Alteration of the Positively Charged D-helix in Human Antithrombin Alteration of the Positively Charged D- Helix in Human Antithrombin

The serpins antithrombin (AT) and protein C inhibitor (PCI) inhibit thrombin in a reaction that is accelerated by heparin (1000-fold and 40-fold respectively). The heparin binding domains of PCI and AT differ. In AT, the D-helix is a major part of the heparin-binding domain, while the H-helix is the heparin-binding domain in PCI. Compared to antithrombin, PCI is a more potent inhibitor of thrombin bound to thrombomodulin (TM). To explore the role of the antithrombin D-helix in this reaction we changed two positively charged residues in the D-helix (K125 and R129) into neutral and negatively charged amino acids by site-directed mutagenesis. These constructs will then be expressed in baculovirus and assayed for their ability to inhibit thrombin in the presence and absence of heparin and TM.