Functional Sialylated O-Glycan to Platelet Aggregation on Aggrus (T1α/Podoplanin) Molecules Expressed in Chinese Hamster Ovary Cells

Aggrus, also called T1α and podoplanin, is a novel platelet aggregation-inducing factor that is expressed in various carcinoma cells. Aggrus/T1α/podoplanin is known to be expressed in lung type I alveolar cells or lymphatic endothelial cells. However, its physiological role has not been clarified. To assess the attribution of glycosylation to Aggrus platelet aggregation activity, recombinant molecules were stably expressed in a series of Chinese hamster ovary (CHO) cell mutants, N-glycan-deficient Lec1, CMP-sialic acid transporter-deficient Lec2, and UDP-galactose transporter-deficient Lec8. A new anti-human Aggrus monoclonal antibody, YM-1, was established to detect the expression of human Aggrus on these CHO cell mutants. Aggrus on Lec1 cells induced platelet aggregation, but those on Lec2 and Lec8 cells did not. Further, the glycans on Aggrus were analyzed by lectin blotting. Aggrus expressed in CHO and Lec1 cells showed Wheat-germ agglutinin, Jacalin, and Vicia villosa lectin bindings. Lectin blotting results indicated that sialylated core 1 structures, sialic acid plus Galβ1,3GalNAc-Ser/Thr, were critical for the platelet aggregation activity. This oligosaccharide structure is known as tumor-associated antigen, which is potentially related to the metastasis process of cancer cells. Aggrus, also called T1α and podoplanin, is a novel platelet aggregation-inducing factor that is expressed in various carcinoma cells. Aggrus/T1α/podoplanin is known to be expressed in lung type I alveolar cells or lymphatic endothelial cells. However, its physiological role has not been clarified. To assess the attribution of glycosylation to Aggrus platelet aggregation activity, recombinant molecules were stably expressed in a series of Chinese hamster ovary (CHO) cell mutants, N-glycan-deficient Lec1, CMP-sialic acid transporter-deficient Lec2, and UDP-galactose transporter-deficient Lec8. A new anti-human Aggrus monoclonal antibody, YM-1, was established to detect the expression of human Aggrus on these CHO cell mutants. Aggrus on Lec1 cells induced platelet aggregation, but those on Lec2 and Lec8 cells did not. Further, the glycans on Aggrus were analyzed by lectin blotting. Aggrus expressed in CHO and Lec1 cells showed Wheat-germ agglutinin, Jacalin, and Vicia villosa lectin bindings. Lectin blotting results indicated that sialylated core 1 structures, sialic acid plus Galβ1,3GalNAc-Ser/Thr, were critical for the platelet aggregation activity. This oligosaccharide structure is known as tumor-associated antigen, which is potentially related to the metastasis process of cancer cells. The concept has been accepted that tumor metastasis is associated with platelet-aggregation activity possessed by human and other mammalian cancer cells (1Honn K.V. Tang D.G. Crissman J.D. Cancer Metastasis Rev. 1992; 11: 325-351Crossref PubMed Scopus (283) Google Scholar, 2Bastida E. Ordinas A. Haemostasis. 1988; 18: 29-36PubMed Google Scholar). A previous study has clarified that membranous 44- and 36-kDa sialoglycoproteins in the cancer cells of mice and humans, respectively, aggregated platelets with no relation to plasma components (3Kato Y. Fujita N. Kunita A. Sato S. Kaneko M. Osawa M. Tsuruo T. J. Biol. Chem. 2003; 278: 51599-51605Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 4Watanabe M. Sugimoto Y. Tsuruo T. Cancer Res. 1990; 50: 6657-6662PubMed Google Scholar, 5Toyoshima M. Nakajima M. Yamori T. Tsuruo T. Cancer Res. 1995; 55: 767-773PubMed Google Scholar). Ectopic expression of these molecules was observed in a variety of cancer cells including colorectal and testicular tumors, in which up-regulated expression was evident (3Kato Y. Fujita N. Kunita A. Sato S. Kaneko M. Osawa M. Tsuruo T. J. Biol. Chem. 2003; 278: 51599-51605Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 6Kato Y. Sasagawa I. Kaneko M. Osawa M. Fujita N. Tsuruo T. Oncogene. 