Compositions and Methods for Modulating Hemostasis

Abstract

Factor Xa variants and methods of use thereof are disclosed.

1 - 44 . (canceled) 
     
     
         45 . A Factor X/Factor Xa zymogen/protease variant which modulates hemostasis. 
     
     
         46 . The variant zymogen protease as claimed in  claim 45 , wherein said variant is a Factor X/Factor Xa variant encoded by SEQ ID NO: 2, wherein nucleotides 1684-1695 of SEQ ID NO: 2 can be any amino acid with the proviso that nucleotides 1684-1886 do not encode Val or Ala. 
     
     
         47 . The variant zymogen/protease of  claim 45  which contains at least one modification in SEQ ID NO: 1 selected from the group consisting of:
 a) Ile at position 16 is Leu, Phe, Asp or Gly; 
 b) Val at position 17 is Leu, Ala, or Gly and 
 c) Asp at position 194 is Asn or Glu. 
 
     
     
         48 . A nucleic acid encoding the variant zymogen/protease of  claim 47 , said nucleic acid optionally encoding an intracellular PACE/Furin cleavage site. 
     
     
         49 . A nucleic acid as claimed in  claim 47  having the sequence of SEQ ID NO: 2, wherein the nucleotides at positions 1684-1695 encode the amino acids selected from the group consisting of Leu-Val-Gly, Gly-Val-Gly, Ile-Ala-Gly, Phe-Val-Gly and Ile-Gly-Gly, said nucleic acid optionally comprising nucleotides at position 2233-2235 which encode an amino acid selected from the group consisting of Asn or Glu. 
     
     
         50 . A variant zymogen/protease encoded by the nucleic acid of  claim 49 . 
     
     
         51 . A pharmaceutical composition comprising the Factor Xa variant of  claim 47  in a biologically compatible carrier. 
     
     
         52 . The nucleic acid of  claim 48  cloned into an expression vector. 
     
     
         53 . The vector of  claim 52 , selected from the group consisting of an adenoviral vector, an adenovirus-associated vector, a retroviral vector, a plasmid, and a lentiviral vector. 
     
     
         54 . A method for treatment of a hemostasis related disorder in a patient in need thereof comprising administration of a therapeutically effective amount of the zymogen/protease of  claim 47  in a biologically acceptable carrier. 
     
     
         55 . The method of  claim 54 , wherein said variant is a pro-coagulant and said disorder is selected from the group consisting of hemophilia A and B, hemophilia A and B associated with inhibitory antibodies, coagulation factor deficiency, vitamin K epoxide reductase Cl deficiency, gamma-carboxylase deficiency, bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); over-anticoagulation treatment disorders, Bernard Soulier syndrome, Glanzman thromblastemia, and storage pool deficiency. 
     
     
         56 . The method of  claim 55 , wherein said coagulation factor is selected from the group consisting of at least one of factor VII, factor IX, factor X, factor XI, factor V, factor XII, factor II, and von Willebrand factor. 
     
     
         57 . The method of  claim 55 , wherein said over-anticoagulation treatment disorder results from administration of heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotics and FXa inhibitors. 
     
     
         58 . The method of  claim 54 , wherein said variant is an anticoagulant and said disorder is selected from the group consisting of thrombosis, thrombocytopenia, stroke, and coagulapathy. 
     
     
         59 . The method of  claim 54 , wherein said the amino acids at positions 16, 17 and 18 in SEQ ID NO: 2 are selected from the group consisting of Leu-Val-Gly, Gly-Val-Gly, Ile-Ala-Gly, Phe-Val-Gly and Ile-Gly-Gly and amino acid at position 194 is selected from the group consisting of Asn and Glu. 
     
     
         60 . The method of  claim 59 , wherein said variant is administered intravenously at least once a day at a dosage between about 10 and 500 pg/kg. 
     
     
         61 . The method of  claim 59 , wherein said variant is administered intravenously at least once a day at a dosage between about 10 and 250 pg/kg. 
     
     
         62 . The method of  claim 59 , wherein said variant is encapsulated in a liposome or mixed with phospholipids or micelles. 
     
     
         63 . A host cell expressing the zymogen/protease variant of  claim 47 . 
     
     
         64 . The variant zymogen/protease of  claim 47 , wherein said variant exhibits lower binding affinity for the active site which can be restored when bound by a suitable co-factor. 
     
     
         65 . The variant of  claim 64 , wherein said co-factor is factor Va.