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 recombinant vitamin K-dependent polypeptide comprising a modified gamma-carboxyglutamic acid (GLA) domain that enhances membrane binding affinity of said polypeptide compared to a native vitamin K-dependent polypeptide, wherein said modified GLA domain comprises an amino acid insertion at the position corresponding to position 4 of SEQ ID NO:5.
2. The polypeptide of claim 1 , wherein a tyrosine residue is inserted at position 4 of SEQ ID NO:5.
3. The polypeptide of claim 1 , wherein said modified GLA domain further comprises substitution of a hydrophobic amino acid residue or a glutamic acid residue at the position corresponding to position 35 of SEQ ID NO:5.
4. The polypeptide of claim 3 , wherein said hydrophobic amino acid residue is a phenylalanine, leucine or isoleucine residue.
5. The polypeptide 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.
6. The polypeptide of claim 5 , wherein a glutamine, a glutamic acid, an aspartic acid, or an asparagine residue is substituted at position 11 of SEQ ID NO:5.
7. The polypeptide of claim 6 , wherein a glutamine residue is substituted at position 11 of SEQ ID NO:5.
8. The polypeptide of claim 1 , wherein said modified GLA domain further comprises an amino acid substitution at the position corresponding to position 33 of SEQ ID NO:5.
9. The polypeptide of claim 8 , wherein a glutamic acid residue is substituted at position 33 of SEQ ID NO:5.
10. The polypeptide of claim 1 , wherein said modified GLA domain further comprises an amino acid substitution at the position corresponding to position 34 of SEQ ID NO:5.
11. The polypeptide of claim 10 , wherein a hydrophobic residue is substituted at position 34 of SEQ ID NO:5.
12. The polypeptide of claim 11 , wherein a phenylalanine, leucine or isoleucine residue is substituted at position 34 of SEQ ID NO:5.
13. The polypeptide of claim 12 , wherein a phenylalanine residue is substituted at position 34 of SEQ ID NO:5.
14. The polypeptide of claim 1 , wherein said modified GLA domain further comprises an amino acid substitution at the position corresponding to position 29 of SEQ ID NO:5.
15. The polypeptide of claim 14 , wherein a phenylalanine or a glutamic acid residue is substituted at position 29 of SEQ ID NO:5.
16. The polypeptide of claim 15 , wherein a phenylalanine residue is substituted at position 29 of SEQ ID NO:5.
17. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier.
18. A method of increasing clot formation in a mammal, comprising administering an amount of the polypeptide of claim 1 effective to increase clot formation in said mammal.
19. A recombinant Factor VII or Factor VIIa polypeptide comprising a modified GLA domain that enhances membrane binding affinity of said polypeptide compared to a native Factor VII or Factor VIIa polypeptide, wherein said modified GLA domain comprises an amino acid insertion at position 4 of SEQ ID NO:3.
20. The polypeptide of claim 19 , wherein a tyrosine residue is inserted at position 4 of SEQ ID NO:3.
21. The polypeptide of claim 19 , wherein said modified GLA domain further comprises substitution of a hydrophobic amino acid residue or a glutamic acid residue at position 34 of SEQ ID NO:3.
22. The polypeptide of claim 21 , wherein said hydrophobic amino acid residue is a phenylalanine, leucine or isoleucine residue.
23. The polypeptide of claim 19 , wherein said modified GLA domain further comprises an amino acid substitution at position 10 of SEQ ID NO:3.
24. The polypeptide of claim 23 , wherein a glutamine, a glutamic acid, an aspartic acid, or an asparagine residue is substituted at position 10 of SEQ ID NO:3.
25. The polypeptide of claim 24 , wherein a glutamine residue is substituted at position 10 of SEQ ID NO:3.
26. The polypeptide of claim 19 , wherein said modified GLA domain further comprises an amino acid substitution at position 32 of SEQ ID NO:3.
27. The polypeptide of claim 26 , wherein a glutamic acid residue is substituted at position 32 of SEQ ID NO:3.
28. The polypeptide of claim 19 , wherein said modified GLA domain further comprises an amino acid substitution at position 33 of SEQ ID NO:3.
29. The polypeptide of claim 28 , wherein a hydrophobic residue is substituted at position 33 of SEQ ID NO:3.
30. The polypeptide of claim 19 , wherein a phenylalanine, leucine or isoleucine residue is substituted at position 33 of SEQ ID NO:3.
31. The polypeptide of claim 19 , wherein a phenylalanine residue is substituted at position 33 of SEQ ID NO:3.
32. The polypeptide of claim 19 , wherein said modified GLA domain further comprises an amino acid substitution at position 28 of SEQ ID NO:3.
33. The polypeptide of claim 32 , wherein a phenylalanine or a glutamic acid residue is substituted at position 28 of SEQ ID NO:3.
34. The polypeptide of claim 33 , wherein a phenylalanine residue is substituted at position 28 of SEQ ID NO:3.
35. A pharmaceutical composition comprising the polypeptide of claim 19 and a pharmaceutically acceptable carrier.
36. A method of increasing clot formation in a mammal, comprising administering an amount of the polypeptide of claim 19 effective to increase clot formation in said mammal.