2004; (in press)Google Scholar). The molecules, designated as Aggrus, belong to a type-I transmembrane sialomucin-like glycoprotein that consisted of an extracellular domain with abundant Ser and Thr residues as potential O-glycosylation sites, a single transmembrane portion, and a short cytoplasmic tail with putative sites for protein kinase C and cAMP phosphorylation (3Kato Y. Fujita N. Kunita A. Sato S. Kaneko M. Osawa M. Tsuruo T. J. Biol. Chem. 2003; 278: 51599-51605Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 7Dobbs L.G. Williams M.C. Gonzalez R. Biochim. Biophys. Acta. 1988; 970: 146-156Crossref PubMed Scopus (218) Google Scholar). Homologous molecules to Aggrus have been identified independently in several mammalians; T1α (7Dobbs L.G. Williams M.C. Gonzalez R. Biochim. Biophys. Acta. 1988; 970: 146-156Crossref PubMed Scopus (218) Google Scholar, 8Rishi A.K. Joyce-Brady M. Fisher J. Dobbs L.G. Floros J. VanderSpek J. Brody J.S. Williams M.C. Dev. Biol. 1995; 167: 294-306Crossref PubMed Scopus (180) Google Scholar), podoplanin (9Breiteneder-Geleff S. Matsui K. Soleiman A. Meraner P. Poczewski H. Kalt R. Schaffner G. Kerjaschki D. Am. J. Pathol. 1997; 151: 1141-1152PubMed Google Scholar), PA2.26 (10Gandarillas A. Scholl F.G. Benito N. Gamallo C. Quintanilla M. Mol. Carcinog. 1997; 20: 10-18Crossref PubMed Scopus (83) Google Scholar), gp40 (11Zimmer G. Klenk H.D. Herrler G. J. Biol. Chem. 1995; 270: 17815-17822Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), RANDAM-2 (12Kotani M. Tajima Y. Osanai T. Irie A. Iwatsuki K. Kanai-Azuma M. Imada M. Kato H. Shitara H. Kubo H. Sakuraba H. J. Neurosci. Res. 2003; 73: 603-613Crossref PubMed Scopus (14) Google Scholar) are isolated from rat alveolar type-I cells in lung, rat glomerular epithelial cell (podocyte), mouse keratinocytes, Madin-Darby canine kidney cells (type I), and glutamatergic neuronal cells in mouse cerebrum, respectively. Podoplanin (gp36) is utilized as a specific marker for lymphatic endothelium in histopathology (13Makinen T. Veikkola T. Mustjoki S. Karpanen T. Catimel B. Nice E.C. Wise L. Mercer A. Kowalski H. Kerjaschki D. Stacker S.A. Achen M.G. Alitalo K. EMBO J. 2001; 20: 4762-4773Crossref PubMed Scopus (675) Google Scholar, 14Petrova T.V. Makinen T. Makela T.P. Saarela J. Virtanen I. Ferrell R.E. Finegold D.N. Kerjaschki D. Yla-Herttuala S. Alitalo K. EMBO J. 2002; 21: 4593-4599Crossref PubMed Scopus (538) Google Scholar). Ramirez and co-workers (15Ramirez M.I. Millien G. Hinds A. Cao Y. Seldin D.C. Williams M.C. Dev. Biol. 2003; 256: 61-72Crossref PubMed Scopus (213) Google Scholar, 16Schacht V. Ramirez M.I. Hong Y.K. Hirakawa S. Feng D. Harvey N. Williams M. Dvorak A.M. Dvorak H.F. Oliver G. Detmar M. EMBO J. 2003; 22: 3546-3556Crossref PubMed Scopus (560) Google Scholar) showed that T1α null mice, generated by a targeted dysfunction of the gene, died at birth from lethal respiratory failure accompanied by immature lymphatic vessel formation (15Ramirez M.I. Millien G. Hinds A. Cao Y. Seldin D.C. Williams M.C. Dev. Biol. 2003; 256: 61-72Crossref PubMed Scopus (213) Google Scholar, 16Schacht V. Ramirez M.I. Hong Y.K. Hirakawa S. Feng D. Harvey N. Williams M. Dvorak A.M. Dvorak H.F. Oliver G. Detmar M. EMBO J. 2003; 22: 3546-3556Crossref PubMed Scopus (560) Google Scholar). T1α/podoplanin conceivably plays an important role in regulating peripheral lung cell proliferation and lymphatic vascular development. In the experiment with respect to the 8F11 antibody that neutralized the platelet aggregating activity, it was evident that the segment of EDXXVTPG in the extracellular domain, designated as platelet aggregation-stimulating domain, was critical for the activity of Aggrus (3Kato Y. Fujita N. Kunita A. Sato S. Kaneko M. Osawa M. Tsuruo T. J. Biol. Chem. 2003; 278: 51599-51605Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). In particular, this motif, which is