The serpin family coagulation protease inhibitor, antithrombin and its glycosaminoglycan activators, heparin and heparan sulfate, comprise a key anticoagulant mechanism which is essential for maintaining hemostasis in vertebrates. Intriguingly, antithrombin has been recently shown to possess antiangiogenic and antitumor functions whose physiologic role remains to be determined. To advance our long-term goal of elucidating the molecular mechanisms by which antithrombin carries out its physiologically relevant functions, we propose to pursue three specific aims: 1) We have previously shown that the molecular determinants which mediate the specificity of heparin-activated antithrombin for blood clotting proteases reside both in an exposed reactive center loop of the serpin and in exosite regions outside the loop. We propose to map these exosite regions on antithrombin and on factor Xa and factor IXa and determine the mechanism by which the exosites mediate heparin rate enhancement of antithrombin reactions with the proteases;2) Antithrombin circulates in an unusual low-activity conformation in which the reactive loop is partially buried in beta-sheet A, but is induced by heparin into a high-activity conformation resembling that of other serpins in which the reactive loop is expelled from the sheet. We propose to identify the molecular determinants of this reactive loop conformational switch and delineate the sequence of molecular events in the heparin binding site which trigger the conformational switch;3) Antithrombin expresses a potent antiangiogenic activity when it undergoes conformational changes which occur spontaneously or are induced by cleavage in the reactive loop and which result in the burial of the reactive loop into beta-sheet A. We will determine the structural basis for the antiangiogenic activity of cleaved, latent and a novel prelatent form of antithrombin. The role of specific endothelial cell binding sites in this activity and whether these binding sites involve a protein receptor and/or specific domains of a heparan sulfate proteoglycan will be determined. The proposed studies are expected to provide increased understanding at the molecular level of the function of a key natural anticoagulant and antiangiogenic blood protein. This understanding is expected to facilitate the rational design of a new generation of anticoagulant and antitumor drugs for the treatment and prevention of cardiovascular diseases and cancer.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
Project #
Application #
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Link, Rebecca P
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Illinois at Chicago
Schools of Dentistry
United States
Zip Code
Yang, Likui; Ding, Qiulan; Huang, Xin et al. (2012) Characterization of the heparin-binding site of the protein z-dependent protease inhibitor. Biochemistry 51:4078-85
Duhan, Urmila (2011) Replacement of Phe274 with conserved residue Tyr274 for reactive center loop expulsion in antithrombin. Clin Appl Thromb Hemost 17:273-8
Huang, Xin; Rezaie, Alireza R; Broze Jr, George J et al. (2011) Heparin is a major activator of the anticoagulant serpin, protein Z-dependent protease inhibitor. J Biol Chem 286:8740-51
Olson, Steven T; Richard, Benjamin; Izaguirre, Gonzalo et al. (2010) Molecular mechanisms of antithrombin-heparin regulation of blood clotting proteinases. A paradigm for understanding proteinase regulation by serpin family protein proteinase inhibitors. Biochimie 92:1587-96
Schedin-Weiss, Sophia; Richard, Benjamin; Olson, Steven T (2010) Kinetic evidence that allosteric activation of antithrombin by heparin is mediated by two sequential conformational changes. Arch Biochem Biophys 504:169-76
Richard, Benjamin; Swanson, Richard; Olson, Steven T (2009) The signature 3-O-sulfo group of the anticoagulant heparin sequence is critical for heparin binding to antithrombin but is not required for allosteric activation. J Biol Chem 284:27054-64
Gettins, Peter G W; Olson, Steven T (2009) Activation of antithrombin as a factor IXa and Xa inhibitor involves mitigation of repression rather than positive enhancement. FEBS Lett 583:3397-400
Gettins, Peter G W; Olson, Steven T (2009) Exosite determinants of serpin specificity. J Biol Chem 284:20441-5
Schedin-Weiss, Sophia; Richard, Benjamin; Hjelm, Rebecka et al. (2008) Antiangiogenic forms of antithrombin specifically bind to the anticoagulant heparin sequence. Biochemistry 47:13610-9
Izaguirre, Gonzalo; Olson, Steven T (2006) Residues Tyr253 and Glu255 in strand 3 of beta-sheet C of antithrombin are key determinants of an exosite made accessible by heparin activation to promote rapid inhibition of factors Xa and IXa. J Biol Chem 281:13424-32

Showing the most recent 10 out of 15 publications