Abstract

Thromboembolic conditions are the leading cause of disability and death in the United States. Platelets play an important role in the pathophysiology of thrombus formation by aiding in the formation and stabilization of the clot. Central to platelet activation is cytoplasmic calcium, which increases after platelet activation through ITAM (Immunoreceptor tyrosine-based activation motif) or GPCR (G-protein-coupled receptor) receptors. Platelet mitochondria rely on cytoplasmic calcium for mitochondrial calcium influx, which is critical for ATP formation and regulation of mitochondrial reactive oxygen species. In other cells, mitochondrial calcium entry is regulated by the multimeric protein complex, mitochondrial calcium uniporter (MCU). Deletion of MCU in other cells leads to various cellular function alterations that are cell-type dependent. In platelets, the role of MCU has not been extensively studied. Thus, determining if MCU exerts a mechanistic role in platelet function is critical for our understanding of platelet activation and thrombosis. Our study aimed to investigate the role of MCU has in platelet activation and thrombosis. Using platelet specific MCU knockout mouse on a C57BL/6 background (MCUplt-/-) as well as wildtype-littermate (MCUplt+/+) controls we demonstrated the MCU is crucial for platelet activation through the immunoreceptor tyrosine activation motif (ITAM) and that this has significant impact in-vivo thrombosis. Mice that lacked platelet MCU showed a reduction in arterial thrombosis (blood clot formation) and brain injury from ischemic stroke. To dissect the molecular mechanism by which MCU impacted thrombus formation we activated MCU KO platelets with various platelet agonists and measured the platelet response using a variety of in vitro and ex vivo techniques. The lack of MCU only affected the platelets response to ITAM receptor activation, and no other receptor, suggesting that the mitochondrial calcium entry mediated by the MCU pathway is selectively involved in ITAM specific signaling. Mechanistically we demonstrated that the MCU specific mechanism of action is through the generation of mitochondrial reactive oxygen species (ROS). ROS aids in the downstream amplification of ITAM signaling that is necessary for platelet activation. Lack of MCU decreases ROS levels leading to decrease signaling and ultimately reduced platelet activation. Interestingly similar effects were observed when human platelets were treated with MCU inhibitors, confirming the relevance of these findings to human physiology. Our results shed light on a new mechanistic pathway for platelet function and could be a target for developing new anti-platelet therapies.

Committee Chair

Robert Campbell

Committee Members

Frederik Denorme; Jorge Di Paola; Laura Schuettpelz; Regina Clemens

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Immunology)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

12-12-2025

Language

English (en)

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