Interaction of thrombin with PAR1 and PAR4 at the thrombin cleavage site

Investigations determined the critical amino acids for alpha-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 alpha-thrombin with a K-m of 28 mu M, a k(cat) of 340 s(-1), and a k(cat)/K-m of 1.2 x 10(7). When the P4 or P2 position was mutated to alanine, PAR1-L38A or PAR1-P40A, respectively, the K-m was unchanged, 29 or 23 mu M, respectively; however, the k(cat) and k(cat)/K-m were reduced in each case. In contrast, when Asp(39) at P3 was mutated to alanine, PAR1-D39A, K-m and k(cat) were both reduced similar to 3-fold, making the k(cat)/K-m the same as that of PAR1-wt exodomain. Recombinant PAR4-wt exodomain was cleaved by alpha-thrombin with a K-m of 61 mu M, a k(cat) of 17 s(-1), and a k(cat)/K-m of 2.8 x 10(5). When the P5 or P4 position was mutated to alanine, PAR4-L43A or PAR4-P44A, respectively, there was no change in the K-m (69 or 56 mu M, respectively); however, the k(cat) 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 K-m but markedly lowered the k(cat) and k(cat)/K-m similar to 35-fold. PAR1-wt exodomain and P4 and P3 mutants were noncompetitive inhibitors of alpha-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 K-i. All PAR4 exodomains were competitive inhibitors of alpha-thrombin. Mutation of P5, P4, or P2 had no effect on the K-i. These investigations show that Leu at P4 in PAR1 or P5 in PAR4 critically influences the kinetics of alpha-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.