AbstractCa2+ entry via Orai1 store-operated Ca2+ 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 Ca2+ 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 Ca2+ -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/Ca2+ signaling and von Willebrand factor release in response to inflammatory stimuli.
The tetraspanins are a superfamily of proteins containing four transmembrane regions that interact with and regulate the trafficking, lateral mobility and clustering of specific ‘partner proteins’. These include signaling receptors, adhesion molecules and metalloproteinases.31 Recently, the first crystal structure of a tetraspanin, CD81, demonstrated a cone-shaped structure with a cholesterol-binding cavity within the transmembranes.4 Molecular dynamics simulations suggest that cholesterol removal causes a dramatic conformational change, whereby the main extracellular region swings upwards.4 This raises the possibility that tetraspanins function as ‘molecular switches’ to regulate partner protein function via conformational change, and suggests that tetraspanins are viable future drug targets.
Tetraspanin Tspan18 was previously studied in chick embryos, in which it stabilizes expression of the homophilic adhesion molecule cadherin 6B to maintain adherens junctions between premigratory epithelial cranial neural crest cells.65 Transcriptional Tspan18 downregulation is required for loss of cadherin 6B expression, breakdown of epithelial junctions, and neural crest cell migration. However, Tspan18 knockdown has no major effect on chick embryonic development.65 The function of Tspan18 in humans or mice has still not been studied.
Store-operated Ca entry (SOCE) through the plasma membrane Ca channel Orai1 is essential for the healthy function of most cell types.7 Loss of SOCE results in severe immunodeficiency that requires a bone marrow transplant for survival. Further symptoms include ectodermal dysplasia and impaired development of skeletal muscle.7 The process of SOCE is biphasic. The first step is initiated following the generation of the second messenger inositol trisphosphate (IP3) from upstream tyrosine kinase or G protein-coupled receptor signaling. IP3 induces the transient release of Ca from endoplasmic reticulum (ER) stores via IP3 receptor channels.8 Depletion of Ca is detected by the ER-resident dimeric Ca-sensor protein STIM1, which then undergoes a conformational change and interacts with Orai1 hexamers in the plasma membrane.109 STIM1 binding induces Orai1 channel opening and clustering via a mechanism that is not fully understood, allowing Ca entry across the plasma membrane.109 The resulting increase in intracellular Ca concentration is relatively large and sustained, sufficient to activate a variety of signaling proteins, including the widely-expressed nuclear factor of activated T-cell (NFAT) transcription factors.8
Endothelial cells line all blood and lymphatic vessels and play a central role in hemostasis and in thrombo-inflammation, in which inflammatory cells contribute to thrombosis.1211 In the thrombo-inflammatory disease deep vein thrombosis, blood flow stagnation induced by prolonged immobility, for example, is the trigger for endothelial cells to exocytose Weibel-Palade storage bodies via a mechanism involving Ca signaling.1413 This releases the multimeric glycoprotein von Willebrand factor (vWF) and the adhesion molecule P-selectin, which recruit platelets and leukocytes, respectively. vWF-bound platelets provide a pro-coagulant surface for activation of clotting factors and thrombin generation, neutrophils release neutrophil extracellular traps, and mast cells release endothelial-activating substances.1715 This series of thrombo-inflammatory events leads to formation of a blood clot which occludes the vein, and can cause death by pulmonary thromboembolism.
The aim of this study was to determine the function of tetraspanin Tspan18 in humans and mice. We found that Tspan18 is highly expressed by endothelial cells, interacts with Orai1, and is required for its cell surface expression and SOCE function. As a consequence, Tspan18-deficient endothelial cells have impaired Ca mobilization and release of vWF upon activation induced by inflammatory mediators, and Tspan18-knockout mice are protected from deep vein thrombosis and myocardial ischemia-reperfusion injury, and have defective hemostasis.
Procedures in Birmingham were approved by the UK Home Office according to the Animals (Scientific Procedures) Act 1986, and those in Würzburg by the district government of Lower Frankonia (Bezirksregierung Unterfranken).
