Summary
Activated protein C (APC) is a serine protease which plays an important role as a
naturally occurring antithrombotic enzyme. APC, which is formed by thrombin-catalyzed
limited proteolysis of the zymogen protein C, functions as an anticoagulant by proteolytic
inactivation of the coagulation cofactors VIIIa and Va. APC is inhibited by several
members of the serpin family as well a by α2-macroglobulin. APC is being developed as a therapeutic for the prevention and treatment
of thrombosis. We have developed an assay to quantify circulating levels of enzymatically
active APC during its administration to patients, in healthy individuals, and in various
disease states. This assay utilizes an EDTA-dependent anti-protein C monoclonal antibody
(Mab) 7D7B10 to capture both APC and protein C from plasma, prepared from blood collected
in an anticoagulant supplemented with the reversible inhibitor p-aminobenzamidine. Mab 7D7B10-derivatized agarose beads are added to the wells of
a 96-well filtration plate, equilibrated with Tris-buffered saline, and incubated
for 10 min with 200 μl of plasma. After washing, APC and protein C are eluted from
the immunosorbent beads with a calcium-containing buffer into the wells of a 96-well
microtiter plate containing antithrombin III (ATIII) and heparin. The amidolytic activity
of APC is then measured on a kinetic plate reader following the addition of L-pyroglutamyl-L-prolyl-L-arginine-p-nitroanilide (S-2366) substrate.
The rate of substrate hydrolysis was proportional to APC concentration over a 200-fold
concentration range (5.0 to 1,000 ng/ml) when measured continuously over a 15 to 30
min time period. The coefficient of variation was 5.9% at 35 ng/ml and 8.8% at 350
ng/ml APC. The sensitivity of the assay could be increased by measuring the amount
of color produced after longer incubation times in the endpoint mode. The measured
APC activity levels were little affected by varying protein C or prothrombin over
the extremes of 0 to 150% of normal plasma concentrations. By constructing the standard
curve in protein C-deficient plasma, the concentration of APC activity in normal pooled
plasma was determined to be 2.8 ng/ml (45 pM), which represents 0.08% of the protein
C concentration. The assay was approximately 50-fold more sensitive than the identical
assay, but using Mab-coated microtiter wells rather than immunosorbent beads as the
capture step.
