BBRC
Biochemical and Biophysical Research Communications 349 (2006) 1301–1307
www.elsevier.com/locate/ybbrc

Inhibition of tumor cell-induced platelet aggregation using
a novel anti-podoplanin antibody reacting with its
platelet-aggregation-stimulating domain
Yukinari Kato a,*, Mika Kato Kaneko a, Atsushi Kuno a, Noboru Uchiyama a,
Koh Amano a, Yasunori Chiba a, Yasushi Hasegawa b, Jun Hirabayashi a,
Hisashi Narimatsu a, Kazuhiko Mishima c, Motoki Osawa daResearch Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Open Space Laboratory C-2,
1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japanb
Department of Surgery, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japanc
Department of Neurosurgery, Saitama Medical School, 38 Morohongo, Moroyama-machi, Iruma-gun, Saitama 350-0495, Japand
Department of Forensic Medicine, Tokai University School of Medicine, Boseidai, Isehara, Kanagawa 259-1193, Japan
Received 27 August 2006
Available online 7 September 2006

Abstract
The mucin-type sialoglycoprotein, podoplanin (aggrus), is a platelet-aggregating factor on cancer cells. We previously described
up-regulated expression of podoplanin in malignant astrocytic tumors including glioblastoma. Its expression was associated with tumor
malignancy. In the present study, we investigated podoplanin expression and platelet-aggregating activities of glioblastoma cell lines.
First, we established a highly reactive anti-podoplanin antibody, NZ-1, which inhibits podoplanin-induced platelet aggregation completely. Of 15 glioblastoma cell lines, LN319 highly expressed podoplanin and induced platelet aggregation. Glycan proﬁling using a
lectin microarray showed that podoplanin on LN319 possesses sialic acid, which is important in podoplanin-induced platelet aggregation. Interestingly, NZ-1 neutralized platelet aggregation by LN319. These results suggest that podoplanin is a main reason for platelet
aggregation induced by LN319. We infer that NZ-1 is useful to determine whether platelet aggregation is podoplanin-speciﬁc or not.
Furthermore, podoplanin might become a therapeutic target of glioblastoma for antibody-based therapy.
 2006 Elsevier Inc. All rights reserved.
Keywords: NZ-1; Podoplanin; Astrocytic tumors; Glioblastoma; Tumor cell-induced platelet aggregation; Lectin array

Recent discovery of lymphatic endothelium markers
such as vascular endothelial growth factor (VEGF) receptors [1] and lymphatic vessel endothelial hyaluronan receptor (LYVE-1) [2] has facilitated identiﬁcation of lymphatic
vessels. However, some of these markers are not expressed
exclusively on lymphatic vessels. A mucin-type transmembranesialoglycoprotein—podoplanin—isahighlyexpressed lymphatic speciﬁc gene in cultured human lymphatic endothelial cells (LECs) [3–5]. In human tissues,
podoplanin expression is apparent only in lymphatic endo*

Corresponding author. Fax: +81 29 861 3191.
E-mail address: yukinari-k@bea.hi-ho.ne.jp (Y. Kato).

0006-291X/$ - see front matter  2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2006.08.171

thelial cells: not in vascular endothelial cells. More recently, human podoplanin has been demonstrated to berecognized speciﬁcally using monoclonal antibody D2-40[4], which was produced originally against M2A antigen
expressed by testicular germ cell tumors [6].
Podoplanin is reportedly expressed in many tumor cells
such as squamous cell carcinoma [4,7–9], malignant mesothelioma [10,11], Kaposi’s sarcoma, angiosarcoma [3],hemangioblastoma [12], testicular seminoma [13], dysgerminoma [4], and brain tumors [14–16]. Recent investigations have reported that podoplanin expression might beassociated with tumor invasion, metastasis, or malignant
progression [9,16].

