Involvement of the Snake Toxin Receptor CLEC-2, in Podoplanin-mediated Platelet Activation, by Cancer Cells

Podoplanin (aggrus), a transmembrane sialoglycoprotein, is involved in tumor cell-induced platelet aggregation, tumor metastasis, and lymphatic vessel formation. However, the mechanism by which podoplanin induces these cellular processes including its receptor has not been elucidated to date. Podoplanin induced platelet aggregation with a long lag phase, which is dependent upon Src and phospholipase Cγ2 activation. However, it does not bind to glycoprotein VI. This mode of platelet activation was reminiscent of the snake toxin rhodocytin, the receptor of which has been identified by us as a novel platelet activation receptor, C-type lectin-like receptor 2 (CLEC-2) (Suzuki-Inoue, K., Fuller, G. L., Garcia, A., Eble, J. A., Pohlmann, S., Inoue, O., Gartner, T. K., Hughan, S. C., Pearce, A. C., Laing, G. D., Theakston, R. D., Schweighoffer, E., Zitzmann, N., Morita, T., Tybulewicz, V. L., Ozaki, Y., and Watson, S. P. (2006) Blood 107, 542–549). Therefore, we sought to evaluate whether CLEC-2 serves as a physiological counterpart for podoplanin. Association between CLEC-2 and podoplanin was confirmed by flow cytometry. Furthermore, their association was dependent on sialic acid on O-glycans of podoplanin. Recombinant CLEC-2 inhibited platelet aggregation induced by podoplanin-expressing tumor cells or lymphatic endothelial cells, suggesting that CLEC-2 is responsible for platelet aggregation induced by endogenously expressed podoplanin on the cell surfaces. These findings suggest that CLEC-2 is a physiological target protein of podoplanin and imply that it is involved in podoplanin-induced platelet aggregation, tumor metastasis, and other cellular responses related to podoplanin. Podoplanin (aggrus), a transmembrane sialoglycoprotein, is involved in tumor cell-induced platelet aggregation, tumor metastasis, and lymphatic vessel formation. However, the mechanism by which podoplanin induces these cellular processes including its receptor has not been elucidated to date. Podoplanin induced platelet aggregation with a long lag phase, which is dependent upon Src and phospholipase Cγ2 activation. However, it does not bind to glycoprotein VI. This mode of platelet activation was reminiscent of the snake toxin rhodocytin, the receptor of which has been identified by us as a novel platelet activation receptor, C-type lectin-like receptor 2 (CLEC-2) (Suzuki-Inoue, K., Fuller, G. L., Garcia, A., Eble, J. A., Pohlmann, S., Inoue, O., Gartner, T. K., Hughan, S. C., Pearce, A. C., Laing, G. D., Theakston, R. D., Schweighoffer, E., Zitzmann, N., Morita, T., Tybulewicz, V. L., Ozaki, Y., and Watson, S. P. (2006) Blood 107, 542–549). Therefore, we sought to evaluate whether CLEC-2 serves as a physiological counterpart for podoplanin. Association between CLEC-2 and podoplanin was confirmed by flow cytometry. Furthermore, their association was dependent on sialic acid on O-glycans of podoplanin. Recombinant CLEC-2 inhibited platelet aggregation induced by podoplanin-expressing tumor cells or lymphatic endothelial cells, suggesting that CLEC-2 is responsible for platelet aggregation induced by endogenously expressed podoplanin on the cell surfaces. These findings suggest that CLEC-2 is a physiological target protein of podoplanin and imply that it is involved in podoplanin-induced platelet aggregation, tumor metastasis, and other cellular responses related to podoplanin. There has been an increasing body of evidence that platelets are involved in cancer metastasis and/or progression (1Nash G.F. Turner L.F. Scully M.F. Kakkar A.K. Lancet Oncol. 2002; 3: 425-430Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 2Gupta G.P. Massague J. J. Clin. Investig. 