Molecular analysis of the pathophysiologicalbinding of the platelet aggregation-inducing factor
podoplanin to the C-type lectin-like receptor CLEC-2
Blackwell Publishing Asia

Yukinari Kato,1,6 Mika Kato Kaneko,1,6 Akiko Kunita,2 Hiromi Ito,1 Akihiko Kameyama,1 Satoshi Ogasawara,1Nana Matsuura,1 Yasushi Hasegawa,1,3 Katsue Suzuki-Inoue,4 Osamu Inoue,4 Yukio Ozaki4 and Hisashi Narimatsu1,51Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology, Open Space Laboratory C-2, 1-1-1 Umezono,
Tsukuba, Ibaraki 305-8568; 2Department of Pathology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033; 3Department ofSurgery, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582; 4Department of Clinical and Laboratory Medicine, Faculty of
Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898 Japan(Received August 5, 2007/Revised September 4, 2007/2nd Revision September 5, 2007/Accepted September 6, 2007/Online publication October 18, 2007)

The mucin-type sialoglycoprotein podoplanin (aggrus) is involved intumor cell-induced platelet aggregation and tumor metastasis.
C-type lectin-like receptor-2 (CLEC-2) was recently identified as an
endogenous receptor of podoplanin on platelets. However, thepathophysiological importance and function of CLEC-2 have notbeen elucidated. Here we clarified the pathophysiological interaction between podoplanin and CLEC-2 in vitro and in vivo. Using
several deletion mutants of CLEC-2 expressed as Fc chimeras,
we first identified an important podoplanin-recognition domain in
CLEC-2. Furthermore, the podoplanin–CLEC-2 interaction was
confirmed using several deletion mutants of podoplanin expressedas Fc chimeras. Not only the disialyl-core1-attached glycopeptide
but also the stereostructure of the podoplanin protein was found tobe critical for the CLEC-2-binding activity of podoplanin. We next
synthesized various glycopeptides of podoplanin that included boththe platelet aggregation-stimulating domain and O-glycan on Thr52.
Interestingly, a disialyl-core1-attached glycopeptide was recognizedspecifically by CLEC-2. Moreover, the anti-podoplanin monoclonal
antibody NZ-1 suppressed both the podoplanin–CLEC-2 interaction
and podoplanin-induced pulmonary metastasis, suggesting that
CLEC-2 is the first pathophysiological receptor of podoplanin tobe identified. In summary, we clarified the molecular interaction
in vitro and in vivo between a platelet aggregation-inducing factor,
podoplanin, and its specific pathophysiological receptor on platelets,
CLEC-2. Podoplanin and CLEC-2 might represent promising therapeutictargets in cancer metastasis. (Cancer Sci 2008; 99: 54–61)

Tumor metastasis is known to be associated with a plateletaggregating activity possessed by human and othermammalian cancers, implying that aggregation is importantin tumor-cell nestling, whereas released growth factors areimportant in angiogenesis or tumor growth.(1) A previous study
has clarified that podoplanin (also known as aggrus), a
membranous sialoglycoprotein in cancer cells of mice (44 kDa)
and humans (36 kDa), aggregates platelets with no relation
to plasma components. (2) Podoplanin is also known to be alymphatic-specific marker,(3) and its expression has beenreported in several tumor cells, including squamous cell
carcinoma,(4 – 6) mesothelioma,(7) testicular seminoma,(8) and brain
tumors,(9,10) although podoplanin is not expressed in gastrointestinal,
pulmonary, or mammary adenocarcinomas, which frequently
undergo metastasis.(7) Recent investigations have reinforced
the notion that the expression of podoplanin in several
tumors is associated with tumor metastasis or progression. (6,10,11)
Podoplanin belongs to the family of type-I transmembranesialomucin-like glycoproteins that comprise an extracellular domain
with abundant Ser and Thr residues as potential O-glycosylationsites, a single transmembrane portion, and a short cytoplasmic
Cancer Sci | January 2008 | vol. 99 | no. 1 | 54–61

