title: Selective disruption of heparin and antithrombin-mediated regulation of human factor IX.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  Combined mutagenesis of FIX was undertaken to selectively disrupt heparin- and antithrombin-mediated regulation of the protease.
  
  
  METHODS
  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.
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  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.
authors: Department of Medicine/Hematology-Oncology, University of Wisconsin-Madison, Madison, WI, USA.
authors: P R
authors: PR
authors: Westmark
authors: Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI, USA.|Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors: P
authors: P
authors: Tanratana
authors: Department of Medicine/Hematology-Oncology, University of Wisconsin-Madison, Madison, WI, USA.
authors: J P
authors: JP
authors: Sheehan
mesh_terms: D000494:Allosteric Regulation; D000925:Anticoagulants / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D000990:Antithrombin III / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects; D005164:Factor IX / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015949:Factor IXa / Q000378:metabolism; D005838:Genotype; D057809:HEK293 Cells; D006207:Half-Life; D002836:Hemophilia B / Q000097:blood* / Q000188:drug therapy / Q000235:genetics; D006493:Heparin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism; D014162:Transfection
chemical_list: D000925:Anticoagulants; C542513:SERPINC1 protein, human; D000990:Antithrombin III; D005164:Factor IX; D006493:Heparin; D015949:Factor IXa; D013917:Thrombin
keywords: antithrombin III; blood coagulation; factor IX; hemophilia B; heparin