CHROMOGENIC SUBSTRATE ASSAY OF EXTRINSIC PATHWAY INHIBITOR (EPI) - LEVELS IN THE NORMAL POPULATION AND RELATION TO CHOLESTEROL

A two-stage chromogenic substrate assay was standardized to measure extrinsic pathway inhibitor (EPI) activity in plasma and serum samples. In the first stage, diluted plasma or serum (0-0.8 %) was incubated with factor VIIa (25 pM), tissue thromboplastin (tissue factor, TF, 1% v/v) with excess binding sites for factor VIIa, and factor Xa (0.8 nM). In the second stage, excess factor X and chromogenic substrate were added as substrate for residual TF/factor VIIa catalytic activity. Heating the samples at 56-degrees-C for 15 min before assay removed > 95 % of the factor VII amidolytic activity of the samples, defibrinated the plasma, and produced only slight reduction of EPI activity. The coefficient of variation for the same sample assayed on different days was 8.7 - 10.6% and the intra-assay coefficient of variation was 5.0%. Addition of anti-EPI immunoglobulin to normal plasma completely abolished the EPI activity of the sample. EPI activity was stable in plasma samples stored at -20-degrees-C, but in serum, some samples lost > 50% activity after 3 months at -70-degrees-C. Median EPI activity of umbilical cord blood was 45% (range 33 - 93%). In a cohort of healthy blood donors (n = 176) EPI activity was significantly correlated with age; the regression line was y = 68% + 0.60x (r = 0.39). The approximated standard deviation for the regression line was 17.9% and the age-adjusted reference limits were determined. Equal levels were seen in males and females. EPI activity was significantly correlated with total cholesterol (r= 0.58), and multiple regression showed that cholesterol was a stronger predictor of EPI activity than age.