A sensitive assay of extrinsic coagulation pathway inhibitor (EPI) in plasma and plasma fractions

We have previously shown that addition of adsorbed plasma to a mixture of TP and FVII reduces the amount of subsequently added FX that can be activated. We now report that this inhibitory effect of plasma is increased dramatically by first incubating TP and FVII with a minor amount of FX. This results in a progressive loss in the ability of TP-FVIIa to convert subsequently added FX to FXa. An assay system quantitating the inhibitory effect of 1 microliter of heated, citrated plasma is described. Optimal inhibition is obtained when the initial amount of FX added is about 0.00125 U, which is 1/16 of the amount used as reagent to measure remaining TP-FVIIa. FVII and FX must be removed from test plasma prior to assay. The inhibitory activity is reduced more by BaSO4 adsorption than by heating plasma to 56 degrees C for 15 minutes. Gel filtration of plasma separates three distinct fractions with inhibitory activity.