THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 278, No. 51, Issue of December 19, pp. 51599 –51605, 2003
Printed in U.S.A.

Molecular Identification of Aggrus/T1␣ as a Platelet
Aggregation-inducing Factor Expressed in Colorectal Tumors*
Received for publication, September 8, 2003, and in revised form, October 1, 2003
Published, JBC Papers in Press, October 1, 2003, DOI 10.1074/jbc.M309935200

Yukinari Kato‡§¶, Naoya Fujita‡¶, Akiko Kunita‡, Shigeo Sato储, Mika Kaneko§, Motoki Osawa§,
and Takashi Tsuruo‡储**
From the ‡Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan,§Department of Experimental and Forensic Pathology, Yamagata University School of Medicine, Yamagata 990-9585,
Japan, and 储Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1, Kami-Ikebukuro,
Toshima-ku, Tokyo 170-8455, Japan

Platelets play an important role in hemostasis, thrombosis, and antimicrobial host defense and are also involved in the induction of inflammation, tissue repair,and tumor metastasis. We have previously characterized the platelet aggregation-inducing sialoglycoprotein(Aggrus/gp44) overexpressed on the surface of tumor
cells. Because a platelet aggregation-neutralizing 8F11
monoclonal antibody that could specifically recognize
Aggrus suppressed tumor-induced platelet aggregation,
we have previously purified Aggrus by 8F11-affinity
chromatography and found that purified Aggrus possessed the ability to induce aggregation of platelets.
Here we show that Aggrus is identical to the T1␣/gp38P/
OTS-8 antigen, the function of which in tumors is unknown. Expression of mouse Aggrus and its human homologue (also known as T1␣-2/gp36) induced platelet
aggregation without requiring plasma components. Using the 8F11 antibody, we identified the highly conserved platelet aggregation-stimulating domain withputative O-glycosylated threonine residues as the critical determinant for exhibiting platelet aggregation-inducing capabilities. We compared the expression level ofhuman aggrus mRNA using an array containing 160
cDNA pair samples derived from multiple human tumorigenic and corresponding normal tissues from individual patients. We found that expression level of aggrus
was enhanced in most colorectal tumor patients. To confirm the protein expression, we generated anti-human
Aggrus polyclonal antibodies. Immunohistochemical
analysis revealed that Aggrus expression was frequently
up-regulated in colorectal tumors. These results suggestthat Aggrus/T1␣ is a newly identified, platelet aggregation-inducing factor expressed in colorectal tumors.

Specific glycoproteins expressed on the surface of platelets
enable the platelets to adhere to receptors exposed in areas ofvascular damage (1). The process of adhesion activates platelet
aggregation, leading to the formation of a platelet plug in thevessel wall. Activated platelets also induce the formation of afibrin clot by carrying coagulation factors and providing a catalytic surface for the major interactions of the coagulation* This study was supported in part by a special grant for Advanced
Research on Cancer from the Ministry of Education, Culture, Sports,
Science and Technology, Japan. The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
¶ Both authors contributed equally to this work.
** To whom correspondence should be addressed. Tel.: ⫹81-3-58417861; Fax: ⫹81-3-5841-8487; E-mail: ttsuruo@iam.u-tokyo.ac.jp.
This paper is available on line at http://www.jbc.org

