The preparation and characterization of novel peptide antagonists to thrombin and factor VIIa and activation of protease-activated receptor 1.
Thrombin and protease-activated receptor 1 (PAR1) activation antagonists were prepared based upon the peptide RPPGF, the angiotensin-converting enzyme breakdown product of bradykinin. A library of 72 peptides consisting of d and/or synthetic amino acids was designed with various substitutions in positions 1 to 5 in Arg-Pro-Pro-Gly-Phe (RPPGF). Two compounds, rOicPGF (TH146) and betaAK2K-4(rOicPGF) (MAP4-TH146), were characterized further. TH146 or MAP4-TH146 completely inhibits threshold gamma-thrombin-induced platelet aggregation at a concentration of 142 +/- 0.05 or 19 +/- 0.06 microM, respectively. TH146 completely inhibits threshold alpha-thrombin-induced washed platelet aggregation at 444 +/- 0.04 microM. TH146 or MAP4-TH146 blocks 2 nM alpha-thrombin-induced fibroblast calcium mobilization with an IC(50) value of 110 or 18 microM, respectively. Furthermore, significant prolongation of the activated partial thromboplastin time, prothrombin time, or thrombin clotting time occurs at 31, 62, or 7.8 microM TH146 and 0.4, 6.25, or 1.56 microM MAP4-TH146, respectively. TH146 and MAP4-TH146 inhibit both alpha-thrombin with a K(i) value of 97 and 49 microM, respectively, and factor VIIa with a K(i) value of 44 and 5 microM, respectively. Both TH146 and MAP4-TH146 specifically bind to the exodomain of recombinant PAR1. MAP4-TH146 (200 microM) completely blocks thrombocytin, a PAR1-activating snake venom protease, without inhibiting the enzyme's active site. TH146 inhibits gamma-thrombin-induced aggregation of mouse platelets, prolongs mouse bleeding times, and delays the time to mouse carotid artery thrombosis. TH146 and MAP4-TH146 inhibit human and mouse platelet aggregation and mouse thrombosis. Analogs of RPPGF are model compounds to develop PAR1 activation antagonists as well as direct inhibitors to thrombin and factor VIIa.
Nieman
Marvin T
MT
Thromgen Inc., Ann Arbor, MI 48109-0640, USA.
Warnock
Mark
M
Hasan
Ahmed A K
AA
Mahdi
Fakhri
F
Lucchesi
Benedict R
BR
Brown
Nancy J
NJ
Murphey
Laine J
LJ
Schmaier
Alvin H
AH
D000818:Animals; D001760:Bleeding Time; D001794:Blood Pressure / Q000187:drug effects; D001920:Bradykinin / Q000031:analogs & derivatives* / Q000097:blood / Q000494:pharmacology* / Q000627:therapeutic use; D004195:Disease Models, Animal; D015942:Factor VIIa / Q000037:antagonists & inhibitors*; D006801:Humans; D051379:Mice; D010446:Peptide Fragments / Q000097:blood / Q000494:pharmacology* / Q000627:therapeutic use; D010455:Peptides / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D013917:Thrombin / Q000037:antagonists & inhibitors*; D013927:Thrombosis / Q000097:blood / Q000517:prevention & control; D010975:Platelet Aggregation Inhibitors
C472709:MAP4-RPPGF; D010446:Peptide Fragments; D010455:Peptides; C079000:bradykinin (1-5); D015942:Factor VIIa; D013917:Thrombin; D001920:Bradykinin; D010975:Platelet Aggregation Inhibitors