tail with putative sites for protein kinase C and cAMP
phosphorylation.(12)We previously showed that the EDxxVTPG segment in theextracellular domain, designated as the platelet aggregationstimulating (PLAG) domain, is critical for the activity of
podoplanin.(2) In particular, this motif, which is highly conserved across species, is triplicated in tandem.(12) In a study oftargeted mutagenesis of podoplanin molecules, we obtained evidence that Thr residues in the PLAG domain play an importantrole in platelet aggregation.(2) Furthermore, the unique characteristics of the Chinese hamster ovary (CHO) mutant cell lines
Lec1 (N-glycan-deficient), Lec2 (CMP-sialic acid transporterdeficient), and Lec8 (UDP-galactose transporter-deficient)revealed that sialylated O-glycan is critical for the platelet
aggregation-inducing activity of podoplanin.(13) Recently, we
purified human podoplanin from the glioblastoma cell line
LN319 and from podoplanin-transfected CHO cells. (14) Using a
lectin microarray, Edman degradation, and matrix-assisted laser
desorption–ionization time-of-flight mass spectrometry (MALDITOF-MS), we showed that podoplanin possesses a disialylcore1 structure at Thr52 in the PLAG domain. It remains to beclarified, however, whether the disialyl-core1 structure at Thr52
is essential for its platelet-aggregating activity. In addition, theminimum unit of glycopeptide necessary for this activity has notbeen identified. Furthermore, several lines of evidence obtainedusing podoplanin knockout mice suggest that podoplanin is crucially involved in lymphatic vessel formation.(15) However, how
podoplanin regulates the formation of lymphatic vessels or thetumor–platelet interaction remains unknown, and identificationof the pathophysiological target with which podoplanin interacts
is ardently awaited among research workers in many scientificfields. Recently, C-type lectin-like receptor-2 (CLEC-2), which
is a non-classical C-type lectin,(16,17) was identified as an endogenous receptor of podoplanin on platelets.(18)In the present work, the specific binding activity between
podoplanin and CLEC-2 was confirmed using several deletionmutants of podoplanin and CLEC-2 expressed as Fc chimeras.
Furthermore, using several glycosyltransferases, we synthesized
glycopeptides including both the PLAG domain and the Oglycan on Thr52 to identify the minimum unit necessary forbinding to CLEC-2. Finally, we verified that the podoplanin–
CLEC-2 interaction is pathophysiologically important in anexperimental metastasis model using an anti-podoplaninantibody, NZ-1.(19)5

To whom correspondence should be addressed.
E-mail: h.narimatsu@aist.go.jp or yukinari-k@bea.hi-ho.ne.jp
These authors contributed equally to this work.

6

doi: 10.1111/j.1349-7006.2007.00634.x© 2007 Japanese Cancer Association

Materials and Methods
Cell lines and stable transfectants. Chinese hamster ovary cells(CHO) transfected with human podoplanin (hPod) were
established as described previously.(2) CHO/hPod cells were
cultured in RPMI-1640 medium (Wako, Tokyo, Japan) supplemented
with 10% heat-inactivated fetal bovine serum (FBS; Sigma, St
Louis, MO, USA), 2 mM l-glutamine (Invitrogen, Carlsbad,
CA, USA), 100 U/mL penicillin, 100 µg/mL streptomycin(Invitrogen), and 1 mg/mL geneticin (G418; Sigma) at 37°C
in a humidified atmosphere of 5% CO2 and 95% air. 293T-REx/
CLEC-2 cells were established as described previously,(20) andwere cultured in Dulbecco’s Modified Eagle’s Medium (Wako)
supplemented with 10% heat-inactivated FBS, 2 mM l-glutamine,
100 U/mL penicillin, 100 µg/mL streptomycin, 100 µg/mL zeocin(Invitrogen), and 5 µg/mL blasticidin (Invitrogen). Expressionof CLEC-2 was induced by the addition of 1 µg/mL doxycycline(Takara Bio, Shiga, Japan) to the medium 24 h before
experimentation. 293T-REx/CLEC-2 cells without doxycycline
were used as a control. The glioblastoma cell line LN319
was donated by Dr Webster K. Cavenee (Ludwig Institute forCancer Research, San Diego, CA, USA) and cultured as
described.(14,19)
Animals. Female BALB/c nude (nu/nu) mice (7 weeks old)
were purchased from Charles River Japan (Kanagawa, Japan).
Animals were housed under pathogen-free conditions. The
Animal Care and Use Committee of the National Institute ofAdvanced Industrial Science and Technology (Tsukuba, Japan)
approved the animal experiments described herein.
Expression and purification of soluble podoplanin and CLEC-2.

cDNA of human podoplanin and human CLEC-2 (AF124841)
containing the extracellular domains of these proteins was
obtained by polymerase chain reaction (PCR) as described
previously.(18) PCR was carried out using HotStarTaq polymerase(Qiagen, Hilden, Germany). The following oligonucleotides
were used as primers (the EcoRV or EcoRI site in the forward
primer and the BglII site in the reverse primer are underlined):hCL2∆N58 (forward CCGATTACACAGCGCAATTACCT, reverse
GAAGATCTAGGTAGTTGGTCCAC), hCL2∆N87 (forward
acgaattcgCAATCAGAACTAAAGGGCAC, reverse [hCL2.R688BglII] acagatctAGGTAGTTGGTCCACCTTGG), hCL2∆N147(forward acgaattcgGAGTACATCAAAGCCAGGAC, reverse
hCL2. R688-BglII), hCL2∆N177 (forward acgaattcgGTTATCTCAGAAAATATGTT, reverse hCL2.R688-BglII), Pod∆C128 (forward
GCGATATCAGAAGGAGCCAGCACAGG, reverse GGCAGATCTTGTTGACAAACCATCTTTC), Pod∆C103 (forward [EcoRIhPod.F1] acgaattcgATGTGGAAGGTGTCAGCTCT, reverse
acagatctGTTTGAGGCTGTGGCGCTTG), Pod∆C80 (forward
EcoRI-hPod.F1, reverse acagatctGATGCGAATGCCTGTTACAC),
and Pod∆C57 (forward EcoRI-hPod.F1, reverse acagatctTTCGCTGGTTCCTGGAGTCA). The PCR products were
purified using a Rapid PCR Purification Kit (Marligen Bioscience,
Ijamsville, MD, USA), digested with EcoRV or EcoRI and
BglII, purified again, and ligated into the pFUSE-hFc2 (IL2ss)
vector (InvivoGen, San Diego, CA, USA), which contains
human IgG Fc after the ligation site and the interleukin 2 signal
sequence before the ligation site to allow secretion of Fc fusion
proteins. CHO cells were transfected with plasmids using
Lipofectamine 2000. For purification of the fusion proteins, themedium was centrifuged, and the supernatant obtained wasapplied to a column of protein A–sepharose (Pierce, Rockford,
IL, USA). After extensive washing with phosphate-bufferedsaline (PBS), the fusion proteins were eluted with 0.1 M glycine
and 0.15 M NaCl (pH 2.8), and then neutralized with 1 M Tris
pH 10.0. The proteins were dialyzed against PBS. The expression
and purity of the proteins were confirmed by sodium
dodecylsulfate–polyacrylamide gel electrophoresis using 10–
20% gradient gels (Wako). Biotinylation of the fusion proteins
Kato et al.

