Platelet adhesion to neutrophil extracellular DNA traps: role in thrombosis

描述（由申请人提供）：中性粒细胞经历细胞死亡途径，导致释放涂有抗菌蛋白的染色质，形成称为中性粒细胞胞外陷阱（NET）的网。NET捕获并杀死微生物。我们的初步结果表明，NET招募和激活血小板。我们有证据表明，细胞外DNA/染色质存在于深静脉血栓形成的病理标本和血栓性疾病患者的血浆中。本申请的中心假设是NET提供支架，其与纤维蛋白一起增加血栓稳定性并促进血栓生长和微粒募集。我们认为，在病理情况下，NET的形成可能会引发血栓形成事件，并有助于条件，如败血症，血栓性微血管病和深静脉血栓形成。此外，我们提出，DNase 1，这使NETs，可以调节血栓形成。我们将在体外和体内血栓形成模型的背景下分析NET。我们将采用具有视频功能的流动室，使用来自基因工程小鼠的细胞。我们建议研究几种血栓形成的小鼠模型，所有这些模型都是在我们实验室中建立的，通过复杂的成像技术可视化体内NET的形成，并研究NET与血栓生长和稳定性的相关性。我们特别热衷于研究NET在新的深静脉血栓形成（DVT）下腔静脉狭窄模型和败血症小鼠模型中的作用。该提案有两个具体目标：1。 研究介导血小板和微粒与NET相互作用的分子机制。 评估NET是否促进纤维蛋白生成。在这个目标中，我们建议在体外研究血小板粘附到NET的分子机制，并解决NET是否促进促凝微粒（MP）和纤维蛋白沉积的招聘。2.研究NETs在体内的形成，并检查NETs与脓毒症诱导的血小板减少症和深静脉血栓形成的生物学相关性。 测量血栓形成患者样本中的NETs生物标志物。在这个目标中，我们建议可视化NET活体显微镜和组织学。我们建议调节NET的生产和循环DNA水平，并观察对败血症和DVT小鼠模型的影响。我们将研究NET作为血栓形成的生物标志物。血栓形成，包括深静脉血栓形成/肺栓塞，现在是美国最大的杀手。我们希望我们的研究将拓宽对血栓形成过程的一般理解，我们可能会发现新的分子靶点来治疗病理性血栓形成。 公共卫生相关性：血栓形成现在是美国最大的杀手。血栓是由血小板粘在一起形成的。白色血细胞影响血栓形成及其稳定性。死亡的白色细胞主动释放DNA，产生大的蜘蛛网状结构，捕获细菌。我们发现血小板与这些网状物结合，血栓中也存在这种网状物。我们建议研究这种DNA支架在血栓形成中的作用，以及使用酶消化DNA作为一种新的抗血栓形成方法的可能性。

DESCRIPTION (provided by applicant): Neutrophils undergo a cell death pathway that results in release of chromatin coated with antimicrobial proteins forming a web called neutrophil extracellular traps (NETs). NETs trap and kill microbes. Our preliminary results show that NETs recruit and activate platelets. We have evidence of extracellular DNA/chromatin presence in pathological specimens from deep vein thrombosis and in plasma of patients with thrombotic disease. The central hypothesis of this application is that NETs provide a scaffold that, together with fibrin, increases thrombus stability and promotes thrombus growth and microparticle recruitment. We propose that the formation of NETs in pathological circumstances could initiate thrombotic events and contribute to conditions such as sepsis, thrombotic microangiopathies and deep vein thrombosis. In addition, we propose that DNase1, which digests NETs, may modulate thrombosis. We will analyze NETs in the context of in vitro and in vivo models of thrombosis. We will employ a flow chamber with video capability using cells from genetically engineered mice. We propose to study several mouse models of thrombosis, all of which are well established in our laboratory, to visualize NETs formation in vivo with sophisticated imaging technology and study the relevance of NETs to thrombus growth and stability. We are particularly keen to investigate the role of NETs in a new inferior vena cava stenosis model of deep vein thrombosis (DVT) and in a mouse model of sepsis. The proposal has 2 specific aims: 1. To study the molecular mechanisms mediating platelet and microparticle interactions with NETs. To evaluate whether NETs promote fibrin generation. In this Aim, we propose to investigate in vitro the molecular mechanisms involved in platelet adhesion to NETs and address whether NETs promote recruitment of pro-coagulant microparticles (MPs) and fibrin deposition. 2. To study NETs formation in vivo and examine the biological relevance of NETs to sepsis-induced thrombocytopenia and to deep vein thrombosis. To measure NETs biomarkers in samples from patients with thrombosis. In this aim, we propose to visualize NETs by intravital microscopy and by histology. We propose to modulate NETs production and circulating DNA levels and see the effects on murine models of sepsis and DVT. We will study NETs as biomarkers in thrombosis. Thrombosis, including deep vein thrombosis/pulmonary embolism is now the biggest killer in the United States. We hope that our studies will broaden understanding of the thrombotic process in general and that we may uncover new molecular targets to treat pathological thrombosis. PUBLIC HEALTH RELEVANCE: Thrombosis is now the biggest killer in the United States. Thrombi are formed by platelets sticking together. White blood cells influence thrombi formation and their stability. Dying white cells actively release DNA producing large spider web-like structures that trap bacteria. We found that platelets bind to these webs and that such webs are present in thrombi. We propose to investigate the role of this DNA scaffold in thrombosis and the possibility of using enzymes to digest the DNA as a new anti- thrombotic approach.