Date of Award

2014

Degree Type

Dissertation

Department

Biological, Geological and Environmental Sciences

First Advisor

McIntyre, Thomas

Subject Headings

Protease inhibitors -- Therapeutic use, Proteins -- Metabolism, Thrombosis -- Treatment, Blood platelets, Ubiquitin, biology, cellular biology, molecular biology, pharmacology

Abstract

Atherothrombotic diseases are responsible for more than 25 of all deaths worldwide. Anti-platelet drugs are the mainstay treatment because of the direct involvement of platelets in the initiation and propagation of thrombosis. However, the currently available anti-platelet drugs, such as antagonists of platelet receptors or of effector systems participating in platelet activation, have their own limitations. A new mode of affecting platelet reactivity may prove to offer unique advantages in a host of clinical settings. Proteasome inhibitors are in clinical use to treat hematologic cancers, but also reduce thrombosis. Whether the proteasome participates in platelet activation or function is opaque since little is known of the proteasome in these terminally differentiated cells. Therefore, I investigated the role of proteasome-mediated proteolysis on platelet function (AIM 1). I find platelets displayed all three primary proteasome protease activities, which MG132 and bortezomib (Velcade®) inhibited. Proteasome substrates are marked by ubiquitin, and platelets contained a functional ubiquitination system that modified the proteome by mono- and poly-ubiquitination. Proteasome inhibition suppressed platelet aggregation by low thrombin concentrations and ristocetin-stimulated agglutination through the GPIb-IX-V complex. Proteasome inhibitor MG132 reduced stimulated spreading and clot retraction. The effects of proteasome inhibitors were not confined to a single receptor as MG132 and bortezomib suppressed thrombin-, ADP-, and LPS-stimulated microparticle shedding. Systemic MG132 strongly suppressed formation of occlusive, platelet-rich thrombi in FeCl3-damaged carotid arteries. Transfusion of platelets treated ex vivo with MG132 and washed prior to transfusion into thrombocytopenic mice also reduced carotid artery thrombosis. The inhibition of the proteasome quells the ultimate step of ubiquitin-mediated protein degradation pathway. Proteasome-mediated degradation is the final common step, however, mult

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