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Y. Kato et al. / Biochemical and Biophysical Research Communications 349 (2006) 1301–1307

Mouse podoplanin/aggrus, a 44-kDa sialoglycoproteinwith platelet aggregation-inducing ability, is expressed on
the surface of mouse colon adenocarcinoma cells [17]. Antibody against mouse podoplanin/aggrus inhibits lung metastasis of NL-17 colon carcinoma cells in vivo [18]. Cloning of
cDNA revealed that human podoplanin is identical tohuman aggrus, a separately isolated protein that can also
induce mouse and human platelet aggregation [19]. Therefore, podoplanin might be involved in tumor cell-induced
platelet aggregation and metastasis. Unique characteristicsof Chinese hamster ovary (CHO) mutant cell lines Lec1,
Lec2, and Lec8 revealed that sialylated O-glycan is criticalfor platelet aggregation-inducing activity [5].
Furthermore, we showed previously that podoplanin
was expressed in astrocytic tumors [16]. In that report, 11
of 43 anaplastic astrocytomas (Grade III in astrocytic
tumors: 25.6%) and in 54 of 115 glioblastomas (Grade IV
in astrocytic tumors: 47.0%), podoplanin was expressedon the surface of anaplastic astrocytoma cells and glioblastoma cells, especially around necrotic areas and proliferating endothelial cells. On the other hand, podoplanin
expression was not observed in diﬀuse astrocytoma (Grade
II in astrocytic tumors: 0/30, 0%). These data suggest that
podoplanin expression might be associated with malignancy of astrocytic tumors. However, platelet-aggregating
activity of podoplanin in glioblastoma and its contribution
to tumor malignancy have not been elucidated because of
the lack of neutralizing antibody of podoplanin-induced
platelet aggregation.
In this study, we produced a novel monoclonal antibody(NZ-1) against human podoplanin by immunizing rats witha platelet-aggregation-stimulating (PLAG) domain of
podoplanin to neutralize podoplanin-induced platelet
aggregation. Using this neutralizing antibody, we candetermine whether platelet aggregation by cancer cells
might be podoplanin-speciﬁc or not. Furthermore, weinvestigated glycan proﬁling of podoplanin on glioblastoma cell lines using a lectin microarray.

aminopterin, and thymidine selection medium supplement (Sigma
Chemical Co.). The culture supernatants were screened by ELISA for thebinding to the synthetic peptide.
Specimens and tissue microarrays. In this study, we used the 642 cases ofhuman tumors (Table 1). Tissue microarrays of brain tumors (132 cases: 22
diﬀuse astrocytoma, 29 anaplastic astrocytoma, and 81 glioblastoma), lungcarcinoma (129 cases: 24 squamous cell carcinomas, 51 adenocarcinomas,
23 large cell carcinomas, and 31 small cell carcinomas), testicular tumors(13 cases), malignant melanoma (37 cases), esophageal squamous cell
carcinoma (65 cases), stomach adenocarcinoma (72 cases), colon adenocarcinoma (66 cases), and rectal adenocarcinoma (57 cases) were purchasedfrom Cybrdi, Inc. (Frederick, MD). Clinical information of patients (age,sex, grade, and pathology diagnosis) is obtainable from its home page.
Furthermore, this study included 11 seminoma patients and 59 lung cancer
patients (28 squamous cell carcinomas and 31 adenocarcinomas) who
underwent surgery during 1990–2003 at Yamagata University Hospital(Yamagata, Japan). Informed consent was obtained from each patient
before specimens were taken. The tumor specimens were ﬁxed routinely in
10% buﬀered formalin for 18–20 h at room temperature and processedusing paraﬃn. Sections (5 lm thick) were cut and attached to poly-L-lysinecoated glass slides. Hematoxylin-eosin was used as a routine staining.
Flow cytometry. Expression levels of human podoplanin were compared for conﬁrmation using ﬂow cytometry. Glioblastomas and transfected CHO cells, which were collected by trypsin-EDTA treatment, wereincubated with NZ-1 (0.1 lg/ml) for 1 h at 4 C. Then the cells wereincubated with Oregon green-conjugated antibodies (Invitrogen Corp.,
Carlsbad, CA), for 30 min. Flow cytometry was performed using FACS
Calibur (Becton–Dickinson).
Western-blot analysis. The cell lines were solubilized with lysis buﬀer(1% Triton in PBS) and electrophoresed under reducing conditions on 10–
20% polyacrylamide gels. The separated proteins were transferred to a
PVDF membrane. After blocking with 4% skim milk in PBS, the membrane was incubated with NZ-1 (a rat monoclonal antibody: 0.1 lg/ml),
D2-40 (a mouse monoclonal antibody, 1/40 diluted; Signet Laboratories,
Inc., Dedham, MA) or anti-b-actin antibody (a mouse monoclonal antibody: 1 lg/ml; Sigma Chemical Co.), and then with peroxidase-conjugatedanti-rat or mouse antibodies (1/1000 diluted; Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, UK) and developed for 1 min with ECL
reagents (Amersham Pharmacia Biotech) using Kodak X-Omat AR ﬁlm.
Immunohistochemistry. Immunohistochemical staining was performedby the avidin-biotinylated immunoperoxidase method. Brieﬂy, 5-lm sections were deparaﬃnized and rehydrated. All the tissues were then exposed
to 3% hydrogen peroxidase for 5 min. NZ-1 (1 lg/ml) was added to theMaterials and methods

