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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
1   Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
2   Canadian Blood Services, Research and Development, Hamilton, Ontario, Canada
,
Varsha Bhakta
2   Canadian Blood Services, Research and Development, Hamilton, Ontario, Canada
,
William P. Sheffield
1   Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
2   Canadian Blood Services, Research and Development, Hamilton, Ontario, Canada
› Author AffiliationsFinancial support: This work was made possible by Grant-In-Aid T5597 from the Heart and Stroke Foundation of Ontario to WPS. JSS was the recipient of a Graduate Fellowship Award from Canadian Blood Services.
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Publication History

Received 15 March 2007

Accepted after resubmission 23 August 2007

Publication Date:
30 November 2017 (online)

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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
 

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