Mechanisms of Arg-Pro-Pro-Gly-Phe inhibition of thrombin

1 Division of Hematology and Oncology, Department of Internal Medicine, University of Michigan, Ann Arbor 48109-0640; 2 Thromgen, Incorporated, Ann Arbor 48104; and 3 Department of Chemistry, Michigan State University, East Lansing, Michigan 48824 Submitted 11 June 2002 ; accepted in final form 3 February 2003 Investigations determined the mechanism(s) by which Arg-Pro-Pro-Gly-Phe (RPPGF) inhibits thrombin-induced platelet activation. High concentrations of RPPGF inhibit thrombin-induced coagulant activity. RPPGF binds to the active site of thrombin by forming a parallel -strand with Ser 214 -Gly 216 and interacts with His 57 , Asp 189 , and Ser 195 of the catalytic triad. RPPGF competitively inhibits -thrombin from hydrolyzing Sar-Pro-Arg-paranitroanilide with a K i = 1.75 ± 0.03 mM. Other mechanisms were sought to explain why RPPGF inhibits thrombin activation of platelets at concentrations below that which inhibits its active site. Soluble RPPGF blocks biotinylated NATLDPRSFLLR of the thrombin cleavage site on protease-activated receptor (PAR)1 from binding to the peptide RPPGC (IC 50 = 20 µM). The soluble recombinant extracellular domain of PAR1 (rPAR1 EC ) blocks biotinylated RPPGF binding to rPAR1 EC (IC 50 = 50 µM) bound to microtiter plates, but rPAR1 EC deletion mutants missing the sequence LDPR or PRSF do not. RPPGF and related forms prevent the thrombin-like enzyme thrombocytin from proteolyzing rPAR1 EC at concentrations that do not block thrombocytin's active site. These studies indicate that RPPGF is a bifunctional inhibitor of thrombin: it binds to PAR1 to prevent thrombin cleavage at Arg 41 and interacts with the active site of -thrombin. protease-activated receptor 1; bradykinin-(1–5); thrombin inhibitor; thrombin receptor Address for reprint requests and other correspondence: A. H. Schmaier, Univ. of Michigan, 5301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0640 (E-mail: aschmaie{at}umich.edu ).