Engineering D-helix of antithrombin in alpha-1-proteinase inhibitor confers antiinflammatory properties on the chimeric serpin

Likui Yang; Peyman Dinarvand; Shabir H. Qureshi; Alireza R. Rezaie

doi:10.1160/TH13-12-1029

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2014; 112(01): 164-175
DOI: 10.1160/TH13-12-1029

Cardiovascular Biology and Cell Signalling

Schattauer GmbH

Engineering D-helix of antithrombin in alpha-1-proteinase inhibitor confers antiinflammatory properties on the chimeric serpin

Likui Yang^*

¹Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri, USA

,

Peyman Dinarvand^*

¹Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri, USA

,

Shabir H. Qureshi

¹Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri, USA

,

Alireza R. Rezaie

¹Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri, USA

› Author Affiliations

Abstract

Summary

Antithrombin (AT) is a heparin-binding serpin in plasma which regulates the proteolytic activity of procoagulant proteases of the clotting cascade. In addition to being an anticoagulant, AT also exhibits antiinflammatory activities when it binds to cell surface heparan sulfate proteoglycans (HSPGs) on the endothelium via its basic residues of D-helix to elicit intracellular signalling responses. By contrast to AT, α₁-proteinase inhibitor (α₁-PI) is a non-heparin-binding serpin that exhibits very slow reactivity with coagulation proteases and possesses no HSPG-dependent antiinflammatory properties. To determine whether the antiinflammatory signaling specificity of AT can be transferred to α₁-PI, we replaced the D-helix of human α₁-PI with the corresponding sequence of human AT and expressed the chimeric serpin α₁-PI/D-helix) in a bacterial expression system. High molecular weight heparin bound to α₁-PI/D-helix and accelerated the inhibition of thrombin by the serpin mutant by a template mechanism reminiscent of the cofactor effect of heparin on inhibition of thrombin by AT. Like AT, α₁-PI/D-helix exhibited antiinflammatory properties in both cellular and animal models. Thus, α₁-PI/D-helix inhibited the barrier-disruptive effect of proinflammatory cytokines and inhibited the activation of nuclear factor-kB transcription factor in lipopolysaccharide-stimulated endothelial cells by a concentration-dependent manner. Furthermore, the chimeric serpin reduced lipopolysaccharide-mediated lethality, elicited a vascular protective effect and inhibited infiltration of activated leukocytes to the peritoneal cavity of mice in an HMGB1-mediated inflammatory model. These results suggest that grafting the D-helix of AT to α₁-PI confers antiinflammatory properties on the serpin and that the chimeric serpin may have therapeutic utility for treating inflammatory disorders.

Keywords

Antithrombin - coagulation inhibitors - endothelial cells - inflammation - SERPINs

Full Text

References

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