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

Abstract

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 Km of 28 microM, a kcat of 340 s-1, and a kcat/Km of 1.2 x 10(7). When the P4 or P2 position was mutated to alanine, PAR1-L38A or PAR1-P40A, respectively, the Km was unchanged, 29 or 23 microM, 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 approximately 3-fold, making the kcat/Km the same as that of PAR1-wt exodomain. Recombinant PAR4-wt exodomain was cleaved by alpha-thrombin with a Km of 61 microM, a kcat of 17 s-1, and a kcat/Km 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 Km (69 or 56 microM, 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 approximately 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 Ki. All PAR4 exodomains were competitive inhibitors of alpha-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 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.

Figure 1

Cleavage of PAR1-wt or mutant exodomains with α-thrombin. Reactions with purified exodomains (3-200 μM) in reaction buffer (10 mM Tris-HCl, 150 mM NaCl, pH 8.0) were initiated by the addition of α-thrombin (0.75 nM for A, C and D or 0.5 nM for B). Acid quenched reactions were resolved on HPLC and initial rates were determined by calculating the peak areas at early time points where the progress curves were linear and compared to a standard curve generated by completely cleaved exodomain. Curve A is α-thrombin cleavage of PAR1-wt exodomain; curve B, PAR1-D39A; curve C, PAR1-L38A; and curve D, PAR1-P40A. The data were fit to the Henri-Michaelis-Menten equation with nonlinear least squares analysis to determine the kinetic constants. Error bars represent the standard deviation and the apparent absence of error bars indicates small standard deviation. Note the change in the scaling for the y-axis in panels B and C.

Figure 2

Cleavage of PAR4-wt or mutant exodomains with α-thrombin. Reactions with purified exodomains (20-400 μM) in reaction buffer (10 mM Tris-HCl, 150 mM NaCl, pH 8.0) were initiated by the addition of α-thrombin (5.0 nM for A, B and C, or 50 nM for D). Acid quenched reactions were resolved on HPLC and initial rates were determined by calculating the peak areas at early time points where the progress curves were linear and compared to a standard curve generated by completely cleaved exodomain. Curve A is α-thrombin cleavage of PAR4-wt exodomain; curve B, PAR4-L43A; curve C, PAR4-P44A; and curve D, PAR4-P46A. The data were fit to the Henri-Michaelis-Menten equation with nonlinear least squares analysis to determine the kinetic constants. Error bars represent the standard deviation and the apparent absence of error bars indicates small standard deviation.

Figure 3

α-Thrombin hydrolysis of Sar-Pro-Arg-pNA in the absence or presence of PAR1-wt or mutant exodomain. α-Thrombin (0.5 nM) was added to Sar-Pro-Arg-pNA (70-1400 μM) in the absence (●) or presence of 5 (○), 10 (▼), 25 (∇) or 50 ( ) μM PAR1-wt exodomain (A), PAR1-L38A (B), PAR1-D39A (C) or PAR1-P40A (D). Initial velocities data were fit to 8 models of enzyme inhibition using a global analysis nonlinear least squares regression analysis to determine the K_i and the type of inhibition (see Methods). Double reciprocal plots (insets) are shown only for graphical representation of the model and were not used to determine the K_i or type of inhibition.

Figure 4

α-Thrombin hydrolysis of Sar-Pro-Arg-pNA in the absence or presence of PAR4-wt or mutant exodomain. α-Thrombin (0.5 nM) was added to Sar-Pro-Arg-pNA (70-1400 μM) in the absence (●) or presence of 50 ( ), 100 (▼), 120 (∇) or 200 (○) μM PAR4-wt (A), PAR4-L43A (B), PAR4-P44A (C), or PAR4-P46A (D). Initial velocities data were fit to 8 models of enzyme inhibition using a global analysis nonlinear least squares regression analysis to determine the K_i and the type of inhibition (see Methods). Double reciprocal plots (insets) are shown only for graphical representation of the model and were not used to determine the K_i or type of inhibition.