Cellular effects of SARS-CoV-2 in mediating thrombotic susceptibility

项目摘要 感染严重急性呼吸综合征新型冠状病毒(SARS-CoV-2)导致新冠肺炎。在……里面 严重情况下，新冠肺炎会导致严重的炎症(细胞因子风暴)，随后是凝血障碍和 血栓前状态，进展为多器官衰竭。包括IL6在内的几种细胞因子都升高了。 此外，一种促炎症的Galectin，Galectin-3(Gal-3)也被发现升高。GAL-3上调IL-6和其他 细胞因子，可以直接激活血小板、中性粒细胞和内皮细胞，并被认为是介导静脉 白介素6在小鼠模型中的血栓形成。越来越多的文献表明，中性粒细胞、血小板和 血管内皮细胞激活是新冠肺炎患者血栓并发症的潜在驱动因素。然而， 炎症、血管细胞激活和炎症之间没有直接的机制联系。 SARS-CoV-2感染期间的血栓形成。我们的目标是定义导致激活的介体 SARS-CoV-2感染过程中的中性粒细胞、血小板和/或内皮细胞及其机制 促进凝血酶生成和血栓形成。在爱荷华大学，我们领导了一项多中心随机 住院标准预防剂量与中剂量依诺肝素的临床试验(RCT)比较 新冠肺炎患者(NCT04360824)并采集血浆样本进行生物标志物和机制研究 学习。鉴于新冠肺炎晚期血栓并发症的激增，我们现在建议招募更多 患者在住院期间每周收集一次连续样本，此后每3个月收集一次，最多3次 好几年了。我们推测新冠肺炎的血栓形成是由IL-6和Gal-3诱导的血管内皮细胞活化介导的 造血和内皮细胞，血栓前状态即使在从病毒中恢复后仍然存在 感染。我们的团队拥有独特的专业知识和资源组合，将在2中解决这一假设 很好地整合了但独立的目标。在目标1中，使用连续收集的患者样本，我们将确定 IL-6、Gal-3和Nets在介导细胞活化和增强凝血酶中的作用机制 新冠肺炎的生成和血栓形成。AIM 2将利用一种新的SARS-CoV-2转基因小鼠模型 感染以确定体内靶向IL6、Gal-3或Nets是否能预防细胞活化、凝血酶 产生和血栓形成。这一提议的优势在于利用了临床样本和一种新的临床前研究 识别细胞激活、凝血酶生成和体内关键机制途径的模型 新冠肺炎中的血栓形成。因此，拟议的研究议程的总体影响非常大，很可能 为降低新冠肺炎血栓负荷提供治疗靶点。

Project Summary Infection with severe acute respiratory syndrome novel corona virus (SARS-CoV-2) causes COVID-19. In severe cases, COVID-19 leads to profound inflammation (“cytokine storm”) followed by coagulopathy and a prothrombotic-state with progression to multiple organ failure. Several cytokines, including IL6 are elevated. Further, a proinflammatory galectin, Galectin-3 (Gal-3) is also found elevated. Gal-3 upregulates IL6 and other cytokines, can directly activate platelets, neutrophils, and endothelial cells, and is known to mediate venous thrombosis via IL6 in a mouse model. A growing body of literature has implicated neutrophil, platelet and endothelial cell activation as potential drivers of thrombotic complications in COVID-19 patients. However, there are no direct mechanistic links established between inflammation, vascular cell activation, and thrombosis during SARS-CoV-2 infection. Our objective is to define the mediators that cause activation of neutrophils, platelets and/or endothelial cells during SARS-CoV-2 infection and their mechanistic roles in promoting thrombin generation and thrombosis. At the University of Iowa, we led a multicenter randomized clinical trial (RCT) comparing standard prophylactic dose to intermediate dose enoxaparin in hospitalized patients with COVID-19 (NCT04360824) and collected plasma samples for biomarkers and mechanistic studies. Given the upsurge in late thrombotic complications of COVID-19, we now propose to recruit additional patients to collect serial samples every week during hospitalization and thereafter every 3 months for up to 3 years. We hypothesize that thrombogenicity in COVID-19 is mediated by IL6- and Gal-3-driven activation of hematopoietic and endothelial cells and that the prothrombotic state persists even after recovery from viral infection. Our team has a unique combination of expertise and resources that will address the hypothesis in 2 well integrated but independent aims. In Aim 1, using serially collected patient’s samples, we will determine the mechanistic role of IL6, Gal-3, and NETs in mediating cellular activation and enhancing thrombin generation and thrombosis in COVID-19. Aim 2 will utilize a novel transgenic mouse model of SARS-CoV-2 infection to determine if targeting IL6, Gal-3, or NETs in vivo protects against cellular activation, thrombin generation and thrombosis. A strength of this proposal is in utilizing clinical samples and a novel preclinical model to identify critical mechanistic pathways for cellular activation, thrombin generation and in vivo thrombosis in COVID-19. Thus, the overall impact of the proposed research agenda is very high and is likely to provide therapeutic targets for decreasing thrombotic burden in COVID-19.