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A functional assay of protein C in human plasma
N Sala, WG Owen and D Collen
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.
Volume 63,
Issue 3,
pp. 671-675,
03/01/1984
Copyright © 1984 by The American Society of Hematology
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