Platelets are defined by their essential roles in hemostasis and the formation of pathological thrombi. Therefore, the major focus of platelet research is focused on identifying and developing safer and more effective anti- thrombotic therapies. A second goal is to have the ability to predict the effectiveness of specific therapies across populations. Pharmacogenomics may allow us to predict who will respond to or be resistant to therapies. In order for this to be successful, there needs to be a clear relationship between specific polymorphisms and physiological output. Finally, the alleles need to be present at high enough frequency to warrant testing prior to the start of therapy. In the current project w will examine common polymorphisms of a platelet receptor (PAR4) to explain the differences in reactivities and response to an antagonist at the molecular level. We will determine the structural rearrangement of PAR4 upon activation by thrombin using structural mass spectrometry (amide hydrogen deuterium exchange (HDX), histidine HDX, and hydroxyl radical foot-printing) with purified PAR4 and PAR4 on cells and platelets. These studies will determine how the tethered ligand influences the overall conformation of the receptor. Pharmacological studies in human platelet show that the PAR4 sequence variants have dramatically different responses. For example, PAR4-120T is hyper-reactive and PAR4-296V is resistant to signaling. We will determine how the PAR4 variants affect the ligand binding site and the transition to the active conformation at the molecular level. Further, PAR4-296V suppresses the activity of other alleles suggesting that it forms dominant negative homodimers. We will examine the physical interaction between PAR4 variants, PAR1 and P2Y12 to determine how these receptors influence signaling pathways. Finally, we will determine how the PAR4 sequence variants influence platelet reactivity in acute coronary syndrome patients on the current standard of care (aspirin and a P2Y12 antagonist) and if the altered platelet reactivity due to PAR4 variants is exacerbated with the PAR1 antagonist vorapaxar. By analyzing the structural rearrangements of PAR4 following activation by thrombin, these studies will provide the first detailed description of the tethered ligand activation mechanism for the PAR family and have the potential to uncover allosteric sites that can be exploited therapeutically. More specifically, understanding how naturally occurring sequence variants influence PAR4's response may allow us to predict the most appropriate antiplatelet therapy for patients depending on their genotype.

Public Health Relevance

The activation of platelets within a blood vessel is a major cause of heart attacks and strokes. Platelets are activated by molecules that bind to specific receptors on the platelet surface. Our research seeks to understand the function of these receptors on the surface by examining their molecular structure. The goal of our research is to identify new therapeutic targets that may lead to agents that prevent unwanted blood clots without the side effect of bleeding.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL098217-07
Application #
9433676
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Kindzelski, Andrei L
Project Start
2010-08-15
Project End
2021-02-28
Budget Start
2018-03-01
Budget End
2019-02-28
Support Year
7
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Sharma, Ruchika; Waller, Amanda P; Agrawal, Shipra et al. (2017) Thrombin-Induced Podocyte Injury Is Protease-Activated Receptor Dependent. J Am Soc Nephrol 28:2618-2630
Bouck, E G; Zunica, E R; Nieman, M T (2017) Optimizing the presentation of bleeding and thrombosis data: Responding to censored data using Kaplan-Meier curves. Thromb Res 158:154-156
Michael, James V; Wurtzel, Jeremy G T; Mao, Guang Fen et al. (2017) Platelet microparticles infiltrating solid tumors transfer miRNAs that suppress tumor growth. Blood 130:567-580
Li, Wei; Nieman, Marvin; Sen Gupta, Anirban (2016) Ferric Chloride-induced Murine Thrombosis Models. J Vis Exp :
Smith, Thomas H; Coronel, Luisa J; Li, Julia G et al. (2016) Protease-activated Receptor-4 Signaling and Trafficking Is Regulated by the Clathrin Adaptor Protein Complex-2 Independent of ?-Arrestins. J Biol Chem 291:18453-64
Nieman, Marvin T (2016) Protease-activated receptors in hemostasis. Blood 128:169-77
Rowley, Jesse W; Chappaz, Stéphane; Corduan, Aurélie et al. (2016) Dicer1-mediated miRNA processing shapes the mRNA profile and function of murine platelets. Blood 127:1743-51
Mumaw, Michele M; de la Fuente, Maria; Arachiche, Amal et al. (2015) Development and characterization of monoclonal antibodies against Protease Activated Receptor 4 (PAR4). Thromb Res 135:1165-71
Nieman, Marvin T (2015) PARtitioning protease signaling. Blood 125:1853-5
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

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