title: Enhancement of heparin cofactor II anticoagulant activity.
abstract: Heparin cofactor II (HCII) is a serpin whose thrombin inhibition activity is accelerated by glycosaminoglycans. We describe the novel properties of a carboxyl-terminal histidine-tagged recombinant HCII (rHCII-CHis(6)). Thrombin inhibition by rHCII-CHis(6) was increased >2-fold at approximately 5 microgram/ml heparin compared with wild-type recombinant HCII (wt-rHCII) at 50-100 microgram/ml heparin. Enhanced activity of rHCII-CHis(6) was reversed by treatment with carboxypeptidase A. We assessed the role of the HCII acidic domain by constructing amino-terminal deletion mutants (Delta1-52, Delta1-68, and Delta1-75) in wt-rHCII and rHCII-CHis(6). Without glycosaminoglycan, unlike wt-rHCII deletion mutants, the rHCII-CHis(6) deletion mutants were less active compared with full-length rHCII-CHis(6). With glycosaminoglycans, Delta1-68 and Delta1-75 rHCIIs were all less active. We assessed the character of the tag by comparing rHCII-CHis(6), rHCII-CAla(6), and rHCII-CLys(6) to wt-rHCII. Only rHCII-CHis(6) had increased activity with heparin, whereas all three mutants have increased heparin binding. We generated a carboxyl-terminal histidine-tagged recombinant antithrombin III to study the tag on another serpin. Interestingly, this mutant antithrombin III had reduced heparin cofactor activity compared with wild-type protein. In a plasma-based assay, the glycosaminoglycan-dependent inhibition of thrombin by rHCII-CHis(6) was significantly greater compared with wt-rHCII. Thus, HCII variants with increased function, such as rHCII-CHis(6), may offer novel reagents for clinical application.
authors.affiliation: Department of Pathology, Center for Thrombosis and Hemostasis, The University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7035, USA.
authors.forename: S J
authors.initials: SJ
authors.lastname: Bauman
authors.forename: F C
authors.initials: FC
authors.lastname: Church
mesh_terms: D000409:Alanine / Q000378:metabolism; D000925:Anticoagulants / Q000494:pharmacology; D000990:Antithrombin III / Q000235:genetics / Q000378:metabolism; D020152:Antithrombin III Deficiency / Q000378:metabolism; D000991:Antithrombins / Q000494:pharmacology; D002268:Carboxypeptidases / Q000494:pharmacology; D043422:Carboxypeptidases A; D003871:Dermatan Sulfate / Q000494:pharmacology; D065427:Factor Xa Inhibitors; D006025:Glycosaminoglycans / Q000378:metabolism / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D015844:Heparin Cofactor II / Q000235:genetics / Q000378:metabolism*; D006629:Hirudins / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000494:pharmacology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012685:Sepharose / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000235:genetics / Q000378:metabolism*; D013917:Thrombin / Q000037:antagonists & inhibitors
chemical_list: D000925:Anticoagulants; D000991:Antithrombins; D065427:Factor Xa Inhibitors; D006025:Glycosaminoglycans; D006629:Hirudins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C062510:hirugen; D003871:Dermatan Sulfate; D015844:Heparin Cofactor II; D000990:Antithrombin III; D006493:Heparin; D012685:Sepharose; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D013917:Thrombin; D008239:Lysine; D000409:Alanine