Interaction of Thrombin with PAR1 and PAR4 at the Thrombin Cleavage Site

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Abstract
Investigations determined the critical amino acids for α-thrombin's interaction with protease-activated receptors 1 and 4 (PAR1 and PAR4, respectively) at the thrombin cleavage site. Recombinant PAR1 wild-type (wt) exodomain was cleaved by α-thrombin with a Km of 28 μM, a kcat of 340 s-1, and a kcat/Km of 1.2 × 107. When the P4 or P2 position was mutated to alanine, PAR1-L38A or PAR1-P40A, respectively, the Km was unchanged, 29 or 23 μM, respectively; however, the kcat and kcat/Km were reduced in each case. In contrast, when Asp39 at P3 was mutated to alanine, PAR1-D39A, Km and kcat were both reduced ∼3-fold, making the kcat/Km the same as that of PAR1-wt exodomain. Recombinant PAR4-wt exodomain was cleaved by α-thrombin with a Km of 61 μM, a kcat of 17 s-1, and a kcat/Km of 2.8 × 105. When the P5 or P4 position was mutated to alanine, PAR4-L43A or PAR4-P44A, respectively, there was no change in the Km (69 or 56 μM, respectively); however, the kcat was lowered in each case (9.7 or 7.7 s-1, respectively). Mutation of the P2 position (PAR4-P46A) also had no effect on the Km but markedly lowered the kcat and kcat/Km ∼35-fold. PAR1-wt exodomain and P4 and P3 mutants were noncompetitive inhibitors of α-thrombin hydrolyzing Sar-Pro-Arg-pNA. However, PAR1-P40A displayed a mixed type of inhibition. Mutation of P4, P3, or P2 had no effect on the Ki. All PAR4 exodomains were competitive inhibitors of α-thrombin. Mutation of P5, P4, or P2 had no effect on the Ki. These investigations show that Leu at P4 in PAR1 or P5 in PAR4 critically influences the kinetics of α-thrombin binding and cleavage of PAR1 and PAR4 exodomains. It also implies that factors other than the hirudin-like binding region on PAR1 exodomain predominate in influencing PAR1 cleavage on cells.

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