Expression and characterization of a novel human recombinant factor IX molecule with enhanced in vitro and in vivo clotting activity
Abstract
Hemophilia B is an inherited X-linked recessive bleeding disorder, due to a defect in human factor IX (FIX). The main treatment for hemophilia B is replacement therapy using FIX concentrates. Prophylactic treatment in severe hemophilia B is very effective but is limited by cost issues. Production of a recombinant FIX (rFIX) with enhanced clotting activity, offering the possibility of fewer infusions and fewer costs with similar efficacy, is one of the current challenges for hemophilia B treatment. The present study focused on an important amino acid sequence known to be involved in the interaction of activated FIX (FIXa) with its cofactor, activated factor VIII (FVIIIa). Using site-directed mutagenesis of glutamate E410 (c240, chymotrypsin numbering), four recombinant FIX-E410 (E410H, A, L and N) mutants were developed and produced by the human hepatoma cell line Huh-7. The in-vitro clotting activity of mutant FIX molecules was 3 to 5-fold higher than wild-type recombinant FIX (FIX-WT). FIX-E410H compound showed the highest in-vitro procoagulant activity. Enhanced specific activity was confirmed using thrombin generation assay. FIX-E410H induced 5.2-fold higher thrombin generation than FIX-WT. In hemophilia B mice, we observed ...Continue Reading
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The C-terminal region of the hepatitis B virus X protein is essential for its stability and function
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Blood Clotting Disorders
Thrombophilia includes conditions with increased tendency for excessive blood clotting. Blood clotting occurs when the body has insufficient amounts of specialized proteins that make blood clot and stop bleeding. Here is the latest research on blood clotting disorders.