highly conserved among species, is tandemly triplicated in gp40 of dog. The occurrence of O-glycosylation to Thr residues was proved by Edman degenerative microsequencing (17Zimmer G. Lottspeich F. Maisner A. Klenk H.D. Herrler G. Biochem. J. 1997; 326: 99-108Crossref PubMed Scopus (38) Google Scholar). Furthermore, in the study of targeted mutagenesis to mouse and human Aggrus molecules, we obtained evidence that glycans on the platelet aggregation-stimulating domain play an important role for platelet aggregation (3Kato Y. Fujita N. Kunita A. Sato S. Kaneko M. Osawa M. Tsuruo T. J. Biol. Chem. 2003; 278: 51599-51605Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 5Toyoshima M. Nakajima M. Yamori T. Tsuruo T. Cancer Res. 1995; 55: 767-773PubMed Google Scholar). The present study examined the association of glycosylation with the platelet aggregating activity; recombinant Aggrus molecules were expressed in a set of three defective cell lines from Pro-5 Chinese hamster ovary (CHO) 1The abbreviations used are: CHO, Chinese hamster ovary; SA, sialic acid; Gal, galactose; WGA, Wheat-germ agglutinin; s-WGA, succinylated WGA; VVA, Vicia villosa lectin; mAb, monoclonal antibody; h, human; m, mouse. cells. Those mutant cells have defects in distinct steps of glycosylation. Lec1 CHO cells lack the glycosyltransferase termed GlcNAc-TI, which produces incomplete intermediates of N-linked carbohydrates (18Stanley P. Caillibot V. Siminovitch L. Cell. 1975; 6: 121-128Abstract Full Text PDF PubMed Scopus (183) Google Scholar, 19Stanley P. Narasimhan S. Siminovitch L. Schachter H. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3323-3327Crossref PubMed Scopus (160) Google Scholar, 20Stanley P. Siminovitch L. Somatic Cell Genet. 1977; 3: 391-405Crossref PubMed Scopus (124) Google Scholar). Lec2 cells are CMP-sialic acid transporter-deficient. In addition, both glycoproteins and glycolipids lack 90% of sialic acids (SA). Lec8 cells exhibit a remarkable reduction of the ability to transport UDP-galactose (Gal) into the Golgi compartment (22Stanley P. Mol. Cell. Biol. 1981; 1: 687-696Crossref PubMed Scopus (56) Google Scholar, 23Deutscher S.L. Hirschberg C.B. J. Biol. Chem. 1986; 261: 96-100Abstract Full Text PDF PubMed Google Scholar). Platelet aggregation-inducing activity was examined in those stably transfected cells. Moreover, the structure of active glycans was examined by the lectin-blot analysis. In addition, a monoclonal antibody to human Aggrus (hAggrus) was obtained by immunizing rat with the synthetic hAggrus peptide. Materials—Lymphatic endothelial cell was purchased from AngioBio (Del Mar, CA). Lung carcinoma cells, NCI-H226, and NCI-H520 were obtained from the American Type Culture Collection (ATCC). The anti-mouse Aggrus rat monoclonal antibody (mAb), 8F11, was prepared previously (24Watanabe M. Okochi E. Sugimoto Y. Tsuruo T. Cancer Res. 1988; 48: 6411-6416PubMed Google Scholar). Mouse anti-human podoplanin (gp36) mAb was obtained from Research Diagnostics Inc. (Flanders, NJ). Anti-FLAG antibody (M2) was purchased from Sigma-Aldrich. Horseradish peroxidase-conjugated secondary antibodies were obtained from DakoCytomation Co., Ltd. (Glostrup, Denmark). Fluorescein-isothiocyanate-conjugated anti-rat IgG was obtained from Cappel (Cochranville, PA). Fluorescein-isothiocyanate-conjugated anti-mouse IgG was from MC Biomedicals (Irvine, CA). Horseradish peroxidase-conjugated streptavidin was obtained from Amersham Biosciences. The following biotinylated lectins were purchased from Vector Laboratories Inc. (Peterborough, UK), concanavalin A, Dolichos biflorus agglutinin, peanuts agglutinin, Ricinus communis agglutinin I, soybean agglutinin, Wheat-germ agglutinin (WGA), succinylated WGA (s-WGA), Griffonia simplicifolia lectin I and II, Sophora japonica agglutinin, Datura stramonium lectin, Jacalin, Lycopersicon esculentum lectin, Solanum tuberosum lectin, Vicia villosa lectin (VVA), Sambucus nigra lectin, and