A cyclic pentapeptide derived from the second EGF-like domain of Factor VII is an inhibitor of tissue factor dependent coagulation and thrombus formation.
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 (Orning 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 microM 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 active-site 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 PN705 I 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.
Orning
Lars
L
Nycomed Imaging AS, Oslo, Norway. lars.orning@no.axis-shield.com
Fischer
Peter M
PM
Hu
Chih-Kao
CK
Agner
Erik
E
Engebretsen
May
M
Husbyn
Mette
M
Petersen
Lizette B
LB
Orvim
Una
U
Llinas
Miguel
M
Sakariassen
Kjell S
KS
D020816:Amino Acid Motifs; D000818:Animals; D000925:Anticoagulants / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D020134:Catalytic Domain / Q000187:drug effects; D002417:Cattle; D002863:Chromogenic Compounds / Q000378:metabolism; D015195:Drug Design; D004789:Enzyme Activation / Q000187:drug effects; D005167:Factor VII / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D005343:Fibrinolytic Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D066298:In Vitro Techniques; D008958:Models, Molecular; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D015842:Serine Proteinase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013925:Thromboplastin / Q000494:pharmacology
D000925:Anticoagulants; D002863:Chromogenic Compounds; D005343:Fibrinolytic Agents; C463960:PN 7051; D010446:Peptide Fragments; D010456:Peptides, Cyclic; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D005167:Factor VII; D013925:Thromboplastin