Function and regulation of constitutive protein translation in platelets

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Project Summary The goals of this proposal are: 1) to elucidate the molecular signaling of protein translation initiation in circulating human platelets and characterize the plasma growth factor- or hormone-driven pathways, map the regenerating human platelet proteome, and determine the roles of constitutive translation in platelet functional reactivity; 2) to determine the roles of ARGONAUTE2 (AGO2, Ago2 in mice) in platelet mRNA, microRNA (miRNA) and protein expression, platelet reactivity, and the specific roles of platelet miRNA-mediated suppression of translation in hemostasis and thrombotic potential. The studies in this proposal will elucidate essential concepts of the molecular physiology of platelet function. In addition, these studies will have profound implications for two areas of widespread clinical significance: alterations in growth factor/hormone homeostasis, and current use and development of pharmacological inhibitors of translation in disease treatment. Surprisingly little attention has been paid to roles of translation in so-called “resting” platelets in circulation, following the overall presumption that translation is of limited importance. In prior studies of “resting” platelet translation, ex vivo platelets were removed from their native plasma environment, and translation steadily came to a halt. We interpret these prior studies to suggest instead that platelets undergo translation constitutively in the presence of plasma, e.g., in circulation, and that this expenditure of molecular resources and cellular energetics is critical for platelet protein homeostasis and functionality. Our strong preliminary data support this hypothesis and we propose a series of innovative approaches to investigate this poorly understood aspect of platelet biology and its functional outcomes. We have shown for the first time that platelets in circulation undergo constitutive translation of a robust proteome due to translation initiation signaling driven by plasma-borne growth factors and hormones, that this process is modulated by platelet miRNAs and AGO2, and that constitutive miRNA-modulated translation in platelets regulates platelet function. We will pursue an array of innovative approaches to test the following hypotheses: Aim 1- 1) “resting” platelets in circulation are translationally active due to ongoing signaling initiated by plasma growth factors and hormones; 2) constitutive translation driven by plasma growth factors maintains platelet protein homeostasis necessary for hemostatic function; Aim 2 - 1) abundant miRNAs and Ago2-mediated RNA inhibition in platelets modulate constitutive translation driven by plasma growth factors and hormones; 2) Ago2/miRNA-mediated suppression maintains platelets in a reactive state necessary to support hemostasis while limiting thrombosis. Together, the outcomes of these studies will significantly expand our understanding of control of platelet function, hemostasis and thrombosis, as well as implications for platelet reactivity and function in growth factor or hormone imbalance in disease, and potential impacts of clinical translation inhibitors on these processes.