The angiotensin converting enzyme breakdown product of bradykinin, bradykinin 1–5 (RPPGF), inhibits thrombin induced human or mouse platelet aggregation by preventing proteolysis of PAR1 and PAR4 by binding adjacent to the thrombin cleavage site and thus preventing activation of these receptors (

Am. J. Physiol.
J. Pharmacol. Exp. Ther.
; FEBS Lett. 579:25–29, 2005). Alanine scanning mutagenesis determined that RPPGF binds to Pro46 at the P2 position of PAR4 to block thrombin cleavage. New studies determined the amino acids required for thrombin to bind and cleave PAR4. Wild type PAR4 exodomain is cleaved by thrombin with a Km of 17 μM, kcat of 3.5 s−1 and kcat/Km of 2x105 M−1 s−1. In contrast, PAR4 exodomain in which the P2 (PAR4-P46A) is changed to alanine, is not efficiently cleaved with 10 nM alpha thrombin. Alteration of PAR4’s P4 position (e.g., PAR4-P44A) does not influence the PAR4’s rate of cleavage. The ability of the PAR4 exodomain to inhibit thrombin proteolysis of H-D-Phe-Pip-pNA was also determined as an independent measure of the thrombin/PAR4 interaction. Wild type PAR4 exodomain and PAR4-P44A are competitive inhibitors of thrombin hydrolysis of the chromogenic substrate with a Ki of 23 ± 6 micromolar and 19.6 ± 4 μM respectively. In contrast, PAR4-P46A and PAR4-P44A/P46A have a Ki > 300 μM nd the nature of the inhibition changes from competitive to noncompetitive. Taken together, these data demonstrate that Pro46 of PAR4 is important for alpha thrombin to bind and orient PAR4 in its active site for efficient cleavage. Further studies examined the role of the combined amino acids in the P4 to P3 positions of PAR4 and PAR1 to contribute to the rate of thrombin cleavage. Chimeric molecules were prepared in which the P4 and P3 positions of PAR4 (ProAla) are replaced with those from PAR1 (LeuAsp) to generate PAR4-LD. The reciprocal chimera was also made (PAR1-PA). PAR4-LD is proteolyzed by 10 nM alpha thrombin more efficiently than PAR4-wt. Alternatively, the rate of PAR1-PA proteolysis by alpha thrombin is less efficient than PAR1-wt but better than PAR4-wt due to the presence of the exosite I binding region in the PAR1 exodomain. However, if the exosite I binding domain is removed from PAR1-PA the rate of cleavage is like that seen with PAR4-wt exodomain. These data indicate PAR1 is cleaved by alpha thrombin at a faster rate than PAR4 due to the amino acids in the P4 and P3 positions as well as the exosite I binding region. In sum, these data demonstrate that the P2 position of PAR4 is the most important amino acid in thrombin binding to the region adjacent to the thrombin cleavage site. However the combined P4 and P3 positions are also important determinants of the rate of receptor cleavage. These data indicate that the amino acids around the thrombin cleavage site of both PAR4 and 1 influence its rate of cleavage. Designing thrombin receptor activation antagonists directed to the thrombin cleavage site on PAR1 and 4 should be effective anti-platelet agents.

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