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 method of increasing clot formation in a mammal, comprising administering an amount of a vitamin K-dependent polypeptide 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 compared to a native vitamin K-dependent polypeptide, and wherein said modified GLA domain comprises a hydrophobic amino acid residue substituted at the position corresponding to position 34 of SEQ ID NO:5.
2. The method of claim 1 , wherein said modified GLA domain further comprises an amino acid substitution at the position corresponding to position 5, 9, 11, 12, 29, 33, 35, or 36 of SEQ ID NO:5.
3. The method of claim 1 , wherein said modified GLA domain further comprises an amino acid substitution at the position corresponding to position 11 of SEQ ID NO:5.
4. The method of claim 3 , wherein said modified GLA domain further comprises an amino acid substitution at the position corresponding to position 29 or 33 of SEQ ID NO:5.
5. The method of claim 4 , wherein a glutamine residue is substituted at the position corresponding to position 11 and a glutamic acid residue is substituted at the position corresponding to position 33 of SEQ ID NO:5.
6. The method of claim 5 , wherein said modified GLA domain further comprises an amino acid substitution at the position corresponding to position 35 of SEQ ID NO:5.
7. The method of claim 6 , wherein a glutamic acid residue is substituted at the position corresponding to position 35 of SEQ ID NO:5.
8. The method of claim 7 , wherein a phenylalanine, leucine, or isoleucine residue is substituted at the position corresponding to position 34 of SEQ ID NO:5.
9. The method of claim 1 , wherein said modified GLA domain further comprises an amino acid substitution at the position corresponding to position 35 of SEQ ID NO:5.
10. A method of increasing clot formation in a mammal, comprising administering an amount of a Factor VII or Factor VIIa polypeptide effective to increase clot formation in said mammal, wherein said Factor VII or Factor VIIa polypeptide comprises a modified GLA domain that enhances membrane binding affinity of said polypeptide compared to a native Factor VII or Factor VIIa polypeptide, and wherein said modified GLA domain comprises a hydrophobic amino acid residue substituted at position 33 of SEQ ID NO:3.
11. The method of claim 10 , wherein said modified GLA domain comprises a phenylalanine, leucine, or isoleucine residue substituted at position 33.
12. The method of claim 10 , wherein said modified GLA domain further comprises an amino acid substitution at position 10.
13. The method of claim 12 , wherein a glutamine, asparagine, glutamic acid, or aspartic acid residue is substituted at position 10.
14. The method of claim 12 , wherein a glutamine residue is substituted at position 10.
15. The method of claim 14 , wherein said modified GLA domain further comprises an amino acid substitution at position 32.
16. The method of claim 15 , wherein a glutamic acid residue is substituted at position 32.
17. The method of claim 16 , wherein said modified GLA domain further comprises an amino acid substitution at position 34.
18. The method of claim 17 , wherein a glutamic acid residue is substituted at position 34.
19. The method of claim 18 , wherein a phenylalanine, leucine, or isoleucine residue is substituted at position 33.
20. The method of claim 19 , wherein a phenylalanine residue is substituted at position 33.
21. The method of claim 17 , wherein said modified GLA domain further comprises an amino acid residue inserted at position 4.
22. The method of claim 21 , wherein a tyrosine residue is inserted at position 4.