Tspan18 mice were generated by Genentech/Lexicon Pharmaceuticals on a mixed genetic background of 129/SvEvBrd and C57BL/6J.18 They were purchased from the Mutant Mouse Regional Resource Center and bred as heterozygotes to generate litter-matched Tspan18 and Tspan18 pairs. Radiation fetal liver chimeric mice were generated as described.19
Anti-epitope tag antibodies were mouse anti-Myc 9B11 and rabbit anti-Myc 71D10 (Cell Signaling Technology), mouse anti-FLAG M2 and rabbit anti-FLAG (Sigma). Other antibodies were mouse anti-human calnexin AF18 (Abcam), rat anti-mouse CD16/32 (BioLegend), CD41 (eBioscience) and panendothelial cell antigen MECA-32 (BD Pharmingen), mouse anti-ERK1/2 and rabbit anti-phospho-ERK1/2 (Cell Signaling Technology) and rabbit anti-human vWF (GE Healthcare). Biotinylated isolectin GS-IB4 glycoprotein was from ThermoFisher Scientific.
The NFAT/AP1-luciferase transcriptional reporter construct has been described previously.2120 pEF6/Myc-His (mock) and pEF6/Myc-His/lacZ were from Invitrogen. N-terminal FLAG-tagged tetraspanin constructs were generated in pEF6/Myc-His as described.2322 pcDNA3.1 Myc-His-tagged human Orai1 and MO70-FLAG-tagged human Orai1 E106Q were from Addgene24 and the dominant-active calcineurin was as described.25
Cell culture and transfections
Detailed information on cell cultures is provided in the Online Supplementary Appendix. Wild-type and IP3 receptor-deficient DT40 chicken B-cell lines,26 and Jurkat human T-cell line, were transfected by electroporation.21 Human embryonic kidney (HEK)-293T (HEK-293 cells expressing the large T-antigen of simian virus 40) and the human HeLa epithelial cell line were transfected using polyethylenimine (Sigma)27 and Lipofectamine 2000 (Invitrogen),28 respectively. Human umbilical vein endothelial cells (HUVEC)29 were transfected with 10 nM Silencer Select siRNA duplexes (Invitrogen) using Lipofectamine RNAiMAX (Invitrogen).
Quantitative real-time polymerase chain reaction
Quantitative real-time polymerase chain reaction (qPCR) was performed using TaqMan assays for Tspan18, Orai1, Orai2, Orai3, 18S and GAPDH.30 Details are available in the Online Supplementary Appendix.
Human umbilical vein endothelial cells were lentivirally transduced with Orai1-Myc as described.31 Details are available in the Online Supplementary Appendix.
Nuclear factor of activated T-cell/AP-1-luciferase transcriptional reporter assay
The NFAT/AP-1-luciferase assay, and β-galactosidase assay to normalize for transfection efficiency, were as described.21
A detailed description of co-immunoprecipitation from transfected HEK-293T cell lysates22 is provided in the Online Supplementary Appendix.
Detailed information is provided in the Online Supplementary Appendix. In brief, cells were prepared as described29 and the Manders’ coefficients (M1 and M2) were used as the co-localization measure.32 Ear vasculature was imaged and quantified as described.3433
Immunohistochemistry was as described;35 details are available in the Online Supplementary Appendix.
Human umbilical vein endothelial cells were loaded with Fluo-4 NW dye according to the manufacturer’s instructions (Molecular Probes). Fluorescence was measured every 3 seconds for 5 minutes using a FlexStation fluorescence reader (Molecular Devices), and thrombin (1 U/mL), histamine (20 μM) or ionomycin (10 μM) were injected after acquiring a baseline for 30 seconds.
ELISA and coagulation time assays
Detailed information is provided in the Online Supplementary Appendix.
Platelet aggregation and adhesion to human umbilical vein endothelial cells
Platelet assays were as described;3736 detailed information is provided in the Online Supplementary Appendix.
In vivo assays
Mouse models were as described;393713 detailed information is in the Online Supplementary Appendix.
