A SIMPLE CHROMOGENIC SUBSTRATE ASSAY OF TISSUE FACTOR PATHWAY INHIBITOR ACTIVITY IN PLASMA AND SERUM

A simple chromogenic substrate assay for the quantitation of tissue fa ctor pathway inhibitor (TFPI) activity in plasma or serum samples was developed. After immobilization on microtiter plates for 20 hours at 4 degrees C, a commercial thromboplastin was incubated for 1 hour at ro om temperature with 1 U/mL of a prothrombin complex concentrate (Protr omplex). After washing, solid-phase Factor Xa activity was measured by a chromogenic substrate (S-2222). Factor Xa generation was progressiv ely inhibited when increasing amounts (1-12 mu L) of heated serum or p lasma, and recombinant TFPI (1-5 ng/mL), were coincubated with Protrom plex. Inhibition by serum or plasma was abolished by anti-TFPI polyclo nal antibodies. Plasma levels of TFPI in 25 healthy volunteers were fo und to be 0.98 +/- 0.19 U/mL (range 0.71-1.52), with an intra- and int er-assay coefficient of variation of 10.7 and 11.1%, respectively. The use of a recombinant human thromboplastin improved the sensitivity an d reproducibility of the assay. Plasma levels of TFPI were found to be normal in 10 women at the end of their pregnancies, in 10 patients re ceiving oral anticoagulant therapy, and in 10 diabetic patients. Signi ficantly higher levels were detected in 10 patients with chronic liver disease and in 10 patients with unexplained juvenile thrombosis. In p atients with cardiovascular disease, a 7-day treatment with subcutaneo us standard heparin increased TFPI activity. The availability of a sim ple and rapid assay to measure TFPI that does not require purified coa gulation proteins may facilitate studies of the pathophysiologic relev ance of this inhibitor.