cascade. Because there exists a clear link between atherosclerotic vascular disease, inflammation, tumor metastasis, andthrombosis (1–3), it is important to identify the mechanisms ofplatelet aggregation that have pathobiologic, prognostic, andtreatment-related relevance. Studies on cancer metastasis
have shown that some human and animal tumor cells possess
platelet aggregation-inducing abilities that correlate with theirmetastatic potential (2, 3). Interactions between tumor cells
and platelets have been considered to facilitate the arrest oftumor cell cluster in the microcirculation with the subsequent
formation of experimental metastasis. However, the molecules
associated with the tumor-induced platelet aggregation have
not yet been identified.
We previously established several clones possessing different
platelet aggregation-inducing capabilities from a mouse colon
adenocarcinoma 26 cell line (4). By generating monoclonal antibodies, we revealed that the established 8F11 monoclonal
antibody inhibited platelet aggregation in vitro (5, 6) and thepulmonary metastasis of highly metastatic clones of mousecolon adenocarcinoma 26 cell line in vivo (7). The 8F11 antibody recognized a cell-surface 44- or 41-kDa sialoglycoprotein(Aggrus) (5, 6) Using 8F11 affinity-column chromatography, we
purified the 44-kDa mouse Aggrus and found that purified
Aggrus itself could induce platelet aggregation with no requirement for plasma components (8). We tried to identify the protein; however, we could not obtain the peptide sequence fromthe purified Aggrus protein because of the abundant carbohydrate chains.
In this study, we identified the 8F11 antibody-reactive
mouse Aggrus as mouse T1␣ antigen (also known as mT1␣/gp38P/OTS-8) (9 –11). Using the platelet aggregation-neutralizing 8F11 antibody, we identified the residue (PLAG1 domain)
critical for platelet aggregation-inducing capability of mouse
Aggrus. Although mouse and human Aggrus (also known as
T1␣-2/gp36) (9, 12) proteins have only 39% amino acid identity,
the PLAG domains are highly conserved. Mutation of threonine
residues in the PLAG domain abolished the platelet aggregation-inducing abilities of human and mouse Aggrus proteins.
Using Cancer Profiling Array II, we found that the expression
level of aggrus was enhanced in colorectal tumor patients. By
generating polyclonal antibodies to human Aggrus/T1␣-2/gp36,
we confirmed the increased human Aggrus/T1␣-2/gp36 protein
expression in colorectal tumors. Therefore, Aggrus is a newly
identified platelet aggregation-inducing factor that is overexpressed in colorectal tumors.
1
The abbreviations used are: PLAG, platelet aggregation-stimulating; AGR, Aggrus; siRNA, small interfering RNA oligonucleotide duplexes; PRP, platelet-rich plasma; CHO, Chinese hamster ovary.

51599

51600Aggrus Possesses Platelet Aggregation-inducing Capability
EXPERIMENTAL PROCEDURES

