,\"publicationDate\":\"2024-08-13\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11321387/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Blood advances\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1182/bloodadvances.2023012391\",\"RegionNum\":1,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"HEMATOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Blood advances","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1182/bloodadvances.2023012391","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"HEMATOLOGY","Score":null,"Total":0}
The combination of Asp519Val/Glu665Val and Lys1813Ala mutations in FVIII markedly increases coagulation potential.
Abstract: A2 domain dissociation in activated factor VIII (FVIIIa) results in reduced activity. Previous studies demonstrated that some FVIII mutants (D519V/E665V and K1813A) with delayed A2 dissociation enhanced coagulation potential. We speculated, therefore, that FVIII encompassing a combination of these mutations might further enhance coagulant activity. The aim was to assess the D519V/E665V/K1813A-FVIII mutation as a gain of function. The FVIII mutants, D519V/E665V/K1813A, D519V/E665V, and K1813A were expressed in a baby hamster kidney cell system, and global coagulation potential of these mutants was compared with wild-type (WT) FVIII in vitro and in hemophilia A mice in vivo. Kinetic analyses indicated that the apparent Kd for FIXa on the tenase assembly with D519V/E665V and D519V/E665V/K1813A mutants were lower, and that the generated FXa for D519V/E665V/K1813A was significantly greater than WT-FVIII. WT-FVIII activity after thrombin activation increased by ∼12-fold within 5 minutes, and returned to initial levels within 30 minutes. In contrast, The FVIII-related activity of D519V/E665V/K1813A increased further with time after thrombin activation, and showed an ∼25-fold increase at 2 hours. The A2 dissociation rate of D519V/E665V/K1813A was ∼50-fold slower than the WT in a 1-stage clotting assay. Thrombin generation assays demonstrated that D519V/E665V/K1813A (0.125 nM) exhibited coagulation potential comparable with that of the WT (1 nM). In animal studies, rotational thromboelastometry and tail-clip assays showed that the coagulation potential of D519V/E665V/K1813A (0.25 μg/kg) was equal to that of the WT (2 μg/kg). FVIII-D519V/E665V/K1813A mutant could provide an approximately eightfold increase in hemostatic function of WT-FVIII because of increased FVIIIa stability and the association between FVIIIa and FIXa.
期刊介绍:
Blood Advances, a semimonthly medical journal published by the American Society of Hematology, marks the first addition to the Blood family in 70 years. This peer-reviewed, online-only, open-access journal was launched under the leadership of founding editor-in-chief Robert Negrin, MD, from Stanford University Medical Center in Stanford, CA, with its inaugural issue released on November 29, 2016.
Blood Advances serves as an international platform for original articles detailing basic laboratory, translational, and clinical investigations in hematology. The journal comprehensively covers all aspects of hematology, including disorders of leukocytes (both benign and malignant), erythrocytes, platelets, hemostatic mechanisms, vascular biology, immunology, and hematologic oncology. Each article undergoes a rigorous peer-review process, with selection based on the originality of the findings, the high quality of the work presented, and the clarity of the presentation.