Tissue factor derived β-hairpin peptides that bind and inhibit FVII activity

The formation of the Tissue Factor(TF):Factor VII(FVII) complex is a pivotal event that initiates coagulation; targeting this early step allows for the prevention of the subsequent cascade amplification driven by positive feedback loops. For this reason, the TF:FVII complex is attracting increasing interest as a potential therapeutic target for regulating the coagulation cascade in a specific and timely manner. In order to generate TF-mimics capable of inhibiting this protein-protein interaction, we have designed four small cyclic peptides that simulate a TF region containing the two antiparallel β-strands: 106–110 (RVFSY) and 123–128 (EPLYEN). These strands are known to interact with FVII at well-known hot spots surrounding residues 365–369. With the aim of obtaining structures as similar as possible to the corresponding region of TF and therefore able to interact better with FVII, the four combinations of proline-proline dipeptides resulting from the four permutations of D-Pro and L-Pro have been introduced between the two strands. These strands have been connected on the opposite side by a disulphide bond in order to stabilise the resulting structures and also make them more resistant to protease action. The two cyclopeptides with the D-Pro-L-Pro and D-Pro-D-Pro moieties adopt β-hairpin-like conformations that recapitulate the structure of the two strands, as demonstrated by CD, NMR and molecular simulation studies. They also bind FVII and inhibit its activity in a Factor X-generating chromogenic assay. The other two peptides are significantly more disordered and are inactive in the same tests. Overall, the data validate the peptide design and confirm the region 365–369 of FVII as a target site for the design of coagulation inhibitors.