Methods and compositions related to mutant Kunitz domain I of TFPI-2
View Patent ↗Disclosed are methods and compositions relating to plasmin inhibition.
1. A KD1 polypeptide comprising:
(i) an amino acid sequence with at least 92% identity to SEQ ID NO:3 and in which arginine is present at position 17,
(ii) an amino acid sequence with at least 92% identity to SEQ ID NO:3 and in which lysine, histidine or arginine is present at position 17, or
(iii) an amino acid sequence with at least 92% identity to SEQ ID NO:3 and in which serine, threonine, asparagine, lysine, histidine or arginine is present at position 17,
wherein the KD1 polypeptide inhibits plasmin activity and has decreased anti-coagulation activity as compared to a wild type KD1 polypeptide.
2. The KD1 polypeptide of claim 1 , which is selected from (a) the KD1 polypeptide of claim 1 wherein the amino acid sequence has at least 93% identity to SEQ ID NO:3, (b) the KD1 polypeptide of claim 1 wherein the amino acid sequence has at least 94% identity to SEQ ID NO:3, (c) the KD1 polypeptide of claim 1 wherein the amino acid sequence has at least 95% identity to SEQ ID NO:3, (d) the KD1 polypeptide of claim 1 wherein the amino acid sequence has at least 96% identity to SEQ ID NO:3, and (e) the KD1 polypeptide of claim 1 wherein the amino acid sequence has at least 97% identity to SEQ ID NO:3.
3. The KD1 polypeptide of claim 1 , wherein at least one of: (a) leucine is present at amino acid sequence position 18, (b) leucine is present at amino acid sequence position 19, (c) arginine is present at amino acid sequence position 31, and (d) leucine is present at amino acid sequence position 34.
4. The KD1 polypeptide of claim 1 , which is selected from (a) the KD1 polypeptide of claim 1 wherein the amino acid sequence has at least 98% identity to SEQ ID NO:3, and (b) the KD1 polypeptide of claim 1 wherein the amino acid sequence has at least 99% identity to SEQ ID NO:3.
5. The KD1 polypeptide of claim 1 , wherein the amino acid sequence is SEQ ID NO:3.
6. The KD1 polypeptide of claim 1 , wherein the amino acid sequence comprises at least one substitution in the sequence set forth in SEQ ID NO:3 that is selected from the group consisting of: a tyrosine to glutamic acid substitution at position 46, a tyrosine to threonine substitution at position 11, an alanine to methionine substitution at position 16, an alanine to glycine substitution at position 16, an alanine to serine substitution at position 16, an aspartic acid to tyrosine substitution at position 10, and an aspartic acid to glutamic acid substitution at position 10.
7. The KD1 polypeptide of claim 6 , wherein the amino acid sequence comprises a tyrosine to glutamic acid substitution at position 46.
8. The KD1 polypeptide of claim 6 , wherein the amino acid sequence comprises a tyrosine to threonine substitution at position 11.
9. The KD1 polypeptide of claim 6 , wherein the amino acid sequence comprises an alanine to methionine substitution at position 16.
10. The KD1 polypeptide of claim 6 , wherein the amino acid sequence comprises an alanine to glycine substitution at position 16.
11. The KD1 polypeptide of claim 6 , wherein the amino acid sequence comprises an alanine to serine substitution at position 16.
12. The KD1 polypeptide of claim 6 , wherein the amino acid sequence comprises an aspartic acid to tyrosine substitution at position 10.
13. The KD1 polypeptide of claim 6 , wherein the amino acid sequence comprises an aspartic acid to glutamic acid substitution at position 10.
14. The KD1 polypeptide of claim 1 which comprises the amino acid sequence set forth in one of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
15. A KD1 polypeptide comprising an amino acid sequence with at least 93% identity to the KD1 amino acid sequence set forth as amino acids 10-67 of SEQ ID NO:1, wherein amino acid 26 of SEQ ID NO:1 is changed from leucine to arginine or lysine, and wherein the polypeptide inhibits plasmin activity and has decreased anti-coagulation activity as compared to a wild type KD1 polypeptide.
