Design and characterization of a1-antitrypsin variants for treatment of contact system-driven thromboinflammation

Abstract

The contact system produces the inflammatory peptide bradykinin and contributes to experimental thrombosis. C1 esterase-inhibitor (C1INH) deficiency or gain-of-function mutations in factor XII (FXII) cause hereditary angioedema, a life-threatening tissue swelling disease. C1INH is a relatively weak contact system enzyme inhibitor. Although a1- antitrypsin (a1AT) does not naturally inhibit contact system enzymes, a human mutation (M358R; a1AT-Pittsburgh) changes it into a powerful broad-spectrum enzyme inhibitor. It blocks the contact system, but also thrombin and activated protein C (APC), making it an unattractive candidate for therapeutic contact system blockade. We adapted the reactive center loop of a1AT-Pittsburgh (AIPR/S) to overcome these obstacles. Two a1AT variants (SMTR/S and SLLR/S) strongly inhibit plasma kallikrein, activated FXII, and plasmin. a1ATSMTR/ S no longer inhibits thrombin, but residually inhibits APC. In contrast, a1AT-SLLR/S residually inhibits thrombin, but no longer APC. Additional modification at the P19 position (S→V) eliminates residual inhibition of thrombin and APC for both variants, while retaining their properties as contact system inhibitors. Both a1AT-SMTR/V and -SLLR/V are superior to C1INH in reducing bradykinin production in plasma. Owing to their capacity to selectively block contact system-driven coagulation, both variants block vascular occlusion in an in vivo model for arterial thrombosis. Furthermore, both variants block acute carrageenan-induced tissue edema in mice. Finally, a1AT-SLLR/V, our most powerful candidate, suppresses epithelial leakage of the gut in a mouse model of colitis. Our findings confirm that redesign of a1AT strongly alters its inhibitory behavior and can be used for the treatment of contact system-mediated thrombosis and inflammation.