Plasmids, siRNA, and Antibodies—Mouse aggrus/mT1␣/gp38P
cDNA (GenBank accession no. AJ297944 or AJ250246) (9, 10) was
obtained by reverse transcription-PCR with NL-17 mRNA as the template. Human aggrus/hT1␣-2/gp36 cDNA (GenBank accession no.
AF030428 or AJ225022) (9, 12) was obtained by PCR using a normal
human lung cDNA library (CosmoBio, Tokyo, Japan) as the template.
The amplified cDNAs were subcloned into a pcDNA3 vector, and a
FLAG epitope tag was added at the COOH terminal. Substitution of theappropriate amino acid codons to alanine codons in aggrus cDNAs was
accomplished using the QuikChange site-directed mutagenesis kit(Stratagene, La Jolla, CA) (13, 14). The mouse aggrus deleted cDNAs
were also ligated into pET-21a vector (Novagen, Darmstadt, Germany),
and the recombinant mouse Aggrus deletion mutant proteins wereexpressed in Escherichia coli. Five small interfering RNA oligonucleotide duplexes (siRNAs) were designed from the mouse aggrus/mT1␣
sequence. In our experiments to suppress mouse Aggrus expression in
NL-17 cells, we used the most effective siRNA from among them. The
coding strands of the siRNA directed to residues 180 –198 of mouse
aggrus cDNA were 5⬘-UGAAUCUACUGGCAAGGCA-3⬘ and 5⬘-UGCCUUGCCAGUAGAUUCA-3⬘, respectively. Non-silencing control siRNA
was purchased from QIAGEN (Valencia, CA). The oligonucleotides had
3⬘ dTdT overhangs. The anti-human Aggrus polyclonal antibody(TT679) was obtained by immunizing rabbits with the synthetic peptide
CEGGVAMPGAEDDVV, corresponding to amino acids 38 –51 of human
Aggrus plus N-terminal cysteine and purified using a column linked tothe peptide. Rat monoclonal 8F11 antibody was purified from ascites
fluid (5). Control rat IgG, anti-FLAG-M2 antibody, and anti-␤-actin
antibody were obtained from Sigma.
Cell Culture Conditions—Chinese hamster ovary (CHO), mouse colon adenocarcinoma NL-14 and NL-17 (4), mouse lymph node stromal
CA-12 (15), mouse osteoblastic MC3T3-E1 (11), and thioglycolate-elicited mouse peritoneal macrophages were cultured at 37 °C in a humidified atmosphere of 5% CO2 and 95% air in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Biocell,
Carson, CA), 2 mM L-glutamine (Invitrogen), and 100 ␮g/ml of kanamycin.
Transient Transfections and Establishment of Stable Transfectants—
Cells were transfected with appropriate amounts of plasmids using Superfect transfection reagent (QIAGEN) or LipofectAMINE 2000 reagent(Invitrogen) according to the manufacturers’ instructions (14). RNA interference was performed by transfecting control or mouse aggrus siRNAplus mock pcDNA3 vector into NL-17 cells (4) using LipofectAMINE 2000
reagent. Stable CHO transfectants or pooled transient CHO transfectants
were selected by cultivating the transfectants in medium containing 1
mg/ml of G-418 (Geneticin), and the expression level of mouse or human
Aggrus was confirmed by immunoblot analysis.
Western Blotting, Immunoprecipitation, and Enzyme-linked Immunosorbent Assay—Western blotting and immunoprecipitation were performed as described previously (14). The synthetic DGMVPPGIEC peptide (wild-type) corresponding to amino acids 39 – 47 of mouse Aggrus
sequence plus C-terminal cysteine and its mutant peptides DGAVPPGIEC (M41A), DGMAPPGIEC (V42A), DGMVAPGIEC (P43A), and
DGMVPAGIEC (P44A) were immobilized on a 96-well plate (mutatedamino acids are shown in bold letters). The reactivity of 8F11 antibody
and control rat IgG to the peptides was estimated by enzyme-linked
immunosorbent assay (16).
Flow Cytometry—Parental CHO or CHO/mAGR cells were harvested
by brief exposure to trypsin. After washing with phosphate-bufferedsaline, cells were treated with 8F11 antibody for 1 h at 4 °C after
treatment with fluorescein isothiocyanate-conjugated anti-rat IgG(Cappel, Cochranville, PA). Fluorescence data were collected using a
FACScalibur flow cytometer (BD Biosciences).
Platelet Aggregation Assay—Mouse platelet-rich plasma (PRP) was
prepared from fresh, heparinized blood drawn from BALB/c mice (8).
Human PRP was obtained from a healthy volunteer. Washed platelets
were separated from PRP by centrifugation at 400 ⫻ g for 5 min,washed, and resuspended in phosphate-buffered saline (8). In someexperiments, CHO/mAGR cells were incubated with 1 mg/ml of 8F11
antibody or normal rat IgG for 20 min on ice before incubation withplatelets (5). The platelet aggregation was monitored using an NKKHEMA Tracer I (Niko Bioscientific Co., Tokyo, Japan) (5, 6).
Reverse Transcription-PCR, PCR, and Cancer Profiling Array Analysis—Reverse transcription-PCR and PCR were performed as described
previously using the mouse aggrus sense (5⬘-CTCAAGCTTCAAGATGTGGACCGTGCCAGTG-3⬘), mouse aggrus antisense (5⬘-GAGGAATTCGGGCGAGAACCTTCCAGAAATC-3⬘), human aggrus sense (5⬘-TGTGGAAGGTGTCAGCTCTG-3⬘), human aggrus antisense (5⬘-TTAGGGC-

GAGTACCTTCCCG-3⬘), mouse ␤-actin sense (5⬘-GATATCGCTGCGCTGGTCGTCGAC-3⬘), mouse ␤-actin antisense (5⬘-CAAGAAGGAAGGCTGGAAAAGAGC-3⬘), human ribosomal protein S9 sense (5⬘-GATGAGAAGGACCCACGGCGTCTGTTCG-3⬘), and human ribosomal protein
S9 antisense (5⬘-GAGACAATCCAGCAGCCCAGGAGGGACA-3⬘) oligonucleotides. We compared the expression level of human aggrus mRNAin the same patient using Cancer Profiling Array II according to the
manufacturer’s instructions (BD Biosciences). Cancer Profiling Array II
consists of 160 cDNA pairs, synthesized from human tumorigenic andcorresponding normal tissues. Each pair was independently normalizedbased on the expression of four housekeeping genes and immobilized inseparate dots (17, 18). The full-length 32P-labeled human aggrus cDNA
was used for hybridization and radioactivity of each dot was visualized
and quantified with a BAS1000 Bio-Imaging Analyzer (Fuji Film,
Tokyo, Japan) (13). The expression level of human aggrus mRNA in theindividual patients was normalized by measuring radioactivity of each
dot after hybridization with 32P-labeled ubiquitin cDNA.
Immunohistochemistry—Fixed and paraffin-embedded colorectal tissue specimens (LandMark Tissue Microarrays, lot 022P15A; Ambion,
Austin, Texas) were deparaffinized, rehydrated, and treated to blockendogenous peroxidase by 3% H2O2 and nonspecific staining by caseincontaining blocking reagent (DAKO Cytomation, Glostrup, Denmark).
TT679 antibody was added (0.01 ␮g/ml) for 18 h at 23 °C, followed byincubation with biotin-conjugated secondary anti-rabbit IgG antibody
and peroxidase-conjugated streptavidin (LSAB2 kit; DAKO Cytomation). Color was developed with 3,3-diaminobenzidine tetrahydrochloride tablet sets (DAKO Cytomation) used for 10 min.
RESULTS