16. The KD1 polypeptide of claim 15 , wherein the amino acid sequence has at least 94% identity to the KD1 amino acid sequence set as amino acids 10-67 of SEQ ID NO:1.
17. The KD1 polypeptide of claim 15 , wherein the amino acid sequence has at least 95% identity to the KD1 amino acid sequence set forth as amino acids 10-67 of SEQ ID NO:1.
18. The KD1 polypeptide of claim 15 , wherein the amino acid sequence has at least 96% identity to the KD1 amino acid sequence as amino acids 10-67 of SEQ ID NO:1.
19. The KD1 polypeptide of claim 15 , wherein the amino acid sequence has at least 97% identity to the KD1 amino acid sequence set forth as amino acids 10-67 of SEQ ID NO:1.
20. The KD1 polypeptide of claim 15 which comprises the amino acid sequence set forth in SEQ ID NO:3.
21. The KD1 polypeptide of claim 15 , wherein the amino acid sequence that has at least 93% identity to the KD1 amino acid sequence set forth as amino acids 10-67 of SEQ ID NO:1 comprises a substitution selected from the group consisting of a tyrosine to glutamic acid substitution at position 55 of SEQ ID NO:1, a tyrosine to threonine substitution at position 20 of SEQ ID NO:1, an alanine to methionine substitution at position 25 of SEQ ID NO:1, an alanine to glycine substitution at position 25 of SEQ ID NO:1, an alanine to serine substitution at position 25 of SEQ ID NO:1, an aspartic acid to tyrosine substitution at position 19 of SEQ ID NO:1, and an aspartic acid to glutamic acid substitution at position 19 of SEQ ID NO:1.
22. The KD1 polypeptide of claim 21 , wherein the amino acid sequence that has at least 93% identity to the KD1 amino acid sequence set forth as amino acids 10-67 of SEQ ID NO:1 comprises a tyrosine to glutamic acid substitution at position 46 of SEQ ID NO:1.
23. The KD1 polypeptide of claim 22 , wherein the amino acid sequence that has at least 93% identity to the KD1 amino acid sequence set forth as amino acids 10-67 of SEQ ID NO:1 comprises a tyrosine to threonine substitution at position 11 of SEQ ID NO:1.
24. The KD1 polypeptide of claim 23 , wherein the amino acid sequence that has at least 93% identity to the KD1 amino acid sequence set forth as amino acids 10-67 of SEQ ID NO:1 comprises an alanine to methionine substitution at position 16 of SEQ ID NO:1.
25. The KD1 polypeptide of claim 24 , wherein the amino acid sequence that has at least 93% identity to the KD1 amino acid sequence set forth as amino acids 10-67 of SEQ ID NO:1 comprises an alanine to glycine substitution at position 16 of SEQ ID NO:1.
26. The KD1 polypeptide of claim 25 , wherein the amino acid sequence that has at least 93% identity to the KD1 amino acid sequence set forth as amino acids 10-67 of SEQ ID NO:1 comprises an alanine to serine substitution at position 16 of SEQ ID NO:1.
27. The KD1 polypeptide of claim 26 , wherein the amino acid sequence that has at least 93% identity to the KD1 amino acid sequence set forth as amino acids 10-67 of SEQ ID NO:1 comprises an aspartic acid to tyrosine substitution at position 10 of SEQ ID NO:1.
28. The KD1 polypeptide of claim 27 , wherein the amino acid sequence that has at least 93% identity to the KD1 amino acid sequence set forth as amino acids 10-67 of SEQ ID NO:1 comprises an aspartic acid to glutamic acid substitution at position 10 of SEQ ID NO:1.
29. The KD1 polypeptide of claim 15 which comprises the amino acid sequence set forth in one of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.