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CARDIOVASCULAR

The Preparation and Characterization of Novel Peptide Antagonists to Thrombin and Factor VIIa and Activation of Protease-Activated Receptor 1

Thromgen, Inc., Ann Arbor, Michigan (M.T.N., M.W., A.A.K.H., A.H.S.); Division of Hematology and Oncology, Departments of Internal Medicine (M.T.N., M.W., F.M., A.H.S.) and Pharmacology (B.R.L.), University of Michigan Medical School, Ann Arbor, Michigan; and Division of Clinical Pharmacology, Departments of Medicine and Pharmacology, Vanderbilt University, Nashville, Tennessee (N.J.B., L.J.M.)

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 AK2K-4(rOicPGF) (MAP4-TH146), were characterized further. TH146 or MAP4-TH146 completely inhibits threshold -thrombin-induced platelet aggregation at a concentration of 142 ± 0.05 or 19 ± 0.06 µM, respectively. TH146 completely inhibits threshold -thrombin-induced washed platelet aggregation at 444 ± 0.04 µM. TH146 or MAP4-TH146 blocks 2 nM -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 -thrombin with a K_i value of 97 and 49 µM, respectively, and factor VIIa with a K_i 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 -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.

Address correspondence to: Dr. Alvin H. Schmaier, University of Michigan, Division of Hematology and Oncology, 5301 MSRB III, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0640. E-mail: aschmaie{at}umich.edu

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