Coupled amidolytic assay for factor VII: Its use with a clotting assay to determine the activity state of factor VII

Abstract

A coupled amidolytic assay for factor VII (VII) has been developed that when used with a clotting assay for VII enables detection of activated VII. In the assay, VII in a test material determines generation of factor x(a) in a mixture of purified factor X, tissue factor, and calcium; factor x(a) is measured with a chromogenic substrate. Factor VII activity in the coupled amidolytic assay (VII(am)) correlated well with VII activity in a 1-stage clotting assay (VII(c)) in 57 healthy subjects, 5 patients with hereditary VII deficiency, and 11 patients with liver disease. Activation of plasma VII by kaolin, clotting, or cold strikingly increased VII(c) but not VII(am) levels. Thus the ratio VII(c)/VII(am) (VII activity ratio) is a measure of VII activation. In 27 warfarin-treated patients the mean VII activity ratio was significantly decreased, reflecting a greater decline in VII(c) than in VII (am). This probably stems from partially carboxylated VII being able to act during the 3-min incubation of the amidolytic assay but unable to act rapidly enough to affect the clotting assay. Measurement of VII(c)/VII(am) should enable evaluation of the activity state of VII in thrombotic disorders and in components for transfusion therapy.