Tumor typeNo. of cases

+++

++

+



Brain tumor
Diﬀuse astrocytoma
Anaplastic astrocytoma
Glioblastoma

132
22
29
81

0
3
23

0
2
8

0
1
5

22
23
45

Lung carcinoma
Squamous cell carcinoma
Adenocarcinoma
Large cell carcinoma
Small cell carcinoma

188
52
82
23
31

2
0
0
0

7
0
0
0

5
0
0
0

38
82
23
31

Esophageal squamous cell
carcinoma
Stomach adenocarcinoma
Colon adenocarcinoma
Rectal adenocarcinoma
Testicular seminoma
Malignant melanoma

65

5

3

6

51

72
66
57
24
38

0
0
0
24
0

0
0
0
0
0

0
0
0
0
0

72
66
57
0
38

Animals and Cell lines. Female SD rats were obtained from Charles
River Japan, Inc. (Kanagawa, Japan). Chinese hamster ovary (CHO),
P3U1, and 15 glioblastoma cell lines (LN18, LN215, LN229, LNZ308,
LN319, LN340, LN428, LN464, U87, U178, U251, U373, A1207, SF763,
and T98G) were obtained from the American Type Culture Collection(ATCC). These cell lines were cultured at 37 C in a humidiﬁed atmosphereof 5% CO2 and 95% air in RPMI 1640 medium (for CHO and P3U1) or
Dulbecco’s modiﬁed Eagle’s medium (DMEM; for glioblastoma cell lines)
supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma
Chemical Co., St. Louis, MO), 2 mM L-glutamine (Gibco Laboratories,
Grand Island, NY), and 100 lg/ml of kanamycin (Sigma Chemical Co.).
Hybridoma production. SD rats were immunized by neck s.c. injectionsof the synthetic peptide EGGVAMPGAEDDVV (hpp3851), corresponding to amino acids 38–51 of human podoplanin with Freund’sComplete Adjuvant (Difco Laboratories, Detroit, MI). One week later,
secondary i.p. immunization was performed. The booster injection wasgiven i.p. 2 days before spleen cells were harvested. The spleen cells werefused with mouse myeloma P3U1 cells using polyethylene glycol (Mr4000); the hybridomas were grown in RPMI medium with hypoxanthine,

Table 1Results of podoplanin staining by NZ-1 (642 cases)
Podoplanin
immunostaining

