Heparin cofactor II (HCII) is a member of the serine protease inhibitor, or serpins, family of proteins. It inhibits thrombin in plasma by providing a bait -like substrate motif on a surface-exposed reactive center loop (RCL), and subsequently forming denaturation-resistant inhibitory complexes with the protease. While thrombin acts a,s a target protease with HCII, neutrophil elastase (NE), another serine protease, reacts with HCII within the RCL, at P6, and inactivates HCII without becoming trapped. The aim of this study was to analyze the effects of mutagenesis of the NE cleavage site within the RCL of HCII to determine the limits of amino acid substitutions that would permit HCII to retain function but be less susceptible to inactivation by NE. PCR was employed to create five amino acid substitutions at P6, as well as; to incorporate an amino-terminal hexahistidine-tag on HCII (H6-HCI1). H6-HCII variants were expressed in an arabinose-inducible manner in E. coli and purified to homogeneity by nickel-chelate and ion exchange chromatography. l jiM H6-HCII was incubated with 200 nM thrombin in the absence and presence of 1 |iM heparin. Wild type H6-HCII reacted with thrombin similarly to plasma-derived HCII, as evaluated by the determination of progressive and heparin-catalyzed second order rate constants. Specifically, progressive second order rate constants of 6.5X104 and 8. IxlO4 M'mirr' and heparin catalyzed rates of 3.5x10 and 2.4x10 M''min ' were obtained for plasma-derived HCII and wild-type recombinant HCII, respectively. Substitutions of tryptophan, serine and phenylalanine for wild-type valine produced similar second order rate constants as wild-type H6-HCII, while cysteine, glutamic acid and leucine P6 mutants lost all thrombin-inhibitory activities. 1 (iM H6-HCII variants were incubated with 20 nM NE and 1 p.M heparin and a novel chromogenic assay was used to quantify residual thrombin inhibitory activities. We determined that the tryptophan, serine and phenylalanine P6 mutants were approximately 10-fold more resistant to NE inactivation compared to their wild-type counterpart. Our results demonstrate for the first time that some amino acid substitutions within this region of the RCL in HCII are tolerated well and that it is possible to produce hardened serpins which maintain thrombin-inhibitory activity, even after exposure to NE. Such proteins might eventually be used to counter the regulation of coagulation that is believed to contribute to the coagulopathy associated with bacterial sepsis.