Unraveling Cytotoxic and Thrombotic Signals in COVID-19

患有严重冠状病毒病（COVID-19）的个体会出现炎症过度，并且在多个血管床中存在血栓形成的高风险。富含纤维蛋白的凝块中断器官灌注导致组织灌注不良和器官衰竭，这是导致COVID-19相关死亡率和发病率的原因。在疫情爆发时，COVID-19的炎症过度和血栓形成的潜在机制尚不清楚。为了寻求新的和改进的治疗方法来对抗大流行病，我们使用患者样本进行了机制，转化研究，体外研究和动物建模，确定了血栓形成的几种机制和潜在的治疗靶点。这些研究产生了COVID-19中血栓炎症的三种新范式，这些发现以多项临床试验的形式迅速转化为临床：项目（1）：COVID-19中的神经细胞外陷阱。COVID炎症的早期报告集中在淋巴细胞和单核细胞的作用。我们在严重COVID-19患者的血液中发现了一种基于嗜中性粒细胞的过度炎症特征，其特征是一种特别的炎症性中性粒细胞死亡形式，DNA被挤出作为粘性的中性粒细胞胞外陷阱（NET）。我们是第一个将旺盛的NET形成确定为严重COVID-19的标志及其与呼吸衰竭、血栓形成和死亡的关联的人。这项工作在COVID-19中性粒细胞过度炎症领域开辟了一条新的研究路线，并已被世界各地的几个团队验证。我们小组和其他人的工作表明，NET增强了炎症和血栓形成，为减轻血栓炎症的多项临床试验奠定了基础。我们为其中许多临床试验提供了额外的专家意见和指导，并直接为NHLBI努力优先考虑针对NIH申办的临床试验免疫调节和血栓形成的新药和治疗方法做出贡献。项目（2）：预测严重COVID-19的生物标志物。在大流行高峰期医院资源紧张的背景下，我们寻求生物标志物来前瞻性地识别呼吸衰竭风险最高的患者，以帮助临床医生适当地分配资源。我们发现，一种称为钙卫蛋白的中性粒细胞胞质钙颗粒蛋白，也称为MRP 8 -14或S100 A8/9，在住院时在血清中测量，能够识别后来发生呼吸衰竭的患者。临床医生无法获得血清钙卫蛋白。我们协助开发了一种血清钙卫蛋白测量测定法，该测定法目前已在欧洲临床用于危重病和其他疾病患者。项目（3）：COVID-19中的病理性自身抗体。COVID-19的凝血病与抗磷脂综合征（APS）的灾难性变体有许多相似之处，APS是一种自身免疫性血栓形成倾向，其特征是循环中的磷脂和磷脂结合蛋白自身抗体。我们的研究首次揭示，一半的COVID-19住院患者至少有一种抗磷脂抗体（aPL）检测呈阳性，这与更差的临床结果相关。我们鉴定了COVID-19血清，特别是来自aPL检测阳性患者的IgG组分在健康人中性粒细胞中产生了高度炎症表型，在静脉血栓形成的动物模型中产生了大量促炎性NET形成和过度凝血。这是第一次描述身体在COVID-19中产生血栓前自身抗体，并导致了对驱动凝血的适应性和免疫反应之间的病理串扰的新研究。我们发现，除了增加NET的形成，自身抗体也屏蔽NET从正常的降解和增强血栓炎症信号。我们发现，COVID患者中的自身抗体以及非COVID脓毒症患者中的自身抗体破坏了正常的稳态内皮表面，并使血液：血管界面在危重病期间倾向于炎症和血栓形成。 这些已发表的和进一步正在进行的研究在推进HHS/NIH应对大流行和改善公共卫生的使命方面发挥了关键作用。

Individuals with severe coronavirus disease (COVID-19) develop hyperinflammation and are at high risk for thrombosis in multiple vascular beds. Interruption of organ perfusion by fibrin-rich clots leads to tissue malperfusion and organ failure that is responsible for the mortality and morbidity associated with COVID-19. At the onset of the pandemic, the underlying mechanisms of hyperinflammation and thrombosis in COVID-19 were unknown. In pursuit of novel and improve therapeutic approaches to combat the pandemic, we undertook mechanistic, translational studies using patient samples, in vitro studies, and animal modeling that identified several mechanisms of thrombosis and potential therapeutic targets. These studies have resulted in three new paradigms of thromboinflammation in COVID-19, and these discoveries have been rapidly translated to the clinic in the form of multiple clinical trials: Project (1): Neutrophil Extracellular Traps in COVID-19. Early reports of inflammation in COVID focused on the role of lymphocytes and monocytes. We discovered a neutrophil-based signature of hyperinflammation in the blood of patients with severe COVID-19, marked by a particularly inflammatory form of neutrophil death by which DNA is extruded as sticky, neutrophil extracellular traps (NETs). We were the first to identify exuberant NET formation as a hallmark of severe COVID-19 and its association with respiratory failure, thrombosis, and death. This work opened a new line of investigation in the field for neutrophil hyperinflammation in COVID-19, and has been validated by several teams across the world. Work from our group and others has shown that NETs potentiate inflammation and thrombosis, laying the foundation for multiple clinical trials to mitigate thromboinflammation. We have provided additional expert input and guidance for many of these clinical trials, and contribute directly to the NHLBI efforts to prioritize new drugs and therapeutic approaches that target immunomodulation and thrombosis for NIH-sponsored clinical trials. Project (2): Biomarkers To Predict Severe COVID-19. In the context of strained hospital resources at the height of the pandemic, we sought biomarkers to prospectively identify patients at the highest risk of respiratory failure in order help clinicians direct resources appropriately. We discovered that a neutrophil cytoplasmic calgranulin called calprotectin, also known as MRP8-14 or S100A8/9 measured in serum at the time of hospitalization was able to identify patients who later developed respiratory failure. Serum calprotectin was not available to clinicians for use. We assisted in the development of a serum calprotectin measurement assay that is now in clinical use in Europe for patients with critical illness and other diseases. Project (3): Pathologic Autoantibodies in COVID-19. The coagulopathy of COVID-19 has many similarities with the catastrophic variant of Antiphospholipid Syndrome (APS), an autoimmune thrombophilia marked by circulating autoantibodies to phospholipids and phospholipid-binding proteins. Our studies were the first to reveal that half of patients hospitalized with COVID-19 test positive for at least one type of antiphospholipid antibody (aPL) which was associated with worse clinical outcomes. We identified that COVID-19 serum, and in particular, the IgG fraction from patients testing positive for aPL created a hyperinflammatory phenotype in healthy human neutrophils, exuberant pro-inflammatory NET formations, and exaggerated clotting in an animal model of venous thrombosis. These were the first description of the body producing prothrombotic autoantibodies in COVID-19, and led to a new line of investigation on the pathologic crosstalk between adaptive and immune responses that drives coagulation. We found that in addition to heightened NET formation, autoantibodies also shield NETs from normal degradation and potentiate thromboinflammatory signaling. We identified that the autoantibodies in patients with COVID and similarly in patients with non-COVID sepsis, disrupt the normal homeostatic endothelial surface, and tip the blood:vessel interface toward inflammation and thrombosis during critical illness. These published and further ongoing studies have been pivotal in advancing the HHS/NIH mission to respond to the pandemic and improve public health.