Y. Kato et al. / Biochemical and Biophysical Research Communications 349 (2006) 1301–1307
sections for 1 h at room temperature. Biotin-conjugated secondary anti-rat
IgG (DakoCytomation, Glostrup, Denmark) was incubated for 30 min atroom temperature followed by the peroxidase-conjugated biotin-streptavidin complex (Vectastain ABC Kit; Vector Laboratories Inc., Burlingame, CA) for 30 min at room temperature. Color was developed using
3,3-diaminobenzidine tetrahydrochloride tablet sets (DakoCytomation)for 3 min. The sections were counterstained with Mayer’s hematoxylin.
Podoplanin expression was semi-quantitatively assessed from the percentage of tumor cells with membrane staining: 0, no staining; +, <10%;++, 10–50%; and +++, >50%.
Quantitative real-time PCR. Total RNAs were prepared from glioblastoma cell lines using an RNeasy mini prep kit (Qiagen Inc., Hilden, Germany). The initial cDNA strand was synthesized using SuperScript III
transcriptase (Invitrogen Corp.) by priming nine random oligomers and an
oligo(dT) primer according to the manufacturer’s instructions. We performed PCR using the human podoplanin sense (5 0 -GGAAGGTGTCAGCTCTGCTC-3 0 ), human podoplanin antisense (5 0 -CGCCTTCCAA
ACCTGTAGTC-3 0 ), human b-actin sense (5 0 -ACTCTTCCAGCCTT
CCTTCCTG-3 0 ), and human b-actin antisense (5 0 -ATCTCCTTCTGC
ATCCTGTCGG-3 0 ) oligonucleotides. Real-time PCR was carried out
using the QuantiTect SYBR Green PCR (Qiagen Inc.). The PCR conditions
were 95 C for 15 min (1 cycle), followed by 40 cycles of 94 C for 15 s, 53 C
for 20 s, 72 C for 10 s for podoplanin, or 94 C for 15 s, 55 C for 25 s, 72 C
for 20 s for b-actin. Subsequently, a melting curve program was applied with
continuous ﬂuorescence measurement. Standard curves for podoplanin andb-actin templates were generated by serial dilution of the PCR products(1 · 108 copies/ll to 1 · 102 copies/ll). The expression levels of podoplanin
were normalized by estimating the quantity of the b-actin transcript.
Platelet aggregation assay by WBA Carna. Heparinized mouse whole
blood (WB) was drawn from BALB/c mice. Platelet aggregation wasmeasured according to the screen ﬁltration pressure method using WBA
Carna (M.C. Medical) [20]. Two hundred microliters each of mouse whole
blood samples and NZ-1 or control rat IgG in four reaction tubes wasstirred at 1000 rpm at 37 C and pre-incubated for 2 min, followed byaddition of 12 ll each of cells (2 · 107 cells/ml). Using a 3.7-mm-diametersyringe containing screen microsieves made of nickel, with 300 openings of
20 · 20 lm2 in a 1-mm-diameter area, WB samples were sucked to detectaggregation pressure at a rate of 200 ll/6.4 s 1–5 min later. The ﬁnal
platelet aggregation pressure of each reaction tube was determined at thepressure rate (%) of a pressure sensor connected to the syringe.
Lectin microarray. Lectin microarray was performed basically as
described by Kuno et al. [21]. Interaction of podoplanin with the lectin
immobilized on the glass slide was detected using biotinylated NZ-1-Cy3streptavidin method to proﬁle glycans of podoplanin. Brieﬂy, podoplaninon LN319 solubilized with 1% Triton X-100 in PBS (PBSTx) was
immunoprecipitated using NZ-1 antibody and then released with 100 ll of
elution buﬀer containing the synthetic peptide, hpp3851, in PBS. Then
10 ng of podoplanin was diluted to 60 ll with PBSTx and applied to thelectin array containing triplicate spots of 43 lectin (see Supplementary
Table 1 and Fig. 2D) into each of 8-divided incubation baths on the glass
slide. After incubation at 20 C for 12 h, the reaction solution was discarded. The glass slide was washed three times with PBSTx; 60 ll of
biotinylated NZ-1 antibody (0.17 lg/ml) in PBS was applied to the arrayand then incubated at 20 C for 3 h. After washing three times with
PBSTx, Cy3-labeled streptavidin (GE Healthcare, UK) was added to thearray and then incubated at 20 C for 30 min. The glass slide was rinsed
with PBSTx and scanned using an evanescent-ﬁeld ﬂuorescence scanner(GTMASScan III; Nippon Laser and Electronics Lab, Nagoya, Japan).

