Thrombomodulin is essential for maintaining quiescence in vascular endothelial cells

Abstract

Thrombomodulin (TM) is a thrombin receptor on endothelial cells that is involved in promoting activation of the anticoagulant protein C pathway during blood coagulation. TM also exerts protective anti-inflammatory properties through a poorly understood mechanism. In this study, we investigated the importance of TM signaling to cellular functions by deleting it from endothelial cells by CRISPR-Cas9 technology and analyzed the resultant phenotype of TM-deficient (TM−/−) cells. Deficiency of TM in endothelial cells resulted in increased basal permeability and hyperpermeability when stimulated by thrombin and TNF-α. The loss of the basal barrier permeability function was accompanied by increased tyrosine phosphorylation of VE-cadherin and reduced polymerization of F-actin filaments at cellular junctions. A significant increase in basal NF-κB signaling and expression of inflammatory cell adhesion molecules was observed in TM−/− cells that resulted in enhanced adhesion of leukocytes to TM−/− cells in flow chamber experiments. There was also a marked increase in expression, storage, and release of the von Willebrand factor (VWF) and decreased storage and release of angiopoietin-2 in TM−/− cells. In a flow chamber assay, isolated platelets adhered to TM−/− cells, forming characteristic VWF–platelet strings. Increased VWF levels and inflammatory foci were also observed in the lungs of tamoxifen-treated ERcre-TMf/f mice. Reexpression of the TM construct in TM−/− cells, but not treatment with soluble TM, normalized the cellular phenotype. Based on these results, we postulate cell-bound TM endows a quiescent cellular phenotype by tightly regulating expression of procoagulant, proinflammatory, and angiogenic molecules in vascular endothelial cells.

Upregulation of pulmonary tissue factor, loss of thrombomodulin and immunothrombosis in SARS-CoV-2 infection

“…In this respect, high thrombomodulin is associated with severe disease here and has predictive value for mortality in COVID-19 infection elsewhere [15,57]. Our results also demonstrated a concomitant increase in plasma sEPCR and thus suggest that endothelial expression of both receptors of the protein C pathway were affected, pointing towards a potentially impaired ability of the endothelium to generate and mediate APC cellular functions which reportedly reduce inflammation, apoptosis, and stabilize endothelial and epithelial barriers [40,41]. While most studies show mild changes in PC levels [4À8], a recent paper reported lower protein C in COVID-19 patients, resulting in an acquired protein C deficiency-like picture that may contribute to development of a thrombogenic state and worsening of patient`s clinical condition [60].…”

“…Whether these changes translate in upregulation of TF protein in the lung, and its cellular distribution remained elusive thus far. Another recent study reported a reduction in pulmonary transcriptional levels of thrombomodulin [39], a major endothelial cell anticoagulant [40,41]. Likewise, it is unknown whether thrombomodulin expression in the endothelial cell of small vessels, where it normally resides [41] is downregulated in the lung of patients who succumbed from the disease.…”

Persistent endotheliopathy in the pathogenesis of long COVID syndrome

“…Thrombomodulin (TM) is an EC surface receptor that facilitates thrombin‐induced activation of protein C on EC surfaces 26 . Recently, Giri et al reported that TM plays a key role in maintaining EC quiescence 27 . Of particular relevance, they showed that VWF expression and secretion was markedly increased in TM‐deficient ECs.…”

“…26 Recently, Giri et al reported that TM plays a key role in maintaining EC quiescence. 27 Of particular relevance, they showed that VWF expression and secretion was markedly increased in TM-deficient ECs.…”

Elevated endothelial dysfunction related biomarker levels indicate the severity and predict the incidence of sepsis

“…Besides, thrombomodulin (TM) is an endothelial cell transmembrane glycoprotein that is only released upon direct endothelial disruption 23 . soluble thrombomodulin (sTM) is released into plasma during in ammation, presumably due to cleavage from endothelial cells by neutrophil-derived enzymes 24,25 .…”