Molecular Identification of A Platelet Aggregation-inducing
Factor—To identify the 8F11 antibody-reactive gp44/mouse
Aggrus (mAGR) protein, we screened cells expressing the protein. We found that mouse osteoblastic MC3T3-E1 (11), lymph
node stromal CA-12 (15), and macrophages expressed mouse
Aggrus protein (Fig. 1A). Several reports suggested that mouse
T1␣ antigen (also known as mT1␣/gp38P/OTS-8) (9 –11) and its
human homologue (T1␣-2/gp36) (9, 12) were expressed in
MC3T3-E1 cells (11) and lymphoid tissues (19). Because of theexpression pattern, we hypothesized that mT1␣, which is required for normal lung cell proliferation and alveolus formationin vivo (20), is identical to an unidentified platelet aggregationinducing factor (Aggrus). We generated pcDNA3 vector containing FLAG-tagged full-length mT1␣ cDNA. When CHO cells
were stably transfected with mT1␣ cDNA (CHO/mAGR), transfected mT1␣ antigen was expressed on the cell surface and wasrecognized by the 8F11 antibody (Fig. 1B). Immunoprecipitation followed by immunoblot analysis revealed that 8F11 antibody recognized the antigen itself (Fig. 1C). We designed fivesiRNAs (21) and found that the siRNA directed to residues
180 –198 of mT1␣ decreased the expression of endogenous
mouse Aggrus in highly metastatic NL-17 and B16-F10 cells(Fig. 1D and data not shown). As we have reported previously(5), protein expression level of gp44/mouse Aggrus in highly
metastatic NL-17 cells was higher than that in poorly metastatic NL-14 cells (Fig. 1E). Semiquantitative PCR analysis
revealed the increased aggrus mRNA expression in NL-17 cells(Fig. 1E). These results indicate that the mT1␣/gp38P/OTS-8
antigen is identical to our previously characterized mouse
Aggrus/gp44 protein.
Aggrus Promotes Aggregation of Human and Mouse Platelets
without Requiring Plasma Components—When CHO/mAGR
cells were incubated with mouse PRP, CHO/mAGR cells, but
not parental CHO cells, induced mouse and human platelet
aggregation (Fig. 2, A and B), and the aggregation was suppressed by pretreatment of the cells with 8F11 antibody (Fig.
2C). Consistent with our previous report (8), plasma components were not required because washed platelets were also
aggregated by incubation with CHO/mAGR (Fig. 2D). We
cloned human aggrus cDNA (hAGR; also known as hT1␣-2 or
gp36) (9, 12) and transiently transfected it into CHO cells(CHO/hAGR). Expression of human Aggrus on CHO cell sur-

Aggrus Possesses Platelet Aggregation-inducing Capability

FIG. 1. Molecular identification of mouse Aggrus. A, the celllysate of lymph node stromal CA-12, osteoblastic MC3T3-E1, and thioglycolate-elicited peritoneal macrophages (m␾) were electrophoresedand immunoblotted with 8F11 antibody. B, flow cytometric analysis of
8F11 reactivity to parental CHO and a stable mouse Aggrus transfectant (CHO/mAGR). C, parental CHO and CHO/mAGR cell lysates wereincubated with protein G agarose that had been conjugated with 8F11
antibody, anti-FLAG antibody, or control rat IgG. After washing, immunoprecipitated proteins were electrophoresed and immunoblottedwith 8F11 antibody. WB, Western blot. D, NL-17 cells were transfectedwith pcDNA3 vector plus non-silencing control siRNA (⫺) or mouse
aggrus (mAGR) siRNA (⫹). Cells were harvested, electrophoresed, andimmunoblotted with 8F11 antibody or anti-␤-actin antibody. E, semiquantitative reverse transcription-PCR analysis and immunoblot analysis of mouse Aggrus or ␤-actin mRNA and protein expression in NL-17
and NL-14 cells.

