A polypeptide corresponding to the extracellular domain of protease-activated receptor 3 (PAR-3) is hydrolyzed by thrombin slowly because of high K-M (>100 muM). However, thrombin is found to bind two PAR-3, one without catalyzing hydrolysis or blocking the active site, while the other is hydrolyzed. In a solvent lacking Na+, hydrolysis of a nitroanilide substrate is enhanced 1.6-fold by addition of PAR-3 polypeptide, with half-saturation at 2.5 muM. In contrast, the fibrinogen clotting activity of thrombin is inhibited completely by PAR-3, with a K-1 of 3 muM. None of the activities of thrombin are affected by addition of 50 muM PAR-4 polypeptide. Thus, PAR-3 in low concentrations binds thrombin in a configuration that blocks the anion-binding exosite but not the catalytic site, while hydrolysis of PAR-3, PAR-4, and other substrates that do not interact with exosite I persists. The allosteric effect of PAR-3 is characteristic of that of Na+. (C) 2003 Elsevier Science (USA). All rights reserved.