was carried out using a SureLINK Chromophoric Biotin
Labeling Kit (KPL, Gaithersburg, MD, USA).
Synthesis of O-linked glycosylated glycopeptides. Human podoplanin
glycopeptide (hpp3854) with a GalNAc residue was purchasedfrom Peptide Institute (Osaka, Japan) and used as an acceptor
substrate. For synthesis of the core1 structure on hpp3854,
25 mM HEPES (pH 7.0) containing 4 mg acceptor substrate,
10 mM MnCl2, and an appropriate concentration of UDP-Gal
was used. A 100-µL volume of purified Core1GalT wasadded to the reaction mixture, which was then incubated at 37°C
for 17 h. The sialylation experiments have been described
previously.(14) For the preparation of hpp3854 with the a sialyl
core1 (NeuAcα2-3Galβ1-3GalNAcα1- or NeuAcα2-6Galβ13GalNAcα1-) residue, 25 mM HEPES (pH 7.0) containing 1 mg
core1-hpp3854, 10 mM MnCl2, and an appropriate concentrationof CMP-NeuAc was used. A 50-µL volume of purified ST3GalI
or ST6GalNAcI enzyme was added to the reaction mixture andincubated at 37°C for 24 h. For the preparation of hpp3854
peptide with the disialyl-core1 (NueAcα2-3Galβ1-3[NeuAcα26]GalNAcα1-) residue, both enzymes were added to the reaction
mixture and incubated at 37°C for 24 h. After removing the resinby filtration using an Ultrafree-MC column (Millipore, Billerica,
MA, USA), the glycopeptides were desalted with a reverse-phasecolumn (Oasis HLB column; Waters, Milford, MA, USA).
Western blot analysis. Proteins were electrophoresed under
reducing conditions on 10 –20% polyacrylamide gels (Wako).
The separated proteins were transferred to a polyvinylidene
fluoride (PVDF) membrane. After blocking with 4% skim
milk in PBS, the membrane was incubated with NZ-1 (a rat
monoclonal antibody; 1 µg/mL), rabbit anti-human Fc (5 µg/mL;
Rockland, Gilbertsville, PA, USA), and then with peroxidaseconjugated anti-rat or anti-rabbit antibodies (1/1000 dilution; Dako,
Glostrup, Denmark) and developed for 1 min with ECL reagents(GE Healthcare UK, Buckinghamshire, UK) using X-Omat
AR film (Kodak, Rochester, NY, USA).
Enzyme-linked immunosorbent assay. Glycopeptides corresponding
to amino acids of the human podoplanin sequence, podoplanin–
Fc chimeras, CLEC-2–Fc chimeras, and antibodies were
immobilized on 96-well plates at 10 µg/mL for 1 h. The polyclonal anti-CLEC-2 antibody was from R & D Systems (Minneapolis,
MN, USA). After blocking with 1% bovine serum albumin(BSA) in PBS, the plates were incubated with NZ-1, biotinylatedNZ-1, biotinylated podoplanin–Fc chimera, or biotinylated
CLEC-2–Fc chimera at 10 µg/mL for 1 h. After washingwith 0.05% Tween 20 in PBS, the plates were incubatedwith peroxidase-conjugated antirat IgG (Dako), or peroxidaseconjugated biotin–streptavidin complex (Vectastain ABC Kit;
Vector Laboratories, Burlingame, CA, USA) for 1 h. After
further washing, the enzymatic reaction was conducted witha substrate solution containing TMB (TMB-Turbo or TMB-Ultra;
Pierce). The reaction was stopped with 1 M H2SO4, and theoptical density was measured at 450 nm with an autoplate
reader. These reactions were carried out using a volume of
50 µL at room temperature.
Flow cytometry. Parental CHO, CHO/hPod, or 293T-REx/
CLEC-2 cells were harvested by brief exposure to 1 mM
ethylenediaminetetraacetic acid. After washing with PBS, thecells were treated with CLEC-2–Fc chimeras or podoplanin–Fc
chimeras for 1 h at 4°C followed by treatment with anti-human
Fc (Rockland) and Oregon green-conjugated anti-rabbit IgG(Invitrogen). Fluorescence data were collected using a FACS
Caliber flow cytometer (BD Biosciences, Barintree, MA, USA).
Mass spectrometry. Each synthetic glycopeptide was analyzed
by MALDI-TOF-MS (Reflex IV; Bruker-Daltonik) in reflectron
negative ion mode. For sample preparation, 0.5 µL matrix
solution (10 mg 2,5-dihydroxybenzoic acid dissolved in 1 mL
30% ethanol) was deposited on the stainless steel target plate
and allowed to dry in air. Next, 0.5 µL of appropriately diluted
Cancer Sci | January 2008 | vol. 99 | no. 1 | 55
© 2007 Japanese Cancer Association