face induced mouse and human platelet aggregation (Fig. 2, A
and B), regardless of poor homology (39% amino acid identitywith mouse Aggrus) between mouse and human Aggrus proteins (9). These results indicate that human and mouse Aggrus
proteins possessed the ability to induce platelet aggregation.
Identification of the 8F11 Recognition Domain in Mouse
Aggrus—The mouse Aggrus-induced mouse platelet aggregation was perfectly suppressed by pretreatment of CHO/mAGR
cells with 8F11 antibody (Fig. 2C). Thus, 8F11 may recognize
the specific epitope of mouse Aggrus that interacts with platelets. To clarify the residues critical for platelet aggregation, we
examined the reactivity of 8F11 antibody to recombinantmouse Aggrus protein expressed in E. coli (Fig. 3, A–C). The
39-DGMVPP-44 residue was identified as essential for 8F11
recognition of recombinant mouse Aggrus protein. To confirm
the result, we transiently transfected the pcDNA3 vector containing mouse aggrus-point mutated cDNAs into CHO cells.
The 8F11 antibody could recognize all point mutants except
G40A, M41A, V42A, and P43A (Fig. 4A). Conversion of glycine-38 to alanine (G38A) decreased the 8F11-binding capability. This result was consistent with the result indicating that
8F11 recognized the synthetic 39-DGMVPPGIE-47 plus C-terminal cysteine but not its point-mutated peptides (Fig. 4B).
Therefore, 39-DGMVPP-44 is the 8F11 recognition domain inmouse Aggrus. We established CHO cells transiently transfected with the pcDNA3 vector containing mouse aggrus-M41A
cDNA (CHO/M41A). CHO/M41A had almost the same platelet
aggregation-inducing ability that CHO/mAGR did (Fig. 4C).
Moreover, the peptide coding 8F11-recognition domain could
not induce platelet aggregation by itself (data not shown).

51601

FIG. 2. Mouse and human Aggrus proteins possessed the ability to induce aggregation of human and mouse platelets. A and B,
the platelet aggregation-inducing capability of parental CHO, a stable
mouse Aggrus transfectant (CHO/mAGR), or a transient human Aggrus
transfectant (CHO/hAGR) was estimated by incubating the cells withmouse PRP (A) or human PRP (B). C, CHO/mAGR cells were preincubated with 1 mg/ml of control rat IgG or 8F11 on ice. After incubationfor 20 min, the platelet aggregation-inducing capability was estimatedby incubating cell suspension with mouse PRP. D, the platelet aggregation-inducing capability of parental CHO or CHO/mAGR was estimated by incubating the cells with washed mouse platelets.

These results suggest that the identified 8F11 recognition domain may not be involved in direct binding to platelets.
Identification of the Platelet Aggregation-stimulating Domain in Aggrus As the Critical Determinant for Exhibiting
Platelet Aggregation-inducing Capabilities—As we have characterized previously (8), carbohydrate chains of mouse Aggrus/
gp44 are involved in the platelet aggregation-inducing capability. We hypothesized that the 8F11 antibody might neutralize
platelet the aggregation-inducing capability of mouse Aggrus
by conformationally interfering with the carbohydrate chainsnear the 8F11 binding domain. We then generated several
mouse aggrus point mutants in which putative O-glycosylated
threonine residues near the 8F11-binding domain were converted to alanine residues. Mutation of these threonine residues affected neither 8F11 binding (Fig. 4A) nor cell surface
expression (data not shown). However, conversion of threonine-34 to alanine (T34A) in mouse Aggrus eliminated theplatelet aggregation-inducing capability (Fig. 5A). Because
threonine followed by proline is likely to be O-glycosylated (22)and Edman microsequencing of the equivalent residues in the
dog homologue of Aggrus revealed gaps at the threonine residues (23), threonine-34 in mouse Aggrus might be O-glycosylated, and the carbohydrate chain was involved in the interaction of the platelets and subsequent platelet aggregation.
Sequence comparison of the residues around threonine-34 inmouse Aggrus and residues around threonine-52 in human,
threonine-34 in rat (24), and threonine-41 or threonine-50 indog (23) homologues showed mouse Aggrus to be highly conserved (Fig. 5B). We therefore termed the residues as PLAG
(platelet aggregation-stimulating) domains. As expected, conversion of threonine-52 to alanine in human Aggrus (T52AhAGR) specifically abolished its platelet aggregation-inducing

