OC 57.5 - Downregulation of Thrombomodulin-Thrombin-Activated Protein C pathway as a mechanism for SARS-CoV-2 induced endotheliopathy and microvascular thrombosis

Background: There is emerging evidence of microvascular thrombosis and thrombotic microangiopathy (TMA) induced by COVID-19, presumably from endothelial injury or “endotheliopathy”. Thrombomodulin (TM) is an endothelial glycoprotein that plays a crucial role as a natural anticoagulant, binding thrombin to activate protein C (PC). TM is shed from endothelial surface during injury. We hypothesize 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.

Aims: To assess: 1) endothelial injury (by soluble TM [sTM] levels) in a cohort of critically-ill COVID-19 pediatric patients; 2) endothelial injury (TM shedding) in vitro by SARS-CoV-2 spike proteins and the subsequent functional consequence in activated PC (APC) levels.

Methods: sTM in plasma samples from SARS-CoV-2 positive patients admitted to Texas Children’s Hospital Pediatric Intensive Care Unit (n=34) and healthy controls (n=38) were measured by ELISA. IRB approval and waiver of informed consent were obtained.

In vitro, confluent glomerular microvascular endothelial cells (GMVECs) were incubated for 24 hours in the presence or absence (control) of purified SARS-CoV-2 spike proteins, S1 and S2. In some experiments, cell lysates were collected, and TM was measured by ELISA; in others, GMVECs were further supplemented with PC and thrombin for 1 hour, followed by supernatant collection for APC measurement by ELISA.

Results: sTM levels were significantly higher in the COVID-19 pediatric patients (p < 0.01) (Fig. 1). In vitro, surface bound TM (Fig 2a) and soluble APC (Fig 2b) were significantly lower in GMVECs after addition of spike proteins (p < 0.05).

Conclusion(s): We provide evidence of endothelial injury in COVID-19 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.