Agarwal S, Cohen C T, Zobeck M, Jacobi P M, Sartain S E
- Texas Children's Hospital, Division of Pediatric Hematology-Oncology, Houston, TX, USA.
- Baylor College of Medicine, Houston, TX, USA.
Thromb Update. 2022 Aug;8:100116. doi: 10.1016/j.tru.2022.100116. Epub 2022 Jul 8.
There is emerging evidence of microvascular thrombosis and thrombotic microangiopathy (TMA) induced by COVID-19, presumably from endothelial injury. Thrombomodulin (TM) is an endothelial glycoprotein that plays a dual role in maintaining healthy endothelium-as a natural anticoagulant by binding thrombin to activate protein C (APC) and a negative regulator of the alternate complement pathway (AP). TM is shed into the plasma as soluble TM (sTM) during endothelial injury. We hypothesize that SARS-CoV-2 spike proteins cause direct microvascular endothelial injury, leading to TM shedding, decreased activation of PC, and consequently, microvascular thrombosis in COVID-19. We conducted this study twofold: 1) , we assessed endothelial injury (by measuring sTM) and AP activation by quantifying Ba (cleavage product of AP component Factor B) in a cohort of critically ill COVID-19 pediatric patients and the implications on clinical outcomes; and 2) we investigated endothelial injury (TM shedding) by SARS-COV-2 spike proteins and the subsequent functional consequence in activated PC (APC) levels and Ba levels. sTM and Ba in plasma samples from SARS-CoV-2 positive patients admitted to Texas Children's Hospital Pediatric Intensive Care Unit (n = 33) and from healthy controls (n = 38) were measured by ELISA. confluent glomerular microvascular endothelial cells (GMVECs) were incubated for 48 h in the presence or absence (control) of purified SARS-CoV-2 spike proteins, S1 and S2. TM from the cell lysates while Ba and APC from the cell supernatants were measured by ELISA. sTM and Ba levels were significantly higher in the COVID-19 pediatric patients compared to healthy controls (p < 0.01 and p < 0.001, respectively). Among the COVID-19 patients, elevated sTM was associated with increased vasopressor use (p = 0.01) and elevated Ba was associated with increased duration of mechanical ventilation (p = 0.04). , surface bound TM and soluble APC were significantly lower in GMVECs after addition of spike proteins (p < 0.05), while Ba was undetectable in both control and spike proteins exposed GMVECs. In conclusion, we provide evidence of endothelial injury in COVID-19 pediatric patients and demonstrate a potential pathway of SARS-CoV-2 induced thrombosis. Decreased surface-bound TM results in lower amount of thrombin-TM complex, hence lesser activation of PC, likely leading to a pro-thrombotic state. These findings in GMVECs could explain the vulnerability of kidneys to COVID-19-induced TMA.
越来越多的证据表明，新型冠状病毒肺炎（COVID-19）可引发微血管血栓形成和血栓性微血管病（TMA），推测这是由内皮损伤所致。血栓调节蛋白（TM）是一种内皮糖蛋白，在维持健康的内皮功能方面发挥双重作用——作为天然抗凝剂，通过结合凝血酶来激活蛋白C（APC），以及作为替代补体途径（AP）的负调节因子。在内皮损伤期间，TM会以可溶性TM（sTM）的形式释放到血浆中。我们假设，严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白会导致直接的微血管内皮损伤，进而导致TM释放、PC激活减少，最终导致COVID-19患者出现微血管血栓形成。我们分两方面开展了这项研究：1）我们评估了一组危重症COVID-19儿科患者的内皮损伤（通过测量sTM）以及通过定量Ba（AP成分B因子的裂解产物）来评估AP激活情况及其对临床结局的影响；2）我们研究了SARS-CoV-2刺突蛋白导致的内皮损伤（TM释放）以及随后对激活的PC（APC）水平和Ba水平的功能影响。通过酶联免疫吸附测定（ELISA）法测量了入住德克萨斯儿童医院儿科重症监护病房的SARS-CoV-2阳性患者（n = 33）和健康对照者（n = 38）血浆样本中的sTM和Ba。将汇合的肾小球微血管内皮细胞（GMVECs）在存在或不存在（对照）纯化的SARS-CoV-2刺突蛋白、S1和S2的情况下孵育48小时。通过ELISA法测量细胞裂解物中的TM，以及细胞上清液中的Ba和APC。与健康对照者相比，COVID-19儿科患者的sTM和Ba水平显著更高（分别为p < 0.01和p < 0.001）。在COVID-19患者中，sTM升高与血管升压药使用增加相关（p = 0.01），而Ba升高与机械通气时间延长相关（p = 0.04）。此外，添加刺突蛋白后，GMVECs表面结合的TM和可溶性APC显著降低（p < 0.05），而在对照和暴露于刺突蛋白的GMVECs中均未检测到Ba。总之，我们提供了COVID-19儿科患者内皮损伤的证据，并证明了SARS-CoV-2诱导血栓形成的潜在途径。表面结合的TM减少导致凝血酶-TM复合物数量减少，从而使PC激活减少，可能导致促血栓形成状态。GMVECs中的这些发现可以解释肾脏对COVID-19诱导的TMA的易感性。
