The activation of platelets is the final common pathway for most ischemic strokes. Acute thrombus formation in the setting of vascular dysfunction and inflammation initiates a cascade of events that culminates in necrotic death of neurons and injury to their supportive structures in the neurovascular unit. However, the signaling pathways that link these events are not well understood. The Rho/Rho-associated coiled-coil forming kinases (ROCK1 and ROCK2) are important regulators of the actin cytoskeleton. Because changes in the actin cytoskeleton underlie platelet aggregation, vascular contractility, and inflammatory cell recruitment, it is likely that the Rho/ROCK pathway will play a central role in ischemic strokes. Accordingly, the overall aim of this proposal is to investigate the role of ROCK isoforms in platelets and to determine how they might contribute to thromboembolic strokes. To achieve this goal, we will target ROCK deletion in platelets using knockout (KO), bone marrow transplantation (BMT), and Cre/loxP technology and will investigate the subsequent loss-of-function of platelet ROCKs in thrombus formation, clot propagation, and focal cerebral ischemia. The results of these proposed studies will hopefully lead to the development of isoform-specific ROCK inhibitors as novel therapies for patients with ischemic strokes. Specific aim 1 will determine the mechanisms by which ROCKs contribute to platelet function and arterial thrombosis. We will test the hypothesis that ROCKs play differential roles in regulating the assembly of the platelet cytoskeleton and mediating platelet function. To determine and compare the effect of ROCK1 and ROCK2 on thrombosis, platelets derived from ROCK1-/- and ROCK2-/- bone marrow transplanted (BMT) mice will be studied for aggregation, adhesion, hetero- and homo-typic aggregate formation, and by direct visualization with electron microscopy after activation with various platelet agonists. Furthermore, we will investigate the potential downstream signaling pathways of ROCKs that regulates platelet actin cytoskeleton and function. Specific aim 2 will determine the pathophysiological consequences of platelet ROCK deletion on thrombus formation and propagation in a clot embolic model of stroke. We will test the hypothesis that ROCKs are critically important for platelet function in vivo, and that platelet deletion of ROCKs confers stroke protection in a mouse model of thrombosis-mediated focal cerebral ischemia. To do this, we will develop platelet-specific ROCK KO mice (ROCK1Plt-/- and ROCK2Plt-/- mice) and utilize (1) a carotid artery injury model for measurement of arterial occlusive thrombosis, (2) an agonist-dependent platelet consumptive model to study micro thrombi formation, and (3) a clot-embolic stroke model using preformed thrombi to determine the ability of a clot to form and adhere to the vasculature, mediate vascular occlusion, and cause cerebral ischemia and infarction.

R01NS070001

2010-02-01

2017-01-31