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

Ca2+ 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.


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
Ferrata Storti Foundation C a 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. Tspan18knockout 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. Histamineor 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 Tspan18knockout 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 the Ca 2+ channel Orai1 and von Willebrand factor release in endothelial cells Introduction
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. [1][2][3] 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. 5,6 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. 5,6 The function of Tspan18 in humans or mice has still not been studied.
Store-operated Ca 2+ entry (SOCE) through the plasma membrane Ca 2+ 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 2+ from endoplasmic reticulum (ER) stores via IP3 receptor channels. 8 Depletion of Ca 2+ is detected by the ER-resident dimeric Ca 2+ -sensor protein STIM1, which then undergoes a conformational change and interacts with Orai1 hexamers in the plasma membrane. 9,10 STIM1 binding induces Orai1 channel opening and clustering via a mechanism that is not fully understood, allowing Ca 2+ entry across the plasma membrane. 9,10 The resulting increase in intracellular Ca 2+ 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 thromboinflammation, in which inflammatory cells contribute to thrombosis. 11,12 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 2+ signaling. 13,14 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. [15][16][17] 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, Tspan18deficient endothelial cells have impaired Ca 2+ 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.

Ethics statement
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).

Mice
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

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.

Lentiviral transduction
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

Co-immunoprecipitation
A detailed description of co-immunoprecipitation from transfected HEK-293T cell lysates 22 is provided in the Online Supplementary Appendix.

Immunofluorescence microscopy
Detailed information is provided in the Online Supplementary Appendix. In brief, cells were prepared as described 29 and the Manders' coefficients (M1 and M2) were used as the co-localization measure. 32 Ear vasculature was imaged and quantified as described. 33,34 Immunohistochemistry Immunohistochemistry was as described; 35 details are available in the Online Supplementary Appendix.

Intracellular Ca 2+
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; 36,37 detailed information is provided in the Online Supplementary Appendix.

In vivo assays
Mouse models were as described; 13,37-39 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 data 40 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 brain 41 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.proteinatlas.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 Ca 2+ 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 2+ 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 2+ 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 Ca 2+ -responsive nuclear factor of activated T-cell signaling in lymphocyte cell lines
To investigate the mechanism by which Tspan18 regulates Ca 2+ 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 2+ signaling downstream of transfected membrane proteins. 21,43 Transfection of a FLAG epitope-tagged Tspan18 expression construct was sufficient to induce robust NFAT/AP-1 activation ( Figure 3A). As controls, five other FLAGtagged 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 2+ signaling, it is maximally activated by combined Ca 2+ signaling and MAPK; Ca 2+ activates NFAT and MAPK activates AP-1. To determine whether Tspan18 activates Ca 2+ signaling, MAPK or both, Tspan18-transfected DT40 cells were stimulated with the Ca 2+ 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 Tcell line ( Figure 3C). Taken together, these data suggest that Tspan18 promotes Ca 2+ 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 Ca 2+ entry channels
To understand the mechanism by which Tspan18 promotes Ca 2+ 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 2+ 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 2+ ( 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 2+ entry via the SOCE channel Orai1, a major entry route for extracellular Ca 2+ 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, [44][45][46] 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 2+ 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

A B C D
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. 22,47 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 Ca 2+ mobilization and Orai1 surface expression requires Tspan18
To determine whether knockdown of Orai1 could phenocopy Tspan18 knockdown, intracellular Ca 2+ mobilization was measured following siRNA-mediated knockdown of Orai1. This resulted in impaired Ca 2+ 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 2+ mobilization ( Figure 5A and B), in agreement with previous studies on Orai proteins in HUVEC. 48,49 Positive control ionomycin treatment gave a sustained intracellular Ca 2+ 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. 1,50,51 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

A B
C D E F G 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, Tspan18knockout 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 littermates, 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 nonhematopoietic 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 nonhematopoietic 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 2+ 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-Tspan18 regulates Orai1 in endothelial cells haematologica | 2019; 104(9) 1899  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.

