Selective disruption of heparin and antithrombin-mediated regulation of human factor IX
Copyright policy )Selective disruption of heparin and antithrombin-mediated regulation of human factor IX
Interaction with antithrombin and heparin regulates distribution, activity, and clearance of factor IXa (FIXa). Hemophilia B prophylaxis targets plasma FIX levels > 1% but neglects extravascular FIX, which colocalizes with antithrombin-heparan sulfate.Combined mutagenesis of FIX was undertaken to selectively disrupt heparin- and antithrombin-mediated regulation of the protease.Human FIX alanine substitutions in the heparin (K126A and K132A) and antithrombin (R150A) exosites were characterized with regard to coagulant activity, plasma thrombin generation, antithrombin inhibition, and plasma half-life.Single or combined (K126A/R150A or K132A/R150A) exosite mutations variably reduced coagulant activity relative to wild-type (WT) for FIX (27-91%) and FIXa (25-91%). Double mutation in the heparin exosite (K126A/K132A and K126A/K132A/R150A) markedly reduced coagulant activity (7-21%) and plasma TG. In contrast to coagulant activity, FIX K126A (1.8-fold), R150 (1.6-fold), and K132A/R150A (1.3-fold) supported increased tissue factor-initiated plasma TG, while FIX K132A and K126A/R150A were similar to WT. FIXa K126A/R150A and K132A/R150A (1.5-fold) demonstrated significantly increased FIXa-initiated TG, while FIXa K132A, R150A, and K126A (0.8-0.9-fold) were similar to WT. Dual mutations in the heparin exosite or combined mutations in both exosites synergistically reduced the inhibition rate for antithrombin-heparin. The half-life of FIXa WT in FIX-deficient plasma was remarkably lengthy (40.9 ±1.4 min) and further prolonged for FIXa R150A, K126A/R150A, and K132A/R150A (> 2 h).Selective disruption of exosite-mediated regulation by heparin and antithrombin can be achieved with preserved or enhanced thrombin generation capacity. These proteins should demonstrate enhanced therapeutic efficacy for hemophilia B.
- University of Wisconsin System United States
- Mahidol University Thailand
- University of Wisconsin–Oshkosh United States
- University of Wisconsin–Madison United States
Models, Molecular, Binding Sites, Genotype, Heparin, Protein Conformation, Antithrombin III, Anticoagulants, Hemophilia B, Factor IXa, Factor IX, Kinetics, Structure-Activity Relationship, HEK293 Cells, Phenotype, Allosteric Regulation, Mutation, Humans, Blood Coagulation, Half-Life, Protein Binding
Models, Molecular, Binding Sites, Genotype, Heparin, Protein Conformation, Antithrombin III, Anticoagulants, Hemophilia B, Factor IXa, Factor IX, Kinetics, Structure-Activity Relationship, HEK293 Cells, Phenotype, Allosteric Regulation, Mutation, Humans, Blood Coagulation, Half-Life, Protein Binding
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