QUANTIFICATION OF CIRCULATING ACTIVATED PROTEIN-C IN HUMAN PLASMA BY IMMUNOASSAYS - ENZYME LEVELS ARE PROPORTIONAL TO TOTAL PROTEIN-C LEVELS

We have developed a simple assay that measures the circulating activat ed protein C (APC) in plasma. The assay requires collection of duplica te blood samples, one in citrate plus heparin and the other in citrate plus inhibitors of the enzyme. In the heparin tube, APC reacts comple tely and irreversibly with its major plasma inhibitors, protein C inhi bitor (PCI) and alpha(1)-antitrypsin (alpha(1)AT), and the complexes f ormed are measured by ELISAs. The amount of circulating APC is calcula ted from the difference between the total amount of complexed APC (sam ple in citrate plus heparin) and the amount of APC complexed in vivo ( sample in citrate plus inhibitor). Over 95% of the APC added to blood collected with heparin was recovered in the assay. The assay can easil y be performed in four hours, and had a detection limit of 0.1 ng/ml A PC. The mean APC level in 18 protein C heterozygous members from seven kindreds was significantly lower (0.6 +/- 0.3 ng/ml) than in 20 healt hy controls (1.1 +/- 0.3 ng/ml) (p < 0.001), whereas the mean level in 10 non-affected members from the kindreds studied was 1.5 +/- 0.3 ng/ ml. In the group of 12 nonanticoagulated heterozygous protein C-defici ent individuals, the three patients with a history of venous thrombosi s had a mean APC level significantly lower than the nine asymptomatic members (p < 0.01), both subgroups showing similar protein C levels. T here was a significant correlation in all groups between the levels of APC and the levels of protein C antigen (r = 0.758, p < 0.0001) and a ctivity (r = 0.745, p < 0.0001), which means that APC circulating leve ls are proportional to protein C levels and suggests that the protein C level is the limiting factor in the rate of protein C activation in vivo.