Molecular Characterization of the Synergistic Effect on TFPI Inhibition By Fusion of Two Inhibitory Peptides

Introduction

Tissue factor pathway inhibitor (TFPI) is a three-Kunitz domain (KD1-3) protease inhibitor that downregulates the extrinsic coagulation pathway. TFPI has a double inhibitory effect; it inactivates factor Xa (FXa) by 1:1 binding via its KD2, and it prevents further FX activation by binding the tissue factor (TF) – factor VIIa (FVIIa) complex via its KD1 and the formation of a quaternary complex. Recently, we demonstrated the crystal structure of a linear TFPI inhibitory peptide composed of 20 amino acids, bound to a TFPI protein composed of N-terminus and KD1. On the other hand, a cyclic TFPI inhibitory peptide of 23 amino acids was shown to co-crystallize with TFPI KD1-KD2. Molecular fusion of the linear and cyclic peptide by an optimized linker sequence would thus target two independent epitopes and combine the antagonistic properties of the two peptides.

Methods

The binding properties of simultaneous interaction of the linear and cyclic peptide with TFPI were studied in Biacore experiments using immobilized human TFPI 1-160 on a CM5 chip. Measurements with the linear or cyclic peptide were done with and without prior saturation of TFPI with the linear peptide and the fusion peptide. The results were confirmed by native-PAGE analysis of peptide/KD1-KD2 mixtures, where the TFPI fragment KD1-KD2 had been incubated with either linear or cyclic peptide or both.

The TFPI inhibitory effect of the linear, cyclic, and fusion peptide was assessed in several TFPI sensitive assays including inhibition of FXa, FX activation by TF/FVIIa, and thrombin generation. Calibrated automated thrombography (CAT) was performed in human hemophilia plasma triggered with low tissue factor. To model a situation of elevated plasma levels of TFPI, the assay was carried out at TFPI concentrations up to 10 nM, which is 40-fold higher than the physiological TFPI plasma concentration.

Results

Biacore binding studies demonstrated that binding kinetics of the cyclic peptide to TFPI 1-160 were not influenced by prior saturation of immobilized TFPI with the linear peptide and vice versa. Prior saturation of immobilized TFPI with the fusion peptide prohibited the linear and cyclic peptide from binding to TFPI, clearly demonstrating the independent binding of the two peptides to different epitopes. By native-PAGE, the linear peptide shifted the KD1-KD2 band completely, whereas the cyclic peptide shifted it only partially. In the presence of both peptides, KD1-KD2 shifted to the highest MW to charge ratio, indicating the formation of a ternary complex consisting of K1-K2, cyclic, and linear peptide.

Although the linear and cyclic peptide inhibited TFPI in functional assays, fusion of the two molecular entities provided the most efficient inhibition of TFPI. This was most evident in assays involving multiple epitopes of TFPI to provide functions such as inhibition of extrinsic FX activation complex and thrombin generation, or at high TFPI concentrations. Thrombin generation assays using of 5- to 40-fold elevated TFPI showed that, separately, the two monomeric peptides are only partial inhibitors, and that a mixture of these peptides led to an improved response. However, molecular fusion of the two entities resulted in the most efficient TFPI neutralization. Thus, a synergistic effect is achieved by linking both peptides. Importantly, thrombin generation compromised by a 40-fold of normal TFPI level is normalized by fusion peptide concentrations as low as 50 nM.

Summary

Based on structural information, we developed a peptide inhibitor composed of two TFPI inhibitory entities. Binding studies support an independent binding mode to non-overlapping binding sites without allosteric cross-talk between binding sites. This introduces synergistic improvement of binding and functional inhibition by bivalent interaction with TFPI. This optimized fusion peptide facilitates efficient TFPI neutralization and resistance to highly increased TFPI levels. Our results further support the use of a fusion peptide in the development of subcutaneous treatment for patients with hemophilia including those with inhibitors.