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

项目摘要 严重急性呼吸综合征新型冠状病毒（SARS-CoV-2）感染导致COVID-19。在 严重的情况下，COVID-19导致严重的炎症（“细胞因子风暴”），随后是凝血功能障碍， 血栓形成前状态，进展为多器官衰竭。包括IL 6在内的几种细胞因子升高。 此外，还发现促炎性半乳糖凝集素半乳糖凝集素-3（Gal-3）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，并已知介导静脉 在小鼠模型中通过IL 6的血栓形成。越来越多的文献表明中性粒细胞、血小板和 内皮细胞活化是COVID-19患者血栓性并发症的潜在驱动因素。然而，在这方面， 在炎症、血管细胞活化和 SARS-CoV-2感染期间的血栓形成。我们的目标是确定引起激活的介质， 中性粒细胞、血小板和/或内皮细胞在SARS-CoV-2感染中的作用及其机制 促进凝血酶生成和血栓形成。在爱荷华州大学，我们进行了一项多中心随机 比较标准预防剂量与中等剂量依诺肝素在住院患者中的临床试验（RCT） COVID-19患者（NCT 04360824），并收集血浆样本进行生物标志物和机制分析 问题研究鉴于COVID-19晚期血栓并发症激增，我们现建议招募额外 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 年我们假设COVID-19中的血栓形成是由IL-6和Gal-3驱动的激活 造血细胞和内皮细胞，并且即使在从病毒感染中恢复后，血栓前状态仍然存在。 感染我们的团队拥有独特的专业知识和资源组合，将解决2中的假设 既有统一的目标，又有独立的目标。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NET在介导细胞活化和增强凝血酶中的机制作用 COVID-19的生成和血栓形成。目的2建立新型SARS-CoV-2转基因小鼠模型 感染，以确定体内靶向IL 6、Gal-3或NET是否保护免受细胞活化、凝血酶 生成和血栓形成。该提案的优势在于利用临床样品和新的临床前研究。 模型，以确定细胞活化，凝血酶生成和体内 新冠肺炎血栓形成因此，拟议研究议程的总体影响非常大，很可能 为降低COVID-19血栓形成负担提供治疗靶点。

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.