Claims
- 1. A preparation of phospholipid scramblase, wherein the protein is approximately 35 kD as measured on a 12.5% SDS-polyacrylamide gel under reducing conditions.
- 2. The preparation of claim 1 wherein the scramblase is human phospholipid scramblase.
- 3. The preparation of claim 1 wherein the protein comprises SEQ ID NO:2.
- 4. The preparation of claim 1 wherein the protein comprises amino acid residues 85-309 of SEQ ID NO:2
- 5. The preparation of claim 1 wherein the scramblase is mouse phospholipid scramblase.
- 6. The preparation of claim 1, wherein the phospholipid scramblase comprises SEQ ID NO:4.
- 7. The preparation of claim 1, wherein the phospholipid scramblase comprises amino acid residues 83-307 of SEQ ID NO:4
- 8. The preparation of claim 5 wherein the protein is isolated from mouse erythrocyte membranes.
- 9. The preparation of claim 1 wherein the protein is produced by cells genetically modified so as to express the protein.
- 10. The preparation of claim 9 wherein the protein is produced by an organism selected from the group consisting of bacteria, insect cells, and yeast.
- 11. A recombinant DNA sequence encoding PL scramblase.
- 12. The sequence of claim 11, wherein the sequence is SEQ ID NO:1.
- 13. The sequence of claim 11, comprising nucleotides 211-1164 of SEQ ID NO:1.
- 14. The sequence of claim 11, comprising nucleotides 463-1137 of sequence ID NO:1.
- 15. The sequence of claim 11, wherein the sequence is SEQ ID NO:3.
- 16. The sequence of claim 11, comprising nucleotides 192-1112 of SEQ ID NO:3.
- 17. The sequence of claim 11, comprising nucleotides 438-1112 of SEQ ID NO:3.
- 18. The sequence of claim 11, wherein the sequence is SEQ ID NO:1 and wherein the sequence is part of a protein expression vector.
- 19. The sequence of claim 11, comprising nucleotides 211-1164 of SEQ ID NO:1 and wherein the sequence is part of a protein expression vector.
- 20. The sequence of claim 11, comprising nucleotides 463-1137 of SEQ ID NO:1 and wherein the sequence is part of a protein expression vector.
- 21. The sequence of claim 11, wherein the sequence is SEQ ID NO:3 and wherein the sequence is part of a protein expression vector.
- 22. The sequence of claim 11, wherein the sequence comprises 192-1112 of SEQ ID NO:3 and wherein the sequence is part of a protein expression vector.
- 23. The sequence of claim 11, comprising nucleotides 438-1112 of SEQ ID NO:3 and wherein the sequence is part of a protein expression vector.
- 24. The sequence of claim 11 wherein the sequence has been modified to prevent post-translational.
- 25. The sequence of claim 24, wherein the mutant phospholipid scramblase comprises a non-functionally equivalent substitution of one or more residues that are phosphorylated by cellular protein kinases, and said residues are selected from the group consisting of Threonine, Serine, or Tyrosine.
- 26. The sequence of claim 24 wherein the sequence comprises a mutation with a non-functional equivalent substitution of residue Thr161 of SEQ ID NO:2; Thr159 of SEQ ID NO:4 or the equivalent residue in the conserved region of another PL scramblase.
- 27. The sequence of claim 24 wherein the sequence comprises at least one non-functional equivalent substitution within residues Asp273-Asp284 of SEQ ID NO:2; Asp271-Asp282 of SEQ ID NO:4 or the equivalent residue in the conserved region of another PL scramblase.
- 28. The sequence of claim 24 wherein the sequence comprises a mutation at Cys297 of SEQ ID NO:2 or the equivalent residue in the conserved region of another PL scramblase.
- 29. A protein encoded by the sequence of claim 11.
- 30. A protein encoded by the sequence of claim 24.
- 31. A protein encoded by the sequence of claim 25.
- 32. A protein encoded by the sequence of claim 26.
- 33. A protein encoded by the sequence of claim 27.
- 34. A protein encoded by the sequence of claim 28.
- 35. An animal genetically engineered to eliminate expression of phospholipid scramblase in all germ line cells.
- 36. The animal of claim 35 wherein the animal is selected from the group consisting of mouse and pig.
- 37. A method of inhibiting expression of the coagulant properties of the plasma membrane of a cell comprising the step of expressing in the cell plasma membrane a mutant PL scramblase, wherein the PL scramblase has a reduced activity in mediating transmembrane movement of plasma membrane phospholipids.
- 38. The method of claim 37 wherein the cell is a part of a tissue or organ.
- 39. The method of claim 37, wherein the mutant phospholipid scramblase comprises a non-functionally equivalent substitution of one or more residues that are normally phosphorylated by cellular protein kinases, and said residues are selected from the group Threonine, Serine, or Tyrosine.
- 40. The method of claim 37, wherein the mutant phospholipid scramblase comprises a non-functionally equivalent substitution at residue Thr161 of SEQ ID NO:2; Thr159 of SEQ ID NO:4 or the equivalent residue in the conserved region of another PL scramblase.
- 41. The method of claim 37, wherein the mutant phospholipid scramblase comprises a non-functionally equivalent substitution of a cysteine residue.
- 42. The method of claim 41 wherein the cysteine residue to be substituted is Cys297 of SEQ ID NO:2; Cys295 of SEQ ID NO:4 or the equivalent residue in the conserved region of another PL scramblase.
- 43. The method of claim 37, wherein the mutant phospholipid scramblase comprises a non-functionally equivalent substitution of at least one of the residues located in the region of Asp273-Asp284 of SEQ ID NO:2;
Asp271-Asp282 of SEQ ID NO:4 or the equivalent residues in the conserved region of another PL scramblase.
