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 #
5R37HL039888-25
Application #
8056029
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
2011-03-01
Budget End
2012-02-29
Support Year
25
Fiscal Year
2011
Total Cost
$392,500
Indirect Cost
Name
University of Illinois at Chicago
Department
Dentistry
Type
Schools of Dentistry
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
Richard, Benjamin; Swanson, Richard; Izaguirre, Gonzalo et al. (2018) Cooperative Interactions of Three Hotspot Heparin Binding Residues Are Critical for Allosteric Activation of Antithrombin by Heparin. Biochemistry 57:2211-2226
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
Gettins, Peter G W; Olson, Steven T (2016) Inhibitory serpins. New insights into their folding, polymerization, regulation and clearance. Biochem J 473:2273-93
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
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

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