Tspan18 is expressed by endothelial cells
A lack of effective antibodies to many tetraspanins is a current problem in the tetraspanin field. This may be due to their relatively small size, high degree of sequence conservation during evolution, and compact 4-transmem- brane structure.4 For example, no Tspan18 antibodies have been published, and commercially-available antibodies are made to Tspan18 peptides and do not detect full-length Tspan18 when rigorously tested (MG Tomlinson, 2019, unpublished manuscript). Therefore, to characterize the Tspan18 expression profile, mouse tissues were tested by qPCR. Tspan18 mRNA was most highly expressed in lung and at lower levels in other tissues (Figure 1A). Analyses of published transcriptomic data40 showed that Tspan18 was most highly expressed by endothelial cells compared to other mouse lung cell types (Figure 1B). Similar analyses of transcriptomic data from mouse brain41 also showed relatively strong endothelial expression of Tspan18 (Figure 1C). Consistent with this, qPCR showed that Tspan18 was expressed by primary HUVEC and the human microvascular endothelial HMEC-1 cell line (Figure 1D). Tspan18 expression was low or absent on most other cell types tested, although peripheral blood leukocytes expressed comparable levels to HUVEC (Figure 1D). In transcriptomic data from the Human Protein Atlas (www.proteinat-las.org), Tspan18 was expressed by most human tissues at a level between 10 and 70 tags per million, but in cell lines was only expressed at 10 or greater tags per million by HUVEC and 8 of the other 64 cell types analyzed.42
Tspan18 is required for Ca2+ signaling in primary human endothelial cells
To investigate Tspan18 function, its expression in HUVEC, which is a widely-used primary human endothelial cell model, was knocked-down using two different siRNA duplexes. Subsequent analyses revealed a 60% reduction in peak Ca elevation in response to the inflammatory mediator thrombin (Figure 2A). A similar defect was observed in response to histamine (Figure 2B). Positive control ionomycin treatment gave a sustained intracellular Ca response in all samples (Figure 2C) and effective knockdown was confirmed by qPCR (Figure 2D). Functionality of thrombin and histamine receptors was confirmed by anti-phospho-ERK1/2 mitogen-activated protein kinase (MAPK) western blotting, as this was not affected by Tspan18 knockdown (Figure 2E).
Tspan18 promotes Ca2+-responsive nuclear factor of activated T-cell signaling in lymphocyte cell lines
To investigate the mechanism by which Tspan18 regulates Ca signaling, a more tractable cell line system was established, namely DT40 cells that are derived from chicken B cells. In this cell line, a transfected NFAT/adapter protein 1 (AP-1) transcriptional luciferase reporter can be used as a readout for Ca signaling downstream of transfected membrane proteins.4321 Transfection of a FLAG epitope-tagged Tspan18 expression construct was sufficient to induce robust NFAT/AP-1 activation (Figure 3A). As controls, five other FLAG-tagged tetraspanins (CD9, CD63, CD151, Tspan32 and Tspan9) were chosen because they represent a diverse range of tetraspanins based on sequence identities.22 These did not induce NFAT/AP-1 activation, despite their substantially higher expression than Tspan18 as assessed by anti-FLAG western blotting (Figure 3A).
Despite the fact that the NFAT/AP-1 promoter can be activated by Ca signaling, it is maximally activated by combined Ca signaling and MAPK; Ca activates NFAT and MAPK activates AP-1. To determine whether Tspan18 activates Ca signaling, MAPK or both, Tspan18-transfected DT40 cells were stimulated with the Ca ionophore ionomycin or phorbol ester PMA to activate the MAPK pathway. PMA synergized with Tspan18 expression in activating NFAT/AP-1, but ionomycin did not (Figure 3B). As a positive control, combined PMA and ionomycin induced relatively strong NFAT/AP-1 activation in the presence or absence of Tspan18 (Figure 3B). The capacity of Tspan18 overexpression to induce NFAT/AP-1 activation was not restricted to DT40 B cells, since similar data were obtained in the human Jurkat T-cell line (Figure 3C). Taken together, these data suggest that Tspan18 promotes Ca signaling and NFAT activation via a mechanism that is common to endothelial cells, B cells and T cells.
Tspan18-induced nuclear factor of activated T-cell activation requires functional Orai1 store-operated Ca2+ entry channels
To understand the mechanism by which Tspan18 promotes Ca signaling, a series of NFAT/AP-1 reporter experiments were conducted in gene-knockout DT40 cells and using inhibitors and a dominant-interfering construct. Firstly, Tspan18-induced NFAT/AP-1 activation was found to be independent of the three IP3 receptors (Figure 3D). IP3 receptors release Ca from ER stores in response to tyrosine kinase and G protein-coupled receptor activation, suggesting that Tspan18 does not operate on these pathways or IP3 receptors themselves. However, Tspan18 did not activate NFAT/AP-1 following chelation of extracellular Ca (Figure 3E), or following treatment with the immunosuppressive drug cyclosporin A (Figure 3F), which prevents NFAT translocation to the nucleus by inhibiting its activatory phosphatase calcineurin. These data suggest that Tspan18 might induce Ca entry via the SOCE channel Orai1, a major entry route for extracellular Ca in non-excitable cells.8 Consistent with this possibility, a dominant interfering form of Orai1 (E106Q), which multimerizes with endogenous Orai1 to yield a non-functional channel,4644 inhibited Tspan18-induced NFAT/AP-1 activation (Figure 3G). As a positive control to confirm that downstream NFAT signaling was still intact in the presence of dominant interfering Orai1, its inhibitory effect was overcome by the expression of an active form of calcineurin (Figure 3G). Therefore, Tspan18 may activate Ca entry through the Orai1 SOCE pathway.
