Fig. 3. Inhibition of thrombin activity. ( Left ) Depiction of normal physiologic inhibition of fluid-phase thrombin by high-molecular-weight (HMW) heparin molecules and the limitations of heparin in inhibiting clot-bound thrombin. The thrombin molecule has three major binding sites:(1) the fibrinogen binding site (Exo I), (2) the fibrinogen catalytic site (Cat Site), and (3) the fibrin binding site (Exo II). After binding of heparin to the antithrombin III (ATIII) molecule via a critical pentasaccharide sequence, a conformational change in the C-terminal portion of the ATIII molecule is induced. Inhibition of the thrombin molecule requires heparin molecules with a critical oligosaccharide chain length of 18 U that serve as a template for the binding of ATIII with thrombin. However, thrombin inhibition via the ATIII–heparin mechanism is limited by availability of the Exo II site, which can also be occupied by fibrin. Although low-molecular-weight (LMW) fractions of heparin induce a conformational change in the C-terminal portion of the ATIII molecule, they cannot serve as a template for ATIII and thrombin because of their short chain length. ( Right ) Depiction of the normal inhibition of clot-bound thrombin by the heparin cofactor II (HCII)—heparin complex and the sites of action of various direct thrombin inhibitors. A minimum chain length of 6 U for the heparin oligosaccacharide is required to activate HCII; however, 20–24-U chain lengths result in a substantially greater thrombin inhibition via HCII. Direct thrombin inhibitors, such as hirudin and hirulog, bind to both the Exo I and the catalytic sites of the thrombin molecule. In contrast, polypeptide aptamers and hirugen bind to the Exo I site, whereas argatroban binds to the fibrinogen catalytic site of thrombin. As modified from Tollefson DM: Insight into the mechanism of action of heparin cofactor II. Thromb Hemostas 1995;74:1209–14. Reprinted with permission.