Assays for Determining Anticoagulant Cofactor Activity

Abstract

Methods are disclosed for determining, in a sample derived from a human, the functional activity of a component of the human blood coagulation system, which activity can be correlated to conversion of a substrate specific for activated Protein C (APC), by measuring in an assay medium containing the sample and a substrate for APC, the conversion of the substrate by APC and correlating the conversion to the functional activity of the component. When the component is anticoagulant Factor V, at least one of exogenous APC, Protein S or an inhibitor of Protein S activity is added to the medium. When the component is Protein C, APC, or Protein S, exogenous anticoagulant Factor V or an inhibitor of anticoagulant activity of Factor V is added to the medium. Methods are also disclosed for diagnosing a blood coagulation/anticoagulation disorder or for determining a predisposition thereto in a human by determining anticoagulant Factor V activity in an assay medium containing a sample derived from the human.

1 - 43 . (canceled)  
   
   
       44 . A method for diagnosing a blood coagulation/anticoagulation disorder or for determining a predisposition thereto in a human, said method comprising: 
 determining anticoagulant Factor V activity in an assay medium containing a sample derived from said human,    wherein an abnormal level of the anticoagulant Factor V activity indicates manifestation of, or predisposition to, said blood coagulation/anticoagulation disorder.    
   
   
       45 . The method of  claim 44 , wherein the assay medium further comprises a substrate for Activated Protein C (APC) and wherein determining anticoagulant Factor V activity comprises measuring conversion of said substrate by APC and correlating said conversion to said anticoagulant Factor V activity.  
   
   
       46 . The method of  claim 44 , wherein said disorder is a thromboembolic disorder.  
   
   
       47 . The method of  claim 46 , wherein said abnormal level is a decreased level.  
   
   
       48 . The method of  claim 44 , wherein said anticoagulant Factor V activity can be correlated to conversion of a substrate specific for activated Protein C (APC), said method further comprising: 
 measuring in said assay medium, containing said sample and a substrate for APC, conversion of said substrate by APC and correlating said conversion to said anticoagulant Factor V activity;    wherein at least one exogenous substance selected from the group consisting of Protein S and an inhibitor of Protein S activity is added to said assay medium.    
   
   
       49 . The method of  claim 44 , wherein the method further comprises adding to the assay medium a blood coagulation factor or a reagent that activates the blood coagulation system via a pathway selected from the intrinsic and extrinsic pathways.  
   
   
       50 . The method of  claim 44 , said method further comprising adding blood coagulation components selected from the group consisting of Factor VII/VIIa, Factor IX, Factor IXa, Factor X/Xa, Factor II, Factor XIa, Factor XIIa and a reagent that serves to introduce an activated coagulation factor.  
   
   
       51 . The method of  claim 50 , wherein said reagent that serves to introduce an activated coagulation factor is selected from the group consisting of a contact activator and a tissue factor.  
   
   
       52 . A method for determining, in a sample derived from a human, a functional activity of anticoagulant Factor V of the human blood coagulation system, which activity can be correlated to conversion of a substrate specific for activated Protein C (APC), said method comprising: 
 measuring in an assay medium containing said sample and a substrate for APC, conversion of said substrate by APC and correlating said conversion to said functional activity of said anticoagulant Factor V;    wherein exogenous APC is added to said assay medium.    
   
   
       53 . The method of  claim 52 , wherein said substrate for APC is selected from the group consisting of Factor Va and Factor VIIIa.  
   
   
       54 . The method of  claim 53 , wherein the sample is derived from an individual on therapy with vitamin K antagonists or otherwise deficient in vitamin K-dependent coagulation factors, and wherein at least one vitamin K dependent coagulation factor in activated or inactivated form is added to the assay medium.  
   
   
       55 . The method of  claim 52 , wherein said method further comprises adding to the assay medium a blood coagulation factor or a reagent that activates the blood coagulation system via a pathway selected from the group consisting of the intrinsic and extrinsic pathways.  
   
   
       56 . The method of  claim 52 , wherein said method further comprises adding blood coagulation components selected from the group consisting of Factor VII/VIIa, Factor IX, Factor IXa, Factor X/Xa, Factor II, Factor XIa, Factor XIIa and a reagent that serves to introduce an activated coagulation factor.  
   
   
       57 . The method of  claim 56 , wherein said reagent that serves to introduce an activated coagulation factor is selected from the group consisting of a contact activator and a tissue factor.  
   
   
       58 . The method of  claim 52 , further comprising a comparison of multiple samples and wherein the functional activity of said exogenous APC added to each sample is essentially constant between said samples.  
   
   
       59 . The method of  claim 58 , wherein a functional excess of said exogenous APC is added to each sample to achieve said essentially constant functional activity.  
   
   
       60 . The method of  claim 58 , wherein an antibody which specifically binds to an epitope associated with anticoagulant activity of Protein S is added to each sample to achieve said essentially constant functional activity.  
   
   
       61 . The method of  claim 52 , further comprising adding to said assay medium a substance selected from the group consisting of Factor VIII and VIIIa.  
   
   
       62 . The method of  claim 52 , wherein said sample is a blood or blood derived sample.  
   
   
       63 . The method of  claim 52 , wherein said determination of said functional activity is used to diagnose a blood coagulation disorder in an individual from which said sample is derived.