Download PDF
Thromb Haemost 2002; 87(01): 13-21
DOI: 10.1055/s-0037-1612937
In Focus
Schattauer GmbH

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

Lars Örning
1   Nycomed Imaging AS, Oslo, Norway; Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA
,
Peter M. Fischer
1   Nycomed Imaging AS, Oslo, Norway; Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA
,
Chih-Kao Hu
1   Nycomed Imaging AS, Oslo, Norway; Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA
,
Erik Agner
1   Nycomed Imaging AS, Oslo, Norway; Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA
,
May Engebretsen
1   Nycomed Imaging AS, Oslo, Norway; Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA
,
Mette Husbyn
1   Nycomed Imaging AS, Oslo, Norway; Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA
,
Lizette B. Petersen
1   Nycomed Imaging AS, Oslo, Norway; Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA
,
Una Ørvim
1   Nycomed Imaging AS, Oslo, Norway; Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA
,
Miguel Llinas
1   Nycomed Imaging AS, Oslo, Norway; Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA
,
Kjell S. Sakariassen
1   Nycomed Imaging AS, Oslo, Norway; Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA
› Author Affiliations
Further Information

Publication History

Received 09 April 2001

Accepted after resubmission 17 October 2001

Publication Date:
13 December 2017 (online)

Also available at
    Permissions and Reprints

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

  • 1 Bom VJJ, Bertina RM. The contributions of Ca2+, phospholipids, and tissue factor apoprotein to the activation of human blood-coagulation factor X by activated factor VII. Biochem J 1990; 265: 327-36.
    CrossrefPubMedGoogle Scholar
  • 2 Ruf W, Rehemtulla A, Morrissey JH, Edgington TS. Phospholipid-independent and -dependent interaction for tissue factor receptor and cofactor functions. J Biol Chem 1991; 266: 2158-66.
    PubMedGoogle Scholar
  • 3 Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: Initiation, maintenance, and regulation. Biochemistry 1991; 30: 10363-70.
    CrossrefPubMedGoogle Scholar
  • 4 Galiani D, Broze Jr GJ. Factor XI activation in a revised model of blood coagulation. Science 1991; 253: 909-11.
    CrossrefPubMedGoogle Scholar
  • 5 Hagen FS, Gray CL, O’Hara P, Grant FJ, Saari GC, Woodbury RG, Hart CE, Insley M, Kisiel W, Kurachi K, Davie EW. Characterization of cDNA coding for human factor VII. Proc Natl Acad Sci USA 1986; 83: 2412-6.
    CrossrefPubMedGoogle Scholar
  • 6 Martin DMA, Boys WG, Ruf W. Tissue factor. Molecular recognition and cofactor function. FASEB J 1995; 09: 852-9.
    CrossrefPubMedGoogle Scholar
  • 7 Banner DW, D’Arcy A, Chene C, Winkler FK, Guha A, Konigsberg WH, Nemerson Y, Kirchhofer D. The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor. Nature 1996; 380: 41-6.
    CrossrefPubMedGoogle Scholar
  • 8 Kirchhofer D, Banner DW. Molecular and structural advances in tissue factor-dependent coagulation. TCM 1997; 07: 316-24.
    PubMedGoogle Scholar
  • 9 Ruf W, Dickinson CD. Allosteric regulation of the cofactor-dependent serine protease coagulation factor VIIa. TCM 1998; 08: 350-6.
    PubMedGoogle Scholar
  • 10 Kemball-Cook G, Johnson DJD, Tuddenham EGD, Harlos K. Crystal structure of active site-inhibited human coagulation factor VIIa (des-Gla). J Struct Biol 1999; 127: 213-33.
    CrossrefPubMedGoogle Scholar
  • 11 Pike ACW, Brzozowski AM, Roberts SM, Olsen OH, Persson E. Structure of human factor VIIa and its implications for the triggering of blood coagulation. Proc Natl Acad Sci USA 1999; 96: 8925-30.
