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

Abstract

SummaryNatural 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.

Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin

“…In both reports, these investigators expressed a truncated form of API M358R in E. coli, one lacking its N-terminal 12 residues [53]. We expressed both API M358R and the LS7-3/P′2L variant, which we designated “API RCL5”, in a hexahistidine-tagged, full length recombinant API protein, finding, that it was less active than API M358R, in our hands by about 13% [30]. Thus, it is uncertain whether or not API variants with increased activity versus thrombin have been synthesized, prior to this study.…”

“…Pre-clinical studies demonstrated further refinements in specificity and activity were required prior to contemplating this protein as a therapeutic agent in individuals prone to overactive coagulation [24]–[26]. Both our laboratory and other groups have made some progress in this regard by using mutagenesis strategies in which additional residues from other serpins were substituted into the API M358R [27]–[30].…”

Reactive Center Loop (RCL) Peptides Derived from Serpins Display Independent Coagulation and Immune Modulating Activities

“…pro-thrombotic) functions of NSP. Serpins are suicide inhibitors forming 1:1 stoichiometric complexes, wherein the protease cleaves the serpin RCL P1-P1Ј scissile bond, forming a complex with the serpin such that both the serpin and protease lose function after binding (1)(2)(3)(4)(5)29). These active peptides may represent a class of anti-inflammatory peptides similar to the highly active expanding classes of defensin peptides.…”

“…This same RCL can insert into the neighboring ␤-sheet A in other serpins in serpinopathies, causing serpin aggregates induced by genetic mutations and causing disease as for anti-thrombin III (SERPIN C1, ATIII), ␣-1 antitrypsin (SERPIN A1, AAT), and neuroserpin (SERPIN I1, NSP). Whereas the amino acid residues in the RCL provide target P1-P1Ј sequences, referred to as a scissile bond, serpins also require the greater part of the protein structure to function with true serpin-protease inhibitory activity (4,5). However, as for other proteins, peptides derived during protein metabolism may act to extend serpin activity beyond the initial suicide-inhibitory function, both increasing and decreasing responses (6 -9).…”

Comparison of mammalian and bacterial expression library screening to detect recombinant alpha-1 proteinase inhibitor variants with enhanced thrombin inhibitory capacity☆

“…While this change elicited a bleeding tendency in the index case (Owen et al, 1983), it sparked interest in using the API scaffold to generate novel protease inhibitors by protein engineering. Directed mutagenesis approaches have met with some success in enhancing the specificity of API for nonphysiological targets, but typically at the cost of elevating the reaction stoichiometry (Dufour et al, 2001;Dufour et al, 2005;Filion et al, 2004;Sutherland et al, 2007). Random mutagenesis approaches involving the screening of hypervariable serpin expression libraries has until recently been employed only in the case of one serpin, plasminogen activator inhibitor 1 (PAI-1) (Pannekoek et al, 1993;Stoop et al, 2001Stoop et al, , 2000van Meijer et al, 1996).…”