Tspan18 interacts with Orai1
To investigate whether Tspan18 interacts with Orai1, transfected epitope-tagged proteins were used because of the lack of effective antibodies to Tspan18. To test for an interaction using co-immunoprecipitation, transfected HEK-293T cells were lyzed in 1% digitonin, a stringent detergent that has been used previously to identify tetraspanin-partner protein interactions.4722 FLAG-tagged Tspan18 co-immunoprecipitated with Myc-tagged Orai1, but five other control tetraspanins did not (Figure 4A). Moreover, Tspan18 and Orai1 co-localized when expressed in HeLa cells, at a level of approximately 90% pixel co-localization when assessed using the Manders’ coefficient (Figure 4B). These data suggest that Tspan18 interacts with Orai1.
Orai1-knockdown endothelial cells have impaired Ca2+ mobilization and Orai1 surface expression requires Tspan18
To determine whether knockdown of Orai1 could phenocopy Tspan18 knockdown, intracellular Ca mobilization was measured following siRNA-mediated knockdown of Orai1. This resulted in impaired Ca mobilization in response to thrombin (Figure 5A) or histamine (Figure 5B). As a control, knockdown of the other Orai family members, Orai2 and Orai3, did not affect Ca mobilization (Figure 5A and B), in agreement with previous studies on Orai proteins in HUVEC.4948 Positive control ionomycin treatment gave a sustained intracellular Ca response in all samples (Figure 5C) and effective knockdown was confirmed by qPCR (Figure 5D-F).
A common mechanism of tetraspanin function is to regulate their partner proteins by facilitating their exit from the endoplasmic reticulum (ER) and trafficking to the cell surface.51501 To determine whether Orai1 localization could be regulated by Tspan18 in this manner, HUVEC were lentivirally transduced with Myc-Orai1 and transfected with control or Tspan18 siRNA duplexes, and Orai1 subcellular localization assessed by confocal microscopy. Orai1 was localized primarily to the cell periphery in control cells, but this was reduced following Tspan18 knockdown, with Orai1 partially co-localized with the ER marker calnexin (Figure 5G). Quantitative analyses showed that approximately 40% of Orai1 was ER-localized in Tspan18 knockdown cells compared to 10-15% in control cells. This partial co-localization could be due to some Orai1 localization in the Golgi and/or trans-Golgi network, as shown by staining close to the nucleus, rather than the more extended perinuclear reticular staining of the ER (Figure 5G). These data support a role for Tspan18 in regulating Orai1 exit from the ER, and/or Golgi, and trafficking to the cell surface.
Tspan18 deficient mice have impaired hemostasis due to a defect in non-hematopoietic cells
To investigate Tspan18 function in vivo, Tspan18-knockout mice were acquired from Genentech/Lexicon Pharmaceuticals. These mice had been generated as part of a library of 472 knockouts,18 but were functionally uncharacterized. Breeding of heterozygotes gave an equal proportion of male and female mice with Mendelian genotype ratios, and the mice bred successfully as homozygote knockouts (data not shown). Furthermore, Tspan18-knockout mice had normal body weights and whole blood cell counts (data not shown).
