Factor Xa and VIIa inhibition by tissue factor pathway inhibitor is prevented by a monoclonal antibody to its Kunitz‐1 domain

Abstract

Background Initiation of coagulation is induced by binding of activated factor VII (FVIIa) to tissue factor (TF) and activation of factor X (FX) in a process regulated by tissue factor pathway inhibitor (TFPI). TFPI contains three Kunitz-type protease inhibitor domains (K1-K3), of which K1 and K2 block the active sites of FVIIa and FXa, respectively. Objective To produce a monoclonal antibody (mAb) directed towards K1, to characterize the binding epitope, and to study its effect on TFPI inhibition. Methods A monoclonal antibody, mAb2F22, was raised against the N-terminal TFPI(1-79) fragment. Binding data were obtained by surface plasmon resonance analysis. The Fab-fragment of mAb2F22, Fab2F22, was expressed and the structure of its complex with TFPI(1-79) determined by X-ray crystallography. Effects of mAb2F22 on TFPI inhibition were measured in buffer- and plasma-based systems. Results mAb2F22 bound exclusively to K1 of TFPI (K ~1 nm) and not to K2. The crystal structure of Fab2F22/TFPI (1-79) mapped an epitope on K1 including seven residues upstream of the domain. TFPI inhibition of TF/FVIIa amidolytic activity was neutralized by mAb2F22, although the binding epitope on K1 did not include the P1 residue. Binding of mAb2F22 to K1 blocked TFPI inhibition of the FXa amidolytic activity and normalized hemostasis in hemophilia human A-like plasma and whole blood. Conclusion mAb2F22 blocked TFPI inhibition of both FVIIa and FXa activities and mapped a FXa exosite for binding to K1. It reversed TFPI feedback inhibition of TF/FVIIa-induced coagulation and restored clot formation in FVIII-neutralized human plasma and blood.

Association of TFPI polymorphisms rs8176592, rs10931292, and rs10153820 with venous thrombosis

“…[13] The TFPI downregulates the tissue factor (TF)-dependent pathway by inhibiting both tissue factor-activated factor VII and activation of factor X, thereby limiting clot growth and preventing prothrombin to thrombin conversion. [14–17] Low TFPI plasma level is associated with increases risk of venous thrombosis. [18] The TFPI is localized on human chromosome 2q and contains 10 exons and 9 introns.…”

Les anticorps thérapeutiques en hémostase

“…Une étude réalisée avec le PF-06741086, une IgG1 spécifique du domaine K2 du TFPI [16] a conduit à des résultats comparables. Un autre anticorps anti-TFPI, le mAb2F22, se fixe exclusivement au domaine K1 du TFPI et interfère avec l'inhibition du facteur VIIa, mais aussi du facteur Xa, en restaurant la formation du caillot [17]. Enfin, un anticorps de sous-classe IgG2 (BAY 1093884) qui se fixe à la fois aux domaines K1 et K2 du TFPI, est en cours d'étude [18].…”

The functional role of the autolysis loop in the regulation of factor X upon hemostatic response

“…Treatment of hemophilia involves increasing the activation rate of FX by replacing the missing coagulation factors upstream of FX 8 or alternatively using bypassing agents 9,10 . Novel bypassing concepts aim to slow down the inactivation of FXa by neutralizing the inhibitory activity of ATIII 11 and TFPI, 12,13 both interventions showing hemostatic potential in hemophilia patients.…”