Inhibitory specificity of the anti-inflammatory myxoma virus serpin, SERP-1

SERP-1 is a myxoma virus-encoded serpin, secreted from infected cells, that is required for virulence and has anti-inflammatory activity. We report that purified recombinant SERP-1 forms SDS-stable complexes with urokinase- type plasminogen activator (uPA), tissuetype plasminogen activator (tPA), plasmin, thrombin, and factor Xa. N-terminal sequencing confirmed Arg³¹⁹- Asn³²⁰ as the site of reaction. Mutation of these residues to Ala-Ala abolished inhibitory activity but had no effect on the specific cleavage at Thr³¹⁵-Leu³¹⁶ seen with elastase and with cathepsin G. Kinetic analysis of the reactions with uPA, tPA, plasmin, thrombin, Xa, and C1s showed second- order rate constants to vary over 3 logs, from k(inh) = 3 x 10⁵ M^-1 s^-1 with thrombin to ~600 M^-1 s^-1 with C1s, while steady-state inhibition constants ranged from K(I) = 10 pM with thrombin to ~100 nM with C1s. Stoichiometries of inhibition varied between SI = 1.4 ± 0.1 for uPA to SI = 13 ± 3 for thrombin. Analysis of the variations in inhibition kinetics shows that when serpins act at low concentrations, comparable with the target protease or with K(I) (as appears likely for SERP-1 in vivo), inhibitory specificity becomes less dominated by k(inh) and is increasingly dependent on partitioning within the branched reaction mechanism and on the lifetime of the inhibited complex.