Background: The tissue factor pathway inhibitor (TFPI) anticoagulant pathway inhibits the initiation of coagulation. Only full-length TFPIα functions as an effective anticoagulant in plasma and its function is greatly enhanced by its cofactor, protein S. TFPI Kunitz domain 3 (K3) plays a key role in the binding, and cofactor function, of protein S. How protein S augments TFPI function at a molecular level, and the physiological importance of this enhancement remains uncertain.

Aims: To generate and characterise anti-human TFPI K3 monoclonal antibodies (mAbs) that inhibit/do not inhibit TFPI-protein S cofactor function.

Methods: Mice were immunised with recombinant human TFPI K3 expressed in insect cells. Hybridoma clones were screened and selected based on mAb affinity, specificity, and inhibitory/non-inhibitory function on the protein S enhancement of TFPI-mediated FXa inhibition.

Results: Following selection of clones with immunoreactivity against human TFPI K3, two antibodies were selected for full characterisation (6H7 & 4D7). Analysis of the binding of these antibodies to either human TFPI K3 or full-length TFPIα revealed high affinity binding (6H7: Kd = 0.3nM (K3), Kd = 0.7nM (TFPI)), (4D7: Kd = 0.6nM (K3), Kd = 1.3nM (TFPI)), and no cross-reactivity with murine TFPI. Using FXa inhibition assays containing TFPI ±protein S, 6H7 completely blocked protein S enhancement of FXa inhibition by TFPI, whereas 4D7 had no effect on protein S cofactor function.

Conclusion(s): These data confirm the importance of the TFPI K3 domain in protein S-dependent enhancement of TFPI function and reveal that distinct epitopes on the K3 are important for the interaction with protein S. These antibodies will pave the way for interrogation of protein S-TFPI cofactor function in human plasma.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/characterisation-of-anti-tfpi-kunitz-domain-3-antibodies-that-selectively-inhibit-protein-s-cofactor-function/