Serpin family protein protease inhibitors function as key regulators of blood coagulation proteases. Two serpins, anti-thrombin and protein Z-dependent protease inhibitor (ZPI), are known to inhibit procoagulant proteases in a manner that is regulated by cofactors and dependent on the functional state of the proteases, but the molecular details of this complex regulatory mechanism are poorly understood. The physiologic importance of these serpins in regulating coagulation proteases is borne out by the observations that knocking out the mouse genes results in embryonic lethality due to a consumptive coagulopathy in the case of anti-thrombin and increased thrombosis when mice are given a thrombotic challenge or are bred on a factor V Leiden background in the case of ZPI. The long-term goal of our studies is to understand how coagulation proteases are regulated by these two critical serpin inhibitors. Our proposed studies seek to build on our prior studies to advance the detailed molecular understanding of this cofactor-dependent regulation. With respect to anti-thrombin, our recent studies have suggested an important revision of the allosteric mechanism of activation of this serpin by heparin in showing that activation is mediated principally by the relief of repulsive interactions with factor Xa and factor IXa and secondarily by the relief of structural constraints of the reactive site that promote reactive site and exosite interactions with the protease. Our proposed studies seek to characterize the molecular determinants of the repulsive interactions, establish how certain mutations can decouple the structural changes accompanying activation to produce an intermediate activated state and to show how an induced-fit mechanism of heparin binding is coupled to allosteric activation. With respect to ZPI, our recent X- ray structure of a complex of ZPI with its cofactor protein, protein Z, has provided new insights into the mechanism by which the cofactor promotes the specific inhibition of membrane-associated factor Xa, despite the unfavorable P1 Tyr of ZPI. Our studies seek to map putative exosites on ZPI for factor Xa and factor XIa, validate the ZPI sites for protein Z binding that have been revealed by the X-ray structure, and assess the role of the unique N-terminal tail of ZPI in the inhibition of membrane-associated factor Xa. For both serpins, gain of function studies in which the serpin scaffold of 11-proteinase inhibitor is used to graft the molecular determinants of exosite interactions in anti-thrombin and of both protein Z and exosite interactions in ZPI will provide a stringent test of the minimal determinants of protease and cofactor recognition by these serpins.)

Public Health Relevance

Cardiovascular diseases resulting in heart attack, stroke and other forms of abnormal clotting are the number one cause of death in humans. These diseases reflect an imbalance in the natural procoagulant and anticoagulant mechanisms that are responsible for hemostasis. Proteins of the serpin superfamily act as critical regulators of this dynamic balance but their molecular mechanisms are poorly understood. Our studies seek to advance understanding of these mechanisms and thereby to improve on currently available anticoagulant therapy. Novel anticoagulant drugs which target serpins and upregulate their normal downregulated function could greatly improve upon currently available anticoagulant drugs which lack specificity and are beset by risks of bleeding .

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 #
5R37HL039888-26
Application #
8231364
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Link, Rebecca P
Project Start
1994-01-01
Project End
2015-02-28
Budget Start
2012-03-01
Budget End
2013-02-28
Support Year
26
Fiscal Year
2012
Total Cost
$388,575
Indirect Cost
$141,075
Name
University of Illinois at Chicago
Department
Dentistry
Type
Schools of Dentistry
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
Maddur, Ashoka A; Swanson, Richard; Izaguirre, Gonzalo et al. (2013) Kinetic intermediates en route to the final serpin-protease complex: studies of complexes of *1-protease inhibitor with trypsin. J Biol Chem 288:32020-35
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
Izaguirre, Gonzalo; Qi, Lixin; Lima, Mary et al. (2013) Identification of serpin determinants of specificity and selectivity for furin inhibition through studies of *1PDX (*1-protease inhibitor Portland)-serpin B8 and furin active-site loop chimeras. J Biol Chem 288:21802-14
Olson, Steven T; Swanson, Richard; Petitou, Maurice (2012) Specificity and selectivity profile of EP217609: a new neutralizable dual-action anticoagulant that targets thrombin and factor Xa. Blood 119:2187-95
Olson, Steven T; Gettins, Peter G W (2011) Regulation of proteases by protein inhibitors of the serpin superfamily. Prog Mol Biol Transl Sci 99:185-240
Chou, Feng-Pai; Xu, Han; Lee, Ming-Shyue et al. (2011) Matriptase is inhibited by extravascular antithrombin in epithelial cells but not in most carcinoma cells. Am J Physiol Cell Physiol 301:C1093-103
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
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) Exosite determinants of serpin specificity. J Biol Chem 284:20441-5
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

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