Inherited defects in single genes contribute significantly in many? people to a predisposition to development of venous thrombosis, which? in turn is a major contributor to the leading killer in industrialized? countries, cardiovascular disease. One of the most important inherited? defects is in the gene for antithrombin. Antithrombin is the principal? inhibitor of the blood coagulation proteinases factor Xa and thrombin? and is regulated by heparin. The long term goal of this proposal is to? achieve an understanding of the molecular basis for defects in? functioning of variant human antithrombins that result in thrombosis.? This will be accomplished through elucidation first of the mechanisms? of heparin activation and proteinase inhibition in normal antithrombin,? and the ways in which mutations or changes in glycosylation alter either? or both of these processes. The general hypotheses are (i) that the? normal functioning of antithrombin can only be understood in terms of? it being a serpin (member of the serine proteinase inhibitor? superfamily) and of consequently being capable of undergoing necessary? and dramatic conformational changes as part of both heparin binding and? activation, and of proteinase inhibition and (ii) that, as a consequence? of the need for antithrombin to fold as a metastable protein and to? undergo conformational change as part of its function, it is prone to? many more defects than other families of protein proteinase inhibitors? which form simple lock-and-key type complexes. The specific areas are:-? (1) To determine the gross structure of the thrombin-antithrombin? complex. (2) To determine the conformational linkage between heparin? binding and expulsion of residues of the reactive center of beta-sheet? A. (3) To test whether the reactive center loop of antithrombin exists? in an equilibrium between less reactive partially-inserted and more? reactive fully loop expelled forms and that heparin activation results? from a shift in this equilibrium. (4) To determine the role of basic? residues in promoting the conformational change in the heparin binding? site that results in expulsion of P15 and P14 residues of the reactive? center loop. (5) To determine the basis for the dysfunction of? naturally occurring human antithrombin variants. (6) To determine? whether antithrombin is fucosylated in cancer and the functional? consequences thereof.
These specific aims will make extensive use of? recombinant antithrombins expressed in mammalian cells that will be? characterized by a combination of spectroscopic, thermodynamic and? kinetic means. For antithrombins that have been activated by mutation,? x-ray crystallography, through collaboration with Dr. Robin Carrell,? will be used.?

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37HL049234-15
Application #
7166831
Study Section
Special Emphasis Panel (NSS)
Program Officer
Link, Rebecca P
Project Start
1993-01-01
Project End
2008-12-31
Budget Start
2007-01-01
Budget End
2008-12-31
Support Year
15
Fiscal Year
2007
Total Cost
$365,835
Indirect Cost
Name
University of Illinois at Chicago
Department
Biochemistry
Type
Schools of Medicine
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
Gettins, Peter G W; Olson, Steven T (2016) Inhibitory serpins. New insights into their folding, polymerization, regulation and clearance. Biochem J 473:2273-93
Dementiev, Alexey; Swanson, Richard; Roth, Ryan et al. (2013) The allosteric mechanism of activation of antithrombin as an inhibitor of factor IXa and factor Xa: heparin-independent full activation through mutations adjacent to helix D. J Biol Chem 288:33611-9
Huang, Xin; Dementiev, Alexey; Olson, Steven T et al. (2010) Basis for the specificity and activation of the serpin protein Z-dependent proteinase inhibitor (ZPI) as an inhibitor of membrane-associated factor Xa. J Biol Chem 285:20399-409
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
Al-Ayyoubi, Maher; Schwartz, Bradford S; Gettins, Peter G W (2007) Maspin binds to urokinase-type and tissue-type plasminogen activator through exosite-exosite interactions. J Biol Chem 282:19502-9
Dobo, Jozsef; Gettins, Peter G W (2004) alpha1-Proteinase inhibitor forms initial non-covalent and final covalent complexes with elastase analogously to other serpin-proteinase pairs, suggesting a common mechanism of inhibition. J Biol Chem 279:9264-9
Backovic, Marija; Gettins, Peter G W (2002) Insight into residues critical for antithrombin function from analysis of an expanded database of sequences that includes frog, turtle, and ostrich antithrombins. J Proteome Res 1:367-73