A SENSITIVE AND FACILE ASSAY FOR THE MEASUREMENT OF ACTIVATED PROTEIN-C ACTIVITY LEVELS INVIVO

Activated protein C (APC) is a serine protease which plays an importan t role as a naturally occurring antithrombotic enzyme. APC, which is f ormed by thrombin-catalyzed limited proteolysis of the zymogen protein C, functions as an anticoagulant by proteolytic inactivation of the c oagulation cofactors VIIIa and Va. APC is inhibited by several members of the serpin family as well a by alpha2-macroglobulin. APC is being developed as a therapeutic for the prevention and treatment of thrombo sis. We have developed an assay to quantify circulating levels of enzy matically active APC during its administration to patients, in healthy individuals, and in various disease states. This assay utilizes an ED TA-dependent anti-protein C monoclonal antibody (Mab) 7D7B10 to captur e both APC and protein C from plasma, prepared from blood collected in an anticoagulant supplemented with the reversible inhibitor p-aminobe nzamidine. Mab 7D7B 10-derivatized agarose beads are added to the well s of a 96-well filtration plate, equilibrated with Tris-buffered salin e, and incubated for 10 min with 200 mul of plasma. Alter 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 contain ing antithrombin III (ATIII) and heparin. The amidolytic activity of A PC is then measured on a kinetic plate reader following the addition o f L-pyroglutamyl-L-prolyl-L-arginine-p-nitroanilide (S-2366) substrate . The rate of substrate hydrolysis was proportional to APC concentrati on over a 200-fold concentration range (5.0 to 1,000 ng/ml) when measu red continuously over a 15 to 30 min time period. The coefficient of v ariation was 5.9% at 35 ng/ml and 8.8% at 350 ng/ml APC. The sensitivi ty of the assay could be increased by measuring the amount of color pr oduced after longer incubation times in the endpoint mode. The measure d APC activity levels were little affected by varying protein C or pro thrombin over the extremes of 0 to 150% of normal plasma concentration s. By constructing the standard curve in protein C-deficient plasma, t he concentration of APC activity in normal pooled plasma was determine d to be 2.8 ng/ml (45 pM), which represents 0.08% of the protein C con centration. The assay was approximately 50-fold more sensitive than th e identical assay, but using Mab-coated microtiter wells rather than i mmunosorbent beads as the capture step.