Serpin family protein protease inhibitors function as key anticoagulant regulators of blood coagulation proteases. Two serpins, antithrombin 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 antithrombin and a prothrombotic phenotype when mice are bred on a factor V Leiden background in the case of ZPI. Our proposed studies seek to build on our prior studies to advance molecular understanding of the cofactor-dependent regulation of blood coagulation proteases by these serpins. With respect to antithrombin, 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 mitigation of repulsive interactions with factor Xa and factor IXa within an antithrombin exosite rather than by provision of an attractive exosite. Our proposed continuation studies seek to characterize the molecular determinants in antithrombin of protease binding exosites and of the allosteric communication network involved in transmitting activating conformational changes from the heparin binding site to the protease binding site. With respect to ZPI, our studies seek to elucidate the physiologic complexes of factor Xa that are targeted by ZPI and its cofactor, protein Z, establish the role of the unique N-terminal tail of ZPI in anticoagulant regulation of factor Xa and discover small molecules that disrupt the ZPI-protein Z complex and downregulate the ZPI anticoagulant mechanism as a potential novel therapy for restoring hemostasis in hemophilia bleeding disorders. Gain of function studies in which the serpin scaffold of ?1-protease inhibitor is used to graft the molecular determinants of the protein Z binding site and protease binding sites onto ZPI will provide a stringent test of the minimal determinants of cofactor and protease recognition by this serpin.

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 suggest novel approaches for treating thrombotic or bleeding disorders.

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 #
5R37HL039888-32
Application #
9438409
Study Section
Special Emphasis Panel (NSS)
Program Officer
Warren, Ronald Q
Project Start
2015-03-01
Project End
2020-02-28
Budget Start
2018-03-01
Budget End
2019-02-28
Support Year
32
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Type
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
Águila, Sonia; Izaguirre, Gonzalo; Martínez-Martínez, Irene et al. (2017) Disease-causing mutations in the serpin antithrombin reveal a key domain critical for inhibiting protease activities. J Biol Chem 292:16513-16520
Huang, Xin; Liu, Baoxin; Wei, Yidong et al. (2017) Lipid oxidation inactivates the anticoagulant function of protein Z-dependent protease inhibitor (ZPI). J Biol Chem 292:14625-14635
Liu, Baoxin; Li, Yong; Luo, Jiachen et al. (2016) Low protein Z plasma level is a risk factor for acute myocardial infarction in coronary atherosclerosis disease patients. Thromb Res 148:25-31
Gettins, Peter G W; Olson, Steven T (2016) Inhibitory serpins. New insights into their folding, polymerization, regulation and clearance. Biochem J 473:2273-93
Huang, Xin; Zhou, Jian; Zhou, Aiwu et al. (2015) Thermodynamic and kinetic characterization of the protein Z-dependent protease inhibitor (ZPI)-protein Z interaction reveals an unexpected role for ZPI Lys-239. J Biol Chem 290:9906-18
Hou, Lifei; Cooley, Jessica; Swanson, Richard et al. (2015) The protease cathepsin L regulates Th17 cell differentiation. J Autoimmun 65:56-63
Roth, Ryan; Swanson, Richard; Izaguirre, Gonzalo et al. (2015) Saturation Mutagenesis of the Antithrombin Reactive Center Loop P14 Residue Supports a Three-step Mechanism of Heparin Allosteric Activation Involving Intermediate and Fully Activated States. J Biol Chem 290:28020-36
Izaguirre, Gonzalo; Aguila, Sonia; Qi, Lixin et al. (2014) Conformational activation of antithrombin by heparin involves an altered exosite interaction with protease. J Biol Chem 289:34049-64
Liu, Yang; Kretz, Colin A; Maeder, Morgan L et al. (2014) Targeted mutagenesis of zebrafish antithrombin III triggers disseminated intravascular coagulation and thrombosis, revealing insight into function. Blood 124:142-50
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

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