- 44. The method of claim 37, wherein the mutant phospholipid scramblase comprises a non-functionally equivalent substitution of at least one residue selected from the group consisting of Asp273, Asp275, Phe277, Ile279, Phe281, and Asp284 of SEQ ID NO:2; Asp271, Asp273, Phe275, Ile277, Phe279, and Asp282 of SEQ ID NO:4 and the equivalent residues in the conserved region of another PL scramblase.
- 45. A method of inhibiting cellular phospholipid scramblase comprising the steps of delivering to the cell an effective amount of a compound that prevents thioacylation of the scramblase and observing an inhibition of scramblase activity.
- 46. The method of claim 45 wherein the compound is an esterase inhibitor.
- 47. The method of claim 45 wherein the compound binds with micromolar or greater affinity to the peptide sequence corresponding to residues Lys290-Cys297 of the PL scramblase SEQ ID NO:2 or the equivalent residues in the conserved region of another PL scramblase.
- 48. The method of claim 47 wherein the compound is selected from the group consisting of antibody, peptide, or nucleotide.
- 49. A method of inhibiting cellular phospholipid scramblase comprising the steps of delivering to the cell and effective amount of a compound that prevents binding of intracellular Ca2+ and observing inhibition of PL scramblase activity.
- 50. The method of claim 49 wherein the compound binds to residues Asp273-Asp284 of SEQ ID NO:2 or the equivalent residues in another PL scramblase.
- 51. The method of claim 50 selected from the group consisting of antibody, peptide, or nucleotide.
- 52. A method of modifying the activity of cellular phospholipid scramblase comprising the steps of delivering to the cell an effect amount of a compound that prevents phosphorylation of the protein and observing change in PL scramblase activity.
- 53. A method for prolonging graft survival of transplanted organs and grafts comprising the step of delivering a modified PL scramblase to an organ perfusate during in vitro organ storage, wherein the modified PL scramblase is modified at a residue selected from the group consisting of Thr161, Asp273 to Asp284, and Cys297 of SEQ ID NO:2 and the equivalent residues in the conserved region of another PL scramblase.
- 54. A method for prolonging graft survival of transplanted organs and grafts comprising the step of delivering to an organ perfusate during in vitro organ storage, a compound that prevents postranslational modification of PL scramblase at a residues selected from the group consisting of Thr161, Asp273 to Asp284, and Cys297 of SEQ ID NO:2 and the equivalent residue in the conserved region of another PL scramblase.
- 55. A method for prolonging the in vivo survival of circulating blood cells comprising the step of preventing surface exposure of plasma membrane phosphatidylserine on the circulating blood cells by delivering a modified PL scramblase within the blood cells, wherein the modified PL scramblase is modified at a residue selected from the group consisting of Thr161, Asp273 to Asp284, and Cys297 of SEQ ID NO:2 and the equivalent residues in another PL scramblase.
- 56. A method for prolonging the in vivo survival of circulating blood cells comprising the step of preventing surface exposure of plasma membrane phosphatidylserine on the circulating blood cells by delivering a compound that prevents postranslational modification of PL scramblase at one or more residues wherein that residue is selected from the group consisting of Thr161, Asp273 to Asp284, and Cys297 of SEQ ID NO:2 and the equivalent residues in the conserved region of another PL scramblase.
- 57. A method for preventing the procoagulant properties of erythrocytes in sickle cell disease comprising the step of delivering a modified PL scramblase in a sickle cell patient, wherein the modified PL scramblase is modified at a residue selected from the group consisting of Thr161, Asp273 to Asp284, and Cys297 of SEQ ID NO:2 and the equivalent residues in the conserved region of another PL scramblase.
- 58. A method for treating autoimmune, thrombotic, thromboemolic, and inflammatory diseases comprising the step of treating a patient with a modified PL scramblase, wherein the modified PL scramblase is modified at a residue selected from the group consisting of Thr161, Asp273 to Asp284, and Cys297 of SEQ ID NO:2 and the equivalent residues in the conserved region of another PL scramblase.
- 59. A method for treating autoimmune, thrombotic, thromboemolic, and inflammatory diseases comprising the step of treating a patient with an inhibitor of PL scramblase selected from the group of compounds that inhibit postranslational modification of the protein at a residue selected from the group consisting of Thr161, Asp273 to Asp284, and Cys297 of SEQ ID NO:2 and the equivalent residues in the conserved region of another PL scramblase.
- 60. The method of claim 58, wherein the disease is selected from disseminated intravascular coagulation, vascular thrombosis, fibrin generation during cardiopulmonary bypass procedures, rheumatoid arthritis, systemic lupus erythematosus, thrombotic thrombocytopenic purpura, heparin-associated thrombosis, and organ transplant rejection.
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of Ser. No. 08/790,186, which claims priority to Serial No. 60/015,385. Both of these applications are incorporated by reference as if fully set forth herein.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] United States Government may have commercial rights under Grant R01 HL36946 from Heart, Lung, & Blood Institute, National Institutes of Health.
Provisional Applications (1)
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Number |
Date |
Country |
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60015385 |
Apr 1996 |
US |
Continuations (2)
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Number |
Date |
Country |
Parent |
09375197 |
Aug 1999 |
US |
Child |
10341845 |
Jan 2003 |
US |
Parent |
08949246 |
Oct 1997 |
US |
Child |
09375197 |
Aug 1999 |
US |
Continuation in Parts (1)
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Number |
Date |
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Parent |
08790186 |
Jan 1997 |
US |
Child |
08949246 |
Oct 1997 |
US |