α₁‐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 ␣ 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...

Modulation of the proteolytic activity of the complement protease C1s by polyanions: implications for polyanion-mediated acceleration of interaction between C1s and SERPING1

“…The k ass value of our α1-(P 4 -P 1 ) mutant lies between the literature value of k ass reported for inhibition of C1s by wild-type SERPING1 (9.5 × 10 4 M −1 • s −1 ) [35,45], and the value reported for the α 1 -P 3 -P 1 mutant (3.5 × 10 3 M −1 • s −1 ) [44]. Given that the structure of our mutant is intermediate between wild-type SERPING1 and the α1-(P 4 -P 1 ) mutant, this suggests that the k ass value obtained is accurate.…”

“…The mutant comprised α 1 -antitrypsin with the P 4 -P 1 residues of the RCL replaced by the P 4 -P 1 residues of the SERPING1 RCL, called α1-(P 4 -P 1 ). This approach was based on an experiment reported by Sulikowski et al [44], where replacement of the P 3 -P 1 residues of the RCL of α 1 -antitrypsin with the sequence LGR resulted in the production of a reasonably effective C1s inhibitor. Our rationale was to produce a chimaeric mutant that would be able to react with C1s, yet be incapable of binding polyanions.…”

Engineering the serpin α1‐antitrypsin: A diversity of goals and techniques

“…135 Similarly, synthetic peptide cleavage studies were leveraged to alter α 1 -AT to PFR at P3-P1, which retained much of α 1 -AT M358R's activity against PKa, but not complement C1s, FXIIa, or APC. 136 Recently, investigators selected P4-P1 0 SMTRS and SLLRS, from the activation peptide of FXII and peptide substrate libraries, respectively, to re-orient α 1 -AT specificity. These variants inhibited PKa, FXIIa, and plasmin, but also retained residual anti-thrombin and anti-APC activities.…”

Therapeutic Approaches in Hereditary Angioedema

“…In a double-blind placebo-controlled dose-escalation study, eight healthy normotensive men exhibited the effects on bradykinin-induced increase in blood flow at forearm level caused by placebo or by icatibant (20,50, and 100 mg/kg) given intravenously. Icatibant inhibited significantly and in a dose-dependent manner the effects of bradykinin [22].…”

“…Another mutation to Ala in the P2 residue made this new mutant to acquire inhibitory activity against C1s [49]. Modification of the amino acid sequence of P3/P2/P1 residues of α1-antitrypsin also yielded some α1-antitrypsin variants able to inhibit potently C1s, kallikrein, or factor XII [50].…”