analyte solution was used to cover the matrix on the target plate
and allowed to dry in air.
Platelet aggregation assay. Heparinized mouse or humanwhole blood was drawn from BALB/c mice or healthy drug-freevolunteers. Platelet aggregation was measured according to thescreen filtration pressure method using WBA Carna (IMI,
Saitama, Japan).(14,19,21) Two hundred microliters each of thewhole-blood samples and NZ-1 or control rat IgG in four
reaction tubes were stirred at 1000 r.p.m. at 37°C and preincubatedfor 2 min, followed by the addition of 12 µL of each of thesamples. Using a 3.7-mm-diameter syringe containing screen
microsieves made of nickel, with 300 openings of 20 × 20 µm2
in a 1-mm-diameter area, whole-blood samples were sucked todetect aggregation pressure at a rate of 200 µL/6.4 s 7–10 min
later. The final platelet aggregation pressure of each reaction
tube was determined at the pressure rate (%) of a pressure
sensor connected to the syringe.
Experimental lung metastasis. CHO/hPod cells were harvested,washed, and resuspended in Hanks’ Balanced Solutions (HBSS;
2.5 × 106 cells/mL).(11) Next, the cells were incubated with150 µg NZ-1, 150 µg control rat IgG, or 100 µg F(ab′)2 of
NZ-1, and inoculated intravenously (2.5 × 105 cells/mouse) into

the lateral tail vein of female BALB/c-nu/nu mice. After20 days, the mice were killed and the surface lung metastatic
foci were counted and measured.
Results
Binding activity of the CLEC-2–Fc chimera to podoplanin. We first
determined the podoplanin-recognition domain of CLEC-2.
Several CLEC-2–Fc N-terminal deletion mutants were produced(Fig. 1a,b), and the binding activities of human podoplaninto these deletion mutants were assayed. Non-classical C-type
lectins such as CLEC-2 are known to possess a C-type lectin-likedomain (CTLD), homologous to the carbohydrate-recognition
domain of classical C-type lectins. As shown in Fig. 1c,
hCL2∆N58 and hCL2∆N87 bound to both LN319 and CHO/
hPod cells, whereas hCL2∆N147 and hCL2∆N177 did not.
Similarly, hCL2∆N58 and hCL2∆N87 were recognized by the
podoplanin–Fc chimera by enzyme-linked immunosorbent assay(ELISA); however, podoplanin–Fc reacted strongly withhCL2∆N58 compared with hCL2∆N87 (Fig. 1d). Furthermore,
we produced several CLEC-2–Fc C-terminal deletion mutants;
however, none of them reacted with podoplanin (data not shown).

Fig. 1. Binding activity of C-type lectin-likereceptor-2 (CLEC-2)–Fc chimeras to podoplanin.
(a) CLEC-2–Fc deletion mutants were produced
as shown in this scheme. (b) Purified CLEC-2–Fc
chimeras (10 or 0.1 µg/lane) were electrophoresedusing 10–20% gels and stained with Coomassie
Brilliant Blue (CBB) or western blotted using anti-Fc.
(c) LN319 and CHO/hPod cells were harvested bybrief exposure to 1 mM ethylenediaminetetraacetic
acid. After washing with phosphate-bufferedsaline (PBS), cells were treated with CLEC-2–Fc
chimeras or PBS for 1 h at 4°C followed bytreatment with antihuman Fc (2 µg/mL) andOregon green-conjugated antirabbit IgG (2 µg/mL). Fluorescence data were collected using by
flow cytometry. (d) CLEC-2–Fc chimeras or an anti-Fc
were immobilized at a concentration of 10 µg/mL
on a 96-well plate. After blocking with 1% bovine
serum albumin (BSA) in PBS, the plates wereincubated with a biotinylated podoplanin–Fc
chimera at 10 µg/mL, followed by incubation with aperoxidase-conjugated biotin–streptavidin complex.
The enzymatic reaction was conducted with asubstrate solution containing TMB (TMB-Ultra).
After the reaction was stopped with 1 M H2SO4,
the optical density was measured at 450 nm with
an autoplate reader. *P < 0.01 compared with
control, by t-test.

