Novel cleaved fragments of fibrinogen

Abstract

The invention provides novel α E CX cleavage fragments of fibrinogen and methods for detecting and purifying these fragments. The method of the invention also includes a diagnostic method for determining fibrinolytic states or atherogenesis in a mammal. Methods of treating disease characterized by fibrinogen metabolism are also disclosed. In addition, the invention also provides monospecific antibodies which are specifically reactive with α E C domain of fibrinogen. Also provided, are DNA and RNA molecules that encode α E CX cleavage fragments of fibrinogen. In addition, the present invention includes a vector and a host cell capable of expressing α E CX cleavage fragments of fibrinogen.

What is claimed is:  
     
         1 . An isolated fragment of fibrinogen comprising an amino acid sequence set forth in SEQ ID NO:1.  
     
     
         2 . An isolated fragment of fibrinogen comprising an amino acid sequence set forth in SEQ ID NO:2.  
     
     
         3 . An isolated fragment of fibrinogen comprising an amino acid sequence set forth in SEQ ID NO:3.  
     
     
         4 . An isolated fragment of fibrinogen comprising an amino acid sequence having about 90% homology to SEQ ID NO:2.  
     
     
         5 . A fragment according to  claim 1 ,  claim 2 ,  claim 3 , or  claim 4 , wherein said fragment has prolonged stability.  
     
     
         6 . A fragment according to  claim 1 , wherein said fragment is conjugated to a carrier.  
     
     
         7 . A fragment according to  claim 1 , in an admixture with a physiologically acceptable diluent.  
     
     
         8 . A fragment according to  claim 2 , wherein said fragment is conjugated to a carrier.  
     
     
         9 . A fragment according to  claim 2 , in an admixture with a physiologically acceptable diluent.  
     
     
         10 . A fragment according to  claim 3 , wherein said fragment is conjugated to a carrier.  
     
     
         11 . A fragment according to  claim 3 , in an admixture with a physiologically acceptable diluent.  
     
     
         12 . A fragment according to  claim 4 , wherein said fragment is conjugated to a carrier.  
     
     
         13 . A fragment according to  claim 4 , in an admixture with a physiologically acceptable diluent.  
     
     
         14 . A method of purifying α E CX fibrinogen, comprising: 
 contacting fibrinogen with a proteolytic enzyme to provide fragments of the fibrinogen;  
 contacting the fragments of fibrinogen with at least one monospecific antibody that binds to an α E C domain of fibrinogen;  
 selectively removing α E CX cleavage fragments of fibrinogen defined by an amino acid sequence set forth in: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or homologs having at least about 90% identity with SEQ ID NO:2 from a sample.  
 
     
     
         15 . A method according to  claim 14 , wherein said proteolytic enzyme is selected from the group consisting of fibrinolytic metalloproteinase, plasmin, u-PA, r-PA, n-PA, t-PA, streptokinase, staphylokinase, and combinations thereof.  
     
     
         16 . A method of purifying α E CX fragments of fibrinogen, comprising: 
 contacting fibrinogen with a proteolytic enzyme to provide fragments of the fibrinogen;  
 contacting the fragments of fibrinogen with at least one monospecific antibody that binds to an α E C domain of fibrinogen;  
 selectively removing α E CX cleavage fragments of fibrinogen defined by an amino acid sequence set forth in: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or homologs having at least about 90% identity with SEQ ID NO:2 from a sample.  
 
     
     
         17 . A diagnostic method for determining a fibrinolytic state or atherogenesis, comprising: 
 contacting fragments of fibrinogen with at least one monospecific antibody that binds to an α E C domain of fibrinogen; and    measuring specific binding of the antibody to α E CX cleavage fragments wherein specific binding of the antibody indicates the presence of α E CX cleavage fragments of fibrinogen in a sample.    
     
     
         18 . A method according to  claim 17 , wherein said method is used to regulate doses of plasminogen activator given to a mammal.  
     
     
         19 . A method according to  claim 17 , wherein said antibody is detectably labeled with a detectable marker moiety.  
     
     
         20 . A method according to  claim 17 , wherein said plasminogen activator is selected from the is selected from the group consisting of u-PA, t-PA, r-PA, n-PA, streptokinase, staphylokinase, and combinations thereof.  
     
     
         21 . A method according to  claim 17 , wherein said method is performed in vivo.  
     
     
         22 . A method according to  claim 17 , wherein said method is performed in a sample from a mammal suffering from a myocardial infarction.  
     
     
         23 . A method according to  claim 17 , wherein said method is used to monitor reperfusion in a mammal with a myocardial infarction.  
     
     
         24 . A method of detecting an α E CX domain of fibrinogen comprising: 
 contacting the fragments of fibrinogen with at least one monospecific antibody that binds to an α E C domain of fibrinogen wherein specific binding of the antibody indicates the presence of α E CX cleavage fragments of fibrinogen; and  
 measuring specific binding of the antibody to the α E  CX cleavage fragments wherein specific binding of the antibody indicates the presence of α E CX cleavage fragments of fibrinogen defined by an amino acid sequence set forth in: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or homologs having at least about 90% identity with SEQ ID NO:2 in a sample.  
 
     
     
         25 . A method according to  claim 24 , wherein the antibody is detectably labeled by conjugation to a detectable moiety.  
     
     
         26 . A method according to  claim 24 , wherein said method is performed in vivo.  
     
     
         27 . A method according to  claim 24 , wherein said method is performed in a mammal suffering from a myocardial infarction.  
     
     
         28 . A nucleic acid, encoding amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or homologs having at least about 90% identity with SEQ ID NO:2, said nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO:5.  
     
     
         29 . A nucleic acid according to  claim 28 , wherein said nucleic acid is isolated natural or synthetic DNA encoding SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or homologs having at least about 90% identity with SEQ ID NO:2.  
     
     
         30 . A nucleic acid according to  claim 28 , wherein said nucleic acid is isolated natural or synthetic RNA encoding SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or homologs having at least about 90% identity with SEQ ID NO:2.  
     
     
         31 . A vector for transfecting a host cell to express a recombinant or heterologous protein, comprising a DNA segment encoding SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or homologs having at least about 90% identity with SEQ ID NO:2, conjugated to a promoter.  
     
     
         32 . A method for making a host cell which expresses a heterologous protein, comprising transfecting the cell with a vector comprising a DNA segment encoding SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or homologs having at least about 90% identity with SEQ ID NO:2, conjugated to a promoter.  
     
     
         33 . A method for treating a mammal suffering from conditions associated with fibrinogen metabolism comprising administering to said mammals an effective amount of a composition comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or homologs having at least about 90% identity with SEQ ID NO:2.  
     
     
         34 . An isolated α E CX fragment of fibrinogen comprising a composition of matter, which composition comprises a band having an apparent molecular weight of from about 34 to about 40 kilodaltons as determined by denaturing non-reducing polyacrylamide gel electrophoresis, said composition detected by a monospecific antibody directed to α E C domain of fibrinogen.  
     
     
         35 . An isolated precursor α E CX fragment of fibrinogen comprising a composition of matter, which composition comprises a band having an apparent molecular weight of from about 36 to about 80 kilodaltons as determined by denaturing non-reducing polyacrylamide gel electrophoresis, said composition detected by a monospecific antibody directed to α E C domain of fibrinogen.