A functional assay of protein C in human plasma.
A three-step spectrophotometric assay was developed for measuring functional protein C (PC) in human plasma. The assay is based on: (1) adsorption of citrated platelet-poor plasma on barium citrate and elution of the vitamin K-dependent factors with EDTA; (2) activation of PC by incubation of the mixture of vitamin K-dependent factors with a complex of thrombin and its endothelial cell cofactor, thrombomodulin; (3) 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: (a) PC-depleted plasma (by immunoadsorption) is inactive; (b) addition of purified PC to PC-depleted plasma reconstitutes its activity; and (c) 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%. It is concluded that the present assay may be used for reliable and accurate estimation of activatable PC in human plasma.
D000327:Adsorption; D001778:Blood Coagulation Disorders / Q000151:congenital / Q000188:drug therapy; D001779:Blood Coagulation Factors / Q000032:analysis*; D001780:Blood Coagulation Tests / Q000379:methods; D002863:Chromogenic Compounds; D001728:Dicumarol / Q000627:therapeutic use; D006023:Glycoproteins / Q000032:analysis* / Q000172:deficiency / Q000378:metabolism; D006801:Humans; D011486:Protein C; D011956:Receptors, Cell Surface / Q000378:metabolism; D018179:Receptors, Thrombin; D013917:Thrombin / Q000378:metabolism
D001779:Blood Coagulation Factors; D002863:Chromogenic Compounds; D006023:Glycoproteins; D011486:Protein C; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D001728:Dicumarol; D013917:Thrombin