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doi: 10.1111/j.1349-7006.2007.00634.x© 2007 Japanese Cancer Association

Fig. 2. Binding activity of podoplanin–Fc chimeras to C-type lectin-like receptor-2 (CLEC-2). (a) Podoplanin–Fc deletion mutants were produced asshown in this scheme. (b) Purified podoplanin–Fc chimeras (10 or 0.1 µg/lane) were electrophoresed using 10–20% gels and either stained with
Coomassie Brilliant Blue (CBB) or western blotted with NZ-1. (c) Expression of CLEC-2 was induced by the addition of 1 µg/mL doxycycline to 293TREx/CLEC-2 cells 24 h before experimentation. Cells were harvested by brief exposure to 1 mM ethylenediaminetetraacetic acid. After washing withphosphate-buffered saline (PBS), doxycycline+ or doxycycline– cells were treated with podoplanin–Fc chimeras for 1 h at 4°C followed by treatment
with antihuman Fc (2 µg/mL) and Oregon green-conjugated antirabbit IgG (2 µg/mL). Fluorescence data were collected using by flow cytometry.
(d) Podoplanin–Fc chimeras, an anti-Fc, or an anti-CLEC-2 polyclonal antibody were immobilized at a concentration of 10 µg/mL on 96-well plates.
After blocking with 1% bovine serum albumin (BSA) in PBS, the plates were incubated with biotinylated NZ-1 or biotinylated CLEC-2–Fc chimera
at 10 µg/mL, followed by incubation with a peroxidase-conjugated biotin–streptavidin complex. The enzymatic reaction was conducted with asubstrate solution containing TMB (TMB-Turbo). After the reaction was stopped with 1 M H2SO4, the optical density was measured at 450 nm with
an autoplate reader. *P < 0.01 compared to control, by t-test.

These results indicate that the stereostructure of CLEC-2 isimportant for recognizing podoplanin-like classical C-type
lectins. Therefore, we used hCL2∆N58 as a recombinant human
CLEC-2–Fc for the binding assay with podoplanin.
Binding activity of the podoplanin–Fc chimera to CLEC-2. We next
produced several types of C-terminal deletion mutants of human
podoplanin as Fc chimeras (Fig. 2a,b) and investigated thebinding activity of CLEC-2 to these deletion mutants. Of thefour types of C-terminal deletion mutant (Pod∆C128, Pod∆C103,
Kato et al.

Pod∆C80, and Pod∆C57), Pod∆C128 and Pod∆C103 bound to
293T/REx/CLEC-2 cells in flow cytometry when CLEC-2 was
induced by doxycycline (Fig. 2c). In contrast, Pod∆C80 and
Pod∆C57 reacted weakly with 293T/REx/CLEC-2 cells. CLEC2 binding to the podoplanin deletion mutants was also examinedby ELISA. As shown in Fig. 2d, Pod∆C128 was recognizedstrongly by CLEC-2-Fc compared with Pod∆C103, and Pod∆C80
and Pod∆C57 were recognized weakly. These results suggestthat not only the PLAG domain but also the C-terminal region
Cancer Sci | January 2008 | vol. 99 | no. 1 | 57
© 2007 Japanese Cancer Association

of podoplanin is necessary for the strong interaction between
podoplanin and CLEC-2, although the PLAG domain is essential
for its platelet-aggregating activity.(2) Alternatively, the stereostructureof podoplanin might be important for its strong binding to
CLEC-2. However, Pod∆C57, which possesses only the PLAG
domain, was shown to be sufficient to bind to CLEC-2; therefore,
we moved on to identification of the minimum glycopeptides of
podoplanin using our glycan-synthetic technology.
Binding activity of podoplanin glycopeptides to CLEC-2. In ourstudy of targeted podoplanin mutagenesis, we previously showedthat Thr residues in the PLAG domain play an important rolein platelet aggregation.(2) However, the minimum unit ofglycopeptide that is necessary for this activity has not been
identified. As shown in Figure 3a, we synthesized five glycopeptides:

