Quantitative clotting assay for activated factor VII
AssigneeOklahoma Medical Research Foundation
InventorJames H. Morrissey
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Abstract
An assay for activated factor VII (factor VIIa) has been developed using truncated tissue factor (tTF), a soluble mutant form of (tTF) that retains the cofactor function of TF toward factor VIIa. Unlike full-length TF, however, tTF appears not to support the conversion of factor VII to VIIa. As a result, the tTF assay for factor VIIa is free from interference from factor VII in the plasma and is therefore specific for factor VIIa. The assay is much simpler than existing assays, because it is a single-stage clotting assay performed almost identically to a prothrombin time assay. It is also considerably more sensitive than current assays for factor VIIa in plasma. Since the tTF assay is calibrated against a factor VIIa standard, it yields an absolute concentration of factor VIIa in ng/ml.
Claims23
A method for measuring the concentration of coagulation factor VIIa in a patient's plasma sample, comprising the steps of:
- (a) mixing a quantity of a plasma sample with a soluble truncated tissue factor, said soluble truncated tissue factor characterized by its inability to bind to phospholipids and its inability to support conversion of factor VII to factor VIIa, wherein said soluble truncated tissue factor consists of amino acids 1-219 of SEQ ID NO:
- 4, to form a first mixture;
- (b) thereafter, adding an effective amount of calcium ions to said first mixture to form a second mixture and to initiate clotting of said second mixture;
- (c) determining a plasma sample clotting time for said second mixture measured from a time of addition of said calcium ions to a time of clot formation;
- and
- (d) comparing said plasma sample clotting time to a standard clotting time determined for factor VIIa of a known concentration to determine the concentration of coagulation factor VIIa in the sample.
A method for measuring the concentration of coagulation factor VIIa in a patient's plasma sample, comprising the steps of:
- (a) mixing a quantity of a plasma sample with a combination of a soluble truncated tissue factor and a phospholipid agent, said soluble truncated tissue factor characterized by its inability to bind to phospholipids and its inability to support conversion of factor VII to factor VIIa, wherein said soluble truncated tissue factor consists of amino acids 1-219 of SEQ ID NO:
- 4, to form a first mixture;
- (b) thereafter, adding an effective amount of calcium ions to said first mixture to form a second mixture and to initiate clotting of said second mixture;
- (c) thereafter, determining a plasma sample clotting time for said second mixture measured from a time of addition of said calcium ions to a time of clot formation;
- and
- (d) comparing said plasma sample clotting time to a standard clotting time determined for factor VIIa of a known concentration to determine the concentration of coagulation factor VIIa in the sample.
A method for measuring the concentration of coagulation factor VIIa in a patient's plasma sample, comprising the steps of:
- (a) mixing a quantity of a plasma sample with a reagent to form a first mixture, said reagent comprising a soluble truncated tissue factor and an effective amount of calcium ions to initiate clotting of said first mixture, said soluble truncated tissue factor characterized by its inability to bind to phospholipids and its inability to support conversion of factor VII to factor VIIa, wherein said soluble truncated tissue factor consists of amino acids 1-219 of SEQ ID NO:
- 4;
- (b) determining a plasma sample clotting time for said first mixture measured from a time of addition of said reagent to a time of clot formation;
- and
- (c) comparing said plasma sample clotting time to a standard clotting time determined for factor VIIa of a known concentration to determine the concentration of coagulation factor VIIa in the sample.
Dependencies are inferred from the claim text.