Results and discussion
Production of a novel monoclonal antibody against
podoplanin
By immunizing rats with platelet-aggregation-stimulating (PLAG) domain of podoplanin, we newly generated a1303

monoclonal antibody, NZ-1, which can speciﬁcally recognize human podoplanin. The NZ-1 stained lymphatic vessels (Fig. 1A), esophageal squamous cell carcinoma(Fig. 1B), and testicular seminoma (Fig. 1C) without antigen retrieval. As summarized in Table 1, using 642 tumors,
NZ-1 stained astrocytic tumors, lung or esophageal squamous cell carcinomas, and testicular seminomas. However,stomach adenocarcinoma, colorectal adenocarcinoma, andmalignant melanoma were not stained by NZ-1. Westernblot analysis showed that NZ-1 recognized two bands:
36-kDa band with a strong signal and 25-kDa band witha weak signal; 25-kDa band might be de- or non-glycosylated form of podoplanin (Fig. 1D). In addition, D2-40,
another anti-human podoplanin antibody, similarly detected 36-kDa band of podoplanin. The NZ-1 binding tohuman podoplanin was neutralized by the synthetic peptide hpp3851, whereas D2-40 binding was not (Fig. 1D).
Furthermore, NZ-1 strongly recognized podoplanin
expressed in podoplanin-transfected CHO cells (CHO/
pod) using ﬂow cytometric analysis (Fig. 1E).
In our previous studies, CHO/pod demonstrated its
capability to induce platelet aggregation of both humans
and mice, whereas CHO did not [19]. We determined the
PLAG domain of podoplanin; PLAG domain was repeated three times [22]. Because NZ-1 antibody was produced
by immunizing PLAG domain, we then checked the inhibitory eﬀect of NZ-1 against podoplanin-induced platelet
aggregation. Results showed that NZ-1 inhibited platelet
aggregation by CHO/pod in a dose-dependent manner;
10 lg of NZ-1 suppressed its platelet aggregation completely (Fig. 1F), whereas control rat IgG did not (data notshown). Furthermore, another anti-podoplanin antibody,
D2-40, did not neutralize the platelet aggregation by
CHO/pod (data not shown), probably because D2-40 did
not recognize PLAG domain including hpp3851(Fig. 1D). Using this neutralizing antibody, it became possible to determine whether the platelet aggregation by cancer cells might be podoplanin-speciﬁc or not.
Expression of podoplanin in glioblastoma cell lines
In a previous study, podoplanin was expressed in malignant astroctytic tumors [16]. We performed Western-blot
and ﬂow cytometry using NZ-1 against 15 glioblastoma celllines to investigate podoplanin expression in glioblastoma
cell lines. Fig. 2A shows that podoplanin was highly
expressed in one glioblastoma cell line, LN319, and slightly
expressed in LN215, LNZ308, U87, U178, U251, andA1207. Mobility shift of podoplanin band was observedin Western-blot, probably because the glycosylation pattern varied among these cell lines. The podoplanin expression in Western-blot was consistent with those of ﬂow
cytometry, except for LN215 (Fig. 2B). The reason why
podoplanin on LN215 was not detected by NZ-1 in ﬂow
cytometry has not been clariﬁed, although it was detectedin Western-blot; podoplanin might not be expressed properly on its cell membrane. Furthermore, real-time PCR