    CrossrefPubMedGoogle Scholar
  • 12 Eigenbrot G, Kirchhofer D, Dennis MS, Santell L, Lazarus RA, Stamos J, Ultsch MH. The factor VII zymogen structure reveals reregistration of 1 strands during activation. Structure 2001; 09: 627-36.
    CrossrefPubMedGoogle Scholar
  • 13 Kavlie A, Wright MS, Stormorken H, Prydz H. Mutations in the factor VII gene of Norwegian FVII deficient patients. Thromb Haemost 1993; 69: 244 (abstr).
    Article in Thieme ConnectPubMedGoogle Scholar
  • 14 Kavlie A, Örning L, Grindflek A, Stormorken H, Prydz H. Characterization of a factor VII molecule carrying a mutation in the second epidermal growth factor-like domain. Thromb Haemost 1998; 79: 1136-43.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 15 Kemball-Cook G, Johnson D, Takamiya O, Banner D, McVey J, Tuddenham E. Coagulation factor VII Gln100Arg. J Biol Chem 1998; 273: 8516-21.
    CrossrefPubMedGoogle Scholar
  • 16 Barstad RM, Stormorken H, Örning L, Stephens RW, Petersen LB, Kierulf P, Sakariassen KS. Reduced thrombus formation in native blood of homozygous factor VII-deficient patients at high arterial wall shear rate. Blood 1994; 84: 3371-7.
    PubMedGoogle Scholar
  • 17 Husbyn M, Örning L, Sakariassen KS, Fischer PM. Peptides corresponding to the second epidermal growth factor-like domain of human blood coagulation factor VII: synthesis, folding and biological activity. J Peptide Res 1997; 50: 475-82.
    PubMedGoogle Scholar
  • 18 Örning L, Stephens RW, Petersen LB, Hamers MJAG, Stormorken H, Sakariassen KS. A peptide sequence from the 2nd EGF-like domain of FVII inhibits TF-dependent FX activation. Thromb Res 1997; 86: 57-67.
    CrossrefPubMedGoogle Scholar
  • 19 Hu CK, Llinás M, Agner KE, Örning L, Sakariassen KS, Fischer PM. Synthesis, biological activity, and solution structures of a cyclic dodecapeptide from the EGF2-domain of blood coagulation factor VII. J Pept Res 2001; 57: 462-72.
    CrossrefPubMedGoogle Scholar
  • 20 Higgins DL, Mann KG. The interaction of bovine factor V and factor V-derived peptides with phospholipid vesicles. J Biol Chem 1983; 258: 6503-8.
    PubMedGoogle Scholar
  • 21 Stephens RW, Örning L, Stormorken H, Hamers MJAG, Petersen LB, Sakariassen KS. Characterisation of cell-surface procoagulant activities using a microcarrier model. Thromb Res 1996; 84: 453-61.
    CrossrefPubMedGoogle Scholar
  • 22 Sakariassen KS, Joss R, Muggli R, Kuhn H, Tschopp TB, Sage H, Baumgartner HR. Collagen type III induced ex vivo thrombogenesis in humans. Role of platelets and leukocytes in deposition of fibrin. Arteriosclerosis 1990; 10: 276-84.
    CrossrefPubMedGoogle Scholar
  • 23 Ørvim U, Barstad RM, Vlasuk G, Sakariassen KS. Effect of selective factor Xa inhibition on arterial thrombus formation triggered by tissue factor/factor VIIa or collagen in an ex vivo model human shear dependent thrombogenesis. Arterioscler Thromb Vasc Biol 1995; 15: 2188-94.
    CrossrefPubMedGoogle Scholar
  • 24 Sakariassen KS, Örning L, Stormorken H. Role of ADP and thromboxanes in thrombus formation in human ex vivo models. Platelets 1997; 08: 385-90.
    CrossrefPubMedGoogle Scholar
  • 25 Sakariassen KS, Muggli R, Baumgartner HR. Measurements of platelet interaction with components of the vessel was in flowing blood. Methods of Enzymology 1989; 169: 37-70.
    PubMedGoogle Scholar
  • 26 Segel IH. Enzyme kinetics. New York, NY: Wiley-Interscience; 1975
    Google Scholar
  • 27 Hertzberg MS, Ben-Tal O, Furie B, Furie BS. Construction, expression and characterization of a chimera of FIX and FX. J Biol Chem 1992; 267: 14759-66.
    PubMedGoogle Scholar