Maackia amurensis lectin I and II. Hybridoma Production—Two Sprague-Dawley rats were immunized with the synthetic peptide, CEGGVAMPGAEDDVV, corresponding to amino acids 38-51 of hAggrus plus an N-terminal cysteine. The spleen cells were fused with mouse myeloma P3U1 cells. Then the culture supernatants of the hybridomas were screened by enzyme-linked immunosorbent assay for the binding activity to the Aggrus peptide. The established mAb was designated YM-1. Immunohistochemistry—YM-1 was added to the deparaffinized and rehydrated specimens for 4 h at 23 °C. Biotin-conjugated secondary anti-rat IgG antibody was added, followed by incubation with peroxidase-conjugated avidin-streptavidin complex (Vectastain ABC Kit, Vector Laboratories Inc.). Color was developed with 3,3-diaminobenzidine tetrahydrochloride tablet sets (DakoCytomation Co. Ltd.) used for 10 min. The human specimens were derived from prostate cancer (25Tian W. Osawa M. Horiuchi H. Tomita Y. Cancer Sci. 2004; 95: 491-495Crossref PubMed Scopus (28) Google Scholar). Production of Aggrus in CHO/Lec Cells—Lec1, Lec2, and Lec8 cells of CHO mutant cell lines were obtained from ATCC (18Stanley P. Caillibot V. Siminovitch L. Cell. 1975; 6: 121-128Abstract Full Text PDF PubMed Scopus (183) Google Scholar, 19Stanley P. Narasimhan S. Siminovitch L. Schachter H. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3323-3327Crossref PubMed Scopus (160) Google Scholar, 20Stanley P. Siminovitch L. Somatic Cell Genet. 1977; 3: 391-405Crossref PubMed Scopus (124) Google Scholar, 21Deutscher S.L. Nuwayhid N. Stanley P. Briles E.I. Hirschberg C.B. Cell. 1984; 39: 295-299Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 22Stanley P. Mol. Cell. Biol. 1981; 1: 687-696Crossref PubMed Scopus (56) Google Scholar, 23Deutscher S.L. Hirschberg C.B. J. Biol. Chem. 1986; 261: 96-100Abstract Full Text PDF PubMed Google Scholar). Cells were cultured at 37 °C in 5% CO2 with α-minimal essential medium (Sigma-Aldrich) containing 10% fetal calf serum (Moregate Biotech, Queensland, Australia), supplemented with l-glutamine (2 mm), l-pro-line (0.04 mg/ml), penicillin (100 units/ml), and streptomycin (100 μg/ml). Human and mouse aggrus cDNAs (AB127958, AJ297944), incorporated with FLAG tag sequence, were subcloned into a vector of pcDNA3 (Invitrogen), which were named as pcDNA-hAGR and pcDNA-mAGR, respectively. Lec1, Lec2, and Lec8 cells were transfected with pcDNA-hAGR and pcDNA-mAGR by a procedure using LipofectAMINE reagent (Invitrogen) as described previously (3Kato Y. Fujita N. Kunita A. Sato S. Kaneko M. Osawa M. Tsuruo T. J. Biol. Chem. 2003; 278: 51599-51605Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Stable production was obtained using a selective culture of the transfectants in a medium containing Geneticin (G418; Sigma-Aldrich) at a concentration of 1.0 mg/ml for a couple of weeks. Flow Cytometry—The expression levels of hAggrus and mouse Aggrus (mAggrus) were confirmed comparatively using by flow cytometry, as described previously (26Kaneko M. Kato Y. Horiuchi H. Osawa M. Immunol. Lett. 2003; 86: 45-51Crossref PubMed Scopus (10) Google Scholar). Briefly, lymphatic endothelial cells, NCIH226 cells, parental CHO cells, and Lec cells, collected by trypsin-EDTA treatment, were incubated with antibodies for 1 h at room temperature, YM-1 culture supernatant, and 8F11 in at a concentration of 1 Then the cells were incubated with antibodies for min. Flow was using Platelet aggregation was examined by the of M. Nakajima M. Yamori T. Tsuruo T. Cancer Res. 1995; 55: 767-773PubMed Google Scholar). Briefly, mouse plasma was prepared from from of of plasma was incubated in a at 37 °C with of cells were Then was at for 10 min. This assay was with a I Co., A was using a at the and Lectin cell were with a for on The consisted of and 1 The supernatants were for at to and Cell containing of protein were prepared for by in 5% 10% lectin blotting cell were with antibody for 1 h at 4 °C. the were was with a by for followed by in were an the were incubated with YM-1 antibody or biotinylated lectins for 1 h at room followed by incubation with peroxidase-conjugated anti-rat IgG or streptavidin to The activity was developed using an Amersham of Aggrus generated a mAb to hAggrus as described in the previous The of YM-1 was confirmed by and flow YM-1 lymphatic vessel of Aggrus expressed on human lymphatic endothelial cells and lung cancer cell were by YM-1 but not mAb This indicated that the YM-1 exhibit for Aggrus that of the Furthermore, flow showed that the YM-1 Platelet by Cells and in lymphatic endothelial and cells also induced platelet aggregation as hAggrus on CHO cells However, lung cell carcinoma which did not did not platelet aggregation not results indicated that the hAggrus also induced platelet Platelet by Lec were generated in the three mutant cells and parental CHO cells by of pcDNA-hAGR and pcDNA-mAGR to assess the physiological of glycosylation on Aggrus The transfectants were established by the of recombinant molecules was comparatively confirmed by flow using the specific antibodies of YM-1 and 8F11 from the of levels of Aggrus were and among these cell the platelet aggregation activity of the established transfectants was both hAggrus and in Lec1 cells induced platelet aggregation as as in parental CHO cells The aggregation was confirmed using cell in three not of induced platelet aggregation are also in as a In aggregation was in Aggrus molecules expressed in Lec2 and Lec8 cells. Lec1 CHO cells are of (18Stanley P. Caillibot V. Siminovitch L. Cell. 1975; 6: 121-128Abstract Full Text PDF PubMed Scopus (183) Google Scholar, 19Stanley P. Narasimhan S. Siminovitch L. Schachter H. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3323-3327Crossref PubMed Scopus (160) Google Scholar, 20Stanley P. Siminovitch L. Somatic Cell Genet. 1977; 3: 391-405Crossref PubMed Scopus (124) Google Scholar). The amino acid of for but did not in Lec1 cells platelet aggregation activity was not associated with this type of Furthermore, transporter-deficient Lec2 cells lack 90% of in both glycoproteins and glycolipids S.L. Nuwayhid N. Stanley P. Briles E.I. Hirschberg C.B. Cell. 1984; 39: 295-299Abstract Full Text PDF PubMed Scopus (198) Google Scholar). of sialylated is in Lec8 cells, is the for in (22Stanley P. Mol. Cell. Biol. 1981; 1: 687-696Crossref PubMed Scopus (56) Google Scholar, 23Deutscher S.L. Hirschberg C.B. J. Biol. Chem. 1986; 261: 96-100Abstract Full Text PDF PubMed Google Scholar). that are by these cell lines on these results and the of on was critical for the of platelet on Aggrus in CHO and Lec type the structure on active hAggrus molecules in the CHO cells, lectin blotting using the was with of the cell I this The hAggrus that was expressed in Lec1 and parental CHO cells with WGA but not The aggregation-inducing molecules at in to the of WGA to Jacalin, which core 1 structure with hAggrus molecules expressed in Lec1, and Lec2 CHO cells. This that active molecules a sialylated core 1 Furthermore, hAggrus from Lec cells was by called Griffonia simplicifolia lectin I, and soybean agglutinin that the on hAggrus from Lec8 was In addition, hAggrus molecules from Lec2 cells showed in with This from the reduction of or of of the lack of 4 the glycosylation of Aggrus expressed in the parental CHO and Lec mutant of lectin to communis agglutinin simplicifolia in a new a for of Aggrus expressed in Lec mutant cells. The structure of is the of the to and residues or by Lec mutant cells at that the oligosaccharide were in generated mAb by immunizing rats with the synthetic Aggrus peptide. has been utilized as a lymphatic endothelium marker in The results of YM-1 indicated that this antibody has to lymphatic endothelium and flow indicated that the hAggrus protein is expressed in the lymphatic endothelial cell and lung cancer cell and Furthermore, these results showed that the YM-1 