Coagulation and complement proteinases are activated in sepsis, and one approach to therapy is to develop proteinase inhibitors that will specifically inhibit these proteinases without inhibiting activated protein C, a proteinase that is beneficial to survival. In this study, we made mutants of the serpin ␣ 1 -PI, designed to mimic the specificity of C1-inhibitor. The P3-P2-P1 residues of ␣1-PI were changed from IPM to LGR and PFR, sequences preferred by C1s and kallikrein, respectively. Inhibition of C1s, kallikrein, factor XIIa, and activated protein C was assessed by SDS-PAGE, and by determination of the k app and SI. , but only minimal inhibition of C1 in a hemolytic assay was observed. Kallikrein, factor XIIa, and activated protein C were inhibited with rates of 382,180 M −1 s −1 , 10,400 M −1 s −1 , and 3500 M −1 s −1 , respectively. ␣ 1 -PI-PFR was a poor inhibitor of C1s, factor XIIa, and activated protein C, but had enhanced reactivity with kallikrein. Changing the P4Ј residue of ␣ 1 -PI-LGR Pro to Glu reduced the activity with C1s, consistent with the idea that C1s requires hydrophobic residues in this region of the serpin for optimal interaction. The data provide insight into the requirements for kallikrein and C1s inhibition necessary for designing inhibitors with appropriate properties for further investigation as therapeutic agents. Keywords:Serpin mutants; kallikrein inhibition; C1 inhibition; serpin reactive center loop C1-inhibitor is a member of the serpin family of proteinase inhibitors with specificity for plasma kallikrein and factor XIIa of the contact system, and C1 of the classical pathway of complement (Davis 1988). These pathways are activated in sepsis, resulting in C1-inhibitor being found either in complex with proteinases or in an inactive cleaved state (Nuijens et al. 1988(Nuijens et al. , 1989. Numerous studies have shown that C1-inhibitor administration can be of therapeutic use in animal models of sepsis and trauma, and can be beneficial in humans in various inflammatory states, although it is clear that more clinical trials are needed to determine the true efficacy and safety of C1-inhibitor (Kirschfink and Nürnberger 1999;Caliezi et al. 2000;Kirschfink and Mollnes 2001). In general, C1-inhibitor appears to reduce complement and contact system activation, reduce hypoxemia, reduce hypotension, and increase survival. C1-inhibitor replacement therapy is also used successfully to treat C1-inhibitor deficiency (hereditary angioedema) (Waytes et al. 1996;Carugati et al. 2001). However, as with all blood products, the risks of transmission of infectious agents remain (De Filippi et al. 1998). In addition, C1-inhibitor itself has properties that make it less than ideal. It readily converts Abbreviations: ␣ 1 -PI, ␣ 1 -proteinase inhibitor; P1, the amino acid at the N-terminal side of the scissile bond in the reactive center loop of the serpin; P2, the amino acid at the N-terminal side of the P1 residue; P3 the amino acid at the N-terminal side of the P2 residue; SI, stoichiomet...

TNK-t-PA is a recombinant mutant of tissue plasminogen activator that has a longer half-life and higher selectivity for fibrin than normal tissue plasminogen activator (t-PA). In addition, it is reported to be serpin resistant because of reduced inhibition by plasminogen activator inhibitor-1. In this study, we have investigated the inhibition of TNK-t-PA by the serpin C1-inhibitor. TNK-t-PA is inhibited with a second-order rate constant of 7.5 per mol/l per s compared with 4.5 per mol/l per s for t-PA. In both cases, the stoichiometry was close to 20, indicating that C1-inhibitor was predominantly a substrate for both forms of t-PA. The formation of cleaved C1-inhibitor was seen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the t-PA-C1-inhibitor (or TNK-t-PA-C1-inhibitor) complex seen on SDS-PAGE and by zymography. Although the rates of inhibition are very slow in vitro, the fact that in vivo formation of the t-PA-C1-inhibitor complex after infusion of t-PA has been well documented suggests that TNK-t-PA will also be inhibited by C1-inhibitor in vivo and, perhaps more importantly, could cause a significant reduction in C1-inhibitor concentration.