The appended tail region of heparin cofactor II and additional reactive centre loop mutations combine to increase the reactivity and specificity of alpha1-proteinase inhibitor M358R for thrombin.
Natural inhibitors of coagulation or inflammation such as the serpins antithrombin (AT), heparin cofactor II (HCII), and alpha(1)-proteinase inhibitor (alpha(1)-PI) can be overwhelmed in thrombosis and/or sepsis. The reactive centre (P1-P1') variant alpha(1)-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 a(1)-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 alpha(1)-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 alpha(1)-PI M358R. Our results predict that this protein may be effective in limiting thrombosis in vivo.
Sutherland
Jason S
JS
Deptartment of Pathology and Molecular Medicine, McMaster University, HSC 4N66, 1200 Main Street West, Hamilton, Ontario, Canada, L8N 3Z5.
Bhakta
Varsha
V
Sheffield
William P
WP
D000818:Animals; D001777:Blood Coagulation; D001780:Blood Coagulation Tests; D015844:Heparin Cofactor II / Q000235:genetics*; D051379:Mice; D020125:Mutation, Missense; D011485:Protein Binding; D013917:Thrombin / Q000378:metabolism*; D000515:alpha 1-Antitrypsin / Q000235:genetics* / Q000378:metabolism / Q000502:physiology
D000515:alpha 1-Antitrypsin; D015844:Heparin Cofactor II; D013917:Thrombin