Platelet adhesion to neutrophil extracellular DNA traps: role in thrombosis

描述(申请人提供)：中性粒细胞经历一条细胞死亡途径，导致染色质释放包被抗菌蛋白，形成一张称为中性粒细胞细胞外陷阱(NETs)的网。蚊帐捕捉并杀死微生物。我们的初步结果表明，Net招募并激活了血小板。我们在深静脉血栓形成的病理标本和血栓性疾病患者的血浆中有细胞外DNA/染色质存在的证据。这一应用的中心假设是，Net提供了一种支架，与纤维蛋白一起，增加了血栓的稳定性，促进了血栓的生长和微粒的募集。我们认为，病理情况下Net的形成可以引发血栓事件，并有助于脓毒症、血栓性微血管病变和深静脉血栓形成等情况。此外，我们认为，消化NETs的DNase1可能调节血栓形成。我们将在体外和体内血栓形成模型的背景下分析NETS。我们将使用一个具有视频功能的流室，使用来自基因工程小鼠的细胞。我们建议研究几种小鼠血栓形成的模型，所有这些都是在我们的实验室建立的，用先进的成像技术在体内可视化Net的形成，并研究Net与血栓生长和稳定性的相关性。我们尤其热衷于研究Nets在深静脉血栓形成(DVT)的新的下腔静脉狭窄模型和脓毒症小鼠模型中的作用。该建议有两个具体目的：1.研究血小板和微粒与Net相互作用的分子机制。评估网络是否能促进纤维蛋白的生成。为此，我们建议在体外研究血小板与Net黏附的分子机制，并探讨Net是否促进促凝剂微粒(MPS)的募集和纤维蛋白的沉积。2.研究Net在体内的形成，探讨Net与脓毒症所致的血小板减少症和深静脉血栓形成的生物学相关性。检测血栓形成患者标本中的Nets生物标志物。在这个目标中，我们建议通过活体显微镜和组织学来可视化网络。我们建议调节Nets的产生和循环DNA水平，并观察其对脓毒症和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总体理解，并可能发现治疗病理性血栓形成的新的分子靶点。

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.