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Y. Kato et al. / Biochemical and Biophysical Research Communications 349 (2006) 1301–1307

Fig. 1. Characterization of NZ-1, a novel monoclonal antibody against podoplanin. (A–C) Immunohistochemical staining with NZ-1 on humanspecimens derived from normal lung (A), esophageal carcinoma (B), and testicular seminoma (C). (D) The cell lysate of CHO-human podoplanin (CHO/
pod) was electrophoresed and immunoblotted using NZ-1 (1 lg/ml), NZ-1 (0.1 lg/ml) + hpp3851 (100 lg/ml), D2-40 (1/40 diluted), or D2-40 (1/40
diluted) + hpp3851 (100 lg/ml). (E) Flow cytometric analyses of NZ-1 to CHO/pod. (F) Platelet aggregation assay. Heparinized mouse whole blood (WB)
was drawn from BALB/c mice. Platelet aggregation was measured using WBA Carna with the screen ﬁltration pressure method. Two hundred microliters
each of mouse whole blood samples and NZ-1 (1, 5, 10, 25, or 50 lg/ml) was pre-incubated for 2 min, followed by the addition of 12 ll each of CHO/pod
cells (2 · 107 cells/ml). Whole blood samples were sucked 1–5 min later to detect aggregation.

analysis was performed to conﬁrm these results (Fig. 2C).
Results showed that podoplanin mRNA was also
expressed highly in LN319 and expressed slightly in
LN215, LNZ308, LN428, U87, U178, U251, U373, andA1207.
Glycan proﬁling of podoplanin on LN319 using lectin
microarray
Previously, podoplanin was expressed stably in a seriesof CHO cell mutants: N-glycan-deﬁcient Lec1, CMP-sialic

acid transporter-deﬁcient Lec2, and UDP-galactose transporter-deﬁcient Lec8 [5]. Podoplanin on Lec1 cells induced
platelet aggregation, but those on Lec2 and Lec8 cells did
not. Furthermore, podoplanin expressed in CHO andLec1 cells showed Wheat-germ agglutinin (WGA) and Jacalin bindings, but not Vicia Villosa lectin (VVA). On theother hand, podoplanin on Lec2 showed VVA and peanut
agglutinin (PNA) binding, but not WGA binding. Lectinblotting results indicated that sialylated core1 structures,
sialic acid + Galb1,3GalNAc-Ser/Thr, were critical forplatelet aggregation activity.

Y. Kato et al. / Biochemical and Biophysical Research Communications 349 (2006) 1301–1307

1305

Fig. 2. (A) Western-blot analyses of glioblastoma cell lines using NZ-1. The cell lysates of 15 glioblastoma cell lines were electrophoresed andimmunoblotted with NZ-1 and anti-b-actin. (B) Flow cytometric analyses of NZ-1 to glioblastoma cell lines. (C) Quantitative analysis of podoplanintranscripts in glioblastoma. First-strand cDNA samples derived from 15 glioblastoma cell lines were used as real-time PCR templates. Respective
expression levels of podoplanin were normalized to that of the b-actin transcript, as described in Materials and methods. (D) Glycan proﬁling of LN319
using lectin microarray. Podoplanin on LN319 immunoprecipitated using NZ-1 antibody was applied to the lectin array containing triplicate spots of 43
lectins (Supplementary Table 1). The podoplanin that was bound to the immobilized lectins was detected using biotinylated NZ-1 antibody and Cy3labeled streptavidin. Positive signals; 24, ABA; 29, Jacalin; 32, ACA; 33, MPA; 34, HPA; 40, MAH; and 41, WGA.

