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

血小板含有许多生物活性分子，包括与炎症反应有关的酶。其中包括12-脂氧合酶（12- lo）酶，它催化膜花生四烯酸生物转化为12-羟基过氧-5,8,10,14-二十碳四烯酸，然后立即还原为12(S)-羟基二十碳四烯酸（12(S)-HETE）。我们实验室产生的数据表明，12-LO表达对于血小板衍生微粒（MPs）的产生是必要的，其调节影响MPs的整体产生。然而，究竟是酶本身还是其产物12(S)-HETE导致了这种表型仍未得到研究，这将是本研究计划的主要焦点。该研究计划的总体目标是研究12-LO表达与血小板衍生MPs产生之间的相关性。具体目的是：1)目的1：在体外研究12-LO表达与血小板源性MPs之间的相关性2)目的2：评估体内12-LO表达与血小板源性MPs之间的相关性******目的1):***血小板在维持人体稳态中发挥重要作用。虽然它们在凝血和防止失血方面的作用得到了更好的认识，但它们也会在细胞外环境中释放MPs。参与血小板活化的一种未被充分认识的酶是12-LO。虽然它的表达及其一些代谢物是凝血酶刺激下血小板聚集所必需的，但它在产生MPs中的作用仍然难以捉摸。由于脂肪酸代谢物在血小板活化和正常止血中起着重要作用，我们假设在体外调节12-LO的表达将对细胞外环境中血小板来源的MPs的释放量产生影响。******目标2):***在体外证明了12-LO表达与血小板衍生产物之间的相关性后，我们将在体内证实这一观察结果。使用表达12-LO（野生型）或血小板型12-LO敲除的小鼠，我们将比较它们产生循环血小板来源MPs的能力。重要的是，这些老鼠在商业上是可以买到的，是可以存活的，目前在我的实验室里。由于我们在体外的初步数据表明，12-LO的表达对血小板来源的MPs的产生很重要，我们相信这一现象将在体内得到证实。******新奇和意义。细胞间通讯是大多数细胞功能的基础。最近的证据表明，细胞来源的MPs在这一过程中起着重要作用。然而，炎症酶在调节细胞源性MPs产生中的作用仍然难以捉摸。该项目旨在建立12-LO（一种被低估的酶）与循环MPs生产之间的联系。总的来说，我的研究计划将提供深入了解血小板衍生MPs生产的基本机制。**

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. **