Methods for the treatment of thrombosis

Abstract

A fibrinolytically active metalloproteinase polypeptide (called “novel acting thrombolytic”) which is useful for blood clot lysis in vivo and methods and materials for its production by recombinant expression are described.

1 . A thrombolytically active variant of SEQ ID NO:5, wherein the sequence of amino acid residues Gln-Gln-Arg at positions 1-3 of SEQ ID NO:5 is substituted with an amino acid that facilitates kex-2 cleavage when the amino acid occurs on the C-terminal side of the kex-2 hydrolysis site, and the remainder of the amino acid sequence of SEQ ID NO:5 is unchanged.  
     
     
         2 . A fusion polypeptide comprising the thrombolytically active variant of  claim 1  fused to a heterologous polypeptide sequence.  
     
     
         3 . A composition comprising the thrombolytically active variant of  claim 1 , and one or more components selected from a pharmaceutically acceptable diluent, a pharmaceutically acceptable preservative, a pharmaceutically acceptable solubilizer, a pharmaceutically acceptable emulsifier, a pharmaceutically acceptable adjuvant, or a pharmaceutically acceptable carrier.  
     
     
         4 . A method for treating thrombosis in a mammal comprising administering locally to a clot in a blood vessel of the mammal a thrombolytically effective amount of the composition of  claim 3 .  
     
     
         5 . The method of  claim 4 , wherein the composition is administered as two or more doses.  
     
     
         6 . The method of  claim 4 , wherein the mammal is a human.  
     
     
         7 . A method for lysing a blood clot comprising contacting the blood clot with a thrombolytically effective amount of the composition of  claim 3 .  
     
     
         8 . The method of  claim 7 , wherein the blood clot is contacted in vivo.  
     
     
         9 . The method of  claim 8 , wherein the blood clot is contacted in vivo in a human.  
     
     
         10 . The method of  claim 7 , wherein the blood clot is contacted in vitro.  
     
     
         11 . A nucleic acid molecule comprising a coding sequence encoding the thrombolytically active variant of  claim 1 .  
     
     
         12 . An expression vector comprising the nucleic acid molecule of  claim 11 , operatively linked to expression regulatory elements.  
     
     
         13 . A host cell comprising the expression vector of  claim 12 .  
     
     
         14 . The host cell of  claim 13 , wherein the cell is a yeast cell.  
     
     
         15 . The host cell of  claim 14 , wherein the yeast is  Pichia pastoris.    
     
     
         16 . A method for producing a recombinant polypeptide comprising providing a population of host cells according to  claim 13  and causing expression of the polypeptide.  
     
     
         17 . A method for producing a recombinant polypeptide comprising providing a population of host cells according to  claim 14  and causing expression of the polypeptide.  
     
     
         18 . A method for producing a recombinant polypeptide comprising providing a population of host cells according to  claim 15  and causing expression of the polypeptide.