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

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 β-strand with Ser214-Gly216 and interacts with His57, Asp189, and Ser195 of the catalytic triad. RPPGF competitively inhibits α-thrombin from hydrolyzing Sar-Pro-Arg-paranitroanilide with a Ki = 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 (IC50 = 20 μM). The soluble recombinant extracellular domain of PAR1 (rPAR1EC) blocks biotinylated RPPGF binding to rPAR1EC (IC50 = 50 μM) bound to microtiter plates, but rPAR1EC deletion mutants missing the sequence LDPR or PRSF do not. RPP...