The expression of the 12-lipoxygenase enzyme as a modulator of platelet-derived microvesicle production

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Platelets contain a number of bioactive molecules, including enzymes implicated in the inflammatory response. Amongst those is the 12-lipoxygenase (12-LO) enzyme that catalyzes the bioconversion of membrane arachidonic acid into 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid, which is then immediately reduced into 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). Data generated by our laboratory has shown that 12-LO expression is necessary for the production of platelet-derived microparticles (MPs) and that its modulation affects the overall productions of MPs. However, whether it is the enzyme itself or its product 12(S)-HETE that is responsible for the phenotype remains uninvestigated and will be the main focus of this research program. The overall aim of the proposed research program is to investigate the correlation between 12-LO expression and the production of platelet-derived MPs. The specific aims are: 1) Aim 1: Investigate, in vitro, the correlation between 12-LO expression and platelet-derived MPs 2) Aim 2: Evaluate, in vivo, the correlation between 12-LO expression and platelet-derived MPs Aim 1): Platelets play an important role in maintaining the homeostasis of the human body. While they are better appreciated for their role in coagulation and preventing blood loss, they also shed MPs in the extracellular milieu. An underappreciated enzyme that participates in platelet activation is the 12-LO. While its expression, and some of its metabolites, are necessary for platelet aggregation upon thrombin stimulation, its role in producing MPs remains elusive. Since fatty acid metabolites play an important role in platelet activation and normal hemostasis, we hypothesize that modulating the expression of 12-LO in vitro will have a consequence on the amount of platelet-derived MPs released in the extracellular milieu. Aim 2): Having demonstrated a correlation between 12-LO expression and platelet-derived production in vitro, we will confirm this observation in vivo. Using mice that express the 12-LO (wild-type) or platelet-type 12-LO knock-out, we will compare their ability to generate circulating platelet-derived MPs. Of importance, these mice are commercially available, viable and currently in my laboratory. Since our preliminary data in vitro have shown that 12-LO expression is important for the production of platelet-derived MPs, we believe that this phenomenon will be confirmed in vivo. Novelty and significance. Intercellular communication is at the basis of most cellular functions. Recent evidence points toward an important role of cell-derived MPs in this process. However, the implication of inflammatory enzymes in regulating the production of cell-derived MPs remains elusive. This program aims to establish a link between the 12-LO, an underappreciated enzyme, and the production of circulating MPs. Overall, my research program will provide insight into the fundamental mechanism of platelet-derived MPs production.