Compositions comprising heterogeneous populations of recombinant human clotting factor xa proteins

Abstract

Compositions are provided comprising recombinant variants of the human clotting Factor Xa. Such compositions include a wide variety of isoforms and post-translational modifications of FXa and are useful for treating subjects in need of hemostasis.

1 - 62 . (canceled) 
     
     
         63 . A method of treating a subject in need of hemostasis comprising administering to said subject a therapeutically effective amount of a pro-coagulant composition comprising:
 a first recombinant FXa variant protein wherein the light chain and heavy chain protein sequences respectively consist of amino acids 1 to 140 and 146 to 386 of the amino acid sequence of SEQ ID NO:1;   and a second recombinant FXa variant protein selected from the group consisting of:   (i) a recombinant FXa variant protein wherein the light chain and heavy chain protein sequences respectively consist of amino acids 1 to 139 and 146 to 386 of the amino acid sequence of SEQ ID NO:1,   (ii) a recombinant FXa variant protein wherein the light chain and heavy chain protein sequences respectively consist of amino acids 1 to 141 and 146 to 386 of the amino acid sequence of SEQ ID NO:1,   (iii) a recombinant FXa variant protein wherein the light chain and heavy chain protein sequences respectively consist of amino acids 1 to 142 and 146 to 386 of the amino acid sequence of SEQ ID NO:1,   (iv) a recombinant FXa variant protein wherein the light chain and heavy chain protein sequences respectively consist of amino acids 1 to 143 and 146 to 386 of the amino acid sequence of SEQ ID NO:1,   (v) a recombinant FXa variant protein wherein the light chain and heavy chain protein sequences respectively consist of amino acids 1 to 140 and 146 to 384 of the amino acid sequence of SEQ ID NO:1,   (vi) a recombinant FXa variant protein wherein the light chain and heavy chain protein sequences respectively consist of amino acids 1 to 141 and 146 to 384 of the amino acid sequence of SEQ ID NO:1,   (vii) a recombinant FXa variant protein wherein the light chain and heavy chain protein sequences respectively consist of amino acids 1 to 142 and 146 to 384 of the amino acid sequence of SEQ ID NO:1; and   (viii) a recombinant FXa variant protein wherein the light chain and heavy chain protein sequences respectively consist of amino acids 1 to 143 and 146 to 384 of the amino acid sequence of SEQ ID NO:1.   
     
     
         64 . The method of  claim 63 , wherein a light chain of at least one of said recombinant FXa variant proteins comprises β-hydroxy Asp 63  and an O-linked hexose. 
     
     
         65 . The method of  claim 63 , wherein a light chain of at least one of said recombinant FXa variant proteins comprises 10 Gla residues. 
     
     
         66 . The method of  claim 63 , wherein a light chain of at least one of said recombinant FXa variant proteins comprises 11 Gla residues. 
     
     
         67 . The method of  claim 63 , wherein a light chain of at least one of said recombinant FXa variant proteins comprises β-hydroxy Asp 63 , an O-linked hexose, and 10 or 11 Gla residues. 
     
     
         68 . A method of treating a subject in need of hemostasis comprising administering to said subject a therapeutically effective amount of a pro-coagulant composition comprising:
 a first recombinant FXa variant protein wherein the light chain and heavy chain protein sequence respectively consist of amino acids 1 to 140 and 146 to 398 of the amino acid sequence of SEQ ID NO:1;   and a second recombinant FXa variant protein selected from the group consisting of:   (i) a recombinant FXa variant protein wherein the light chain and heavy chain protein sequence respectively consist of amino acids 1 to 139 and 146 to 398 of the amino acid sequence of SEQ ID NO:1,   (ii) a recombinant FXa variant protein wherein the light chain and heavy chain protein sequence respectively consist of amino acids 1 to 141 and 146 to 398 of the amino acid sequence of SEQ ID NO:1,   (iii) a recombinant FXa variant protein wherein the light chain and heavy chain protein sequence respectively consist of amino acids 1 to 141 and 146 to 399 of the amino acid sequence of SEQ ID NO:1,   (iv) a recombinant FXa variant protein wherein the light chain and heavy chain protein sequence respectively consist of amino acids 1 to 142 and 146 to 398 of the amino acid sequence of SEQ ID NO:1,   (v) a recombinant FXa variant protein wherein the light chain and heavy chain protein sequences respectively consist of amino acids 1 to 140 and 146 to 399 of the amino acid sequence of SEQ ID NO:1, and   (vi) a recombinant FXa variant protein wherein the light chain and heavy chain protein sequences respectively consist of amino acids 1 to 142 and 146 to 399 of the amino acid sequence of SEQ ID NO:1.   
     
