Abstract
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 βAK2K-4(rOicPGF) (MAP4-TH146), were characterized further. TH146 or MAP4-TH146 completely inhibits threshold γ-thrombin-induced platelet aggregation at a concentration of 142 ± 0.05 or 19 ± 0.06 μM, respectively. TH146 completely inhibits threshold α-thrombin-induced washed platelet aggregation at 444 ± 0.04 μM. TH146 or MAP4-TH146 blocks 2 nM α-thrombin-induced fibroblast calcium mobilization with an IC50 value of 110 or 18 μM, respectively. Furthermore, significant prolongation of the activated partial thromboplastin time, prothrombin time, or thrombin clotting time occurs at 31, 62, or 7.8 μM TH146 and 0.4, 6.25, or 1.56 μM MAP4-TH146, respectively. TH146 and MAP4-TH146 inhibit both α-thrombin with a Ki value of 97 and 49 μM, respectively, and factor VIIa with a Ki value of 44 and 5 μM, respectively. Both TH146 and MAP4-TH146 specifically bind to the exodomain of recombinant PAR1. MAP4-TH146 (200 μM) completely blocks thrombocytin, a PAR1-activating snake venom protease, without inhibiting the enzyme's active site. TH146 inhibits γ-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.
Footnotes
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This work was supported by Grant #1607 from the Michigan Economic Development Corporation and National Institutes of Health Grant HL61981 (to A.H.S.), HL04445 (to L.J.M.), and HL65193 (to N.J.B).
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doi:10.1124/jpet.104.069229.
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ABBREVIATIONS: PAR, protease-activated receptor; RPPGF, Arg-Pro-Pro-Gly-Phe; TH146, rOicPGF; MAP4-TH146, βAK2K4(rOicPGF); PRP, platelet-rich plasma; APTT, activated partial thromboplastin time; PT, prothrombin time; TCT, thrombin clotting time; FX, factor X; RWJ-58259, (αS)-N-[(1S)-3-amino-1-[[(phenylmethyl)amino]carbonyl]propyl]-α-[[[[[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrroli-dinylmethyl)-1H-indazol-6-yl]amino]carbonyl]amino]-3,4-difluorobenzenepropanamide.
- Received March 30, 2004.
- Accepted June 21, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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The Preparation and Characterization of Novel Peptide Antagonists to Thrombin and Factor VIIa and Activation of Protease-Activated Receptor 1