51602Aggrus Possesses Platelet Aggregation-inducing Capability

FIG. 3. Identification of the 8F11 antibody-recognition domain
in mouse Aggrus. A and B, the indicated FLAG-tagged mouse aggrus
deleted cDNAs were ligated into pET-21a vector, and the recombinantmouse Aggrus proteins were expressed in E. coli, electrophoresed, andimmunoblotted with 8F11 antibody or anti-FLAG antibody. WB, Western blot. C, amino acid sequences of mouse Aggrus deletion mutants
used in these experiments and their 8F11 reactivity are shown schematically (solid bars, strong binding; gray bar, weak binding; open bars,
no binding).

capability (Fig. 5C). These results indicate that the PLAG
domain (especially O-glycosylated threonine residues) is critical for platelet aggregation.
Expression of Aggrus in Colorectal Tumors—Mouse aggrus
mRNA expression was observed in parental mouse colon adenocarcinoma 26 and its sublines, and the expression levels of
aggrus mRNA in these cells were higher than that in normalmouse colon tissue (data not shown). Thus, we estimated thehuman aggrus expression in human tumors using Cancer Profiling Array II. In this array, normalized cDNA pairs from 160
primary tumor tissues and the corresponding normal tissues
from individual patients were immobilized on nylon membrane(17, 18). Human aggrus expression increased in almost all
intestinal tumors (colon, rectum, and small intestine) compared with corresponding normal tissues (Fig. 6A). After hybridization with 32P-labeled ubiquitin cDNA, human aggrus
expression in each patient was normalized. The average expression ratio of human aggrus in colon (n ⫽ 10), rectum (n ⫽7), and small intestine (n ⫽ 10) tumor patients was about 2.8,
3.2, and 3.9, respectively (Fig. 6B). To confirm the result, we
performed PCR with matched cDNA pairs from colon adenocarcinoma patient 5 and small intestine adenocarcinoma patient 2, whose cDNAs were immobilized in Cancer Profiling
Array II (Fig. 6A, circled). Semiquantitative PCR analysis revealed that human aggrus expression was clearly enhanced inboth tumor tissues (Fig. 6C). In contrast, human aggrus expression in lung, breast, and ovary varied from patient topatient (Fig. 6, A and B), and the average expression ratio inlung, breast, and ovary was about 1.6, 0.94, and 1.1, respectively. Human aggrus expression in adenocarcinoma of theintestine was higher than that in colon adenomas or small
intestine carcinoid tumors (data not shown). Therefore, human
aggrus expression might be specific for carcinoma. We obtained
similar results using Cancer Profiling Array I (data not shown).

FIG. 4. Confirmation of amino acid residues recognized by theplatelet aggregation-neutralizing 8F11 antibody. A, CHO cells
were transiently transfected with a pcDNA3 vector containing none
(Mock), wild-type (WT), or the indicated mutant mouse aggrus cDNAs.
The cell lysates were immunoblotted with 8F11 or anti-FLAG antibody.
WB, Western blot. B, the synthetic DGMVPPGIEC peptide (WT) corresponding to amino acids 39 – 47 of mouse Aggrus sequence plus Cterminal cysteine and its mutant peptides were immobilized on a 96well plate, as described under “Experimental Procedures.” Thereactivity of 8F11 antibody and control rat IgG was estimated byenzyme-linked immunosorbent assay. Each point represents a mean ⫾S.D. of triplicate determinations. C, the platelet aggregation-inducing
activity of parental CHO, a stable mouse Aggrus transfectant (CHO/
mAGR), or a transient M41A-mouse Aggrus transfectant (CHO/M41A)
was estimated by incubating the cells with mouse PRP.

To confirm the increased human Aggrus protein expressionin colorectal tumors, we generated polyclonal antibodies tohuman Aggrus (TT679) because the 8F11 antibody cannot recognize human Aggrus (Fig. 7A). The generated TT679 antibody
recognized the 36-kDa human Aggrus protein and did notcross-react with other protein species (data not shown). Strong
staining was seen in human specimens derived from colon
adenocarcinoma tissues (Fig. 7, B and C) but not in samples
from normal colon tissues (Fig. 7, D and E). These results
indicate that expression level of Aggrus is enhanced in mostcolorectal tumor patients.
DISCUSSION

Several reports suggested that tumor-induced platelet aggregation is an early step in the development of a metastatic lesion(2, 3). Ultrastructural studies have also demonstrated that

