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 comprising a modified gamma-carboxyglutamic acid (GLA) domain that enhances membrane binding affinity of said vitamin K-dependent 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 one to five amino acid substitutions, wherein the proline residue at position 10 of SEQ ID NO:3 or SEQ ID NO:4 is substituted with a glutamine, aspartic acid, or glutamic acid residue in the modified GLA domain.
2. The polypeptide of claim 1 , wherein the proline residue at position 10 is substituted with a glutamine residue in the modified GLA domain.
3. The polypeptide of claim 1 , wherein the modified GLA domain comprises a substitution at position 28 of SEQ ID NO:3 or SEQ ID NO:4.
4. The polypeptide of claim 1 , wherein a phenylalanine residue is substituted at position 28 in the modified GLA domain.
5. The polypeptide of claim 4 , wherein the proline residue at position 10 is substituted with a glutamine residue in the modified GLA domain.
6. The polypeptide of claim 1 , wherein the modified GLA domain comprises a substitution at position 32 of SEQ ID NO:3 or SEQ ID NO:4.
7. The polypeptide of claim 6 , wherein a glutamic acid residue is substituted at position 32 in the modified GLA domain.
8. The polypeptide of claim 7 , wherein the proline residue at position 10 is substituted with a glutamine residue in the modified GLA domain.
9. The polypeptide of claim 6 , wherein an aspartic acid residue is substituted at position 32 in the modified GLA domain.
10. The polypeptide of claim 9 , wherein the proline residue at position 10 is substituted with a glutamine residue in the modified GLA domain.
11. The polypeptide of claim 1 , wherein the modified GLA domain comprises a substitution at position 11 of SEQ ID NO:3 or SEQ ID NO:4.
12. The polypeptide of claim 11 , wherein the proline residue at position 10 is substituted with a glutamine residue in the modified GLA domain.
13. A composition comprising a pharmaceutically acceptable carrier and a vitamin K-dependent polypeptide, wherein the vitamin K-dependent polypeptide comprises a modified GLA domain that enhances membrane binding affinity of the vitamin K-dependent polypeptide relative to a corresponding native vitamin K-dependent polypeptide, wherein said modified GLA domain comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4 with two amino acid substitutions, wherein a glutamine residue is substituted at amino acid position 10 of SEQ ID NO:3 or SEQ ID NO:4 in the modified GLA domain, and wherein a glutamic acid residue is substituted at position 32 of SEQ ID NO:3 or SEQ ID NO:4 in the modified GLA domain.
14. A composition comprising a pharmaceutically acceptable carrier and a vitamin K-dependent polypeptide, wherein said vitamin K-dependent polypeptide comprises a modified GLA domain that enhances membrane binding affinity of said vitamin K-dependent 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 one to five amino acid substitutions, wherein the proline residue at position 10 of SEQ ID NO:3 or SEQ ID NO:4 is substituted with a glutamine, aspartic acid, or glutamic acid residue in the modified GLA domain.
15. The composition of claim 14 , wherein said pharmaceutical composition further comprises soluble tissue factor.
16. The composition of claim 14 , wherein the proline residue at position 10 is substituted with a glutamine residue in the modified GLA domain.
17. The composition of claim 14 , wherein the modified GLA domain comprises a substitution at position 28 of SEQ ID NO:3 or SEQ ID NO:4.
18. The composition of claim 17 , wherein a phenylalanine residue is substituted at position 28 in the modified GLA domain.
19. The composition of claim 18 , wherein the proline residue at position 10 is substituted with a glutamine residue in the modified GLA domain.
20. The composition of claim 17 , wherein the modified GLA domain comprises a substitution at position 32 of SEQ ID NO:3 or SEQ ID NO:4.
21. The composition of claim 20 , wherein a glutamic acid residue is substituted at position 32 in the modified GLA domain.
22. The composition of claim 21 , wherein the proline residue at position 10 is substituted with a glutamine residue in the modified GLA domain.
23. The composition of claim 20 , wherein an aspartic acid residue is substituted at position 32 in the modified GLA domain.
24. The composition of claim 23 , wherein the proline residue at position 10 is substituted with a glutamine residue in the modified GLA domain.
25. The composition of claim 14 , wherein the modified GLA domain comprises a substitution at position 11 of SEQ ID NO:3 or SEQ ID NO:4.
26. The composition of claim 25 , wherein the proline residue at position 10 is substituted with a glutamine residue in the modified GLA domain.