sepharose and Antithrombin-III-Deficiency

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.

Topics: Alanine; Anticoagulants; Antithrombin III; Antithrombin III Deficiency; Antithrombins; Carboxypeptidases; Carboxypeptidases A; Dermatan Sulfate; Factor Xa Inhibitors; Glycosaminoglycans; Heparin; Heparin Cofactor II; Hirudins; Humans; Kinetics; Lysine; Mutagenesis, Site-Directed; Peptide Fragments; Protein Binding; Recombinant Proteins; Sepharose; Serine Proteinase Inhibitors; Thrombin

A method for large-scale production of a pasteurized antithrombin III (AT III) concentrate for therapeutic use has been adapted from published methods. It includes the following steps: (1) batchwise adsorption onto heparin-Sepharose from plasma depleted of cryoprecipitate and prothrombin complex; (2) chromatographic elution at high salt concentration; (3) pasteurization for 10 h at 60 degrees C in the presence of added citrate ion; (4) desalting on Sephadex G-25, and (5) sterile filtration and freeze-drying. Seven batches prepared in this manner gave a mean yield of 269 U AT III/kg plasma. The product passed all the usual animal safety and pyrogenicity tests and has been used successfully in several courses of treatment of congenital deficiencies.

Topics: Adsorption; Antithrombin III; Antithrombin III Deficiency; Hot Temperature; Humans; Sepharose; Sterilization