All ETDs from UAB

Advisory Committee Chair

Victor M Darley-Usmar

Advisory Committee Members

Scott W Ballinger

John C Chatham

James F George

Aimee L Landar

Document Type

Dissertation

Date of Award

2015

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The principal function of platelets is to regulate hemostasis, and dysfunction of platelet aggregation or activation can lead to either excessive thrombotic or hemorrhagic complications. It has been shown that the ability to generate ATP and exposure to oxidative stress can affect platelet thrombotic potential. While platelet metabolism has been extensively studied, the importance of substrate availability and metabolic plasticity in regulating aggregation under physiological or pathological conditions is not well understood. In this dissertation, we examined the reliance of platelets on different metabolic substrates, the importance of ATP production for aggregation in response to thrombin stimulation, metabolic stress following exposure to lipid peroxidation products, and the metabolic consequences of storage in the blood bank. We found that the platelet metabolic program was malleable and that loss of one substrate could be compensated for another during aggregation. Flexibility in substrate utilization is imperative for platelet function in the wound microenvironment, which is both inflammatory and deprived of nutrients. Inflammatory and oxidative stress environments are capable of generating lipid peroxidation products, such as 4-hydroxy-2-nonenal (4-HNE), which are known to affect platelet aggregation. In these studies we demonstrate that addition of exogenous 4-HNE inhibited aggregation, decreased mitochondrial function, and resulted in a compensatory increase in glycolysis. Identification of proteins modified by alkyne HNE (4-HNE with terminal alkyne), by mass spectrometry, revealed essential targets in the activation cascade, such as proteins involved in regulating metabolism, adhesion, cytoskeletal reorganization, aggregation and vesicular transport, demonstrating the pleiotropic effects of 4-HNE on platelet aggregation. Platelet transfusions are a lifesaving treatment for thrombocytopenia, however, platelet storage results in progressive decline in its quality, including decreased platelet aggregability and metabolism, which is collectively termed the storage lesion. We showed that, compared to freshly isolated platelets, stored platelets have a decrease in mitochondrial function driven by an increase in proton leak. This uncoupling was attenuated by albumin, consistent with a fatty acid driven mechanism of mitochondrial uncoupling. Taken together, these findings delineate the metabolic regulation of platelet aggregation and have important implications for the design of interventions to influence the thrombotic function of platelets.

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