Crystal Structure Of Tissue Factor Pathway Inhibitor (TFPI) Kunitz Domain 1 and 2 In Complex With An Inhibitory Cyclic Peptide,

TFPI is a Kunitz-type protease inhibitor which efficiently regulates the extrinsic coagulation pathway. It is composed of three flexible linked Kunitz-type domains (KD) where KD1 and KD2 are involved in efficient inhibition of TF/FVIIa and FXa. TFPI inhibition has been shown to improve coagulation and hemostasis in hemophilia models in vitro and in vivo.

Recombinant KD1-KD2 (residues 22-150) produced by E. coli and complexed to JBT-B5, a cyclic peptide composed of 23 amino acids, was co-crystallized in 20% w/v PEG6000 and 50mM imidazole, pH8.0. JBT-B5 binding to TFPI was verified by BiaCore experiments with TFPI immobilized on a chip surface. Functional inhibition of TFPI by JBT-B5 was tested in model assays including TFPI inhibition of FXa, FX activation by TF/FVIIa, inhibition of TFPI released upon platelet activation, and by global hemostatic assays including calibrated automated thrombography in FVIII-inhibited plasma and rotational thomboelastometry (ROTEM) using FVIII-inhibited whole blood. Inhibition of cell surface TFPI was analyzed in an FX activation assay performed on HUVECs.

TFPI KD1-KD2 bound to JBT-B5 formed a crystal containing two independent complexes in the asymmetric unit. The complexes belong to the orthorhombic spacegroup P212121 and diffracted to a maximum resolution of 1.95 Å. To our knowledge, this is the first TFPI structure consisting of KD1, KD2 and their linker. The KD1-KD2 structure is fully defined in the electron density. Both domains show a Kunitz-type structure, where only ∼1/3 of the structure is engaged in secondary structure elements. These are two short α-helical elements at Ser24-Ala27(KD1)/Asp95-Phe98(KD2) (α1/α3) and Leu69-Met75/Leu140-Glu148 (α2/α4) and a two-stranded β sheet comprising Met39-Asn45/Ile110-Asn116 (β1/β3) and Arg49-Ile55/Lys120-Lys126 (β2/β4). These elements form the topological framework that is stabilized by the three canonical disulfide bonds involving Cys26-Cys76, Cys35-Cys59, and Cys51-Cys72 in KD1 and Cys95-Cys147, Cys106-Cys130, and Cys122-Cys143 in KD2. The 23mer TFPI inhibitor, JBT-B5, is sandwiched between the two Kunitz domains of TFPI and assumes a β-hairpin-like structure. It can be segmented into (i) a two-stranded β sheet comprising Tyr2-Ala8 and Thr17-Phe23; (ii) and a long β-turn loop comprising Met9-Met16. The β-sheet is stabilized by a disulfide bridge (Cys7 and Cys18) and a hydrophobic zipper comprising the side chains of Tyr3, Trp5 and Trp20. Strikingly, JBT-B5 locks KD1-KD2 in a distinct conformational state in which both Kunitz-domains are related via a two-fold symmetry and reactive center loops (RCL) from each KD are forced to opposite sides. The interactions between KD1-KD2 and JBT-B5 are extensive, as are the intramolecular interactions within JBT-B5. Analyzing the interactions between KD1-KD2 and JBT-B5 with the PISA server resulted in a total interaction surface of 1340Å2. More than 2/3 of the interaction surface consists of a hydrophobic anchor in JBT-B5 which interact with residues spread all over TFPI including KD1, KD2 and their linker. In addition, several polar interactions stabilize the KD1-KD2/JBT-B5 complex, explaining JBT-B5´s exlusive binding to human TFPI. Interaction studies revealed high affinity binding to TFPI (K_D: 0.5nM). The highly complex and extensive interaction of JBT-B5 with TFPI translates to a highly efficient inhibition of recombinant TFPI and;TFPI released from activated platelets which is demonstrated in model assays on FXa (EC₅₀: 1.3nM) and FXa generation by TF/FVIIa (EC₅₀: 0.2nM). HUVE cell-based FX activation and global hemostasis assays such as thrombin generation in hemophilia plasma (EC₅₀: 4nM) and ROTEM in FVIII-inhibited whole blood confirm the inhibition of all physiologic TFPI forms.

For the first time, a TFPI structure comprising KD1, KD2 and their linker in complex with a TFPI- inhibitory cyclic peptide was solved. This structure provides atomic details explaining the inhibitory mode of action of this efficient TFPI antagonist, and will guide the design of efficient inhibitors for use in hemophilia treatment.