P.J. Noy et al.
1900 haematologica | 2019; 104(9) Figure 6. Tspan18-knockout mice have a hemostasis defect due to the absence of Tspan18 expression by non-hematopoietic cells. (A) Tail bleeding assays were performed by amputating 3 mm tail tips of anesthetized mice and the weight of blood lost was measured. The mice were Tspan18 +/+ , Tspan18 -/-, or lethally irradiated Tspan18 -/-or Tspan18 +/+ mice reconstituted with fetal liver cells from Tspan18 +/+ or Tspan18 -/-embryos. Each symbol represents one animal. All data were analyzed by Fisher's exact test. *P<0.05; ***P<0.001. Note that bleeding was stopped by cauterizing the tails of some mice, because of regulations limiting the amount of blood loss on our Home Office License. (B and C) Washed platelets from Tspan18 +/+ or Tspan18 -/-mice were activated with 3 μg/mL collagen (B) or 0.3 U/mL thrombin (C), and aggregation was measured by light transmission with stirring. Quantitated percentage aggregation each minute is shown. Error bars represent the Standard Error of Mean (SEM) from at least three pairs of litter-matched mice. (D and E) Plasma samples from Tspan18 +/+ and Tspan18 -/-mice were subjected to a prothrombin time test with human placental thromboplastin (D) and an activated partial thromboplastin time test with purified soy phosphatides with ellagic acid (E). Error bars represent SEM from four pairs of litter-matched mice. (F) Immunohistochemistry was used to show a grossly normal vasculature in Tspan18 +/+ and Tspan18 -/-mice formalin-fixed paraffin-embedded 5 μm sections from kidney and pancreas, using the MECA32 anti-mouse panendothelial cell antibody. Images are representative of three pairs of litter-matched mice. (G) Confocal microscopy was used to show a grossly normal vasculature in Tspan18 +/+ and Tspan18 -/-mice ears, by staining anterior ear tissue with biotinylated isolectin GS-IB4 glycoprotein followed by Alexa647-conjugated streptavidin. Images are representative of three pairs of litter-matched mice. ImageJ quantitation of 3 fields of view (500 x 500 pixels) per mouse showed a mean total vessel length of 11131±1271 pixels for Tspan18 +/+ and 11684±283 pixels for Tspan18 -/-(n=3; error represents SEM). mins: minutes. Tspan18 +/+ and Tspan18 -/-mice were anesthetized, and the abdominal aorta exposed and mechanically injured through a single firm compression with forceps. Blood flow was subsequently monitored with a Doppler flowmeter to calculate the time until complete occlusion of the vessel. Each symbol represents one animal. (C) Tspan18 +/+ and Tspan18 -/-mice were anesthetized and the mesentery was exteriorized through an abdominal incision. Platelets were fluorescently labeled with Dylight 488-conjugated anti-GPIX derivative. Small mesenteric arterioles were exposed to FeCl 3 -induced chemical injury via topical application. Time to appearance of the first thrombi was recorded (left), and the time until complete occlusion of the vessel was measured using fluorescence intravital microscopy (right). Each symbol represents one animal. (D) Tspan18 +/+ and Tspan18 -/-mice were anesthetized and surgery performed to stenose the inferior vena cava. After 48 hours (h), thrombus length (left) and weight (right) were measured. Each symbol represents one animal and horizontal bars represent the median. *P<0.05. All data in (A-D) were analyzed by one-way ANOVA with Dunnett's multiple comparisons test. (E) Myocardial ischemia-reperfusion injury was induced in the left ventricle of the beating heart of anesthetized mice by occluding the left anterior descending artery for 45 min with a suture. Reperfusion was instigated for 2 h by removal of the ligature, after which the organ was harvested. Frozen sections were analyzed for the presence of platelets in the microcirculation by immunofluorescence microscopy. Three litter-matched pairs of Tspan18 +/+ and Tspan18 -/-mice were used, with three sections per mouse and five images analyzed per section. Each symbol represents one image. For each image, the integrated density value was calculated as a representation of the total number of platelets (left), and the average aggregate size was also calculated (right). Error bars represent the SEM and data were analyzed by Mann-Whitney test. ****P<0.0001.

A B
C D E

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 FeCl 3 ( Figure 8C), which is also a platelet-driven model, 38 but shows reduced platelet deposition and thrombus formation in the complete absence of vWF. 53,54 In a deep vein thrombosis thrombo-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, 55,56 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.

Discussion
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 2+ 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, 1,50,51 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 2+ -responsive NFAT activation, when over-expressed.
At the cell surface, tetraspanins can regulate the lateral diffusion and clustering of their partner proteins. 2,3 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 2+ 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 2+ mobilization, consistent with other studies. 48,49 The inflammatory mediators thrombin and histamine activate G protein-coupled receptors to induce downstream Ca 2+ mobilization and the release of vWF from Weibel-Palade bodies. 14 Consistent with impaired Ca 2+ 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. 55,56 The hemostasis defect still needs to be explained, because although a tail bleeding phenotype has been demonstrated in endothelial-specific vWFknockout mice, these animals also had low plasma vWF, 58,59 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 2+ -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.