antibody the The present study utilized the of of CHO mutant cell lines to the sialylated was critical for the platelet aggregation-inducing activity of of expressed in CHO cell lines showed an to those in the carcinoma cells M. T. H. R. G. J. J. T. Biochem. J. 2003; PubMed Google Scholar), that recombinant Aggrus in CHO cells, which platelet aggregation, be as as glycosylation in cancer cells. the physiological of the activity the that Aggrus is expressed in tumor cells indicated that glycosylation of Aggrus is to the activity of platelet aggregation metastasis (3Kato Y. Fujita N. Kunita A. Sato S. Kaneko M. Osawa M. Tsuruo T. J. Biol. Chem. 2003; 278: 51599-51605Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 6Kato Y. Sasagawa I. Kaneko M. Osawa M. Fujita N. Tsuruo T. Oncogene. 2004; (in press)Google Scholar). Lectin showed that active Aggrus molecules expressed in CHO cells a sialylated core 1 structure This structure was with those in the PA2.26 F.G. Gamallo C. S. Quintanilla M. J. Cell Sci. PubMed Google Scholar) and gp40 (17Zimmer G. Lottspeich F. Maisner A. Klenk H.D. Herrler G. Biochem. J. 1997; 326: 99-108Crossref PubMed Scopus (38) Google Scholar). In to sialylated and called and antigen, respectively, sialylated and core 1 are also known as tumor-associated E. J. Pathol. PubMed Scopus Google Scholar, Cancer Metastasis Rev. 2004; PubMed Scopus Google Scholar). are in membranous and plasma components in cancer has been as a for the of and other the is and In cells, expressed on of and core F.G. G. J. H. J. U. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. V. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the glycans of expressed in to of the core structure with I. J. C. J. J. Biochem. 1995; PubMed Scopus Google Scholar, F.G. F. J. J. Biochem. PubMed Scopus Google Scholar, S. F.G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). this of Aggrus in carcinoma cells is to be distinct from T1α/podoplanin in cells in glycosylation. However, Aggrus in lymphatic endothelial cells and Aggrus in showed the in and both cells induced platelet aggregation The of Aggrus from were indicated in previous with mouse (3Kato Y. Fujita N. Kunita A. Sato S. Kaneko M. Osawa M. Tsuruo T. J. Biol. Chem. 2003; 278: 51599-51605Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, F.G. Gamallo C. S. Quintanilla M. J. Cell Sci. PubMed Google Scholar). The glycosylation or that were by are to the structure on Aggrus molecules from cancer cells and cells, the in the platelet aggregation-stimulating domain, to the glycosylation has an important with the platelet aggregation activity of Aggrus or not. in has that the is to various physiological as the J. L. R. Y. Stanley P. PubMed Scopus Google Scholar). in highly conserved plays essential in a variety of A series of with CHO cells, including Lec2 and have been for the glycosylation of J. L. R. Y. Stanley P. PubMed Scopus Google Scholar, J. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). is that to on factor of J. L. R. Y. Stanley P. PubMed Scopus Google Scholar, K. L. H. S. PubMed Scopus Google Scholar). The in structure binding K. L. H. S. PubMed Scopus Google Scholar, C. G. A. G. Cell Biol. PubMed Scopus Google Scholar). CHO cells, we that sialylated glycans on Aggrus platelet is to that the in glycosylation platelet aggregation-inducing activity of Aggrus in on that sialylated glycans on Aggrus induced platelet aggregation, Aggrus with a on the platelet have the association of Aggrus with three lectins of and which are expressed in platelets P. A. A. H. H. Haemostasis. 2003; PubMed Scopus Google Scholar). However, no evidence has been obtained by blotting and flow to not In the it be important to which on platelets is to the activity aggregation by In we generated a new antibody, YM-1. Then we showed that the of on is essential for platelet aggregation by membranous Aggrus important in of the that the platelet aggregation activity is related to tumor