Lipid peroxidation- and pyroptosis-induced tissue factor activation in pathogen-induced blood coagulation

项目摘要/摘要 组织因子(TF)是凝血级联反应的主要启动者，受到仔细的调节，以防止 异常的凝血激活。然而，包括细菌和病毒感染在内的病理条件会导致 血管内转铁蛋白表达，并导致血栓形成。由于转铁蛋白诱导的血栓形成是急性 心肌梗死、缺血性中风和肺栓塞，提高了对心肌梗死机制的理解 病理性TF激活可能导致新的治疗靶点。上睑下垂是炎症细胞死亡的一种形式， 在细菌性脓毒症中驱动TF介导的血管内凝血激活。新出现的研究也表明 炎性小体在SARS-CoV-2感染中的激活，但其在TF激活中的作用尚不清楚。我们的预赛 研究表明，SARS-CoV-2及其辅助蛋白ORF3A可诱导T细胞因子活化。 需要TMEM16F的磷脂酰丝氨酸(PS)依赖机制，类似于PS依赖的TF激活 患有上睑下垂。在目标1中，我们将研究ORF3a诱导的TF激活是否由炎症小体驱动。 介导性下垂。脂质过氧化及其高活性的最终产物，如4-羟基-2-壬烯醛(HNE) 参与各种形式的细胞程序性死亡，包括上睑下垂。然而，海涅的角色，最 在脂类过氧化过程中产生的稳定和有毒的反应醛，在上睑下垂相关的TF和 凝血激活尚不清楚。我们的初步数据表明，HNE诱导PS依赖的TF激活 在脂多糖诱导的巨噬细胞中，并引起小鼠血管内凝血激活。然而，完整的 HNE诱导PS外化和TF激活的机制尚不清楚。在目标2中，我们将使用 用化学遗传学方法研究HNE体外通过下垂诱导组织因子活化的机制 在活体内。尽管脂质过氧化在细菌性脓毒症的细胞死亡和凝血激活中起着核心作用， 一种治疗靶向的酶，负责无节制的脂质过氧化和产生 病理水平的反应性自由基，如HNE尚不清楚。在目标3中，我们将使用转基因小鼠 缺乏脂质过氧化和HNE形成来研究Tf依赖的病理性凝血激活 以及脓毒症期间的血栓形成。这些研究的成功完成将有助于勾勒出一条共同的道路 参与各种病原性感染的病理性转铁蛋白的激活，并将有助于识别特定的 具有治疗靶向性的酶，可减弱疾病中的转铁蛋白活性。

Project Summary/Abstract Tissue factor (TF), the primary initiator of the blood coagulation cascade, is carefully regulated to prevent aberrant coagulation activation. However, pathological conditions including bacterial and viral infection induce intravascular TF expression and lead to thrombosis. As TF-induced thrombosis is a major cause of acute myocardial infarction, ischemic stroke, and pulmonary embolism, improved understanding of the mechanisms of pathological TF activation may lead to new therapeutic targets. Pyroptosis, a form of inflammatory cell death, drives TF-mediated intravascular coagulation activation in bacterial sepsis. Emerging studies also implicate inflammasome activation in SARS-CoV-2 infection, but its role in TF activation is unknown. Our preliminary studies demonstrate that SARS-CoV-2 and specifically its accessory protein ORF3A induce TF activation in a phosphatidylserine (PS)-dependent mechanism that requires TMEM16F, similar to PS-dependent TF activation in pyroptosis. In Aim 1, we will investigate whether ORF3a-induced TF activation is driven by inflammasome- mediated pyroptosis. Lipid peroxidation and its highly reactive end products such as 4-hydroxy-2-nonenal (HNE) are involved in various forms of programmed cell death including pyroptosis. However, the role of HNE, the most stable and toxic reactive aldehyde produced during lipid peroxidation, in pyroptosis-associated TF and coagulation activation is not known. Our preliminary data showed that HNE induces PS-dependent TF activation in LPS-primed macrophages and causes intravascular coagulation activation in mice. However, the complete mechanism by which HNE induces PS externalization and TF activation is not known. In Aim 2, we will use chemical genetic approaches to dissect the mechanism of HNE-induced TF activation via pyroptosis in vitro and in vivo. Although lipid peroxidation plays a central role in cell death and coagulation activation in bacterial sepsis, a therapeutically targetable enzyme responsible for the unbridled lipid peroxidation and generation of pathological levels of reactive radicals such as HNE is not known. In Aim 3, we will use genetically modified mice deficient in lipid peroxidation and HNE formation to investigate TF-dependent pathologic coagulation activation and thrombosis during sepsis. A successful completion of these studies will help delineate a common pathway involved in pathologic TF activation across varied pathogenic infections and will also help identify a specific therapeutically targetable enzyme to attenuate TF activation in disease.