The present study investigated glycan proﬁling of
podoplanin on LN319 using the lectin microarray (seeFig. 2D and Supplementary Table 1) with the detection
method by biotinylated NZ-1 antibody and Cy3-labeledstreptavidin. Fig. 2D shows that podoplanin on LN319
reacted strongly with core1 ± sialic acid binders (Agaricus
bisporus agglutinin (ABA), Jacalin, Amaranthus caudatus
agglutinin (ACA), and Maclura pomifera agglutinin(MPA)), sialo-mucin binders (Maackia amurensis hemagglutinin (MAH) and WGA), and alpha-GalNAc binder(Helix pomatia agglutinin (HPA)); it did not react withcore1 binders (Bauhinia purpurea alba lectin (BPL) and
PNA). On the other hand, in the case of asialo-podoplanintreated with sialidase A, the signals were observed on BPL
and PNA spots instead of the loss of signals on MAH andWGA spots; HPA signals were increased after treatment ofsialidase A (data not shown). These results indicate that

podoplanin on LN319 also possesses disialyl-T antigen,sialyl-T antigen, and/or sialyl-Tn antigen, and have theplatelet-aggregating activity. Although a signiﬁcant signal
was observed on HPA spot, another Tn antigen binder,
VVA, did not react with podoplanin. We recently conﬁrmed that HPA reacts with Tn antigen much stronger
than VVA on lectin microarray (data not shown). These
data suggest that sialyl-Tn antigens on podoplanin
expressed in LN319 might be partially de- or nonsialylated.
Platelet aggregation by glioblastoma cell lines
Tumor cells can activate platelets via several pathways:
tumor cell-induced thrombin generation through a
coagulation pathway [23], releasing ADP [24], thromboxane A2 (TXA2) [25], MMP-2 [26], and a membranous pro-

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Y. Kato et al. / Biochemical and Biophysical Research Communications 349 (2006) 1301–1307sity) and Dr. I. Sasagawa (Yamagata Tokusyukai Hospital) for providing us lung carcinoma and seminoma
samples.
Appendix A. Supplementary data
Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.bbrc.
2006.08.171.
References

Fig. 3. Platelet aggregation assay. Heparinized mouse whole blood (WB)
was drawn from BALB/c mice. Platelet aggregation was measured using
WBA Carna with the screen ﬁltration pressure method. Two hundred
microliters each of mouse whole blood samples and NZ-1 or control IgG(0, 10, 25, or 50 lg/ml) was stirred at 1000 rpm at 37 C, and preincubated for 2 min, followed by the addition of 12 ll each of LN319 cells(2 · 107 cells/ml). Whole blood samples were sucked 2 min later to detectaggregation. Data are means ± SD of three independent experiments.

tein Aggrus/podoplanin [19]. In our previous studies,
CHO/pod was able to induce platelet aggregation, whereas
CHO did not [19]. Fig. 2 shows that LN319 extraordinarily
expressed podoplanin among 15 glioblastoma cell lines.
Therefore, we investigated platelet-aggregating activity by
LN319. Results showed that LN319 was able to induce
platelet aggregation; this aggregation was inhibited by
NZ-1 antibody in a dose-dependent manner, whereas control rat IgG did not (Fig. 3). These results indicate thatplatelet aggregation by LN319 was attributable to highexpression of podoplanin on its cell membrane.
In summary, we produced a novel anti-podoplanin antibody that can speciﬁcally neutralize podoplanin-induced
platelet aggregation. This neutralizing antibody enableddetermination of whether platelet aggregation induced bycancer cells is podoplanin-speciﬁc or not. Using this antibody, we demonstrated that glioblastoma cells expressed
podoplanin and possess high platelet-aggregating activity;
they might be involved in tumor thrombosis. Podoplanin
might become a promising therapeutic target for antibody-based therapy.
Acknowledgments
This study was supported in part by a grant for
Research Fellowships from the Japanese Society for the
Promotion of Science for Young Scientists, Japan(Y. Kato), by the Kanae Foundation for Life and Sociomedical Science (Y. Kato), by the Osaka Cancer Research
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