Modified vitamin k-dependent polypeptides

Abstract

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 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 an amino acid substitution selected from a hydrophobic amino acid residue or a glutamic acid residue at position 34, wherein amino acid positions of the Factor VII or Factor VIIa polypeptide are numbered according to SEQ ID NO:3. 
     
     
         2 . The polypeptide of  claim 1 , wherein a phenylalanine, leucine or isoleucine residue is substituted at position 34. 
     
     
         3 . The polypeptide of  claim 1 , wherein a glutamic acid residue is substituted at position 34. 
     
     
         4 . The polypeptide of  claim 1 , further comprising an amino acid substitution at position 10. 
     
     
         5 . The polypeptide of  claim 4 , wherein a glutamine, asparagine, glutamic acid, or aspartic acid residue is substituted at position 10. 
     
     
         6 . The polypeptide of  claim 5 , wherein a glutamine residue is substituted at position 10. 
     
     
         7 . The polypeptide of  claim 1 , further comprising an amino acid substitution at position 32. 
     
     
         8 . The polypeptide of  claim 7 , wherein a glutamic acid residue is substituted at position 32. 
     
     
         9 . The polypeptide of  claim 1 , further comprising an amino acid substitution at position 28. 
     
     
         10 . The polypeptide of  claim 9 , wherein a phenylalanine or a glutamic acid residue is substituted at position 28. 
     
     
         11 . The polypeptide of  claim 10 , wherein a phenylalanine residue is substituted at position 28. 
     
     
         12 . The polypeptide of  claim 1 , further comprising an insertion at position 4. 
     
     
         13 . The polypeptide of  claim 12 , wherein a tyrosine or glycine residue is inserted at position 4. 
     
     
         14 . (canceled) 
     
     
         15 . An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide of  claim 1 . 
     
     
         16 - 22 . (canceled) 
     
     
         23 . The polypeptide of  claim 1 , wherein the Factor VII or Factor VIIa polypeptide further comprises a glutamine residue substituted at position 10 and a glutamic acid residue substituted at position 32. 
     
     
         24 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an amount of Factor VII or Factor VIIa polypeptide effective to increase clot formation, 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 an amino acid substitution selected from a hydrophobic amino acid residue at position 34, wherein amino acid positions of the Factor VII or Factor VIIa polypeptide are numbered according to SEQ ID NO:3. 
     
     
         25 . (canceled) 
     
     
         26 . A method for treating a bleeding disorder in a patient, said method comprising administering the pharmaceutical composition of  claim 24  to said patient. 
     
     
         27 - 32 . (canceled) 
     
     
         33 . An isolated mammalian host cell that expresses a Factor VII or Factor VIIa polypeptide, said 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 an amino acid substitution selected from a hydrophobic amino acid residue or a glutamic acid residue at position 34, wherein amino acid positions of the Factor VII or Factor VIIa polypeptide are numbered according to SEQ ID NO:3. 
     
     
         34 - 40 . (canceled) 
     
     
         41 . 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 relative to a corresponding native Factor VII or Factor VIIa polypeptide, said modified GLA domain comprising an amino acid substitution selected from a hydrophobic amino acid residue or a glutamic acid residue at position 34, wherein amino acid positions of the Factor VII or Factor VIIa polypeptide are numbered according to SEQ ID NO:3. 
     
     
         42 - 48 . (canceled) 
     
     
         49 . A method for producing a Factor VII or Factor VIIa polypeptide having a modified GLA domain comprising an amino acid substitution selected from a hydrophobic amino acid residue or a glutamic acid residue at position 34, wherein amino acid positions of the Factor VII or Factor VIIa polypeptide are numbered according to SEQ ID NO:3, the method comprising (a) providing a culture of the mammalian host cell of  claim 33  under conditions which permit expression of the polypeptide, and (b) recovering the polypeptide. 
     
     
         50 - 56 . (canceled)