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- CHis6). Thrombin inhibition by rHCII-CHis6 was increased >2-fold at ~5 μg/ml heparin compared with wild-type recombinant HCII (wt-rHCII) at 50-100 μg/ml heparin. Enhanced activity of rHCII-CHis6 was reversed by treatment with carboxypeptidase A. We assessed the role of the HCII acidic domain by constructing amino-terminal deletion mutants (Δ1-52, Δ1-68, and Δ1-75) in wt-rHCII and rHCII-CHis6. Without glycosaminoglycan, unlike wt-rHCII deletion mutants, the rHCII-CHis6 deletion mutants were less active compared with full-length rHCII-CHis6. With glycosaminoglycans, Δ1-68 and Δ1-75 rHCIIs were all less active. We assessed the character of the tag by comparing rHCII-CHis6, rHCII-CAla6, and rHCII-CLys6 to wt-rHCII. Only rHCII-CHis6 had increased activity with heparin, whereas all three mutants have increased heparin binding. We generated a carboxylterminal 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-CHis6 was significantly greater compared with wt-rHCII. Thus, HCII variants with increased function, such as rHCII-CHis6, may offer novel reagents for clinical application.