Coagulation proteases, in addition to their role in the regulation of blood coagulation, can modulate intracellular signaling events by activating a subfamily of G-protein coupled receptors named protease-activated receptors (PARs) expressed on the cell surface of various organs. In vitro studies have indicated that while the anticoagulant protease, activated protein C (APC), in complex with endothelial protein C receptor (EPCR) elicits a barrier protective response via activation of PAR-1 in vascular endothelial cells, thrombin elicits a barrier disruptive response via the activation of the same receptor. APC has been approved by the FDA for treatment of severe sepsis. It has been hypothesized that the activation of PAR-1 by APC on endothelial cells is responsible, at least partially, for its protective properties in preventing organ failure (i.e., lung and heart) which is caused by septic shock in severe sepsis. Thus, it is highly important to understand how the activation of PAR-1 by either APC or thrombin elicits paradoxical protective and disruptive cellular responses, respectively. We recently discovered that the activation of PAR-1 by thrombin can also elicit barrier protective cellular responses if EPCR is occupied by the Gla-domain of protein C. We demonstrated that the unoccupied EPCR together with PAR-1 are both associated with caveolin-1 within lipid-rafts of endothelial cells. Under these conditions, PAR-1 appears to signal via Gq and/or G12/13 subfamily of G-proteins, and thus thrombin activation of PAR-1 leads to disruptive cellular responses. However, we discovered that the occupancy of EPCR by protein C results in dissociation of EPCR from caveolin-1, thereby recruiting PAR-1 to protective pathways by likely coupling it to Gi-protein. Based on our recent preliminary data, we hypothesize that the occupancy of EPCR by protein C can also change the signaling specificity of PAR-2. Thus, it appears that the cell surface occupancy of EPCR determines the signaling specificity of activated PAR-1 and PAR-2 not the proteases that are cleaving them. To understand the PAR-dependent signaling specificity of coagulation proteases, we have prepared several mutant proteases and receptors and set up appropriate in vitro and in vivo models to investigate the following four Specific Aims.
In Aim 1, we will investigate the PAR-1- and PAR- 2-dependent signaling specificity of coagulation proteases under conditions in which endothelial cells have been pre-incubated with vitamin K-dependent coagulation zymogens so that to occupy EPCR or other potential receptors by their natural ligands.
In Aim 2, we will investigate the contribution of the Gla-domain of vitamin Kdependent coagulation proteases to the recognition and cleavage specificity of PARs localized into the lipidrafts.
In Aim 3, we will develop novel APC variants which have improved PAR-1-dependent protective signaling properties, but have reduced anticoagulant activities.
In Aim 4, we will characterize the cardioproperties of the improved APC variants or variants possessing only signaling activity or only anticoagulant activity in a mouse ischemia/reperfusion injury model.

Public Health Relevance

The studies of this application will focus on the mechanism by which coagulation proteases activate a subfamily of G-protein coupled receptors called protease-activated receptors to alter the properties of vascular endothelial cells. The activation of these receptors by coagulation proteases is known to be important for the regulation of a range of (patho) physiological processes including coagulation, inflammation and angiogenesis. Understanding this role of coagulation proteases can lead to development of a new generation of therapeutic drugs for controlling thrombotic and inflammatory disorders, including heart attack and severe sepsis.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL101917-03
Application #
8230684
Study Section
Special Emphasis Panel (ZRG1-VH-G (02))
Program Officer
Link, Rebecca P
Project Start
2010-04-01
Project End
2014-02-28
Budget Start
2012-03-01
Budget End
2013-02-28
Support Year
3
Fiscal Year
2012
Total Cost
$371,521
Indirect Cost
$89,677
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

Showing the most recent 10 out of 24 publications