Assay For Tissue Factor And Factor Vlla

Abstract

The invention provides assays for detecting and quantitating tissue factor and factor VIIa in simple and complex biological systems. The assays are performed by detecting and/or measuring the tissue factor cofactor activity and factor VIIa enzymatic activity using aminonaphthalene-based fluorogenic substrates.

1 .- 14 . (canceled) 
   
   
       15 . A method for determining factor VIIa (fVIIa) enzymatic activity in a sample suspected to contain fVIIa, comprising:
 (a) combining fVIIa and a molar excess of TF to produce a TF/fVIIa enzyme complex; and   (b) detecting the enzymatic activity of the complex using a fluorogenic substrate or a chromogenic substrate.   
   
   
       16 . The method of  claim 15 , wherein the substrate is a compound of the formula: 
     
       
         
         
             
             
         
       
       or a pharmaceutically acceptable non-toxic salts thereof; wherein
 R 1  is hydrogen, straight or branched chain lower alkyl having 1-6 carbon atoms optionally substituted with C 1 -C 6  alkoxy, straight or branched chain alkenyl having 2-8 carbon atoms, straight or branched chain alkynyl having 2-8 carbon atoms, cycloalkyl having 3-7 carbon atoms, alkylcycloalkyl where the alkyl portion has 1-6 carbon atoms, cycloalkylakyl where the alkyl portion has 1-6 carbon atoms, or phenylalkyl where the alkyl portion is straight or branched chain alkyl having 1-6 carbon atoms, or a group of the formula 
 
     
     
       
         
         
             
             
         
       
       
         
           R 5  represents hydrogen or an amino acid side chain; and 
           R 4  is hydroxy, C 1 -C 6  alkoxy, an amino acid or a peptide residue; 
         
         R 2  is hydrogen, straight or branched chain lower alkyl having 1-6 carbon atoms, straight or branched chain alkenyl having 2-8 carbon atoms, straight or branched chain alkynyl having 2-8 carbon atoms, cycloalkyl having 3-7 carbon atoms, alkylcycloalkyl where the alkyl portion has 1-6 carbon atoms, or phenylalkyl where the alkyl portion is straight or branched chain alkyl having 1-6 carbon atoms, or a group of the formula 
       
     
     
       
         
         
             
             
         
       
       
         
           R 5  represents hydrogen or an amino acid side chain; and 
           R 4  is hydroxy, C 1 -C 6  alkoxy, an amino acid or peptide residue; or 
         
         NR 1 R 2  forms a nitrogen heterocycle; and 
         R 3  is an amino acid or a peptide residue. 
       
     
   
   
       17 . The method of  claim 15 , where the substrate is a chromogenic substrate. 
   
   
       18 . The method of  claim 17 , where the chromogenic substrate is a pNA-based substrate. 
   
   
       19 . The method of  claim 15 , further comprising (c) generating a numerical value associated with the enzymatic activity of the sample and (d) comparing the numerical value with a standard curve. 
   
   
       20 . The method of  claim 19 , where the curve is generated by quantifying fVIIa-dependent enzymatic activity of the TF/fVIIa complex in samples with known concentrations of fVIIa. 
   
   
       21 . The method of  claim 19 , wherein the fVIIa is native human factor VIIa or recombinant human factor VIIa. 
   
   
       22 . The method of  claim 19 , wherein the fVIIa source is plasma, tissue extract, cell extract or recombinant material. 
   
   
       23 . The method of  claim 19 , wherein the TF is native human tissue factor or recombinant human tissue factor. 
   
   
       24 . The method of  claim 19 , wherein the TF source is a synthetic or naturally derived thromboplastin. 
   
   
       25 . The method of  claim 19 , wherein the fVIIa and the TF are not of human origin. 
   
   
       26 . The method of  claim 19 , wherein the concentration of fVIIa is from 0.1 pM to 1 mM. 
   
   
       27 . The method of  claim 19 , wherein the reaction mixture contains divalent metal ion or a metal ion chelator. 
   
   
       28 . The method of  claim 27 , wherein the divalent metal ion is calcium ion, manganese ion or magnesium ion. 
   
   
       29 . The method of  claim 27 , wherein the metal ion chelator is ethylenediamimetetraacetic acid (EDTA) or ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA).