CLEC-2 is a newly characterized C-type lectin-like receptor, which has been shown to mediate platelet activation and aggregation upon binding to rhodocytin, a snake venom protein [1
]. CLEC-2 has also been shown to enhance infectivity of HIV-1 produced in 293T cells [5
]. This effect was not mediated by the viral envelope protein implying that a protein from the 293T cells was captured during viral budding and was responsible for the interaction of the virus with CLEC-2. These data suggested the presence of an endogenous, surface-bound ligand for CLEC-2 on 293T cells. We used recombinant CLEC-2 protein to confirm that 293T cells express a ligand for the receptor. This recombinant CLEC-2 was used to pull down potential ligand molecules from cell lysates and identified a candidate ligand of 36kDa that corresponds in size to podoplanin. We have shown that 293T and other CLEC-2 binding cells used in this study express podoplanin and that anti-podoplanin antibody inhibits both binding to and activation of CLEC-2 by 293T cells. A direct interaction between podoplanin and CLEC-2 was formally demonstrated by co-immunoprecipitation and surface plasmon resonance experiments, which determined the affinity to be 24.5μM.
These results are consistent with a report that certain cancer cells express podoplanin on their surface, which allows them to interact with CLEC-2 [13
]. The study by Suzuki-Inoue et al sought to identify a receptor for podoplanin and demonstrated that Src inhibitors blocked podoplanin-induced signalling through CLEC-2. Our study defines the signalling in CLEC-2 that is triggered by podoplanin and demonstrates that it is mediated through the YXXL motif in the cytoplasmic domain of CLEC-2. We have further demonstrated that glycosylation of CLEC-2 is not required for this interaction. There is good evidence that HIV infects renal cells, that these cells can produce infective HIV virions and that renal cells express podoplanin which may be incorporated into the budding virion [6
]. The present study has demonstrated that podoplanin binds directly to CLEC-2 with an affinity in the micromolar range, so inhibitors of this interaction may help prevent dissemination of HIV-1 by platelets.
Podoplanin was originally identified on a range of cells including airway epithelia, fibroblasts, keratinocytes, osteoblasts and renal tubular epithelial cells [17
]. Podoplanin is also expressed at high levels on lymphatic endothelial cells [21
] and on certain tumour cells, where it has been shown to activate platelets [16
]. In the renal glomerulus, podoplanin helps maintain the structure of podocytes, required for efficient glomerular filtration [15
]. Podocytes are not normally in contact with blood, but acute renal damage could expose them to blood, allowing interaction between CLEC-2 on platelets and podoplanin on podocytes. Physiologically, this would trigger platelet activation and formation of a haemostatic plug, thereby preventing further leakage into the collecting ducts, and possibly helping in renal repair through release of growth factors. However, this could also trigger intrarenal and especially intraglomerular thrombosis, which can be observed in renal diseases when there is renal epithelial damage, including during renal transplant rejection.
Inhibitors of the podoplanin-CLEC-2 interaction have potential use as novel anti-platelet agents. The significance of the interaction of podoplanin and CLEC-2 is unclear as the majority of cells that express podoplanin, with the exception of tumour cells undergoing metastasis, do not normally contact platelets. As with certain other C-type lectin-like receptors, it is possible that the function of CLEC-2 is primarily in defence, and specifically in this to prevent excessive blood loss and to respond to infection following contact with podoplanin. Further studies will provide information on the physiological and pathophysiological significance of the interaction of podoplanin and CLEC-2.