  • 28 Forberg E, Huhmann I, Jimenez-Boj E, Watzke HH. The impact of Glu102Lys on factor X function in a patient with a doubly homozygous factor X deficiency (GLA14Lys and Glu102Lys). Thromb Haemost 2000; 83: 234-8.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 29 Weiss HJ, Turitto VT, Baumgartner HR, Nemerson Y, Hoffman T. Evidence for the presence of tissue factor activity on subendothelium. Blood 1989; 73: 968-75.
    PubMedGoogle Scholar
  • 30 Jang IK, Gold HK, Leinbach RC, Fallon JT, Collen D, Wilcox JN. Antithrombotic effect of a monoclonal antibody against tissue factor in a rabbit model of platelet-mediated arterial thrombosis. Artioscler Thromb 1992; 12: 948-54.
    PubMedGoogle Scholar
  • 31 Salatti JA, Anton P, Nemerson Y, Sakariassen KS. Modulation of procoagulant activity of extracellular endothelial matrix by anti-tissue factor antibody and the synthetic peptide Arg-Gly-Asp-Val. Experiments with flowing non-anticoagulated human blood. Blood Coagul Fibrinol 1993; 04: 881-90.
    CrossrefPubMedGoogle Scholar
  • 32 Barstad RM, Hamers MJAG, Kierulf P, Westvik AB, Sakariassen KS. Procoagulant human monocytes mediate tissue factor/factor VIIa-dependent platelet-thrombus formation when exposed in flowing non-anticoagulated human blood. Arterioscler Thromb Vasc Biol 1995; 15: 11-6.
    CrossrefPubMedGoogle Scholar
  • 33 Ørvim U, Roald HE, Stephens RW, Roos N, Sakariassen KS. TF induced coagulation triggers platelet-thrombus formation as efficiently as fibrillar collagen at arterial blood flow conditions. Arterioscler Thromb 1994; 14: 1977-83.
    PubMedGoogle Scholar
  • 34 Kirchhofer D, Tschopp TB, Baumgartner HR. Active-site blocked factors VIIa and IXa differentially inhibit fibrin formation in a human ex vivo thrombosis model. Arterioscler Thromb Vasc Biol 1995; 15: 1098-106.
    CrossrefPubMedGoogle Scholar
  • 35 Ørvim U, Barstad RM, Örning L, Petersen LB, Ezban M, Hedner U, Sakariassen KS. Antithrombotic efficacy of inactivated active site recombinant factor VIIa is shear dependent in human blood. Arterioscler Thromb Vasc Biol 1997; 17: 3049-56.
    CrossrefPubMedGoogle Scholar
  • 36 Golino P, Ragni M, Cirillo P, D’Andrea D, Scognamiglio A, Ravera A, Buono C, Ezban M, Corcione N, Vigorito F, Condorelli M, Chiarello M. Antithrombotic effects of recombinant human, active site-blocked factor VIIa in a rabbit model of recurrent arterial thrombosis. Circ Res 1998; 82: 39-46.
    CrossrefPubMedGoogle Scholar
  • 37 Harker LA, Hanson SR, Kelly AB. Antithrombotic benefits and hemorrhagic risks of direct thrombin inhibitors. Thromb Haemost 1995; 74: 464-72.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 38 Hellebust H, Bergseth S, Örning L. Expression of the second epidermal growth factor-like domain of human factor VII in Escherichia coli. J Biotech 1998; 66: 203-10.
    CrossrefPubMedGoogle Scholar
  • 39 Fang CH, Lin TC, Guha A, Nemerson Y, Konigsberg WH. Activation of factor X by factor VIIa complexed with human-mouse tissue factor chimeras requires human exon 3. Thromb Haemost 1996; 76: 361-8.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 40 Kelley RF, Costas KE, O’Connell MP, Lazarus RS. Analysis of the factor VIIa binding site on human tissue factor: effects of tissue factor mutations on the kinetics and thermodynamics of binding. Biochemistry 1995; 34: 10383-92.
    CrossrefPubMedGoogle Scholar
  • 41 Padmanabhan K, Padmanabhan KP, Tulinsky A, Park CH, Bode W, Huber R, Blankenship DT, Cardin AD, Kisiel W. Structure of human des(1-45) factor Xa at 2.2 Å resolution. J Mol Biol 1993; 232: 947-66.
    CrossrefPubMedGoogle Scholar