Tspan18-knockout mice were first evaluated for a hemostasis phenotype using a tail bleed assay. Most Tspan18-knockout mice bled more than wild-type litter-mates, demonstrating a significant disruption to hemostasis (Figure 6A). Some Tspan18-knockout mice did not bleed excessively (Figure 6A), indicating that the bleeding phenotype was variable. This variability could be due to genetic modifier loci, as demonstrated in mice deficient for the platelet collagen/fibrin receptor GPVI.52 To determine whether impaired hemostasis was due to loss of Tspan18 from hematopoietic or non-hematopoietic cells, tail bleeding assays were performed on irradiated fetal liver chimeric mice. These demonstrated that the bleeding phenotype was due to Tspan18 loss from non-hematopoietic cells (Figure 6A), and suggests that a platelet defect is not responsible. Consistent with this, Tspan18-knockout platelets aggregated normally in response to collagen (Figure 6B) or thrombin (Figure 6C). Furthermore, prothrombin time and activated partial thromboplastin time tests showed that coagulation was similar for wild-type and Tspan18-knockout plasma (Figure 6D and E). These data suggest that the impaired hemostasis phenotype is due to a defect in a non-hematopoietic vascular cell type such as the endothelial cell. This did not appear to be due to any observable structural defects in the vasculature, because immunohistochemistry analyses of blood vessels in organs such as kidney and pancreas were similar for wild-type and Tspan18-knockout mice (Figure 6F), as were immunofluorescence analyses of blood vessels in the ear (Figure 6G).
Tspan18 and Orai1 are required for endothelial release of von Willebrand factor in response to inflammatory mediators
Endothelial cell stimulation by inflammatory agonists induces vWF release from Weibel-Palade bodies via a process that involves Ca signaling.14 To investigate whether Tspan18 could be required for vWF release, HUVEC were subjected to Tspan18 knockdown and stimulated in culture medium with thrombin or hista mine. Soluble vWF release, as detected by ELISA, was reduced by approximately 90% compared to control cells (Figure 7A). This was corroborated by immunofluorescent staining of vWF that showed minimal release of Weibel-Palade bodies after Tspan18 knockdown (Figure 7B). Similar to Tspan18, Orai1 knockdown reduced Weibel-Palade body release after thrombin stimulation, but knockdown of Orai2 or Orai3 had no effect (Figure 7C). Furthermore, Tspan18 knockdown reduced platelet adhesion to a thrombin-activated HUVEC monolayer by 85-90% (Figure 7D). These data support a role for Tspan18 and Orai1 in vWF release and platelet capture following endothelial cell activation by inflammatory mediators.
Tspan18-knockout mice have impaired histamine-induced release of endothelial von Willebrand factor and impaired thrombo-inflammatory responses
To determine whether Tspan18 has a role in vWF release in vivo, Tspan18-knockout mice were intra-peritoneally injected with histamine, and plasma vWF levels were analyzed by ELISA. Induced plasma vWF release was reduced by approximately 50% in the absence of Tspan18 (Figure 8A). Basal plasma vWF was normal in Tspan18-knockout mice (Figure 8A), indicating a requirement for Tspan18 in regulated, but not basal, vWF release.
To investigate the role of Tspan18 in thrombosis, two arterial thrombosis models and two thrombo-inflammatory models were used. In a platelet-driven aorta injury arterial thrombosis model,38 no difference in time to complete occlusion of the vessel between Tspan18-knockout and wild-type littermate control mice was observed (Figure 8B). Similarly, there was no thrombosis defect in mesenteric arterioles following application of FeCl3 (Figure 8C), which is also a platelet-driven model,38 but shows reduced platelet deposition and thrombus formation in the complete absence of vWF.5453 In a deep vein thrombosis throm-bo-inflammatory model that is dependent on endothelial vWF,13 thrombus length and weight were reduced by approximately 60% in the Tspan18-knockout mice, compared to wild-type littermate controls (Figure 8D). Moreover, 4 of 9 Tspan18-knockout mice failed to develop a thrombus compared to 100% thrombus formation in wild-type mice (Figure 8D). Macroscopically, thrombi from Tspan18-knockout mice had similar red and white parts to those from wild-type mice (data not shown). Finally, in a vWF-dependent myocardial ischemia-reperfusion thrombo-inflammatory model,5655 platelet deposition and aggregate size in the microcirculation were reduced by approximately 50% (Figure 8E). The reduction in severity in the two thrombo-inflammatory models is consistent with the requirement of Tspan18 for endothelial vWF release in response to inflammatory mediators.
We have discovered that Tspan18 is expressed by endothelial cells and interacts with the SOCE channel Orai1. Tspan18-knockdown endothelial cells had reduced Orai1 expression at the cell surface and impaired Ca signaling. This is consistent with the established role of tetraspanins in interacting with specific partner proteins in the ER, and promoting their trafficking to the cell surface,51501 albeit via mechanisms that are yet to be defined. Tspan18 is not particularly related to any of the other 32 mammalian tetraspanins,22 suggesting that it may be unique amongst tetraspanins in regulating Orai1. Indeed, none of the five tetraspanins that were selected as controls interacted with Orai1, or induced Ca-responsive NFAT activation, when over-expressed.