2004; 114: 1691-1693Crossref PubMed Scopus (114) Google Scholar). Several studies have reported on tumor cell-induced platelet activation, implicating that aggregation serves for tumor cell nestling, whereas released growth factors serve for angiogenesis or tumor growth. Aggrus, a sialoglycoprotein on the surface of cancer cells, which was later found to be identical to podoplanin, induces platelet aggregation (3Kato Y. Fujita N. Kunita A. Fujita N. Tsuruo T. Osawa M. J. Biol. Chem. 2003; 278: 51599-51605Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 4Kaneko M. Kato Y. Kunita A. Fujita N. Tsuruo T. Osawa M. J. Biol. Chem. 2004; 279: 38838-38843Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 5Kaneko M.K. Kato Y. Kameyama A. Ito H. Kuno A. Hirabayashi J. Kubota T. Amano K. Chiba Y. Hasegawa Y. Sasagawa I. Mishima K. Narimatsu H. FEBS Lett. 2007; 581: 331-336Crossref PubMed Scopus (91) Google Scholar). Podoplanin/aggrus is a type-I transmembrane sialomucin-like glycoprotein that consists of an extracellular domain with abundant Ser/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. Fujita N. Tsuruo T. Osawa M. J. Biol. Chem. 2003; 278: 51599-51605Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 6Dobbs L.G. Williams M.C. Gonzalez R. Biochim. Biophys. Acta. 1988; 970: 146-156Crossref PubMed Scopus (218) Google Scholar). Increased expression of podoplanin/aggrus was observed in various tumor cells, including squamous cell carcinomas (7Schacht V. Dadras S.S. Johnson L.A. Jackson D.G. Hong Y.K. Detmar M. Am. J. Pathol. 2005; 166: 913-921Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar, 8Kato Y. Kaneko M. Sata M. Fujita N. Tsuruo T. Osawa M. Tumour. Biol. 2005; 26: 195-200Crossref PubMed Scopus (190) Google Scholar), testicular seminoma (9Kato Y. Sasagawa I. Kaneko M. Osawa M. Fujita N. Tsuruo T. Oncogene. 2004; 23: 8552-8556Crossref PubMed Scopus (136) Google Scholar), and brain tumors (10Mishima K. Kato Y. Kaneko M.K. Nakazawa Y. Kunita A. Fujita N. Tsuruo T. Nishikawa R. Hirose T. Matsutani M. Acta Neuropathol. (Berl.). 2006; 111: 563-568Crossref PubMed Scopus (120) Google Scholar, 11Mishima K. Kato Y. Kaneko M.K. Nishikawa R. Hirose T. Matsutani M. Acta Neuropathol. (Berl.). 2006; 111: 483-488Crossref PubMed Scopus (209) Google Scholar, 12Kato Y. Kaneko M.K. Kuno A. Uchiyama N. Amano K. Chiba Y. Hasegawa Y. Hirabayashi J. Narimatsu H. Mishima K. Osawa M. Biochem. Biophys. Res. Commun. 2006; 349: 1301-1307Crossref PubMed Scopus (176) Google Scholar). Recent investigations have indeed reinforced the notion that podoplanin/aggrus expression might be associated with tumor metastasis or malignant progression (11Mishima K. Kato Y. Kaneko M.K. Nishikawa R. Hirose T. Matsutani M. Acta Neuropathol. (Berl.). 2006; 111: 483-488Crossref PubMed Scopus (209) Google Scholar, 13Yuan P. Temam S. El-Naggar A. Cancer. 2006; 107: 563-569Crossref PubMed Scopus (257) Google Scholar). Podoplanin is also expressed abundantly on glomerular epithelial cells (podocytes), after which the sialoglycoprotein was named, type I lung alveolar cells, and lymphatic endothelial cells (7Schacht V. Dadras S.S. Johnson L.A. Jackson D.G. Hong Y.K. Detmar M. Am. J. Pathol. 2005; 166: 913-921Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar, 14Breiteneder-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). Podoplanin-deficient mice die at birth due to respiratory failure and have defects in lymphatic, but not blood vessel pattern formation, which are associated with diminished lymphatic transport, congenital lymphedema, and dilation of lymphatic vessels, suggesting that it is crucially involved in lymphatic vessel formation (15Schacht 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). However, how podoplanin regulates the formation of lymphatic vessels or tumor platelet interaction has remained totally unknown, and the identification of its pathophysiological target to which podoplanin interacts has been ardently awaited among research workers in a number of scientific fields. In this study, we sought to identify the target molecule for podoplanin on the platelet membrane since platelet aggregation can be used as a marker for their interaction. For this end, we investigated the characteristics of podoplanin-induced platelet aggregation and compared them with those of well defined platelet stimulators. In the process of physiologic thrombus formation with platelet aggregation, the first step is platelet interaction with exposed collagen fibers at sites of vessel injury (16Nieswandt B. Watson S.P. Blood. 