The activity of coagulation proteases is regulated primarily by the serine protease inhibitors (serpins) in plasma. Antithrombin (AT) is a major serpin that regulates the activity of proteases of both intrinsic and extrinsic pathways. AT, however, is a weak inhibitor of coagulation proteases unless it is activated by heparin- like glycosaminoglycans that line the microvasculature. This is the basis for the extensive use of heparin for prophylaxis and treatment of venous thrombosis. Heparin activates AT by binding to a basic exosite on the serpin and inducing a conformational change on the reactive center loop, thereby facilitating the optimal recognition of AT by the coagulation proteases. Heparin can also bind to the basic exosite of a coagulation protease to hold AT and the protease in one complex, thereby facilitating the recognition by a bridging mechanism. AT also exhibits anti-inflammatory activity, however, heparin eliminates this activity of the serpin. Protein Z (PZ)-dependent protease inhibitor (ZPI) is another serpin which, unlike AT, has a narrower target specificity, thus being capable of inhibiting only factors IXa (fIXa), Xa (fXa) and XIa (fXIa). While the ZPI inhibition of both fIXa and fXIa is PZ-independent, inhibition of fXa requires PZ as the cofactor. Unlike the relatively well-studied mechanism of the cofactor function of heparin, the mechanism by which PZ functions as a cofactor to dramatically promote the ZPI inhibition of fXa has not been investigated. The overall objective of this proposal is to understand how these two serpins regulate the catalytic activities of coagulation proteases in the absence and presence of their respective cofactors. Thus, we propose to prepare several AT, ZPI, PZ and coagulation protease mutants to investigate 1) the mechanism by which heparin promotes the AT inactivation of coagulation proteases;2) the mechanism of anti-inflammatory activities of wild-type AT and an AT mutant that can inhibit fXa but not other coagulation proteases using cellular models, 3) the mechanism by which ZPI specifically inhibits its target proteases;and 4) the mechanism by which PZ functions as a cofactor to promote the inactivation of fXa by ZPI. These studies will utilize enzyme kinetics, fluorescence spectroscopy, plasmon resonance, and competitive binding methods to provide critical information about kinetic and thermodynamic constants that define the biological properties and the mechanism of interaction of the two serpins with their target molecules. These studies can lead to design and development of specific therapeutic drugs that may potentially be useful for treatment of thrombotic and inflammatory disorders. The proposed studies in this application will utilize enzyme kinetics, fluorescence spectroscopy, plasmon resonance, and competitive binding methods to provide critical information about the kinetic and thermodynamic constants that define the specific interaction of plasma inhibitors with their target clotting enzymes. Understanding the mechanism by which the natural plasma inhibitors regulate the activity of the clotting enzymes can lead to design and development of specific therapeutic drugs and inhibitors that may potentially be useful for treatment of abnormalities (thrombosis and hemorrhage) in blood.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL062565-13
Application #
8008762
Study Section
Special Emphasis Panel (ZRG1-HEME-C (02))
Program Officer
Link, Rebecca P
Project Start
1999-04-01
Project End
2012-06-30
Budget Start
2011-01-01
Budget End
2012-06-30
Support Year
13
Fiscal Year
2011
Total Cost
$330,750
Indirect Cost
Name
Saint Louis University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
050220722
City
Saint Louis
State
MO
Country
United States
Zip Code
63103
Roy, Ram Vinod; Ardeshirylajimi, Abdolreza; Dinarvand, Peyman et al. (2016) Occupancy of human EPCR by protein C induces β-arrestin-2 biased PAR1 signaling by both APC and thrombin. Blood :
Ding, Qiulan; Yang, Likui; Zhao, Xiaoqing et al. (2016) Paradoxical bleeding and thrombotic episodes of dysprothrombinaemia due to a homozygous Arg382His mutation. Thromb Haemost :
Hassanian, S M; Ardeshirylajimi, A; Dinarvand, P et al. (2016) Inorganic polyphosphate promotes cyclin D1 synthesis through activation of mTOR/Wnt/β-catenin signaling in endothelial cells. J Thromb Haemost 14:2261-2273
Hassanian, S M; Dinarvand, P; Smith, S A et al. (2015) Inorganic polyphosphate elicits pro-inflammatory responses through activation of the mammalian target of rapamycin complexes 1 and 2 in vascular endothelial cells. J Thromb Haemost 13:860-71
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
Lu, Qiuya; Yang, Likui; Manithody, Chandrashekhara et al. (2015) Expression and Characterization of Gly-317 Variants of Factor IX Causing Variable Bleeding in Hemophilia B Patients. Biochemistry 54:3814-21
Dinarvand, Peyman; Hassanian, Seyed Mahdi; Weiler, Hartmut et al. (2015) Intraperitoneal administration of activated protein C prevents postsurgical adhesion band formation. Blood 125:1339-48
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
Rezaie, Alireza R (2014) Protease-activated receptor signalling by coagulation proteases in endothelial cells. Thromb Haemost 112:876-82

Showing the most recent 10 out of 95 publications