hpp3854 (corresponding to amino acids 38–54 of human
podoplanin) + GalNAc, hpp3854 + core1, hpp3854 + NeuAcα23 core1, hpp3854 + NeuAcα2-6 core1, and hpp3854 + disialylcore1 using our glycan-synthetic technology. The glycans
synthesized were confirmed by mass spectrometry (data notshown). We then investigated the binding activity of CLEC-2 tothese glycopeptides by ELISA. As a result, hpp3854 + disialylcore1 reacted strongly with CLEC-2 compared with hpp3854 +GalNAc, whereas hpp3854 + core1, hpp3854 + NeuAcα2-3
core1, and hpp3854 + NeuAcα2-6 core1 showed no difference
to hpp3854 + GalNAc, although weak reactions were observed(Fig. 3b). These results indicate that only one glycan on Thr52,
the disialyl-core1 structure, is enough for recognition of CLEC2. We also investigated the platelet-aggregating activity of theseglycopeptides. However, none of these glycopeptides induced
platelet aggregation (data not shown), suggesting that only
glycopeptide-binding to CLEC-2 is not enough to activate platelet.
Platelet-aggregation assay for the podoplanin–Fc chimera. We
previously showed that endogenous or recombinant podoplaninproteins purified from LN319 glioblastoma cell lines or CHO/
hPod cells, respectively, induce platelet aggregation.(14) Here, weinvestigated the platelet-aggregating activity of the podoplanin–
Fc chimeras. As shown in Fig. 4, none of the podoplanin–Fc
deletion mutants induced platelet aggregation at a dose of
10 µg/mL. In contrast, after preincubation of podoplanin–Fc withan anti-human Fc antibody, platelet aggregation was induced by
Pod∆C128, but not by Pod∆C103, Pod∆C80, or Pod∆C57. At
a dose of 100 µg/mL, not only Pod∆C128 but also Pod∆C103
induced platelet aggregation. These results indicate that bothcrosslinking of the podoplanin–Fc chimera and strong binding
to CLEC-2 are necessary for inducing platelet aggregation.
Inhibition of podoplanin-induced platelet aggregation by CLEC-2–
Fc deletion mutants. Of the four CLEC-2–Fc deletion mutants,

hCL2∆N58 and hCL2∆N87 were found to react with podoplanin;
therefore, we investigated whether these CLEC-2–Fc deletionmutants could inhibit podoplanin-induced platelet aggregation.
We found that hCL2∆N58 strongly and hCL2∆N87 moderately
inhibited podoplanin-induced platelet aggregation, whereas
hCL2∆N147 and hCL2∆N177 did not (Fig. 5). This result
indicates that CLEC-2 is a physiological ligand of podoplanin.

Fig. 3. Binding activity of podoplanin glycopeptide to C-type lectinlike receptor (CLEC)-2. (a) Podoplanin glycopeptides were synthesized
as shown in this scheme. (b) The synthetic glycopeptides were
immobilized at a concentration of 10 µg/mL on 96-well plates. After
blocking with 1% bovine serum albumin (BSA) in phosphate-bufferedsaline, the plates were incubated with biotinylated CLEC-2–Fc at 10 µg/
mL, followed by a peroxidase-conjugated biotin–streptavidin complex.
The enzymatic reaction was conducted with a substrate solution
containing TMB (TMB-Ultra). After the reaction was stopped by 1 M
H2SO4, the optical density was measured at 450 nm. *P < 0.01 comparedto hpp3851 + GalNAc, by t-test.

58

Fig. 4. Platelet aggregation assay of podoplanin–Fc chimeras. Platelet
aggregation was measured using WBA Carna with the screen filtrationpressure method. Two hundred microliters each of mouse whole blood
samples were preincubated for 2 min, followed by the addition of
12 µL each of podoplanin–Fc chimeras or podoplanin–Fc chimeras
preincubated with anti-Fc antibodies (10 µg/mL) for 30 min on ice.
Whole blood samples were sucked 10 min later to detect aggregation.

doi: 10.1111/j.1349-7006.2007.00634.x© 2007 Japanese Cancer Associationwas lower in mice injected with CHO/hPod + NZ-1 thanin those injected with CHO/hPod + control IgG (Fig. 7c).
Macroscopically, we did not observe metastatic foci in the liver,kidney, spleen, colon, or ovary in any of the mice (data notshown). Furthermore, we investigated whether F(ab′)2 of NZ-1
could also suppress podoplanin-induced pulmonary metastasis
in the same model. As shown in Fig. 7a, F(ab′)2 of NZ-1 also
suppressed pulmonary metastasis. The number of metastatic
lung nodules in mice injected with CHO/hPod + F(ab′)2 of NZ1 was significantly lower than that in CHO/hPod + control IgG
in the same way with whole antibody of NZ-1 (P < 0.01;
Fig. 7b). Lung weight was also lower in mice injected with
CHO/hPod + F(ab′)2 of NZ-1 than in those injected with
CHO/hPod + control IgG (Fig. 7c). These results indicate thatcompared with control IgG, NZ-1 significantly inhibited
pulmonary metastasis, suggesting that the podoplanin–CLEC-2
interaction is pathophysiologically critical in podoplanininduced platelet aggregation and metastasis.
Fig. 5. Inhibition of podoplanin-induced platelet aggregation by Ctype lectin-like receptor-2 (CLEC-2)–Fc deletion mutants. Heparinizedhuman whole blood (WB) was drawn from healthy drug-free volunteers.
Platelet aggregation was measured using WBA Carna with the screen
filtration pressure method. Two hundred microliters each of human WB
samples were preincubated for 2 min, followed by the addition of 12 µL
each of podoplanin–Fc chimera (Pod∆C128; 2 µg/mL) preincubatedwith anti-Fc antibodies (2 µg/mL) and CLEC-2-Fc chimeras (10 µg/mL)
for 30 min on ice. WB samples were sucked 7 min later to detectaggregation. Black arrow, sucked blood; blue arrow, unsucked blood;arrowhead, clogged filter. Data are mean ± SD of three independent
experiments. *P < 0.01, **P < 0.05 compared to control, by t-test.

Inhibition of the podoplanin–CLEC-2 interaction. CLEC-2–Fc
inhibited podoplanin-induced platelet aggregation (Fig. 5). As
shown in Fig. 6a, the Pod∆C128–CLEC-2 interaction wasdiminished by NZ-1 in flow cytometry. Furthermore, theinteraction between Pod∆C57 and CLEC-2 was inhibitedcompletely by NZ-1 (Fig. 6b). In ELISA, NZ-1 also partiallyinhibited the Pod∆C128–CLEC-2 interaction (data not shown).
In contrast, NZ-1 completely inhibited podoplanin-induced
platelet aggregation,(19) suggesting that the podoplanin–CLEC-2
interaction might be pathophysiologically important in vivo.

Inhibition of podoplanin-induced metastasis by NZ-1 or F(ab′′)2of NZ-1. We then investigated whether NZ-1 could suppress

podoplanin-induced pulmonary metastasis in an experimental
metastasis model. Injection of CHO/hPod cells together with
control IgG led to development of multiple lung metastatic foci(Fig. 7a). The number of metastatic lung nodules in mice
injected with CHO/hPod + NZ-1 was significantly lower thanthat in CHO/hPod + control IgG (P < 0.01; Fig. 7b). Consistent
with this decrease in the number of metastatic foci, lung weight

Discussion

The immunogloblin superfamily and G protein-coupled receptors
are known to stimulate platelet aggregation, but CLEC-2, a Ctype lectin-like receptor, is a completely new class of plateletaggregating receptor.(17,22) Of interest, CLEC-2 binds to its
ligand in an unusual manner. C-type lectins usually need Ca2+ tobind to ligand; however, CLEC-2 does not need Ca2+ to bind toligand (Figs 2,3) as one of non-classical C-type lectins.(16)
CLEC-2, with a single immunoreceptor tyrosine-based
activation motif (ITAM), activates Syk directly, whereas most Ctype lectins, such as MDL-1 and DCAR, couple with Syk
indirectly via association with ITAM-bearing adaptor proteinssuch as FcRγ and DAP12.(17,23) The powerful stimulatory activityof CLEC-2 on platelets suggests that it may play an importantrole in vivo. Recently, CLEC-2 was identified as a novel endogenous receptor of podoplanin on the cell surface.(18) However, thepathophysiological importance and function of CLEC-2 have notbeen elucidated. In the present study, the importance of the
podoplanin–CLEC-2 interaction has been clarified in vitro andin vivo.
Here, we first investigated the interaction between podoplanin
and CLEC-2 in detail. As shown in Fig. 3, only a disialyl-core1
on Thr52 plus the PLAG domain (hpp3854 + disialyl-core1)
was recognized by CLEC-2. Of the four podoplanin–Fc deletionmutants, Pod∆C128 reacted most strongly with CLEC-2 andinduced platelet aggregation. In contrast, the others boundweakly to CLEC-2 and did not induce platelet aggregation(Fig. 4). Moreover, hpp3854 + disialyl-core1 also did not induce
platelet aggregation (data not shown), suggesting that strong
binding to CLEC-2 may be essential for inducing platelet
aggregation.

Fig. 6. Inhibition of the podoplanin–C-type lectinlike receptor-2 (CLEC-2) interaction by NZ-1. (a)Expression of CLEC-2 was induced by the addition of
1 µg/mL doxycycline to 293T-REx/CLEC-2 cells 24 h
before experimentation. Cells were harvested bybrief exposure to 1 mM ethylenediaminetetraacetic
acid. After washing with phosphate-buffered saline(PBS), cells were treated with NZ-1 (100 µg/mL) +Pod∆C128 (1 µg/mL), Pod∆C128 alone, or PBS for 1 h
at 4°C followed by treatment with anti-human Fc(2 µg/mL) and Oregon green-conjugated anti-rabbit
IgG (2 µg/mL). (b) 293T-REx/CLEC-2 cells were treatedwith NZ-1 (100 µg/mL) + Pod∆C57 (10 µg/mL),
Pod∆C57 alone, or PBS for 1 h at 4°C followed
by treatment with anti-human Fc (2 µg/mL) andOregon green-conjugated anti-rabbit IgG (2 µg/mL).

Kato et al.

