(768.2) In Vitro Screening Identifies TRPV4 and PAR1 as Targets for Endothelial Barrier Stabilization in COVID-19

Poster Board Number: E583

Laura Michalick (Charité - Universitätsmedizin Berlin, DZHK (German Centre for Cardiovascular Research), partner site Berlin), Bertina Mandzimba-Maloko (Charité - Universitätsmedizin Berlin), Shima Hamedi (Charité - Universitätsmedizin Berlin), Melanie Dohmen (Charité - Universitätsmedizin Berlin, Max Delbrück Center for Molecular Medicine), Markus Brack (Charité - Universitätsmedizin Berlin), Sabrina Schulz (Charité - Universitätsmedizin Berlin), Felix Behrens (Charité - Universitätsmedizin Berlin, DZHK (German Centre for Cardiovascular Research), partner site Berlin, Berlin Institute of Health (BIH)), Szandor Simmons (Charité - Universitätsmedizin Berlin, DZHK (German Centre for Cardiovascular Research), partner site Berlin), Holger Müller-Redetzky (Charité - Universitätsmedizin Berlin), Norbert Suttorp (Charité - Universitätsmedizin Berlin, German Center for Lung Research (DZL)), Florian Kurth (Charité - Universitätsmedizin Berlin), Victor Corman (Charité - Universitätsmedizin Berlin, German Center for Infection Research (DZIF)), Andreas Hocke (Charité - Universitätsmedizin Berlin, German Center for Lung Research (DZL)), Martin Witzenrath (Charité - Universitätsmedizin Berlin, German Center for Lung Research (DZL)), Stefan Hippenstiel (Charité - Universitätsmedizin Berlin, German Center for Lung Research (DZL)), Wolfgang Kuebler (Charité - Universitätsmedizin Berlin, St. Micheals Hospital, University of Toronto, German Center for Lung Research (DZL), DZHK (German Centre for Cardiovascular Research), partner site Berlin)

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 Ca2+ 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 O2 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 Ca2+ 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 Ca2+ 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.

This study is supported by BMBF (PROVID), BIH (Translational Vascular Biomedicine) DFG (SFB-TR84), and the German Centre for Cardiovascular Research (DZHK).