2003; 102: 449-461Crossref PubMed Scopus (915) Google Scholar). Platelet adhesion and aggregation on collagen fibers and the subsequent stable clot formation are an integrated process that involves several platelet receptors and agonists such as ADP, thromboxane A2, and coagulation factors including thrombin. One of the major and powerful receptors involved herein is a collagen receptor, glycoprotein VI-FcRγ chain complex (GPVI) 2The abbreviations used are: GPVIglycoprotein VISykspleen tyrosine kinaseSLP-76SH2 domain-containing leukocyte protein of 76 kDaPLCγ2phospholipase Cγ2CLEC-2C-type lectin-like receptor 2CHOChinese hamster ovaryIL2ssinterleukin 2 signal sequencePBSphosphate-buffered salineFITCfluorescein isothiocyanateLEClymphatic endothelial cellhhumanmmouserrecombinant.. The signal transduction pathway related to GPVI encompasses a number of intracellular signaling molecules, such as tyrosine kinases Src, spleen tyrosine kinase (Syk), an adapter protein, SH2 domain-containing leukocyte protein of 76 kDa (SLP-76), and phospholipase Cγ2 (PLCγ2) (16Nieswandt B. Watson S.P. Blood. 2003; 102: 449-461Crossref PubMed Scopus (915) Google Scholar). We have recently identified a novel class of platelet activation receptor, C-type lectin-like receptor 2 (CLEC-2), which belongs to a non-classical C-type lectin, as a receptor on the platelet membrane for a platelet-aggregating snake venom, rhodocytin (17Suzuki-Inoue K. Fuller G.L. Garcia A. Eble J.A. Pohlmann S. Inoue O. Gartner T.K. Hughan S.C. Pearce A.C. Laing N. T. Y. Watson S.P. Blood. 2006; 107: PubMed Scopus Google Scholar). CLEC-2 activation on protein tyrosine phosphorylation including a tyrosine kinase Src, an adapter protein and in platelets in a to GPVI (17Suzuki-Inoue K. Fuller G.L. Garcia A. Eble J.A. Pohlmann S. Inoue O. Gartner T.K. Hughan S.C. Pearce A.C. Laing N. T. Y. Watson S.P. Blood. 2006; 107: PubMed Scopus Google Scholar, K. Y. M. Y. Y. Y. T. T. K. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the powerful of CLEC-2 on platelets that it an in a physiological of the novel signaling receptor has not been identified to date. glycoprotein spleen tyrosine kinase SH2 domain-containing leukocyte protein of 76 kDa phospholipase Cγ2 C-type lectin-like receptor 2 hamster 2 signal lymphatic endothelial cell a of podoplanin-induced platelet aggregation, we found that the mode of platelet activation induced by podoplanin was reminiscent of the Therefore, we sought to evaluate whether CLEC-2 serves as a physiological counterpart for podoplanin. Association between CLEC-2 and podoplanin was confirmed by flow cytometry. CLEC-2 inhibited platelet aggregation induced by podoplanin-expressing tumor cells or lymphatic endothelial These findings suggest that podoplanin platelet aggregation by with CLEC-2 and that the interaction between podoplanin and CLEC-2 regulates various cellular responses related to podoplanin. and mice in by and for and mice and for and the of was by Y. T. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). The of GPVI was a and Y. T. M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). cell that expressed CLEC-2 a protein was by of S. J. G. A. J.A. J. 2003; PubMed Scopus Google Scholar). podoplanin and podoplanin podoplanin was was tyrosine was Src kinase and its collagen type was was was Platelet was by the in the of and was to the of blood was blood was which by by and of acid and platelets by as to activation the K. Inoue O. J. Watson S.P. Blood. 2003; 102: PubMed Scopus (84) Google Scholar). of platelets in K. Inoue O. J. Watson S.P. Blood. 2003; 102: PubMed Scopus (84) Google at a cell of and hamster cells The podoplanin with was a of as M. Kato Y. Kunita A. Fujita N. Tsuruo T. Osawa M. J. Biol. Chem. 2004; 279: 38838-38843Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). and was as K. D. Y. Y. T. Inoue O. M. M.C. Watson S.P. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). cells an with or and by expression of podoplanin was confirmed by flow podoplanin as in and was expressed a protein in cell and as S. J. G. A. J.A. J. 2003; PubMed Scopus Google Scholar). CLEC-2 expression was induced by the of to the cells used for cell cells, and stable of podoplanin to these cells as M. Kato Y. Kunita A. Fujita N. Tsuruo T. Osawa M. J. Biol. Chem. 2004; 279: 38838-38843Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). podoplanin with the a of and cells with by a a in a cell was by K. for and as Y. Kaneko M.K. Kuno A. Uchiyama N. Amano K. Chiba Y. Hasegawa Y. Hirabayashi J. Narimatsu H. Mishima K. Osawa M. Biochem. Biophys. Res. Commun. 2006; 349: 1301-1307Crossref PubMed Scopus (176) Google Scholar). cell of was the for cells in and endothelial cells and to the and of of CLEC-2 CLEC-2 podoplanin and podoplanin the extracellular of these by their as was The used as in the and the in the CLEC-2 CLEC-2 podoplanin podoplanin The by a with and and to the or the which 2 signal the to of and and after the sites, The of CLEC-2 and podoplanin to the of CLEC-2 and podoplanin to the The was The was by and cells with the K. D. Y. Y. T. Inoue O. M. M.C. Watson S.P. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In the of the the was by For the of the the was and the was to a of protein with the by by The Platelet or platelets at by cell rhodocytin, 2 or Platelet aggregation was by with the of an aggregation for at at The was with for or was with platelets for at In cell with of the for at to The of cell and that of in platelet and was as K. Inoue O. J. Watson S.P. Blood. 2003; 102: PubMed Scopus (84) Google Scholar). platelets with to platelet with cells with podoplanin for the by the of Platelet and was as The was with by and by tyrosine platelets with by by the of by and by tyrosine in with of the first or of the cells with rhodocytin and for at with those cells with and with of with for cells a and Platelet by Podoplanin on a Src and but on investigated the characteristics of podoplanin-induced platelet aggregation and compared them with those of well defined platelet stimulators. or podoplanin was with cells, and their surface expression was confirmed by a flow but not aggregation of platelets with a long lag suggesting that aggregation is induced by podoplanin but not by other in podoplanin inhibited platelet aggregation, whereas podoplanin used as a not platelet aggregation induced by podoplanin (3Kato Y. Fujita N. Kunita A. Fujita N. Tsuruo T. Osawa M. J. Biol. Chem. 2003; 278: 51599-51605Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 12Kato Y. Kaneko M.K. Kuno A. Uchiyama N. Amano K. Chiba Y. Hasegawa Y. Hirabayashi J. Narimatsu H. Mishima K. Osawa M. Biochem. Biophys. Res. Commun. 2006; 349: 1301-1307Crossref PubMed Scopus (176) Google Scholar, Y. M. T. S. T. Tsuruo T. Res. Google the of podoplanin on platelet Platelet aggregation induced by was inhibited by the Src kinase but not by its We also confirmed that cells with podoplanin aggregation of which was inhibited by but not by not with the to protein tyrosine phosphorylation was observed podoplanin-induced platelet aggregation This was in the of to platelet aggregation, but in protein tyrosine phosphorylation was also observed in the of the not podoplanin platelet aggregation was also We extracellular domain of podoplanin expressed as a domain protein and platelets with the the that the interacts with a receptor, we used which also aggregation suggesting that podoplanin, but not cellular is responsible for platelet The long lag the of formation and Src platelet activation is reminiscent of platelet activation induced by collagen GPVI or by the snake rhodocytin K. Y. M. Y. Y. Y. T. T. K. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), but not receptor, platelet aggregation which is not dependent on Src Therefore, we first investigated the that podoplanin platelets by to platelets in chain Podoplanin or rhodocytin, but not induced platelet aggregation in mice in the chain complex suggesting that GPVI is not a target protein for podoplanin. We to whether podoplanin platelets by the mechanism to a receptor on the platelet membrane for rhodocytin, we have recently identified a novel class of platelet activation receptor, C-type lectin-like receptor 2 (CLEC-2), which belongs to a non-classical C-type (17Suzuki-Inoue K. Fuller G.L. Garcia A. Eble J.A. Pohlmann S. Inoue O. Gartner T.K. Hughan S.C. Pearce A.C. Laing N. T. Y. Watson S.P. Blood. 2006; 107: PubMed Scopus Google Scholar). signaling molecule for platelet aggregation CLEC-2 (17Suzuki-Inoue K. Fuller G.L. Garcia A. Eble J.A. Pohlmann S. Inoue O. Gartner T.K. Hughan S.C. Pearce A.C. Laing N. T. Y. Watson S.P. Blood. 