At the cell surface, tetraspanins can regulate the lateral diffusion and clustering of their partner proteins.32 A question that arises from the present study is whether Tspan18 regulates Orai1 clustering at the endothelial cell surface. Interestingly, a unimolecular coupling model of Orai1 activation was recently proposed, whereby one molecule of a STIM1 dimer is sufficient to induce opening of the Orai1 hexamer channel.10 This would enable the other STIM1 molecule in the dimer to cross-link with a second Orai1 hexamer and form a lattice of clustered Orai1 channels. The degree of cluster formation could dictate the kinetics of channel activation and could concentrate Ca influx to particular regions of the plasma membrane.10 This could affect the extent to which downstream effectors are activated. Tetraspanins have been reported to exist as nanodomains of approximately ten tetraspanins of a single type,57 therefore Tspan18 may cluster Orai1 into pre-formed nanodomains, so modulating Orai1 lattice formation by STIM1. This may provide a means by which endothelial cells fine-tune SOCE and downstream functional responses. It remains to be determined whether Tspan18 also regulates Orai2 and Orai3, but we found no role for these Orai family members in inflammatory mediator-induced HUVEC Ca mobilization, consistent with other studies.4948
The inflammatory mediators thrombin and histamine activate G protein-coupled receptors to induce downstream Ca mobilization and the release of vWF from Weibel-Palade bodies.14 Consistent with impaired Ca signaling, Tspan18-knockout endothelial cells had impaired inflammatory mediator-induced vWF release in vitro and in vivo. In contrast, basal release of vWF appeared to be normal, because basal plasma vWF levels were unaffected in Tspan18-knockout mice. We hypothesize that impaired vWF release, in response to inflammatory mediators, explains the in vivo phenotypes observed in Tspan18-knockout mice. The protection from deep vein thrombosis is consistent with the central role of vWF in this disease.13 Furthermore, the reduced platelet deposition in the microcirculation during myocardial ischemia-reperfusion injury is consistent with the role of vWF in this process.5655 The hemostasis defect still needs to be explained, because although a tail bleeding phenotype has been demonstrated in endothelial-specific vWF-knockout mice, these animals also had low plasma vWF,5958 unlike Tspan18 knockouts. The tail bleeding assay measures blood loss following excision of the tip of the tail, which contains the two lateral veins, the dorsal vein, and the ventral artery. We speculate that endothelial cells in the veins and artery, adjacent to the site of excision, are activated and release vWF via Ca-dependent signaling. The vWF may trap platelets, facilitating their aggregation and preventing excessive blood loss from the site of tail injury. Therefore, our data suggest that acute release of vWF adjacent to a site of injury might be important for hemostasis, at least for some types of injury. Finally, the lack of a phenotype in the two arterial thrombosis models is consistent with the importance of platelets in these models,38 and we found no defect in aggregation in vitro for Tspan18-knockout platelets.
In summary, we have identified Tspan18 as a novel regulator of endothelial Orai1 and SOCE. Our in vivo data show that Tspan18 regulates inflammation-induced vWF release but not basal release, and promotes hemostasis and thrombo-inflammatory processes but not arterial thrombosis.
We are grateful to Carl Blobel, Chris Bunce, Dean Kavanagh, Neil Morgan and Steve Publicover for their helpful comments on this project. We thank the Birmingham Biomedical Sciences Unit for maintaining mice, and the Birmingham Advanced Light Microscope Facility for imaging expertise.
- Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/104/9/1892
- FundingThis work was funded by a British Heart Foundation Project Grant (PG/13/92/30587) which supported PJN, Biotechnology and Biological Sciences Research Council PhD Studentships which supported RLG and EJH, British Heart Foundation PhD Studentships which supported DC, JSR and CZK (FS/05/048, FS/12/79/29871 and FS/18/9/33388), a British Heart Foundation Senior Fellowship (FS/08/062/25797) to MGT which also supported JY, a Biotechnology and Biological Sciences Research Council Project Grant (BB/P00783X/1) which supported NH and a Medical Research Council New Investigator Award (RRAK10717) which supported MGT. SPW is a British Heart Foundation Chair (CH03/003).
- Received March 26, 2018.
- Accepted December 19, 2018.
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