     
         69 . The method of  claim 68 , wherein a light chain of at least one of said recombinant FXa variant proteins comprises β-hydroxy Asp 63  and an O-linked hexose. 
     
     
         70 . The method of  claim 68 , wherein a light chain of at least one of said recombinant FXa variant proteins comprises 10 Gla residues. 
     
     
         71 . The method of  claim 71 , wherein a light chain of at least one of said recombinant FXa variant proteins comprises 11 Gla residues. 
     
     
         72 . The method of  claim 68 , wherein a light chain of at least one of said recombinant FXa variant proteins comprises β-hydroxy Asp 63 , an O-linked hexose, and 10 or 11 Gla residues. 
     
     
         73 . The method of  claim 68 , wherein a heavy chain of at least one of said recombinant FXa variant proteins comprises one core-1 O-linked glycan. 
     
     
         74 . The method of  claim 73 , wherein the core-1 O-linked glycan is mono-sialylated. 
     
     
         75 . The method of  claim 73 , wherein the core-1 O-linked glycan is di-sialylated. 
     
     
         76 . The method of  claim 68 , wherein a heavy chain of at least one of said recombinant FXa variant proteins comprises two core-1 O-linked glycans. 
     
     
         77 . The method of  claim 76 , wherein both core-1 O-linked glycans are mono-sialylated. 
     
     
         78 . The method of  claim 76 , wherein both core-1 O-linked glycans are di-sialylated. 
     
     
         79 . The method of  claim 76 , wherein one of said core-1 O-linked glycans is mono-sialylated and the second of said core-1 O-linked glycans is di-sialylated. 
     
     
         80 . A method of treating a subject in need of hemostasis comprising administering to said subject a therapeutically effective amount of a pro-coagulant composition comprising:
 a first recombinant FXa variant protein wherein the light chain and heavy chain protein sequences respectively consist of amino acids 1 to 141 and 146 to 399 of the amino acid sequence of SEQ ID NO:1;   and a second recombinant FXa variant protein selected from the group consisting of:   (i) a recombinant FXa variant protein wherein the light chain and heavy chain protein sequences respectively consist of amino acids 1 to 140 and 146 to 399 of the amino acid sequence of SEQ ID NO:1; and   (ii) a recombinant FXa variant protein wherein the light chain and heavy chain protein sequences respectively consist of amino acids 1 to 141 and 146 to 398 of the amino acid sequence of SEQ ID NO:1.   
     
     
         81 . The method of  claim 80 , wherein a light chain of each of said first and second recombinant FXa variant proteins comprises β-hydroxy Asp 63 , an O-linked hexose, and at least 10 Gla residues. 
     
     
         82 . The method of  claim 81 , wherein a light chain of each of said first and second recombinant FXa variant proteins comprises β-hydroxy Asp 63 , an O-linked hexose, and 11 Gla residues. 
     
     
         83 . The method of  claim 80 , wherein a heavy chain of each of said first and second recombinant FXa variant proteins comprises two core-1 O-linked glycans. 
     
     
         84 . The method of  claim 83 , wherein both core-1 O-linked glycans are di-sialylated.