Inhibition of Human α-Thrombin with Small Covalent Inhibitors and r-Hirudin

Thrombin is a key serine protease involved in the blood cascade. Two small molecular inhibitors, 4-nitrophenyl diethylphosphate (paraoxon) and 4-nitrophenyl 2-propyl methylphosphonate (MPNP), covalently bond to the active-site Ser to form a mimic of the tetrahedral intermediate found in thrombin-catalyzed reactions. The pH dependence of their inhibition of human α-thrombin at 25.0 + 0.1C was determined. The pKa1 and pKa2 for paraoxon are 7.8 ± 0.1 and 9.3 ± 0.2 respectively, and for MPNP are 8.0 ± 0.1 and 8.6 ± 0.2 respectively. The pKas may be attributed to the role of His57 and Leu16 respectively in catalysis. The maximal second order rate constant, ki/Ki, for the inhibition of human α-thrombin was determined to be 0.47 ± 0.05 Ms with paraoxon and 6.2 ± 0.1 Ms with MPNP. Hirudin, found naturally in Hirudo medicinalis, is a slow and tight binding inhibitor of thrombin, interacting noncovalently over a large area. This study focused on the dependence of this interaction on the presence of Na and Cl ions at an ionic strength () of 0.3 M. Kinetic measurements were conducted spectrophotometrically in the presence of the chromophoric substrate, H-D-Phe-Pip-Arg-4-nitroanilide. The dissociation constant and second-order rate constant, when fitted, for the inhibition of human -thrombin with r-hirudin at pH ~ 8.1 and 25.0 + 0.1 C are: (0.47 + 0.15) pM and (4.8 ± 0.1) x 10 Ms in NaCl; (2.8 ±0.1) pM and (3.49 + 0.02) x 10 Ms in Na acetate; (13.8 ± 0.2) pM and (8.2 ± 0.4) x 10 M s in choline Cl; (12 ± 3) pM in choline acetate. The solvent isotope effect, KH/KD, for Ki is 0.26 + 0.15 in NaCl; 0.72 + 0.02 in Na acetate; 2.1 + 0.5 in choline acetate; and 2.59 + 0.03 in choline chloride. This suggests that the association of hirudin with thrombin is accompanied with changes in hydrogen bonding as a function of Na and Cl ions.