Modified vitamin K-dependent polypeptides
The invention provides vitamin K-dependent polypeptides with enhanced membrane binding affinity. These polypeptides can be used to modulate clot formation in mammals. Methods of modulating clot formation in mammals are also described.
1. A vitamin K-dependent polypeptide that increases clot formation comprising a modified GLA domain that enhances membrane binding affinity of said polypeptide relative to a corresponding native vitamin K-dependent polypeptide, wherein said modified GLA domain comprises the sequence of SEQ ID NO:3 or SEQ ID NO:4 with two, three, four, or five substitutions, wherein said substitutions comprise a glutamine residue substituted at amino acid position 10 and amino acid substitutions at one or more amino acid positions selected from positions 28, 32, 33, and 34 of SEQ ID NO: 3 or SEQ ID NO: 4.
2. The polypeptide of claim 1 , wherein said modified GLA domain comprises an amino acid substitution at position 32.
3. The polypeptide of claim 2 , wherein said modified GLA domain comprises a glutamic acid residue substituted at position 32.
4. The polypeptide of claim 1 , wherein said modified GLA domain comprises an amino acid substitution at position 34.
5. The polypeptide of claim 2 , wherein said modified GLA domain further comprises an amino acid substitution at position 34.
6. The polypeptide of claim 3 , wherein said modified GLA domain further comprises an amino acid substitution at position 34.
7. The polypeptide of claim 6 , wherein said modified GLA domain further comprises a glutamic acid residue substituted at position 34.
8. The polypeptide of claim 1 , further comprising a tyrosine or glycine residue inserted at position 4.
9. The polypeptide of claim 2 , further comprising a tyrosine or glycine residue inserted at position 4.
10. The polypeptide of claim 1 , wherein said modified GLA domain comprises the amino acid substitutions at two or more amino acid positions selected from positions 28, 32, 33, and 34.
11. The polypeptide of claim 1 , wherein said modified GLA domain comprises an amino acid substitution at position 33.
12. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier.
13. A method for increasing clot formation in a mammal, comprising administering an amount of a vitamin K-dependent polypeptide that is effective to increase clot formation in said mammal, wherein said vitamin K-dependent polypeptide comprises a modified GLA domain that enhances membrane binding affinity of said polypeptide relative to a corresponding native vitamin K-dependent polypeptide, wherein said modified GLA domain comprises the sequence of SEQ ID NO:3 or SEQ ID NO:4 with two, three, four, or five substitutions, wherein said substitutions comprise a glutamine residue substituted at amino acid position 10 and amino acid substitutions at one or more amino acid positions selected from positions 28, 32, 33, and 34 of SEQ ID NO: 3 or SEQ ID NO: 4.
14. A method for treating a hemophilia in a subject, comprising administering an amount of a vitamin K-dependent polypeptide that is effective to treat hemophilia in the subject, wherein said vitamin K-dependent polypeptide comprises a modified GLA domain that enhances membrane binding affinity of said polypeptide relative to a corresponding native vitamin K-dependent polypeptide, wherein said modified GLA domain comprises the sequence of SEQ ID NO:3 or SEQ ID NO:4 with two, three, four, or five substitutions, wherein said substitutions comprise a glutamine residue substituted at amino acid position 10 and amino acid substitutions at one or more amino acid positions selected from positions 28, 32, 33, and 34 of SEQ ID NO: 3 or SEQ ID NO: 4.