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 factor 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 room temperature with 1 U/mL of a prothrombin complex concentrate (Protromplex). After washing, solid-phase Factor Xa activity was measured by a chromogenic substrate (S-2222). Factor Xa generation was progressively inhibited when increasing amounts (1-12 microL) of heated serum or plasma, and recombinant TFPI (1-5 ng/mL), were coincubated with Protromplex. Inhibition by serum or plasma was abolished by anti-TFPI polyclonal antibodies. Plasma levels of TFPI in 25 healthy volunteers were found to be 0.98 +/- 0.19 U/mL (range 0.71-1.52), with an intra- and inter-assay coefficient of variation of 10.7 and 11.1%, respectively. The use of a recombinant human thromboplastin improved the sensitivity and 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 receiving oral anticoagulant therapy, and in 10 diabetic patients. Significantly higher levels were detected in 10 patients with chronic liver disease and in 10 patients with unexplained juvenile thrombosis. In patients with cardiovascular disease, a 7-day treatment with subcutaneous standard heparin increased TFPI activity. The availability of a simple and rapid assay to measure TFPI that does not require purified coagulation proteins may facilitate studies of the pathophysiologic relevance of this inhibitor.