title: Hemostatic agents of broad applicability produced by selective tuning of factor Xa zymogenicity.
abstract: There is a clinical need to develop safe therapeutic strategies to mitigate bleeding. Previously, we found that a novel zymogen-like factor Xa variant (FXa-I16L) was effective in correcting the coagulation defect in hemophilic mice. Here we expand the mutational framework to tune the FX(a) zymogen-like state. Alteration of FXa zymogenicity yields variants (V17M, I16L, I16M, V17T, V17S, and I16T) with a wide range (≤1000-fold) of reduced function toward physiologic substrates and inhibitors. The extent of zymogen-like character, including resistance to antithrombin III, correlates well with plasma half-life (<2 minutes to >4 hours). Importantly, biologic function, including that of the most zymogen-like variant (FXa-I16T), was greatly enhanced when bound to FVa membranes. This resulted in improvement of clotting times and thrombin generation in hemophilic plasma. The FXa variants were remarkably effective in mouse injury models. In these systems, the data show that the more active the protease, the more difficult it is to overcome the protective mechanism of circulating inhibitors to achieve a therapeutic benefit. Depending on the treatment situation, the more zymogen-like variants (V17S and I16T) were most useful when given before injury whereas variants exhibiting greater activity but shorter half-lives (I16L and I16M) were most effective when administered after injury. This new class of FXa variants provides a useful and flexible platform for selectively bioengineering biologic function and half-life to target different clinical bleeding scenarios.
authors.affiliation: The Children's Hospital of Philadelphia, The Center for Cell and Molecular Therapeutics, and Division of Hematology, Department of Pediatrics, The University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA.
authors.forename: Lacramioara
authors.initials: L
authors.lastname: Ivanciu
authors.affiliation: The Children's Hospital of Philadelphia, The Center for Cell and Molecular Therapeutics, and Division of Hematology, Department of Pediatrics, The University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA.
authors.forename: Rodney M
authors.initials: RM
authors.lastname: Camire
mesh_terms: D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015951:Factor Xa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006207:Half-Life; D006467:Hemophilia A / Q000097:blood / Q000188:drug therapy; D006490:Hemostatics / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000627:therapeutic use; D051379:Mice; D008807:Mice, Inbred BALB C; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D011516:Prothrombin / Q000378:metabolism; D013925:Thromboplastin / Q000737:chemistry
chemical_list: D004792:Enzyme Precursors; D006490:Hemostatics; D050505:Mutant Proteins; D011516:Prothrombin; D013925:Thromboplastin; D015951:Factor Xa