A new rapid automated clotting assay enables measurement of residual TFPI activity in human plasma

Introduction Tissue factor pathway inhibitor (TFPI) inhibits initiation of coagulation with Kunitz domain 1 (K1) binding FVIIa in complex with tissue factor (TF), Kunitz domain 2 (K2) binding FXa and Kunitz domain 3 (K3) binding Protein S and enhancing inhibition of FXa-activated prothrombinase. Anti-TFPI antibodies can enhance coagulation, therefore, have been used as prophylactic therapy in hemophilia A and B with or without inhibitors. The effect of a panel of mAbs directed against TFPI K1, K2 and K3 was tested using a new rapid automatable clotting assay measuring the activity of TFPI. Method Normal Human Plasma (ISTH #Lot 5) and Hemophilia A Plasma (HA pool), spiked with increasing concentration of anti-TFPI mAbs (a K1 mAb, 2 K2 mAbs with different TFPI affinity, and a K3 mAb; 0.1-1000 nM; Novo Nordisk A/S), were diluted with excess of TFPI deficient plasma. A low amount of TF, cephalin and Ca 2+ trigger clotting, measured for 5 min. The clotting time is compared with a calibraton curve (mix of normal human plasma and TFPI-depleted plasma) and% TFPI activity calculated. This design allows automation on coagulation analyzers. Results Adding either of the anti-TFPI mAbs to Normal or HA plasma resulted in a concentration-dependent reduction of TFPI activity ([ Fig. 1 ], [ Fig. 2 ]). Similar concentration-response was seen for the K1 mAb, the high-affinity K2 mAb and the K3 mAb, while higher concentration of the low affinity K2 mAb was needed to inhibit TFPI activity [ 1 ] [ 2 ] [ 3 ]. Fig. 1 Results for TFPI activity (%) using the TFPI Assay for for ISTH# 5 plasma samples spiked with anti-TFPI antibodies. Note Conclusion Antibodies to TFPI K1, K2 and K3 domain inhibited TFPI activity in a concentration-dependent manner, demonstrating that the TFPI clotting assay can measure the effect of neutralizing antibodies binding either of the Kunits domains. Overall, the results confirm the rapid and automated TFPI clotting assay performance to detect TFPI activity including involvement of all three Kunitz-domains. Furthermore, the results suggest that the assay may be used to detect the functional effect of specific anti-TFPI antibodies in citrated plasma samples. Fig. 2 Results for TFPI activity (%) using the TFPI Assay for HA Pooled plasma samples spiked with anti-TFPI antibodies Publication History Article published online: 17 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany