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Introduction

In 2000, Colonna et al. performed a bioinformatics study and identified the CLEC-2 gene Clec1B on chromosome 12 in the Dectin-1 gene cluster. The CLEC-2 gene is located near the genes for six other C-type lectin receptors and was identified as a novel member of the C-type lectin-like superfamily (Colonna et al. 2000). Originally, CLEC-2 mRNA was found in the liver and on some haematopoietic cells, in particular dendritic cells (DCs) and natural killer cells in human and mice (Colonna et al. 2000). Later, in 2006, CLEC-2 mRNA and CLEC-2 protein expression was found to be abundant in the megakaryocyte/platelet lineage, liver sinusoidal endothelial cells and liver Kupffer cells, although expression in liver has yet to be independently confirmed (Chaipan et al. 2006; Suzuki-Inoue et al. 2006; Senis et al. 2007). In 2009, CLEC-2 was further shown to be expressed on subsets of murine myeloid cells and dendritic cells under basal conditions as well as on a variety of leukocyte subsets in response to inflammatory stimuli (Kerrigan et al. 2009; Acton et al. 2012). However, a recent study identified a much more restrictive expression profile, demonstrating CLEC-2 expression to be localised on platelets and on a subset of circulating inflammatory dendritic cells, which was lost upon entry into secondary lymphoid organs (Lowe et al. 2015a, b). The discrepancy in regard to expression to haematopoietic subsets is explained by off-target actions of the available antibodies. Investigation of the function of CLEC-2 in platelets was facilitated by its identification as a receptor for the snake venom rhodocytin using affinity chromatography and mass spectrometry (Suzuki-Inoue et al. 2006). CLEC-2 is a type II membrane protein with an extracellular carbohydrate-like recognition domain, a transmembrane domain and a cytoplasmic tail that is essential for downstream signalling. The CLEC-2 cytosolic tail has a hemi (or half) immunoreceptor tyrosine-based activation motif (ITAM) which consists of a single YXXL sequence which is phosphorylated by Src and Syk tyrosine kinases. CLEC-2 has two well-characterised ligands: an exogenous ligand, rhodocytin, and an endogenous ligand, podoplanin. In addition, it has been shown to be activated by the sulphated sugar, fucoidan, which also activates the platelet collagen receptor GPVI.

Podoplanin is a transmembrane glycoprotein that is widely expressed outside of the vasculature under physiological conditions including on kidney podocytes, alveolar type 1 epithelial cells, lymphatic endothelial cells (LECs), cerebral choroid plexus and many epithelial beds as well as being induced in subsets of haematopoietic cells. The expression of podoplanin on LECs is crucial for prevention of mixing of the blood and lymphatic vasculatures during development through its interaction with platelet CLEC-2 (Bertozzi et al. 2010; Herzog et al. 2013; Lowe et al. 2015a, b). On kidney podocytes, the absence of podoplanin was suggested to impair blood filtration, resulting in the presence of proteins in the urine (Koop et al. 2008). However, with the generation of transgenic mice with targeted deletion of podoplanin on kidney podocytes, the function of podoplanin in the kidney, both physiologically and under inflammatory conditions, is now questionable and requires further investigation (Rayes and Watson, unpublished). Podoplanin has been shown to be upregulated under pathologic conditions, such as inflammation, cancer and autoimmune disease on inflammatory macrophages, tumour cells and T-helper 17 (Th17) cells, respectively, and in development.

The role of podoplanin and CLEC-2 has been studied in a variety of disorders. It is important to note that the beneficial or detrimental effect of the CLEC-2-podoplanin interaction seems to be disease-dependent. Upregulation of podoplanin on macrophages and tumour cells induces platelet aggregation and thrombosis, whereas the expression of podoplanin on TH17 cells alters TH17 cell function and survival and improves recovery in a mouse model of multiple sclerosis (spontaneous experimental autoimmune encephalomyelitis) on a susceptible background.

In this chapter, CLEC-2 signalling pathways and the role of CLEC-2 in developmental pathways and in both thrombotic and non-thrombotic disorders will be discussed.

CLEC-2 Signalling

(Hem)ITAM Receptors in Platelets

As mentioned above, the C-type lectin receptor CLEC-2 is a glycoprotein receptor and a member of the (hem)ITAM family of receptors, which includes other receptors such as the B-cell antigen receptor, the low affinity immune receptor FcγRIIa and the major platelet signalling collagen receptor, GPVI/FcRγ chain (referred to as GPVI). An ITAM consists of two YXXL sequences (single amino acid code) separated by 6–12 amino acids. HemITAM receptors such as CLEC-2 differ as they contain only one YXXL sequence in their intracellular tails (Fuller et al. 2007). CLEC-2 signalling is additionally dependent on a conserved triacidic amino acid sequence immediately upstream of the hemITAM motif, which is also a feature of all but one of the other five known hemITAM-containing receptors (Hughes et al. 2013; Ruckrich and Steinle 2013). CLEC-2 is expressed as a dimer, thus providing two YXXL sequences (Fig. 1).

Fig. 1
figure 1

Graphical representation of the proximal events in CLEC-2 mediated platelet activation. Upon receptor engagement, the hemITAM contained within the intracellular tail of CLEC-2 becomes phosphorylated by Syk and possibly also by Src family kinases. HemITAM phosphorylation allows recruitment of Syk via its SH2 domains, allowing downstream phosphorylation of LAT and the formation of the LAT signalosome. The result of this signalosome formation is the mobilisation of intracellular Ca2+ via recruitment and activation of PLCγ2

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(Hem)ITAM Signalling

Upon receptor engagement via endogenous or exogenous ligands, the tyrosine residues contained within ITAM domains become targets of phosphorylation via Src family kinases (SFKs) such as Src and Lyn (Senis et al. 2014). A key difference between CLEC-2 and traditional ITAM signalling is that the hemITAM motif appears to be more reliant upon spleen tyrosine kinase (Syk) than SFKs for initial phosphorylation, with SFKs being important for activation of Syk and for signalling further downstream (Spalton et al. 2009; Severin et al. 2011). Upon platelet activation by agonists such as rhodocytin or podoplanin, the hemITAM sequence is phosphorylated, allowing recruitment and docking of the tandem SH2 domain-containing kinase Syk. Once recruited to the phosphorylated hemITAM via its SH2 domains, Syk undergoes a conformational change, moving from an auto-inhibited to an active conformation. In this active conformation, Syk undergoes further auto- and trans-phosphorylation. The latter is mediated by SFKs, allowing for full activation of Syk and phosphorylation of several downstream proteins. One of the most important of these is LAT, an adapter protein containing several tyrosine residues that undergo phosphorylation. LAT has no intrinsic activity and serves as the core of the ‘LAT signalosome’, allowing the docking and activation of many of the effectors of this pathway including Grb-2, Gads, SLP-76, PI3-K, Btk and PLCγ2 (Suzuki-Inoue et al. 2006; Fuller et al. 2007). PLCγ2 is generally considered to be the most important, giving rise to formation of inositol 1,4,5-trisphosphate (IP3) and 1,2-dacylglycerol (DAG) which mobilise Ca2+ and activate protein kinase C, respectively. These changes culminate in the exocytosis of intracellular granules, such as α- and dense granules, as well as inside-out activation of platelet integrins including αIIbβ3 (Hughes et al. 2015; Zheng et al. 2015). The release of ADP from dense granules and formation of thromboxane A2, made de novo from membrane phospholipids, play critical positive feedback roles in activation of CLEC-2 in human platelets (Pollitt et al. 2010). CLEC-2 activation in human is also dependent on the Rho GTPase, Rac (Pollitt et al. 2010). This contrasts with activation by the collagen ITAM receptor GPVI which shows a reduced rather than complete dependency on the role of the two feedback messengers and Rac.

Functional Roles of CLEC-2 Signalling

Platelet activation and aggregation is a multistep process that involves different receptors and signalling pathways with the aim of prevention of excessive blood loss following vessel injury. CLEC-2 is expressed on platelets with an average copy number of 2000 receptor per platelet. Several studies have investigated a possible role of CLEC-2 in haemostasis and thrombus formation using various mouse models. CLEC-2-deficient platelets aggregate normally in response to classical platelet agonists but not to rhodocytin. Moreover, CLEC-2-deficient platelets have normal adhesion and spreading on extracellular matrices including collagen, laminin, von Willebrand factor and fibrinogen (Suzuki-Inoue et al. 2010). Several groups have investigated the consequence of CLEC-2 deficiency in a tail bleeding assay (haemostasis) or laser, photochemical or FeCl3 thrombosis model with contrasting results. Using an anti-CLEC-2 antibody to deplete CLEC-2 from the platelet surface in vivo, May et al. (2009) showed a prolonged bleeding time and profound defect in arterial thrombus formation following FeCl3 injury (May et al. 2009). Using irradiated mice reconstituted with Clec-2−/− foetal liver, Suzuki-Inoue et al. (2010) showed a reduction in thrombus formation following photochemical injury but no significant change in tail bleeding times (Suzuki-Inoue et al. 2010). In contrast, Hughes et al. (2010) did not observe a significant change in tail bleeding time or aggregation on collagen at high shear using radiation chimeric mice reconstituted with CLEC-2-deficient bone marrow, and additionally were unable to demonstrate phosphorylation of CLEC-2 under flow conditions, providing indirect evidence that it is not activated (Hughes et al. 2010). The reason for the discrepancy between the various studies remains unclear. A recent study showed a profound defect in thrombus formation in mice double deficient in GPVI and CLEC-2 suggesting possible compensatory roles of the two (hem)ITAM receptors (Bender et al. 2013), although this which must involve adhesion and signalling as a similar result is not seen in mice deficient in Syk. Overall the consensus is that CLEC-2 has a minor role (at best) in haemostasis, consistent with the absence of an identified ligand in the vasculature, and that its primary role is to support many of the non-canonical roles for platelets that are beginning to emerge.

CLEC-2 in Development

Blood-Lymphatic Vascular Separation Defect in CLEC-2-Deficient Mice

Two decades ago, mice deficient in the downstream CLEC-2 signalling molecule, Syk, were shown to develop subcutaneous haemorrhages in the skin and oedema at mid-gestation (Cheng et al. 1995; Turner et al. 1995). A similar phenotype was observed in mice deficient in the adaptor protein SLP-76 (Clements et al. 1998; Pivniouk et al. 1998; Clements et al. 1999) or PLC-γ2 (Wang et al. 2000). The development of haemorrhages and oedema was later shown to result from aberrant connections between the blood and lymphatic vasculatures causing the appearance of blood-filled lymphatic vessels and impaired lymphatic drainage, respectively (Abtahian et al. 2003; Ichise et al. 2009). Support for the involvement of CLEC-2 in this phenotype came following the characterisation of mice deficient in its endogenous ligand, podoplanin, which were also shown to harbour blood-filled lymphatic vessels in the skin and gut at birth (Schacht et al. 2003), a phenotype later identified in CLEC-2-deficient embryos in mid-gestation (Bertozzi et al. 2010; Suzuki-Inoue et al. 2010; Finney et al. 2012). Both CLEC-2- and podoplanin-deficient mice experience almost 100 % perinatal mortality that is thought to be caused by defective lymphatic function and fluid retention leading to a failure to inflate the lungs (Schacht et al. 2003; Finney et al. 2012). Furthermore, the mice that survived for sufficient time to suckle developed chylous ascites, further indicative of impaired lymphatic function (Abtahian et al. 2003; Finney et al. 2012; Hess et al. 2014).

Identifying the Causative CLEC-2-Expressing Cell That Underlies the Defects in Lymphatic Development

The mechanism by which podoplanin and CLEC-2 prevent the mixing of the blood and lymphatic vasculatures during development remains controversial. A role for podoplanin specifically on the lymphatic endothelium has been confirmed by the presence of blood-filled lymphatic vessels in mice with a Tie-2-Cre-mediated deletion of podoplanin or the glycosyltransferase enzyme, T-synthase, which is important for the glycosylation of the podoplanin extracellular domain (Fu et al. 2008; Herzog et al. 2013). The interacting CLEC-2-expressing cell type has been investigated through a series of lineage tracing studies that initially pinpointed Syk/SLP-76 activity in a circulating endothelial progenitor (Abtahian et al. 2003; Sebzda et al. 2006) but which was later shown to be a circulating cell of the myeloid lineage (Bohmer et al. 2010). Using a series of transgenic mice, a role for Syk in macrophages, T- and B-lymphocytes, was eliminated, while the loss of CLEC-2, Syk or SLP-76 in the megakaryocyte/platelet lineage (generated by crossing floxed homozygotes to a PF4-Cre transgenic) was shown to be indispensable for blood-lymphatic vascular separation (Bertozzi et al. 2010; Finney et al. 2012).

A role for CLEC-2 on platelets/megakaryocytes is supported by the presence of blood-filled lymphatic vessels following the selective ablation of this lineage using diphtheria toxin (again achieved using a PF4-Cre transgenic), through treatment with aspirin in utero or in mice that lack functional megakaryocytes due to a deficiency in the transcription factor Meis-1 (Carramolino et al. 2010; Uhrin et al. 2010). Furthermore, the contribution of platelet-specific effects over megakaryocytes is supported by the development of blood-filled intestinal lymphatic vessels in irradiated mice reconstituted with Syk-, SLP-76-, PLCγ2- or CLEC-2-deficient bone marrow, where the number of circulating megakaryocytes is negligible (Ichise et al. 2009; Bertozzi et al. 2010; Finney et al. 2012).

Unravelling the Molecular Mechanism of the Impairment in Lymphatic Development

Initially it was proposed that a direct interaction between platelets and LECs at the cardinal vein was critical to ‘clot off’ the primary budding lymphatic structures and prevent the formation of blood-lymphatic vascular connections (Bertozzi et al. 2010; Uhrin et al. 2010; Hess et al. 2014). However, high-resolution microscopy studies of developing embryos have failed to identify any connection between the blood and lymphatic vasculatures at this stage (Hagerling et al. 2013). Furthermore, blood-filled lymphatic vessels can be seen in irradiated adult mice reconstituted with CLEC-2- or Syk-deficient bone marrow where the lymphatic system is already established (Bertozzi et al. 2010; Finney et al. 2012). It was recently proposed that a thrombus, formed through podoplanin-induced CLEC-2 activation on platelets, forms at the joining of the thoracic duct and the subclavian vein to prevent backflow of blood into the lymphatic system during development and throughout adulthood (Hess et al. 2014). However, an important role for thrombus formation is disputed by the absence of a blood-lymphatic vascular separation defect in mice lacking the major platelet integrin αIIbβ3 (Uhrin et al. 2010).

It has also been proposed that one or more of the bioactive molecules or growth factors that are abundant in platelet dense, α- and lysosomal secretory granules and released upon their activation could contribute towards influencing lymphatic endothelial cell behaviour. So far, there has been little evidence to support this mechanism since patients or mice with defective dense- or α-granule secretion syndromes known as Hermansky-Pudlak syndrome or grey platelet syndrome, respectively, have not been described to exhibit lymphatic defects (e.g. Deppermann et al. 2013). Platelets additionally store large concentrations of the bioactive lipid sphingosine-1-phosphate (S1-P), which have been shown to have potent effects on endothelial cell migration, but while the loss of S1-P in mice is associated with widespread haemorrhaging and embryonic lethality, no phenotype of blood-lymphatic mixing was observed (Liu et al. 2000).

Other potential mechanisms include the direct interaction between platelet-CLEC-2 and podoplanin on LECs, which has been shown to inhibit LEC migration (Finney et al. 2012). It is proposed that these inhibitory signals are critical to arrest the growth of budding lymphatic vessels and prevent the formation of aberrant connections (anastomoses) with the blood vascular endothelium (Finney et al. 2012). The ability of CLEC-2 to cluster its receptor podoplanin on the surface of LECs was later demonstrated. This interaction supports platelet adhesion and is also proposed to inhibit signalling by podoplanin in LECs as shown in other cell types (Pollitt et al. 2014). The presence of anastomoses between veins and lymphatic veins has been described by many groups, including direct visualisation of mixing of the two vasculatures in mice with a platelet-specific loss of functional Syk (Hughes et al. 2015).

Other Developmental Functions of CLEC-2

Podoplanin was identified on fibroblastic reticular cells (FRCs) of the spleen over two decades ago and has since been shown to have several important roles in the organisation and function of lymphoid compartments (Farr et al. 1992; Yu et al. 2007). Recently, a number of these functions have been linked to CLEC-2 including trafficking of CLEC-2-expressing DCs to lymph nodes which is dependent on podoplanin expression on lymphatic endothelium and on FRCs (Acton et al. 2012). In addition, CLEC-2 has been shown to play a role in maintaining the integrity of high-endothelial venules within lymph nodes through its interaction with podoplanin on FRCs (Herzog et al. 2013). In this study, mice with a platelet/megakaryocyte-specific loss of CLEC-2 (i.e. PF4-Cre transgenic CLEC-2fl/fl) developed blood-filled lymph nodes, and while this does not influence the primary immune response, it impairs the regulation of acquired immune responses (Herzog et al. 2013; Benezech et al. 2014). A much more severe phenotype is observed following constitutive loss of CLEC-2 or podoplanin where pups are born with a complete absence of mesenteric and inguinal lymph nodes due to a defect in lymph node maturation during late embryogenesis (Peters et al. 2011; Benezech et al. 2014). Significantly, lymph nodes are present in PF4-CLEC-2-deficient mice indicating that the developmental defect is not due to loss of platelet activation. This suggests a potential role for CLEC-2 outside of the platelet lineage during development and a second critical role for platelet-derived CLEC-2 in maintaining lymph node vascular integrity post-development.

A role for CLEC-2 in maintaining vascular integrity has been described in the developing brain. Podoplanin expression was shown to be restricted to the choroid plexus and ependymal lining of the ventricles at E16.5, but has since been seen to be widely expressed throughout the neuroepithelium between E10.5 and E12.5 (Williams et al. 1996; Schacht et al. 2003; Lowe et al. 2015a, b). Haemorrhages were first reported in the midbrain parenchyma of CLEC-2-deficient embryos at E12.0 (Tang et al. 2010). While severe cerebral haemorrhaging had been described in T-synthase-deficient embryos at E12.0, only recently were podoplanin-deficient embryos characterised and shown to exhibit extensive cerebral haemorrhaging between E11.5 and E12.5 (Lowe et al. 2015). Developing cerebral blood vessels in CLEC-2- and podoplanin-deficient mice were visibly tortuous and prone to haemorrhage at E10.5 caused by aberrant associations between the endothelium and the surrounding mural cells. CLEC-2-induced platelet aggregation is implicated in the haemorrhaging as bleeding is also seen in αIIb-deficient embryos. Thus, activation of platelets by podoplanin-CLEC-2 leads to platelet aggregation and sealing of gaps in developing vessels. It remains unclear whether this also contributes to the altered patterning of vessels that is seen before the onset of haemorrhage.

CLEC-2- and podoplanin-deficient mice experience respiratory failure at birth due to the inability to inflate their lungs (Ramirez et al. 2003; Finney et al. 2012). This is likely secondary to impairment in lymphatic function leading to fluid retention. In addition, it has been speculated that this may also be due to loss of podoplanin on alveolar type I cells, although as yet there is no direct evidence for this (Millien et al. 2006).

Podoplanin-deficient mice have been reported to exhibit myocardial pathology, where hypoplasia in the myocardium is proposed to result from impaired development of the epicardium (Mahtab et al. 2008). However, no such phenotype has been reported in CLEC-2-deficient mice, suggesting podoplanin plays a functional role independent of CLEC-2 in cardiac development.

CLEC-2 Beyond Haemostasis: The Beneficial and Detrimental Role of the Interaction of CLEC-2 and Podoplanin

In the last few years, increasing evidence for a role of platelets in the regulation of inflammation, infection and cancer has emerged in human disease and mouse models. Platelets are now recognised as having multiple roles in inflammation and infection, including the ability to regulate leukocyte functions, such as secretion of granular contents and production of reactive oxygen species (ROS) from neutrophils (Gros et al. 2015), while the formation of a thrombus inside a blood vessel supports innate immunity through pathogen recognition and containment (Engelmann and Massberg 2013). Many of these new roles for platelets have been shown to involve CLEC-2 and podoplanin (Fig. 2).

Fig. 2
figure 2

Possible beneficial and detrimental roles of platelet CLEC-2/podoplanin interactions in normal physiology and in disease

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The Role of CLEC-2-Podoplanin in Inflammatory Diseases

Multiple studies have shown the selective role of CLEC-2-podoplanin interaction in inflammatory and non-inflammatory conditions. The beneficial or detrimental effect depends largely on the inflammatory state and the nature of the platelet-leukocyte interaction. Indeed, in a mouse model of multiple sclerosis (MS), an autoimmune inflammatory disorder of the central nervous system (CNS), podoplanin expression on a subset of CD4+ cells, TH17 cells, alters TH17 expansion and survival resulting in an increased rate of resolution of the inflammation (Peters et al. 2015). This beneficial effect of podoplanin was abrogated by a global deletion of CLEC-2 showing that the interaction between CLEC-2 and podoplanin mediates the resolution of the inflammation. However, whether CLEC-2 on platelets, DCs or possibly B-cells (where expression is controversial) contributes to this effect remains unclear. Significantly, upregulation of podoplanin was also observed in human patients with active MS plaque but not in glioblastoma multiforme (GBM), an aggressive malignant primary brain tumour (Nylander et al. 2015) indicating this upregulation is disease-specific. Thus, podoplanin-CLEC-2 interaction seems to modulate the inflammatory response in MS infiltrates, and this effect depends on the presence of a specific microenvironment.

It has been suggested that platelets contribute to several stages of atherosclerosis, including initiation, lesion growth and thrombus formation upon plaque disruption. Interestingly, the level of expression of podoplanin on smooth muscle cells and macrophages in atherosclerotic lesions from human patients correlates with the severity of the lesion, although the significance of this remains to be determined (Hatakeyama et al. 2012).

CLEC-2 expression has been reported in synovium of patients with rheumatoid arthritis (RA), a chronic systemic inflammatory disease leading to progressive destruction of the articular cartilage and bone (Del Rey et al. 2014). Importantly, the interaction of synovial fibroblasts with platelet CLEC-2 has been shown to lead to an increase in the proinflammatory cytokines IL6 and IL8 (Del Rey et al. 2014). CLEC-2 expression on microvesicles is also increased in RA patients (Gitz et al. 2014). It has been speculated that the abundant levels of microvesicles in RA may induce potent pro-inflammatory effects on the synovial fluid (Boilard et al. 2010; Del Rey et al. 2014). Moreover, podoplanin expression is increased in areas of inflammation, and synovial fibroblast activation and synovial transformation (Ekwall et al. 2011; Miyamoto et al. 2013; Del Rey et al. 2014).

