FUNCTIONAL AND IMMUNOLOGICAL METHODS FOR THE MEASUREMENT OF HUMAN TISSUE FACTOR PATHWAY INHIBITOR

Recent publications have addressed the topic of an important regulator of the extrinsic pathway, the tissue factor pathway inhibitor (TFPI). Tissue factor-dependent reactions are regulated by TFPI, a member of a superfamily of proteins, homologous to aprotinin (Kunitz). An indirect assay has been developed to quantitate levels of TFPI in human plasma by measuring its ability to inhibit FVIIa-TF activity (Actichrome(R) TFPI). A sandwich ELISA (Imubind(R) Total TFPI), which recognizes all forms of TFPI (e.g. lipid associated, carboxytruncated TFPI, as well as recombinant and native TFPI) was used to compare antigenic and functional TFPI levels. The Actichrome(R) TFPI kit exhibited no detectable response to other clotting factors tested, in their normal and pathologic ranges. Mean circulating levels of TFPI were 55 ng/ml and 61 ng/ml using the activity assay and the ELISA, respectively. The difference might be attributed to inactive forms of TFPI that are detected by the ELISA. Intra-assay and inter-assay variation (% CV) was less than 5% for three samples of known concentration were assayed in replicates of 20. The total TFPI ELISA has a sensitivity of 0.3 ng/ml for serum and plasma samples, assayed at a 20-fold dilution. Specificities of both the capture and detection antibodies were confirmed via Western blot analysis visualizing bands at 34 kDa and 21 kDa, respectively, for full length and truncated TFPI. In conclusion, two assays which quantitate circulating levels of TFPI have been developed and optimized: the Actichrome(R) TFPI Activity Assay and the Imubind(R) Total TFPI ELISA. These assays are specific, sensitive and accurate for TFPI levels in human biological fluids (e.g. cell culture supernatants and plasma). These two kits, when applied to human plasma, reveal subtle differences.