A FUNCTIONAL ASSAY OF PROTEIN-C IN HUMAN-PLASMA

  • 1 January 1984
    • journal article
    • research article
    • Vol. 63  (3) , 671-675
Abstract
A 3-step spectrophotometric assay was developed for measuring functional protein C (PC) in human plasma. The assay is based on: adsorption of citrated platelet-poor plasma on barium citrate and elution of the vitamin K-dependent factors with EDTA; activation of PC by incubation of the mixture of vitamin K-dependent factors with a complex of thrombin and its endothelial cell cofactor, thrombomodulin; addition of antithrombin III and heparin to the system to inhibit thrombin and other coagulation enzymes generated during incubation and measurement of the activated PC with a synthetic (chromogenic) substrate. The assay appears to be specific for PC because: PC-depleted plasma (by immunoadsorption) is inactive; addition of purified PC to PC-depleted plasma reconstitutes its activity; and no enzymatic activity is generated in the absence of the thrombin-thrombomodulin complex. Mixtures of a normal plasma pool with PC-depleted plasma yielded an amount of enzymatic activity proportional to the fraction of normal plasma. Using this as a standard curve, the amount of PC in the plasma of 23 normal subjects was 97% .+-. 15%. The within-assay coefficient of variation was 3.5% and the between-assay coefficient 6.5%. A linear correlation (r = 0.86) was found between PC as measured with the functional assay and with a radioimmunoassay. In 3 patients with congenital PC deficiency, the functional PC level was 37% .+-. 9% and the antigen level 64% .+-. 11%. The present assay may be used for reliable and accurate estimation of activatable PC in human plasma.