Tspan18 is a novel regulator of the Ca²⁺ channel Orai1 and von Willebrand factor release in endothelial cells

Abstract

Ca 2+ entry via Orai1 store-operated Ca 2+ channels in the plasma membrane is critical to cell function, and Orai1 loss causes severe immunodeficiency and developmental defects. The tetraspanins are a superfamily of transmembrane proteins that interact with specific ‘partner proteins’ and regulate their trafficking and clustering. The aim of this study was to functionally characterize tetraspanin Tspan18. We show that Tspan18 is expressed by endothelial cells at several-fold higher levels than most other cell types analyzed. Tspan18-knockdown primary human umbilical vein endothelial cells have 55-70% decreased Ca 2+ mobilization upon stimulation with the inflammatory mediators thrombin or histamine, similar to Orai1-knockdown. Tspan18 interacts with Orai1, and Orai1 cell surface localization is reduced by 70% in Tspan18-knockdown endothelial cells. Tspan18 overexpression in lymphocyte model cell lines induces 20-fold activation of Ca 2+ -responsive nuclear factor of activated T cell (NFAT) signaling, in an Orai1-dependent manner. Tspan18-knockout mice are viable. They lose on average 6-fold more blood in a tail-bleed assay. This is due to Tspan18 deficiency in non-hematopoietic cells, as assessed using chimeric mice. Tspan18-knockout mice have 60% reduced thrombus size in a deep vein thrombosis model, and 50% reduced platelet deposition in the microcirculation following myocardial ischemia-reperfusion injury. Histamine- or thrombin-induced von Willebrand factor release from endothelial cells is reduced by 90% following Tspan18-knockdown, and histamine-induced increase of plasma von Willebrand factor is reduced by 45% in Tspan18-knockout mice. These findings identify Tspan18 as a novel regulator of endothelial cell Orai1/Ca 2+ signaling and von Willebrand factor release in response to inflammatory stimuli.

Tspan18 is a novel regulator of thrombo-inflammation

“…In the current absence of an effective Tspan18 antibody, we used RT-PCR to demonstrate Tspan18 expression in human endothelial cells, but not most other cell types. This was supported by our analyses of publicly available RNA-Seq data from mouse lung and brain, which showed that Tspan18 is most highly expressed in endothelial cells of these organs [6]. Analysis of single-cell RNA-Seq data from 20 mouse organs provides further support, since 9 of the 14 most highly Tspan18-expressing cell types are endothelial cells (Fig.…”

“…We further showed that Tspan18 knockdown in primary human umbilical vein endothelial cells (HUVECs) impairs Ca 2+ signaling by 55-70% following stimulation with the GPCR agonists histamine or thrombin, similar to Orai1 knockdown [6]. Orai1 surface localisation is 70% reduced following Tspan18 knockdown, suggesting a role for Tspan18 in trafficking Orai1 from the ER.…”

“…In addition to their thrombo-inflammatory phenotypes, Tspan18-knockout mice have a haemostasis defect, as detected by increased blood loss in a tail bleeding assay [6]. Consistent with a role for Tspan18 in endothelial cells, this bleeding phenotype is the result of Tspan18 deletion from non-haematopoietic cells only; this was determined using wild-type/Tspan18-knockout fetal liver chimeric mice [6]. The underlying mechanism remains unclear, since there is no evidence that an acute release of VWF from endothelial cells following injury is critical for haemostasis.…”

“…VWF is constitutively released at a relatively low level by WPB exocytosis, but can be acutely exocytosed in larger quantities via a mechanism involving Ca 2+ signaling following endothelial cell activation [5]. We have recently discovered that tetraspanin Tspan18 is important for endothelial cell Ca 2+ signaling, acute VWF release, and thrombo-inflammation, by interacting with the store-operated Ca 2+ entry channel Orai1 and promoting its trafficking to the cell surface [6]. This review will describe our current knowledge of Tspan18 expression and function, and will speculate on the molecular mechanism by which Tspan18 regulates Orai1.…”

From Classical Laboratory Parameters to Novel Biomarkers for the Diagnosis of Venous Thrombosis

“…Other recent biomarkers have been or are currently explored for the diagnosis or venous thrombosis and they may be grouped into those with limited or no signal, mixed results or strong results, but they require further validation before considering their use in clinical practice [7,142,143] (Table 4). Although many of these biomarkers are very promising, currently, they cannot be used to make decisions in the management of patients with venous thromboembolism because they do not have an established cut-off point in order to validate them, and most of them have not been used in clinical trials.…”