A Cyclic Pentapeptide Derived from the Second EGF-Like Domain of Factor VII Is an Inhibitor of Tissue Factor Dependent Coagulation and Thrombus Formation

Summary

We have previously reported the finding of a cyclic dodecapeptide representing loop I of the second EGF-like domain of FVII, which inhibited TF-dependent FX activation (Örning et al. 1997). The biological activity was localized to the tripeptide motif, Glu-Gln-Tyr. We have now synthesized a cyclic analog of this motif, Cys-Glu-Gln-Tyr-Cys (PN7051), evaluated its anticoagulant and antithrombotic properties and performed a detailed structural characterization of the peptide.

PN7051 is a dose-dependent inhibitor of TF-dependent FX activation and coagulation of plasma with IC₅₀ values of 10 ± 2 µM and 1.3 ± 0.2 mM, respectively. It shows inhibitory efficacy on acute thrombus formation in an ex vivo model of human thrombosis using native blood. Fibrin deposition, platelet-fibrin adhesion, platelet-thrombus formation, and thrombin-antithrombin complex formation were all inhibited by PN7051 at IC₅₀ values between 0.3 and 0.7 mM. The cyclic peptide is a non-competitive inhibitor of FX activation with no significant activesite effects on FXa or FVIIa, indicating it affects FVII/TF/FX complex formation and function. Studies on the structure activity relationship revealed that Gln³-Tyr⁴, but not Glu² were of importance for inhibition. In line with biological results, NMR measurements of PN7051 suggested that the Gln and Tyr residues configure a structural feature that contributes to the anticoagulant activity. Modeling of the Glu⁹⁹Gln¹⁰⁰Tyr¹⁰¹ motif in FVII and comparison with the solution structure of PN7051 suggest that the cyclic pentapeptide exerts its antithrombotic effect by interfering with the docking of Tyr¹⁰¹ into a hydrophobic pocket in the catalytic domain thereby disrupting an essential interaction between the second EGF-like and the catalytic domains of FVII.

Keywords

Factor VII - antithrombotic - ex vivo

• References

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