STUDY OBJECTIVE: Endothelial dysfunction and increased microvascular permeability are hallmarks of severe COVID-19. At present, the underlying mechanisms of endothelial barrier failure in COVID-19 remain elusive. Here, we show that increased thrombin activity in plasma from severe COVID-19 patients activates endothelial protease-activated receptor (PAR1), which mediates barrier failure by triggering TRPV4-mediated Ca(2+) influx in lung microvascular endothelial cells. METHODS: Citrate plasma was sampled as part of the Pa-COVID-19 cohort study (ethics approval EA2/066/20) from patients with severe COVID-19 (high flow O(2) or mechanically ventilated; WHO severity score: 5-7) COVID-19. Plasma samples were diluted to 10% (v/v) in cell culture medium without FCS and tested for their ability to disrupt barrier integrity of primary human pulmonary microvascular endothelial cells (HPMEC) monolayers by electrical cell-substrate impedance sensing (ECIS), immunofluorescence for endothelial VE-cadherin and F-actin, western blot analyses of PAR-1 cleavage, and real-time Ca(2+) imaging. Plasma from healthy donors served as control. RESULTS: COVID-19 plasma had elevated thrombin activity while levels of antithrombin III, a key anti-coagulant with thromboprotective function were decreased. COVID-19 plasma caused endothelial barrier dysfunction as measured by ECIS and gap formation in HPMEC monolayers. Endothelial barrier disruption and endothelial Ca(2+) influx in response to COVID-19 plasma could be blocked by selective antagonists targeting thrombin (Argatroban), its receptor PAR1 (SCH79797), or TRPV4 (HC-067047). CONCLUSION: Here, we identify a novel signaling axis involving thrombin, its receptor PAR1, and TRPV4 as mechanism for increased microvascular permeability in COVID-19. Targeting this signaling axis in endothelial barrier failure may provide a promising adjunctive therapy in COVID-19.
- Michalick, L.
- Mandzimba-Maloko, B.
- Hamedi, S.
- Dohmen, M.
- Brack, M. C.
- Schulz, S.
- Behrens, F.
- Simmons, S.
- Müller-Redetzky, H.
- Suttorp, N.
- Kurth, F.
- Corman, V. M.
- Hocke, A. C.
- Witzenrath, M.
- Hippenstiel, S.
- Kuebler, W. M.
