PB1123 - Measurement of activated factor X based on complex formation with tissue factor pathway inhibitor

Background: Low molecular weight peptide substrates allow the sensitive measurement of activated coagulation factor X (FXa) but may show deficits in selectivity. The addition of specific inhibitors increases the assay’s selectivity but at the same time its complexity.

Aims: Solid phase-bound tissue factor pathway inhibitor (TFPI) is used to measure FXa by formation of the immobilized inhibitor-protease complex and its immunological detection by an FX-specific antibody. The work was funded by Baxalta Innovations GmbH, part of Takeda.

Methods: TFPI (R&D Systems, 10 µg/mL in PBS) was coated to Maxisorp F96 plates at 4°C overnight. After a washing step with PBS containing 0.05% Tween 20 (PBST), plates were blocked with PBST containing 0.5% skimmed dry milk, 3 mM EDTA and 120 KIE aprotinin: This buffer was used also as dilution buffer. Purified FXa (Haematologic Technologies) was used as the assay standard. The TFPI-FXa complex formed was detected with an anti-FX IgG peroxidase conjugate (CoaChrom) using SureBlue (KPL) for the peroxidase measurement.

Results: The six-point calibration curve ranged from 0.03 to 1.04 ng/mL. Addition of Rivaroxaban caused complete signal inhibition (50% inhibition at 177 ng/mL). None of the six purified proteases thrombin, FVIIa, FXIa, FXIIa, kallikrein, and plasmin showed any significant alteration of the concentration-response curve obtained for purified FXa. Analysis of a prothrombin complex concentrate demonstrated that an excess of FX zymogen did not result in a significant signal. In contrast, despite the complexity of the protein matrix of an activated prothrombin complex concentrate (aPCC) the concentration-response curves obtained for aPCC was highly parallel to that obtained for the purified FXa standard. Inter-run precision determined in six separate measurements for four aPPC lots ranged from 3.7% to 5.4%.

Conclusion(s): The TFPI-FXa complex formation assay described enables for a sensitive, robust, and reliable FXa measurement with high selectivity for FXa.