The appended tail region of heparin cofactor II and additional reactive centre loop mutations combine to increase the reactivity and specificity of α1-proteinase inhibitor M358R for thrombin

Jason S. Sutherland; Varsha Bhakta; William P. Sheffield

doi:10.1160/TH07-03-0197

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2007; 98(05): 1014-1023
DOI: 10.1160/TH07-03-0197

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

The appended tail region of heparin cofactor II and additional reactive centre loop mutations combine to increase the reactivity and specificity of α₁-proteinase inhibitor M358R for thrombin

Jason S. Sutherland

¹Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada

²Canadian Blood Services, Research and Development, Hamilton, Ontario, Canada

,

Varsha Bhakta

²Canadian Blood Services, Research and Development, Hamilton, Ontario, Canada

,

William P. Sheffield

¹Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada

²Canadian Blood Services, Research and Development, Hamilton, Ontario, Canada

› Author Affiliations

Abstract

Summary

Natural inhibitors of coagulation or inflammation such as the serpins antithrombin (AT), heparin cofactor II (HCII), and 1-proteinase inhibitor (α₁-PI) can be overwhelmed in thrombosis and/or sepsis. The reactive centre (P1-P1) variant α₁-PI M358R inhibits not only procoagulant thrombin but also anticoagulant activated protein C (APC). We previously described HAPI M358R, comprising a fusion of HCII residues 1–75 to the N-terminus of a1-PI M358R that yielded increased anti-thrombin, but not anti-APC activity. We hypothesized that further alterations to the HAPI M358R reactive centre loop would yield additional refinements in specificity. The reactions with thrombin or APC of recombinant α₁-PI M358R variants with or without the HCII extension were characterized electrophoretically and kinetically. Their extension of clotting times and inhibition of fibrin-bound thrombin were measured, and the survival of HAPI M358R in mice was determined. Replacing the P7-P3 and P2’ residues of HAPI M358R with AT residues reduced APC inhibition rates by 140-fold, but those of thrombin less than two-fold;substituting the P16-P2 and P2’-P3’ residues of HAPI M358R with HCII residues reduced APC inhibition rates by 180-fold, but those of thrombin 10.5-fold. Fused variants extended thrombin clotting times more effectively than unfused inhibitors, were at least as effective at inhibiting clot-bound thrombin, and remained intact in the murine circulation. The combination of modifications inside and outside the RCL resulted in a 1,360-fold increase in selectivity of HAPI M358R (AT P7-P3/P2’) for thrombin versus APC relative to α₁-PI M358R. Our results predict that this protein may be effective in limiting thrombosis in vivo.

Keywords

Serpins - thrombin - APC - recombinant proteins - kinetics

Full Text

References

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