The purpose of the proposed studies is to determine how PAR1 and PAR4 interact to mediate thrombin signaling in platelets. Specifically, the project will identify the PAR1-PAR4 interaction interface and determine how the anionic region on PAR4 exodomain contributes to platelet activation in vivo in the presence of PAR1. The long-term goal is to identify potential targets for anti-platelet therapies that do not pose a risk for bleeding by understanding how PAR1 and PAR4 interact with one another to mediate thrombin signaling for platelet activation.
Specific Aim 1 will characterize a blocking antibody to PAR4's anionic region and determine the role of this region in platelet activation in vivo. The proposed studies will first test the hypothesis that the anionic region on PAR4 is a potential target for anti-platelet therapy. An antibody to PAR4s anionic region, CAN12, blocks human and mouse platelet activation. Since CAN12 blocks 1-thrombin-induced human platelet aggregation, studies will determine its mechanism by examining influence of CAN12 on PAR1 activation when PAR1 is co- expressed with PAR4. In addition, studies will test the hypothesis that the anionic region of PAR4 is critical for PAR4 activation in vivo using transgenic animals expressing mouse PAR4 with mutations in the anionic cluster (mPAR4-AAA), mouse PAR1 or both under the control of the GPIb1 promoter. The transgenic animals will be compared in ex vivo platelet function assays, thrombosis assays and tail bleeding times.
Specific Aim 2 will determine the importance of PAR1 and PAR4 interaction on the surface of cells for efficient activation by thrombin. We will determine the regions on PAR1 and PAR4 required for homodimer and heterodimer formation using Bioluminescence Resonance Energy Transfer (BRET) and cysteine crosslinking. The cysteine crosslinking studies will use an inducible system to examine homodimers and heterodimers over a wide range of expression levels to ensure that interactions are occurring in a physiological range and are not an artifact of overexpression. The BRET and crosslinking studies will be verified with bimolecular fluorescence complementation (BiFC) studies. Finally, we will test the hypothesis that PAR1 enhances PAR4 activation by physically interacting. The proposed studies will examine the interactions between PAR1 and PAR4 on the molecular level to determine how these receptors communicate with one another to mediate thrombin signaling in platelets. These studies have the potential to identify general mechanisms of GPCR interactions to mediate a range of signals that may be transferred to other GPCRs found on the platelet.

Public Health Relevance

A major cause of heart attacks and strokes is the development of a platelet rich thrombus in a blood vessel. Thrombin is the most potent platelet activator by binding and cleaving protease activated receptors. The overall goal of this proposal is to identify the contact sites between the thrombin-protease activated receptor complexes which will provide insights for drug design for antiplatelet therapies that do not increase bleeding.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL098217-04
Application #
8478172
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Kindzelski, Andrei L
Project Start
2010-08-15
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
4
Fiscal Year
2013
Total Cost
$258,946
Indirect Cost
$94,012
Name
Case Western Reserve University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Arachiche, Amal; de la Fuente, MarĂ­a; Nieman, Marvin T (2014) Platelet specific promoters are insufficient to express protease activated receptor 1 (PAR1) transgene in mouse platelets. PLoS One 9:e97724
Mumaw, M M; de la Fuente, M; Noble, D N et al. (2014) Targeting the anionic region of human protease-activated receptor 4 inhibits platelet aggregation and thrombosis without interfering with hemostasis. J Thromb Haemost 12:1331-41
Arachiche, Amal; Mumaw, Michele M; de la Fuente, Maria et al. (2013) Protease-activated receptor 1 (PAR1) and PAR4 heterodimers are required for PAR1-enhanced cleavage of PAR4 by *-thrombin. J Biol Chem 288:32553-62
Arachiche, Amal; de la Fuente, Maria; Nieman, Marvin T (2013) Calcium mobilization and protein kinase C activation downstream of protease activated receptor 4 (PAR4) is negatively regulated by PAR3 in mouse platelets. PLoS One 8:e55740
de la Fuente, Maria; Noble, Daniel N; Verma, Sheetal et al. (2012) Mapping human protease-activated receptor 4 (PAR4) homodimer interface to transmembrane helix 4. J Biol Chem 287:10414-23
Adams, Gregory N; LaRusch, Gretchen A; Stavrou, Evi et al. (2011) Murine prolylcarboxypeptidase depletion induces vascular dysfunction with hypertension and faster arterial thrombosis. Blood 117:3929-37