The plasma protein, antithrombin, and its glycosaminoglycan activator, heparin, act as essential anticoagulant regulators of blood clotting proteinases. Inherited mutations which cause a loss of antithrombin inhibitory function or which block heparin activation of this function are thus associated with an increased risk of thrombosis . Moreover, complete antithrombin deficiency in man is unknown and produces embryonic lethality in mice. The long term goal of this proposal is to provide a complete understanding of the anticoagulant function of antithrombin and heparin at the molecular level and elucidate their contribution to hemostasis. Our hypotheses are: i) antithrombin's specificity for inhibiting multiple procoagulant proteinases with different substrate specificities arises from secondary interaction sites or exosites outside of the main reactive loop region of the inhibitor used for binding proteinases; ii) three basic residues of antithrombin are principally responsible for binding heparin at an allosteric site through an induced-fit mechanism and the action of these residues is coupled to global conformational changes leading to inhibitor activation; iii) limited conformational changes in or near the 325-375 region which are not essential for inhibitor function are responsible for the expression of the anti-angiogenic activity of antithrombin. These hypotheses will be tested by the following specific aims: l) We will elucidate the structural determinants of antithrombin's specificity for different clotting proteinases and assess how these determinants are expressed upon heparin activation; 2) We will determine the relative importance of antithrombin residues responsible for binding heparin and how they cooperate to induce antithrombin into an activated state with high heparin affinity and rapid reactivity with clotting proteinases; and 3) We will characterize the conformational differences between antithrombin forms with and without anti-angiogenic activity and localize the serpin structure which mediates this activity. These studies will utilize site-directed mutagenesis together with thermodynamic and kinetic characterization of variant antithrombins to elucidate the relationship between antithrombin structure and function. These studies are expected to increase understanding of natural molecular defects in antithrombin and to provide a rational basis for the design of novel antithrombotic and antiangiogenic agents through the engineering of modified antithrombins or heparin mimetics.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Hematology Subcommittee 2 (HEM)
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Link, Rebecca P
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University of Illinois at Chicago
Schools of Dentistry
United States
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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

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