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