title: Full or partial substitution of the reactive center loop of alpha-1-proteinase inhibitor by that of heparin cofactor II: P1 Arg is required for maximal thrombin inhibition.
abstract: The abundant plasma protein alpha(1)-proteinase inhibitor (alpha(1)-PI) physiologically inhibits neutrophil elastase (NE) and factor XIa and belongs to the serine protease inhibitor (serpin) protein superfamily. Inhibitory serpins possess a surface peptide domain called the reactive center loop (RCL), which contains the P1-P1' scissile peptide bond. Conversion of this bond in alpha(1)-PI from Met-Ser to Arg-Ser in alpha(1)-PI Pittsburgh (M358R) redirects alpha(1)-PI from inhibiting NE to inhibiting thrombin (IIa), activated protein C (APC), and other proteases. In contrast to either the wild-type or M358R alpha(1)-PI, heparin cofactor II (HCII) is a IIa-specific inhibitor with an atypical Leu-Ser reactive center. We examined the effects of replacement of all or part of the RCL of alpha(1)-PI with the corresponding parts of the HCII RCL on the activity and specificity of the resulting chimeric inhibitors. A series of 12 N-terminally His-tagged alpha(1)-PI proteins differing only in their RCL residues were expressed as soluble proteins in Escherichia coli. Substitution of the P16-P3' loop of alpha(1)-PI with that of HCII increased the low intrinsic antithrombin activity of alpha(1)-PI to near that of heparin-free HCII, while analogous substitution of the P2'-P3' dipeptide surpassed this level. However, gel-based complexing and quantitative kinetic assays showed that all mutant proteins inhibited thrombin at less than 2% of the rate of alpha(1)-PI (M358R) unless the P1 residue was also mutated to Arg. An alpha(1)-PI (P16-P3' HCII/M358R) variant was only 3-fold less active than M358R against IIa but 70-fold less active against APC. The reduction in anti-APC activity is desired in an antithrombotic agent, but the improvement in inhibitory profile came at the cost of a 3.5-fold increase in the stoichiometry of inhibition. Our results suggest that, while P1 Arg is essential for maximal antithrombin activity in engineered alpha(1)-PI proteins, substitution of the corresponding HCII residues can enhance thrombin specificity.
authors.affiliation: Department of Pathology and Molecular Medicine, McMaster University, HSC-4N66, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada.
authors.forename: Marc L
authors.initials: ML
authors.lastname: Filion
authors.forename: Varsha
authors.initials: V
authors.lastname: Bhakta
authors.forename: Laurie H
authors.initials: LH
authors.lastname: Nguyen
authors.forename: Peter S
authors.initials: PS
authors.lastname: Liaw
authors.forename: William P
authors.initials: WP
authors.lastname: Sheffield
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000737:chemistry*; D045744:Cell Line, Tumor; D015844:Heparin Cofactor II / Q000235:genetics*; D006801:Humans; D007457:Iodine Radioisotopes / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011486:Protein C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017433:Protein Structure, Secondary / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*
chemical_list: D007457:Iodine Radioisotopes; D011486:Protein C; D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; D015844:Heparin Cofactor II; D001120:Arginine; D013917:Thrombin