2006; 107: PubMed Scopus Google Scholar), we first whether is for podoplanin-induced platelet Podoplanin as well as rhodocytin induced platelet aggregation in type mice but not in mice the other induced platelet aggregation in mice the have that podoplanin-induced platelet aggregation has a to that of rhodocytin, we to a that CLEC-2 is a physiological counterpart for podoplanin. Podoplanin Platelet by with investigated the of CLEC-2 to podoplanin-expressing We extracellular domain of CLEC-2 expressed as a domain protein protein of GPVI and is used as a cells with or podoplanin. a confirmed podoplanin expression on the cells or cells with or and was with but not to podoplanin-expressing cells, suggesting that CLEC-2 with podoplanin. of podoplanin to cells was cells, we used cells, in which CLEC-2 was expressed the of a The of to cells induces surface expression of as with a to the receptor in and but not its protein of CLEC-2 and to the cell that podoplanin is a physiological of We also confirmed that the extracellular domain of podoplanin expressed as a domain protein but not its to cell not of podoplanin-expressing cells with but not with inhibited podoplanin-induced platelet aggregation not platelet aggregation by other agonists podoplanin or rhodocytin These findings confirmed that podoplanin platelet aggregation by with on in Podoplanin for to belongs to non-classical C-type lectin-like which a C-type lectin-like domain to a domain but the for and M. J. J. PubMed Scopus Google Scholar). the of to podoplanin to podoplanin was expressed on type cells or acid cells which of sialic acid in and M. Kato Y. Kunita A. Fujita N. Tsuruo T. Osawa M. J. Biol. Chem. 2004; 279: 38838-38843Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 5Kaneko M.K. Kato Y. Kameyama A. Ito H. Kuno A. Hirabayashi J. Kubota T. Amano K. Chiba Y. Hasegawa Y. Sasagawa I. Mishima K. Narimatsu H. FEBS Lett. 2007; 581: 331-336Crossref PubMed Scopus (91) Google Scholar). we reported M. Kato Y. Kunita A. Fujita N. Tsuruo T. Osawa M. J. Biol. Chem. 2004; 279: 38838-38843Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), podoplanin expressed on cells to platelet aggregation, expression of the in and was confirmed by flow a and with these was associated with podoplanin expressed on but not with that expressed on These us to that the of sialic acid on O-glycans of podoplanin is for interaction between CLEC-2 and podoplanin. of cells with rhodocytin podoplanin to the cells suggesting that the sites of to identification of the CLEC-2 is Podoplanin on the of or Platelet platelet aggregation in and was induced by cells that to podoplanin by we investigated whether tumor cell which podoplanin, platelet aggregation by with cell and a cell podoplanin and platelet aggregation Y. Kaneko M.K. Kuno A. Uchiyama N. Amano K. Chiba Y. Hasegawa Y. Hirabayashi J. Narimatsu H. Mishima K. Osawa M. Biochem. Biophys. Res. Commun. 2006; 349: 1301-1307Crossref PubMed Scopus (176) Google Scholar, M. Y. Tsuruo T. Res. 1988; Google Scholar). We confirmed surface expression of podoplanin in and and platelet aggregation by the cell and of or with but not with inhibited platelet aggregation induced by the cell and These findings suggest that podoplanin that is endogenously expressed in tumor cells also induces platelet activation interaction with Podoplanin is expressed in lymphatic endothelial cells, but not in endothelial cells, and is used as a marker of lymphatic (7Schacht V. Dadras S.S. Johnson L.A. Jackson D.G. Hong Y.K. Detmar M. Am. J. Pathol. 2005; 166: 913-921Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar, 14Breiteneder-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). We investigated whether lymphatic endothelial cells which endogenously podoplanin, platelets with We confirmed that expressed podoplanin and platelet aggregation M. Kato Y. Kunita A. Fujita N. Tsuruo T. Osawa M. J. Biol. Chem. 2004; 279: 38838-38843Abstract Full Text Full Text PDF PubMed Scopus (84) Google The platelet aggregation was inhibited by but not by suggesting that podoplanin expressed on is also to platelet aggregation by with the physiological of the interaction between platelet CLEC-2 and podoplanin expressed on to be The of podoplanin has been in tumor (11Mishima K. Kato Y. Kaneko M.K. Nishikawa R. Hirose T. Matsutani M. Acta Neuropathol. (Berl.). 2006; 111: 483-488Crossref PubMed Scopus (209) Google and lymphatic vessel formation (15Schacht 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). the identification of the podoplanin receptor a to of cellular the for its physiologic has not been is well that platelets are by the and CLEC-2 belongs to a non-classical C-type lectin, which is a class of CLEC-2 platelets the novel signaling in which a single in its cytoplasmic tail a for platelet activation (17Suzuki-Inoue K. Fuller G.L. Garcia A. Eble J.A. Pohlmann S. Inoue O. Gartner T.K. Hughan S.C. Pearce A.C. Laing N. T. Y. Watson S.P. Blood. 