The Transferable Tail:  Fusion of the N-Terminal Acidic Extension of Heparin Cofactor II to α₁-Proteinase Inhibitor M358R Specifically Increases the Rate of Thrombin Inhibition

Abstract

The conversion of the reactive center bond of the serpin alpha1-proteinase inhibitor (alpha1-PI, also known as alpha1-antitrypsin) from Met-Ser to Arg-Ser decreases the rate at which it inhibits neutrophil elastase and endows it with the ability to inhibit thrombin and activated protein C (APC). Another serpin, heparin cofactor II (HCII), contains a unique N-terminal extension that binds thrombin exosite 1. We fused residues 1-75 of HCII to the N-terminus of alpha1-PI M358R, forming an HCII-alpha1-PI chimera (HAPI M358R). It inhibited alpha-thrombin 21-fold faster than alpha1-PI M358R, with second-order rate constants of 2.3 x 10(8) M(-1) min(-1) versus 1.1 x 10(7) M(-1) min(-1), respectively. When gammaT-thrombin, which lacks an intact exosite 1, was substituted for alpha-thrombin, the kinetic advantage of HAPI M358R over alpha1-PI M358R was reduced to 9-fold, whereas APC and trypsin, proteases lacking exosite 1-like regions, were inhibited only 1.3- and 2-fold more rapidly by HAPI M358R than by alpha1-PI M358R, respectively. Maximal enhancement of alpha1-PI M358R activity required the acidic residues found between HCII residues 55 and 75, because no enhancement was observed either by fusion of residues 1-54 alone or by fusion of a mutated HCII acidic extension in which all Glu and Asp residues between positions 55 and 75 were neutralized by mutation. Fusing residues 55-75 to alpha1-PI M358R yielded a relative rate enhancement of only 6-fold, suggesting a need for the full tail region to achieve maximal enhancement. Our results suggest that transfer of the N-terminal acidic extension of HCII to alpha1-PI M358R enhanced its inhibition of thrombin by conferring the ability to bind exosite 1 on HAPI M358R. This enhancement may aid in efforts to tailor this inhibitor for therapeutic use.

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

“…We have previously reported that API made in this soluble, intracellular bacterial expression system, exhibits SI values of 2–3[29], [30], [41]. Others employing different bacterial expression systems, some in which recombinant API was renatured from inclusion bodies, have reported SI values closer to unity [55], [56].…”

“…The rate of reaction of recombinant API proteins was quantified by determining the second order rate constant (k 2 ) of thrombin inhibition in a discontinuous assay, under pseudo first order conditions, as in previous studies from this laboratory [29], [40], [41], as was the stoichiometry of inhibition (SI). The SI is the number of serpin molecules required to inhibit one protease molecule, and was determined as previously described, by reacting various ratios of thrombin with API proteins, plotting the ratio versus the residual thrombin activity, and using linear regression to extrapolate to zero [29], [40], [41].…”

The complete N-terminal extension of heparin cofactor II is required for maximal effectiveness as a thrombin exosite 1 ligand

“…Previous work from this laboratory showed that fusion of residues 1-75 of HCII to the N-terminus of another thrombin inhibitory serpin, the M358R “Pittsburgh” variant of α 1 -proteinase inhibitor (α 1 -PI, also known as α 1 -antitrypsin) increased the rate of thrombin inhibition in the absence of GAGs by 21-fold [19]. The rate advantage of the HAPI M358R fusion protein over unfused α 1 -PI M358R was reduced to 6-fold when residues 55-75 were substituted for the entire N-terminal acidic extension, and eliminated altogether either when all acidic residues between 55-75 were neutralized by mutation in the full 1-75 transferred extension, or when residues 1-54 alone were employed for fusion [19].…”

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

“…5. Lysates containing API M358R exhibited a gradual increase in thrombin-bound API reactivity over 60 min, while two API M358R variants with additional mutations previously shown to enhance their rate of thrombin inhibition by 2- (Scott et al, 2014) to 19-fold (Sutherland et al, 2006) exhibited more rapid rates of thrombin binding that plateaued over time, and API T345R/M358R exhibited minimal, background levels of binding (Fig. 5).…”

“…Recombinant API proteins were characterized kinetically as thrombin inhibitors by determination of their pseudo-first order rate constant under appropriate order conditions (≥10-fold molar excess of API over thrombin in a two-stage assay) and division of that value by the initial API concentration to yield the second order rate constants (k 2 ) (Filion et al, 2004;Sheffield et al, 2012). Reaction stoichiometries (i.e., stoichiometries of inhibition, SI) were also determined as previously described (Sutherland et al, 2006).…”