Risk of arterial thrombosis, including myocardial infarction (MI) and stroke, is increased in the setting of systemic disease states associated with chronic inflammation, including cancer, diabetes, atherosclerosis and obesity. Inappropriate platelet activation is a driving mechanism of thrombosis in these settings and recent studies suggest that mechanisms of platelet activation in diseased states may be different from those in normal healthy conditions. Dissecting these novel mechanisms is the central goal of this multi-PI proposal. Recent published and preliminary studies showed that endogenous danger signals well known to be generated during diseased states, including oxidized low density lipoprotein, advanced glycated proteins, cell-derived microparticles, and S100A peptides all interact with a specific platelet receptor, CD36, to initiate intracellular signals that promote platelet activation and thrombosis. Furthemore platelets were found to express the MAP kinase ERK5, a known sensor of reactive oxygen species (ROS). Platelet ERK5 was then shown to act as a redox switch responsive to extracellular ROS under ischemic conditions, promoting platelet activation and enhancing myocardial damage during MI; and ERK5 was found to be activated downstream of CD36 in response to oxLDL-mediated ROS generation. Platelet specific deletion of ERK5 ameliorated platelet activation and the pro-thrombotic state associated with hyperlipidemic oxidant stress. We thus hypothesize that ERK5 serves as a central ?node? in pathologic platelet activation, responding to receptor-mediated intracellular signals triggered by CD36 and non- receptor mediated extracellular signals (ROS) mediated by tissue ischemia, through both its signaling and protein regulation activities.
Specific aim 1 will test the hypothesis that specific ROS generated by CD36 signaling maintains platelets in a pro-activated state via activation of ERK5. Human In vitro and mouse in vivo studies will use genetic models, diet-induced disease models, and highly specific CD36 ligands to identify critical cell membrane partners necessary for CD36-mediated ERK5 activation, as well as downstream effectors of ROS and ERK5 in platelets; and to determine how ERK5 signaling integrates with ?classic? pathways of platelet activation to promote thrombosis.
Aim 2 will test the hypothesis that ERK5 regulates platelet protein expression by modulating platelet protein translation and/or protein ubiquitination. In vitro and in vivo models will be used to determine whether changes in platelet protein expression in response to ROS are dependent on protein synthesis, degradation, or both.
Aim 3 will determine mechanisms by which platelet ERK5 activation in the setting of tissue ischemia and extracellular ROS increases tissue and organ dysfunction. Genetic and pharmacologic approaches and in vivo models of MI and oxidant stress will be used. By understanding mechanisms of platelet ERK5 activation and downstream pathways these studies will provide insights into `dysregulated' platelet function in pathologic conditions and ischemic environments that may lead to new therapeutic targets and better understanding of why current therapies based on platelet function in healthy conditions are inadequate.

Public Health Relevance

Inappropriate activation of blood platelets, the main cellular initiator of thrombosis (blood clots), is a major contributor to heart attacks and strokes; and activated platelets also to promote heart damage after a heart attack. This project seeks to identify and characterize the mechanisms by which blood and tissue abnormalities generated after heart attacks, or during chronic disease states, such as diabetes, atherosclerosis, and cancer, alter platelet function by activating a specific intracellular signaling protein known as ERK5. Identification of new pathways by which platelets respond to environmental stress offers new sites of therapeutic interventions.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Ochocinska, Margaret J
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Medical College of Wisconsin
Internal Medicine/Medicine
Schools of Medicine
United States
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