Interaction of activated protein C with serpins

The inhibition of activated protein C by six different serine protease inhibitors (serpins) that have arginine residues in the P1 position has been investigated. Micromolar concentrations of C1-inhibitor failed to inhibit the enzyme, and it was inhibited only slowly by antithrombin III with an association rate constant (kass.) of 0.15 M-1.s-1. The kass. values for the other serpins tested (protease nexin I, protein C inhibitor, and mutants of alpha 1-antichymotrypsin and alpha 1-antitrypsin with P1 arginine residues) were at least 1000-fold higher, with P1-Arg-alpha 1-antitrypsin (kass. = 7 x 10(4) M-1.s-1) being the most effective inhibitor. The inhibition with these four serpins appeared to be reversible, with inhibition constants in the nanomolar range. The relatively high value of kass. for protease nexin I (5 x 10(3) M-1.s-1) suggested that it may be involved in the control of activated protein C on the surface of platelets where protein nexin I is present at relatively high concentrations. The value of kass. for protease nexin I, protein C inhibitor and antithrombin III showed a bell-shaped dependence on heparin concentration. At optimal concentrations, heparin accelerated the rate of inhibition by protease nexin I, protein C inhibitor and antithrombin III by 44-, 18- and 13-fold respectively. The kinetic constants for the inhibition of thrombin were also determined, and in all cases the serpins were more effective inhibitors of thrombin. Comparison of the sequences of the active-site regions of activated protein C and thrombin suggested that the more hydrophobic active site of thrombin may be more favourable for interactions with serpins.