alpha(1)-Proteinase inhibitor mutants with specificity for plasma kallikrein and C1s but not C1

Abstract

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 alpha(1)-PI, designed to mimic the specificity of C1-inhibitor. The P3-P2-P1 residues of alpha1-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. alpha(1)-PI-LGR inhibited C1s with a rate of 7790 M(-1)s(-1), 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. alpha(1)-PI-PFR was a poor inhibitor of C1s, factor XIIa, and activated protein C, but had enhanced reactivity with kallikrein. Changing the P4' residue of alpha(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.

Fig. 1.

Alignment of the P3-P5′ residues of C1-inhibitor, α₁-proteinase inhibitor (α₁-PI), and the α₁-PI-LGR, α₁-PI-PFR, and α₁-PI-LGR-P4′E mutants. The mutated residues are highlighted.

Fig. 2.

SDS-PAGE (10% acrylamide, nonreduced) of α₁-proteinase inhibitor (α₁-PI)-LGR, α₁-PI-PFR, and α₁-PI-LGR-P4′E, and their reactions with C1s, kallikrein, factor XIIa, and activated protein C, as indicated. Each lane contained 5 μg of proteinase and/or inhibitor. The mixtures of proteinase and inhibitor were incubated for 30 min at 37°C prior to stopping the reaction by addition of sample buffer.