The proteolytic activity of coagulation proteases is regulated by the serine protease inhibitors (serpins) in plasma. Anti-thrombin (AT) is a major serpin that regulates the proteolytic activity of coagulation proteases of both intrinsic and extrinsic pathways. AT is a relatively weak inhibitor of coagulation proteases unless it binds to the heparin-like glycosaminoglycans lining the microvasculature or therapeutically administered for prophylaxis and treatment of venous thrombosis. In addition to its anticoagulant function through the direct inhibition of procoagulant coagulation proteases, AT also elicits potent anti-inflammatory activities when it binds to vessel wall glycosaminoglycans, thereby eliciting intracellular signaling responses in vascular endothelial cells. Protein Z (PZ)-dependent protease inhibitor (ZPI) is another serpin which, unlike AT, exhibits a narrower protease specificity, thus capable of inhibiting only factors Xa (fXa) and XIa (fXIa). While the ZPI inhibiton of fXIa is independent of a protein cofactor, its inhibition of fXa requires PZ as the cofactor. Recent results have indicated that heparin can bind to an unknown site on ZPI to promote its reactivity with both fXa and fXIa. Unlike the relatively well-studied mechanism of the cofactor function of heparin in enhancing the AT inhibition of coagulation proteases, less is known about mechanisms through which PZ and heparin function as cofactors to accelerate the ZPI inhibition of its target proteases. The overall objective of this project is to understand how these two serpins regulate the catalytic activities of coagulation proteases in the absence and presence of their respective cofactors and how AT elicits protective intracellular signaling responses. We propose to prepare several AT, ZPI, PZ and coagulation protease mutants 1) to investigate the mechanism by which AT elicits anti-inflammatory signaling activities in response to proinflammatory cytokines using cellular models;2) to investigate the anti-inflammatory and cardioprotective properties of AT derivatives having altered anticoagulant and signaling properties in a mouse left anterior descending coronary artery (LAD) ischemia/reperfusion injury model;3) to investigate the mechanism by which ZPI interacts with its target proteases;and 4) to investigate the mechanism by which PZ and heparin function as cofactors to promote the inactivation of fXa and fXIa by ZPI. These studies will utilize cellular and animal models as well as basic biochemistry, kinetics and molecular biology methods to provide critical information as to how AT elicits protective intracellular signaling responses and how ZPI interacts with protein and polysaccharide cofactors to inhibit its target proteases.

Public Health Relevance

The proposed studies of this application will provide new insight into mechanisms through which the two plasma inhibitors, anti-thrombin and protein Z-dependent protease inhibitor, regulate the anticoagulant and anti-inflammatory pathways. The use of anti-thrombin as an anti-inflammatory molecule is associated with increased risk of bleeding. The studies of this application can lead to design and development of specific anti-thrombin molecules with improved therapeutic profiles which may be useful for treatment of thrombotic, inflammatory and cardiovascular disorders.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL062565-14A1
Application #
8372327
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Link, Rebecca P
Project Start
1999-04-01
Project End
2016-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
14
Fiscal Year
2012
Total Cost
$389,111
Indirect Cost
$112,194
Name
Saint Louis University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
050220722
City
Saint Louis
State
MO
Country
United States
Zip Code
63103
Ma, Yina; Wang, Jinli; Gao, Junjie et al. (2015) Antithrombin up-regulates AMP-activated protein kinase signalling during myocardial ischaemia/reperfusion injury. Thromb Haemost 113:338-49
Hassanian, Seyed Mahdi; Dinarvand, Peyman; Rezaie, Alireza R (2014) Adenosine regulates the proinflammatory signaling function of thrombin in endothelial cells. J Cell Physiol 229:1292-300
Lu, Qiuya; Yang, Likui; Manithody, Chandrashekhara et al. (2014) Molecular basis of the clotting defect in a bleeding patient missing the Asp-185 codon in the factor X gene. Thromb Res 134:1103-9
Qureshi, Shabir H; Lu, Qiuya; Manithody, Chandrashekhara et al. (2014) Characterization of the protein Z-dependent protease inhibitor interactive-sites of protein Z. Biochim Biophys Acta 1844:1631-7
Rezaie, Alireza R (2014) Protease-activated receptor signalling by coagulation proteases in endothelial cells. Thromb Haemost 112:876-82
Dinarvand, Peyman; Hassanian, Seyed Mahdi; Qureshi, Shabir H et al. (2014) Polyphosphate amplifies proinflammatory responses of nuclear proteins through interaction with receptor for advanced glycation end products and P2Y1 purinergic receptor. Blood 123:935-45
Yang, L; Dinarvand, P; Qureshi, S H et al. (2014) Engineering D-helix of antithrombin in alpha-1-proteinase inhibitor confers antiinflammatory properties on the chimeric serpin. Thromb Haemost 112:164-75
de Oliveira, Andreia da Silva; Yang, Likiu; Echevarria-Lima, Juliana et al. (2014) Thrombomodulin modulates cell migration in human melanoma cell lines. Melanoma Res 24:11-9
Ding, Qiulan; Yang, Likui; Hassanian, Seyed Mahdi et al. (2013) Expression and functional characterisation of natural R147W and K150del variants of protein C in the Chinese population. Thromb Haemost 109:614-24
Wang, J; Wang, Y; Wang, J et al. (2013) Antithrombin is protective against myocardial ischemia and reperfusion injury. J Thromb Haemost 11:1020-8

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