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† Academic Article uri icon

  •  
  • Overview
  •  
  • Research
  •  
  • Identity
  •  
  • Additional Document Info
  •  
  • View All
  •  

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.

publication date

  • September 2006

has subject area