Cancer Sci | January 2008 | vol. 99 | no. 1 | 59
© 2007 Japanese Cancer Association

Fig. 7. Inhibition of podoplanin-induced metastasis by NZ-1.
(a) Chinese hamster ovary cells transfected with human podoplanin(CHO/hPod) were harvested, washed, and resuspended in HBSS
(2.5 × 106 cells/mL). Then, the cells were incubated with 150 µg NZ-1,
150 µg control rat IgG, or 100 µg F(ab′)2 of NZ-1 and inoculated
intravenously (2.5 × 105 cells/mouse) into lateral tail vein of female
BALB/c-nu/nµ mice (five mice in each group). After 20 days, the mice
were killed, and surface lung metastatic foci were counted andmeasured. Detailed numbers of (b) the lung metastasis nodules and (c)the weight of the lung in each mouse are shown. Data are mean ± SD.
*P < 0.01, by t-test.

Using several CLEC-2–Fc deletion mutants, the CTLD of the
C-type lectin family was consistent with the podoplanin-recognition
domain (Fig. 1a,b). These results indicate that the interaction
between the CTLD of CLEC-2 and a disialyl-core1 in the PLAG
domain of podoplanin is the most critical association in
podoplanin-induced platelet aggregation. Previous structure andmutational binding analysis of CLEC-2 also suggested that its
endogenous ligand is likely to be a protein with a predominantly
negatively charged binding surface.(24) Our data, showing thatthe disialyl-core1 structure of podoplanin is essential for binding to CLEC-2, supports this suggestion because sialic acid isnegatively charged.
60

A recombinant podoplanin–Fc fusion protein stimulated more
powerful platelet aggregation when it was crosslinked by antiFc antibodies; Pod∆C128 could induce platelet aggregation at aconcentration of only 0.2 µg/mL (data not shown). Previously,
Fuller et al. reported that an anti-CLEC-2 antibody also induced
platelet aggregation.(22) Furthermore, we previously showedthat podoplanin-expressing CHO cells strongly induce platelet
aggregation in spite of their low expression of podoplanin.(19)These findings suggest that clustering of CLEC-2 might generate
activation signals and platelet aggregation, although the
podoplanin–CLEC-2 interaction was observed using even a
monomeric glycopeptide of podoplanin (Fig. 3). Furthermore,
not only the PLAG domain–CTLD interaction but also thestereostructure of both podoplanin and CLEC-2 might beessential for the platelet-aggregating activity.
We showed that podoplanin on the surface of tumor cells induces
platelet aggregation by interacting with CLEC-2, because
CLEC-2–Fc inhibited podoplanin-induced platelet aggregation(Fig. 5). A study using several clones possessing different
metastatic abilities from a mouse colon adenocarcinoma 26 cellline revealed that a highly metastatic clone expresses more
podoplanin and induces platelet aggregation to a greater
extent,(25) although podoplanin is not expressed in human colon
adenocarcinoma. (7,19) Moreover, an anti-mouse podoplaninantibody (8F11) that inhibits podoplanin-induced platelet aggregation
suppressed lung colonization of mouse colon adenocarcinoma.(26)We also showed that an anti-human podoplanin antibody (NZ-1),
which neutralized platelet aggregation, inhibited the experimental
metastasis of CHO/hPod cells (Fig. 7). The podoplanin–CLEC-2
interaction was diminished by NZ-1 (Fig. 6a); however, theinteraction was not inhibited completely even at a dose of 1 mg/
mL of NZ-1 (data not shown), indicating that CLEC-2 might beable to recognize sialic acid on threonine residues other thanThr52, because podoplanin is highly glycosylated. Indeed, theinteraction between Pod∆C57 and CLEC-2 was inhibited completely by NZ-1, because Pod∆C57 possesses only one disialylcore1 (Fig. 6b). In the experimental metastasis model, F(ab′)2
fragments of NZ-1 also suppressed pulmonary metastasis(Fig. 7), suggesting that suppression of tumor metastasis by NZ1 is not only dependent on antibody dependent cell-mediatedcytotoxity, but is also caused by neutralization of the interaction
between podoplanin and CLEC-2. These findings, taken
together, suggest that CLEC-2 is a critical pathophysiologicalligand of podoplanin in vivo, and that podoplanin-induced
platelet aggregation through CLEC-2 is one of the importantmechanisms of tumor metastasis, although it might be true inlimited human tumors. The importance of the podoplanin–
CLEC-2 interaction has been elucidated for the first time to ourknowledge in a pathophysiological condition.
In conclusion, we have clarified the molecular interaction
between a platelet aggregation-inducing factor, podoplanin, andits specific receptor on platelets, CLEC-2, and have shown thata glycopeptide including only one disialyl-core1 structure isnecessary for the binding of podoplanin to CLEC-2. It is noteworthy that a glycopeptide containing only one disialyl-core1 isfunctional and essential for platelet aggregation-inducing
activity. The interaction between podoplanin and CLEC-2 may
regulate tumor invasion and metastasis. Therefore, podoplanin
and CLEC-2 might represent promising therapeutic targets incancer metastasis.
Acknowledgments
This study was supported in part by the Medical Glycomics Project
supported by the New Energy and Industrial Technology Development
Organization, and by a grant for Research Fellowships from the Japan
Society for the Promotion of Science for Young Scientists, Japan(Y. Kato).

doi: 10.1111/j.1349-7006.2007.00634.x© 2007 Japanese Cancer Association

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