Modified annexin proteins and methods for their use in organ transplantation and in prevention of thrombosis and reperfusion injury

Abstract

Modified annexin proteins, including a homodimer of human annexin V, are provided. Methods for their use, such as to prevent thrombosis without increasing hemorrhage and to attenuate ischemia-reperfusion injury (IPI), are also provided. The modified annexins bind phosphatidylserine (PS) on cell surfaces, thereby preventing the assembly of the prothromkinase complex. The modified annexin decreases the binding of leukocytes and platelets during post-ischemic reperfusion, thereby restoring microvascular blood flow and decreasing organ damage.

1 - 28 . (canceled)  
     
     
         29 . A linker peptide comprising at least one glycine-serine sequence, wherein the linker peptide couples an annexin protein to a second polypeptide.  
     
     
         30 . The linker peptide of  claim 29 , wherein the peptide comprises a formula selected from the group consisting of Ser-Gly-(Xaa) n -Gly-Ser, Arg-Ser-Gly-(Xaa) n -Gly-Ser-Thr, Gly-Ser-(Xaa) n -Ser-Gly, and Gly-Ser-(Xaa) n -Ser-Gly-Lys-Leu, 
 wherein (Xaa) can be any amino acid and n is any number from at least 2 to at least 32.    
     
     
         31 . The linker peptide of  claim 30 , wherein the peptide comprises an amino acid sequence of at least 20 amino acids.  
     
     
         32 . The linker peptide of  claim 30 , wherein the peptide comprises an amino acid sequence of at least 15 amino acids.  
     
     
         33 . The linker peptide of  claim 30 , wherein the peptide comprises an amino acid sequence of at least 14 amino acids.  
     
     
         34 . The linker peptide of  claim 30 , wherein (Xaa), comprises Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro.  
     
     
         35 . The linker peptide of  claim 29 , wherein the peptide comprises 2 glycine-serine sequences separated by an amino acid sequence of at least about 26 amino acids.  
     
     
         36 . The linker peptide of  claim 29 , wherein the peptide comprises 2 glycine-serine sequences separated by an amino acid sequence of at least about 8 amino acids.  
     
     
         37 . The linker peptide of  claim 29 , wherein the peptide comprises the 14-amino acid sequence Arg-Ser-Gly-Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro-Gly-Ser-Thr.  
     
     
         38 . The linker peptide of  claim 29 , wherein the peptide comprises the 12-amino acid sequence Gly-Ser-Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro-Ser-Gly.  
     
     
         39 . The linker peptide of  claim 29 , wherein the peptide comprises the 14-amino acid sequence Gly-Ser-Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro-Ser-Gly-Lys-Leu.  
     
     
         40 . The linker peptide of  claim 29 , wherein the annexin protein is selected from the group consisting of annexin I, annexin II, annexin III, annexin V, and annexin VIII.  
     
     
         41 . The linker peptide of  claim 29 , wherein the second polypeptide is selected from the group consisting of an annexin, an Fc portion of an immunoglobulin, a peptide ligand, a monoclonal antibody, and a polyclonal antibody.  
     
     
         42 . The linker peptide of  claim 29 , wherein the second polypeptide is an annexin protein selected from the group consisting of annexin I, annexin II, annexin III, annexin V, and annexin VIII.  
     
     
         43 . A pharmaceutical composition comprising a linker peptide, wherein the linker peptide couples an annexin protein to a second polypeptide.  
     
     
         44 . The pharmaceutical composition of  claim 43 , wherein the annexin protein is selected from the group consisting of annexin I, annexin II, annexin III, annexin V, and annexin VIII.  
     
     
         45 . The pharmaceutical composition of  claim 43 , wherein the second polypeptide is selected from the group consisting of an annexin, an Fc portion of an immunoglobulin, a peptide ligand, a monoclonal antibody, and a polyclonal antibody.  
     
     
         46 . The pharmaceutical composition of  claim 45 , wherein the second polypeptide is an annexin protein selected from the group consisting of annexin I, annexin II, annexin III, annexin V, and annexin VIII.  
     
     
         47 . The pharmaceutical composition of  claim 43 , wherein the composition comprises an annexin dimer, an annexin heterodimer, an annexin homodimer, an annexin heterotetramer, or an annexin protein connected to an additional protein.  
     
     
         48 . The pharmaceutical composition of  claim 43 , wherein the composition comprises a peptide linked annexin homodimer.  
     
     
         49 . The pharmaceutical composition of  claim 43 , wherein the composition comprises a peptide linked annexin heterodimer.  
     
     
         50 . The pharmaceutical composition of  claim 43 , wherein the linker peptide comprises a 14-amino acid sequence Arg-Ser-Gly-Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro-Gly-Ser-Thr.  
     
     
         51 . The pharmaceutical composition of  claim 43 , wherein the linker peptide comprises a 12-amino acid sequence Gly-Ser-Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro-Ser-Gly.  
     
     
         52 . The pharmaceutical composition of  claim 43 , wherein the linker peptide comprises a 14-amino acid sequence Gly-Ser-Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro-Ser-Gly-Lys-Leu.  
     
     
         53 . A pharmaceutical composition comprising a linker peptide, wherein the linker peptide couples a first annexin protein to a second annexin protein and wherein the linker peptide comprises a 12-amino acid sequence Gly-Ser-Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro-Ser-Gly.  
     
     
         54 . A pharmaceutical composition comprising a linker peptide, wherein the linker peptide couples a first annexin protein to a second annexin protein and wherein the linker peptide comprises a 14-amino acid sequence Gly-Ser-Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro-Ser-Gly-Lys-Leu.