2006; 107: PubMed Scopus Google Scholar, G.L. Williams J.A. Eble J.A. Pohlmann S. K. Y. Watson S.P. Pearce A.C. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). the powerful of CLEC-2 on platelets that it an in However, its physiological has not been identified to date. In this study, we identified the physiological for the podoplanin and which to be a receptor and a for and the for research on the of the interaction. of extracellular domain of CLEC-2 to podoplanin-expressing cells was confirmed by flow the extracellular domain of podoplanin associated with the cell of podoplanin-expressing cells with CLEC-2 inhibited podoplanin-induced platelet aggregation suggesting that interaction between CLEC-2 and podoplanin is responsible for podoplanin-induced platelet We sought to the mode of interaction and the mechanism of podoplanin-induced platelet aggregation We found that podoplanin expressed in acid cells which of sialic acid in and to with CLEC-2 and was to platelet aggregation These suggest that sialic acid on O-glycans of podoplanin is for to on the and of CLEC-2 also that an is to be a protein with a surface J. K. Eble J.A. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). to be with this since sialic acid is of podoplanin to cells was inhibited by rhodocytin suggesting that target the in In to podoplanin, does not to be for rhodocytin to CLEC-2 since rhodocytin has potential its acid and are to the mechanism of interaction between podoplanin and on the and of CLEC-2 that to CLEC-2 is to by a major of CLEC-2 upon J. K. Eble J.A. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). podoplanin to CLEC-2 the cytoplasmic signaling of several CLEC-2 Recombinant podoplanin expressed as a protein induced powerful platelet aggregation and protein tyrosine phosphorylation podoplanin expressed on the surface of cells a protein, CLEC-2 podoplanin expressed on the of cells, which not be to the be due to podoplanin the in the of podoplanin and/or sialic acid on them to this studies are to on this The findings in this that podoplanin on the surface of tumor cells induces platelet aggregation by with on the of several a cell that a expressed podoplanin and induces platelet aggregation to a M. Y. Tsuruo T. Res. 1988; Google Scholar). that podoplanin-induced platelet aggregation lung of Y. M. T. S. T. Tsuruo T. Res. Google Scholar). These suggest that podoplanin-induced platelet activation CLEC-2 is of the of tumor The of interaction not be to tumor platelets a number of factors that are in including endothelial growth growth and Y. 2006; PubMed Scopus Google Scholar). factors released platelets with on the platelet to the process of tumor tumor growth or metastasis J. D.G. S.C. S. J. J. S. A. 2006; PubMed Scopus Google Scholar). of the interaction between CLEC-2 and podoplanin be a target to tumor growth and the interaction between podoplanin and platelet CLEC-2 in physiological to be elucidated since podoplanin is not expressed in endothelial cells in blood However, it be that podoplanin is expressed in and upon it to thrombus formation by platelets CLEC-2 this is the the interaction between CLEC-2 and podoplanin is an as this thrombus formation physiological This is We found that podoplanin on the surface of lymphatic endothelial cells also induced platelet The physiological of this to be elucidated since lymphatic vessels the physiologic are not with However, it be of or Podoplanin-deficient mice have defects in lymphatic vessel pattern formation (15Schacht 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). signaling molecules, and blood and lymphatic A. R. A. B. Jackson D.G. V. 2003; PubMed Scopus Google Scholar), are not in which that and by of blood and in platelets are for podoplanin-induced platelet activation by CLEC-2 (17Suzuki-Inoue K. Fuller G.L. Garcia A. Eble J.A. Pohlmann S. Inoue O. Gartner T.K. Hughan S.C. Pearce A.C. Laing N. T. Y. Watson S.P. Blood. 2006; 107: PubMed Scopus Google Scholar). we have evidence to it is to that podoplanin-induced platelet activation CLEC-2 regulates formation of lymphatic In we that a novel platelet activation receptor, is the physiological counterpart for podoplanin, and their interaction induces platelet The sialic acid on O-glycans of podoplanin to be for its by The interaction between CLEC-2 and podoplanin tumor and it be related to the formation of lymphatic We are to Morita, K. and for and for the of cell and is expressed to and for