Aggrus Possesses Platelet Aggregation-inducing Capability

51603

FIG. 5. Identification of PLAG domain in Aggrus protein. A,
CHO cells were transiently transfected with mouse aggrus cDNA carrying the indicated point mutations. Their platelet aggregation-inducing capabilities were estimated by incubating the cells with mouse PRP.
B, alignment of the amino acid sequence of Aggrus homologues aroundthe PLAG domain. Identical amino acids are denoted by white letters on
black background. An asterisk indicates the possible O-glycosylationsite. C, CHO cells were transiently transfected with T52A- or T55Ahuman aggrus cDNA (CHO/hAGR-T52A or CHO/hAGR-T55A, respectively). Their platelet-aggregation inducing capabilities were estimatedby incubating the cells with mouse PRP.

platelets seem to enhance the development of arrested tumor
emboli into a secondary metastatic colony (25, 26). Althoughseveral inhibitors of platelet aggregation have been reported toretard tumor metastasis in certain animal models (27–29), thetumor adhesive glycoproteins have not yet been identified.
We previously established several clones possessing different
metastatic abilities from a mouse colon adenocarcinoma 26 cellline (4). Among these clones, a highly metastatic clone, NL-17,
was found to exhibit a high platelet aggregation-inducing activity, although a poorly metastatic clone, NL-14, had marginalplatelet aggregation-inducing capability (28, 30). Therefore,
the ability to induce platelet aggregation was related to themetastatic potential. To identify the platelet aggregation-inducing molecule(s) associated with tumor metastasis, we generated monoclonal antibodies by immunizing rats with NL-17
cells. One of the established antibodies, 8F11, exhibited theplatelet aggregation-neutralizing activity in vitro and the inhibitory activity of lung colonization in vivo (5, 7). The 8F11
antibody recognized a 44-kDa membrane sialoglycoprotein(mouse Aggrus/gp44) on NL-17 cells (5). Consistent with theplatelet aggregation-inducing capabilities of NL-17 and NL-14
cells (28, 30), the expression level of 8F11 antibody-reactive
mouse Aggrus in NL-17 cells was higher than that in NL-14
cells (Fig. 1E). We previously succeeded in purifying the mouse
Aggrus from NL-17 cells (8) and found that the purified mouse

FIG. 6. Human aggrus expression in colorectal, rectal, andsmall intestinal tumors. A and B, human aggrus expression in tumor(T) and normal (N) tissues in the same patient was assessed by hybridizing 32P-labeled human aggrus cDNA to Cancer Profiling Array II. The
expression level of human aggrus in the individual patients was normalized by measuring radioactivity of each dot after hybridization with
32
P-labeled ubiquitin cDNA. The radioactivity of each dot was visualized (A) and quantified (B). Red bar, average of the human Aggrus
expression ratio in each tissue (B). C, semiquantitative PCR analysis ofhuman aggrus and ribosomal protein S9 expressions in colon adenocarcinoma patient 5 and small intestine adenocarcinoma patient 2 (indicated by circles in A).

51604Aggrus Possesses Platelet Aggregation-inducing Capability

FIG. 7. Human Aggrus protein expression in colon adenocarcinoma. A, CHO cells were transiently transfected with a pcDNA3 vector
containing the indicated mouse or human aggrus cDNAs. The cell lysates were immunoblotted with 8F11 antibody or anti-human Aggrus antibody(TT679). B–E, Immunohistochemical staining with anti-human Aggrus antibody (TT679) on human specimens derived from colon adenocarcinoma(B and C) compared with specimens derived from normal colon (D and E) tissues.

