Pulmonary endothelial NEDD9 and the prothrombotic pathophenotype of acute respiratory distress syndrome due to SARS-CoV-2 infection.

The pathobiology of in situ pulmonary thrombosis in acute respiratory distress syndrome (ARDS) due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is incompletely characterized. In human pulmonary artery endothelial cells (HPAECs), hypoxia increases neural precursor cell expressed, developmentally downregulated 9 (NEDD9) and induces expression of a prothrombotic NEDD9 peptide (N9) on the extracellular plasma membrane surface. We hypothesized that the SARS-CoV-2-ARDS pathophenotype involves increased pulmonary endothelial N9. Paraffin-embedded autopsy lung specimens were acquired from patients with SARS-CoV-2-​​​​​​ARDS ( = 13), ARDS from other causes ( = 10), and organ donor controls ( = 5). Immunofluorescence characterized the expression of N9, fibrin, and transcription factor 12 (TCF12), a putative binding target of SARS-CoV-2 and known transcriptional regulator of . We performed RNA-sequencing on normal HPAECs treated with normoxia or hypoxia (0.2% O) for 24 h. Immunoprecipitation-liquid chromatography-mass spectrometry (IP-LC-MS) profiled protein-protein interactions involving N9 relevant to thrombus stabilization. Hypoxia increased TCF12 messenger RNA significantly compared to normoxia in HPAECs in vitro (+1.19-fold,  = 0.001; false discovery rate = 0.005), and pulmonary endothelial TCF12 expression was increased threefold in SARS-CoV-2-ARDS versus donor control lungs ( < 0.001). Compared to donor controls, pulmonary endothelial N9-fibrin colocalization was increased in situ in non-SARS-CoV-2-ARDS and SARS-CoV-2-ARDS decedents (3.7 ± 1.2 vs. 10.3 ± 3.2 and 21.8 ± 4.0 arb. units,  < 0.001). However, total pulmonary endothelial N9 was increased significantly only in SARS-CoV-2-ARDS versus donor controls (15 ± 4.2 vs. 6.3 ± 0.9 arb. units,  < 0.001). In HPAEC plasma membrane isolates, IP-LC-MS identified a novel protein-protein interaction between NEDD9 and the β-subunit of the αβ-integrin, which regulates fibrin anchoring to endothelial cells. In conclusion, lethal SARS-CoV-2-ARDS is associated with increased pulmonary endothelial N9 expression and N9-fibrin colocalization in situ. Further investigation is needed to determine the pathogenetic and potential therapeutic relevance of N9 to the thrombotic pathophenotype of SARS-CoV-2-ARDS.