The Role of CLEC-2 and Podoplanin in Infection

There is increasing recognition of the importance of platelets in bacterial and viral infections (Cox et al. 2011; Chabert et al. 2015). Severe and sometimes life-threatening bleeding can occur in certain viral infections, with thrombocytopenia often arising from viral-induced platelet destruction, consumption or sequestration. Platelet-viral interactions are complex and will depend on the species of the virus and/or the platelet environment. One of the most important viral agents that CLEC-2 associates with is the human immunodeficiency virus (HIV-type 1). Zucker-Franklin et al. (1990) were the first group to visualise HIV internalisation by megakaryocytes and platelets (Zucker-Franklin et al. 1990). HIV-1-infected cells release HIV-1 virions expressing podoplanin on their capsules that allow HIV-1 to interact with CLEC-2 and DC-SIGN on platelets. This interaction mediates HIV-1 capture and facilitates the spread of infection (Chaipan et al. 2006; Chaipan et al. 2010). Encouragingly, a CLEC-2-specific antiserum reduced HIV-1 transmission by platelets by approximately 50 % (Chaipan et al. 2006). Podoplanin depletion only diminished CLEC-2-dependent HIV transmission in B-THP cells, a Raji B-cell line, suggesting that podoplanin incorporation for HIV spread is dependent on podoplanin expression in the targeted cell for HIV infection (Chaipan et al. 2010). Therefore, incorporation of a second, as yet unrecognised, CLEC-2 ligand may be responsible for CLEC-2-dependent capture of T-cell-derived and peripheral blood mononuclear cell-derived viruses, facilitating CLEC-2-driven HIV transmission (Ozaki et al. 2009; Chaipan et al. 2010; Lowe et al. 2015a, b).

In a mouse model of salmonella infection, Hitchcock et al. (2015) have recently reported that upregulation of podoplanin in the liver leads to an inflammation-driven occlusive thrombosis. Significantly, the venous thrombosis was substantially abrogated in the absence of CLEC-2 on platelets or in the absence of macrophages without affecting the time course of the bacteraemia. The upregulation of podoplanin in the liver is driven by a TLR4- and INFγ-dependent inflammation. Given that CLEC-2 does not have a major role in haemostasis, this highlights the C-type lectin-like receptor as a novel target in some forms of thromboinflammatory disease.

The Role of CLEC-2 and Podoplanin in Cancer

A potential role of podoplanin in cancer metastasis has also emerged. Podoplanin is upregulated in a wide range of cancer types such as colorectal adenocarcinoma (Kato et al. 2003), testicular germ cell tumours (Kato et al. 2004), squamous cell carcinomas of the lung, cervix, oral cavity, larynx and mesothelioma and in tumours of the central nervous system (Kato et al. 2005; Kimura and Kimura 2005; Schacht et al. 2005; Shibahara et al. 2006; Rodrigo et al. 2010). Tumour cells can interact with platelets to form heterogeneous aggregates which protect the invasive cells from shear stress and immunological assault (Gay and Felding-Habermann 2011). It is speculated that the coating of platelets allows the tumour cells to evade the immune system as well as supporting adherence to the vessel wall. The antihuman podoplanin antibody (MS-1 mAb) has been shown to reduce platelet aggregation in vitro and pulmonary metastasis in vivo (Takagi et al. 2013). This highlights podoplanin as a promising target for developing novel antitumour and anti-metastatic agents.

Take-Home Messages

  • CLEC-2 interaction with podoplanin induces platelet activation and aggregation.

  • During development, CLEC-2-podopanin interaction is crucial for blood/lymphatic vessel separation.

  • Post-development, CLEC-2-podoplanin interaction maintains the integrity of high endothelial venules in lymph nodes.

  • CLEC-2-podoplanin is not crucial for haemostasis.

  • Detrimental role of CLEC-2-podoplanin: HIV propagation, salmonella-induced liver thrombosis and cancer metastasis.

  • Beneficial role of CLEC-2-podoplanin inhibition of T-cell expansion and survival in multiple sclerosis.

Conclusion

Although CLEC-2 and podoplanin have been intensively studied in the last decade, many questions concerning their roles remain unanswered, most notably the roles outside of the vasculature. Anti-CLEC-2 or anti-podoplanin drugs may have a beneficial effect in many diseases, in particular in inflammatory disorders. Additional studies are required to fully understand the mechanism underlying the role of CLEC-2 and podoplanin in physiology and pathology.