Aggrus itself possessed the ability to induce platelet aggregation. These data indicate that 8F11 antibody-reactive Aggrus isa platelet aggregation-inducing factor expressed on tumors.
We could not obtain the peptide sequence from the purified
mouse Aggrus. Thus, we searched cells expressing mouse
Aggrus. We found that mouse Aggrus was expressed on thesurface of mouse osteoblastic MC3T3-E1 cells (Fig. 1A). Because the 38-kDa mouse T1␣/gp38P/OTS-8 antigen was originally isolated from MC3T3-E1 cells (11), we hypothesized thatmouse Aggrus was identical to mouse T1␣/gp38P/OTS-8. Fourlines of evidence support this assumption. First, the 8F11 antibody recognized CHO cells that had been transfected withmouse T1␣/gp38P/OTS-8 cDNA (Fig. 1A); second, the immunoprecipitated FLAG-tagged mouse T1␣/gp38P/OTS-8 protein
was recognized by 8F11 antibody (Fig. 1C); third, siRNA directed to mouse T1␣/gp38P/OTS-8 decreased the 8F11 antibody-reactive endogenous mouse Aggrus expression in NL-17(Fig. 1D); and fourth, expression of mouse T1␣/gp38P/OTS-8 on
CHO cell surface induced platelet aggregation with no requirement for plasma components (Fig. 2). We observed that proteins recognized by 8F11 antibody in different mouse cell lines
vary in their electrophoretic motility (Fig. 1A). We also previously reported that 8F11 antibody recognized a 44-kDa protein
in colon adenocarcinoma sublines (5) and a 41-kDa protein in
B16 melanoma sublines (6). The siRNA to aggrus decreased theexpression level of both 44-kDa Aggrus in NL-17 cells (Fig. 1D)
and 41-kDa Aggrus in B16-F10 cells (data not shown). Therefore, we think these variations are caused by differences inpost-translational modification.
The identified mouse Aggrus was first identified as an earlyresponse protein (OTS-8) that was induced by phorbol ester inosteoblastic MC3T3-E1 cells (11). Mouse Aggrus has also beenreported as a cell-surface antigen (gp38/gp38P) that is expressed in type-I thymus epithelial cells and in peripheral
lymphoid tissues (10, 19). Human, rat, and dog homologues(E11 antigen/podoplanin/RT140/PA2.26/gp36/gp40) have been
already cloned (9, 12, 23, 24, 31) and have been reported to bemolecules expressed by various epithelial and nonepithelial
cell types. The molecules have putative extracellular and transmembrane domains and a short cytoplasmic tail with putative
protein kinase C and cAMP phosphorylation sites. Sequence
analysis indicates that Aggrus does not share common domains
with other protein families of known function that predict its
function. Thus, the physiological role of Aggrus has not yet
been determined. Several reports suggested that Aggrus could
be a regulator of fluid transport or a receptor for the influenza

C virus (9, 32). Our findings indicate that Aggrus functions as
an adhesion molecule that promotes platelet aggregation inpathological conditions.
A striking feature of Aggrus proteins is their extraordinarily
high content of serine and threonine residues that might beO-glycosylated. In dog Aggrus/gp40, 14 serine and threonine
residues were identified by Edman degradation to be modified
by O-glycosylation (23). Because our data (Fig. 4) suggested
that 8F11 antibody might neutralize mouse Aggrus-induced
platelet aggregation by conformationally interfering the carbohydrate chains near the 8F11-binding domain, we generated
several mouse aggrus point mutants in which putative O-glycosylated threonine residues around the 8F11-binding domain.
We revealed that putative O-glycosylated threonine-34 ofmouse Aggrus is a critical residue for its platelet aggregationinducing capability (Fig. 5). This result is consistent with ourprevious report (8) that sialylated carbohydrate chains ofAggrus/gp44 were involved in their platelet aggregation-inducing capabilities. Although human and mouse Aggrus proteins
have only 39% amino acid identity (9), the residues aroundthreonine-34 of mouse Aggrus are highly conserved (Fig. 5B).
Mutation of the equivalent threonine-52 in human Aggrus
abolished its platelet aggregation-inducing activity (Fig. 5C).
We also cloned rat homologue of Aggrus/RT140/E11/podoplanin(33–35) and found that it could induce platelet aggregation(data not shown). Mutation of the threonine-34 to alanine in rat
Aggrus abolished its platelet aggregation-inducing capability(data not shown). Thus, the PLAG domain is identified as
critical residues for their platelet aggregation-inducing capability. Because heavily O-glycosylated proteins were identified
as counter receptors for selectins (36, 37), it is possible that
Aggrus binds to selectins for triggering platelet aggregation.
We also observed increased aggrus mRNA expression inmost patients with intestinal tumors (Fig. 7, B–E). This result
was consistent with the result that aggrus mRNA expression incolon adenocarcinoma 26 sublines was higher than that innormal mouse colons. Interestingly, the aggrus expression in
adenocarcinoma of the intestine was higher than that in colon
adenomas or small intestine carcinoid tumors (data notshown). Therefore, human aggrus expression might be specificfor carcinoma and was strongly correlated with tumor
progression.
Our results indicate that Aggrus is a newly identified platelet aggregation-inducing factor expressed on the surface ofseveral tumors. Because platelet aggregation is associated withthe development of arrested tumor emboli into a secondaryAggrus Possesses Platelet Aggregation-inducing Capability
metastatic colony, therapies aimed at neutralizing Aggrus
function could prove successful in inhibiting tumor metastasis.
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