Platelet Aggregation-stimulating Domain Research Articles

Podoplanin is indispensable for cell motility and platelet-induced epithelial-to-mesenchymal transition-related gene expression in esophagus squamous carcinoma TE11A cells

BackgroundThe transmembrane glycoprotein podoplanin (PDPN) is upregulated in some tumors and has gained attention as a malignant tumor biomarker. PDPN molecules have platelet aggregation-stimulating domains and, are therefore, suggested to play a role in tumor-induced platelet activation, which in turn triggers epithelial-to-mesenchymal transition (EMT) and enhances the invasive and metastatic activities of tumor cells. In addition, as forced PDPN expression itself can alter the propensity of certain tumor cells in favor of EMT and enhance their invasive ability, it is also considered to be involved in the cell signaling system. Nevertheless, underlying mechanisms of PDPN in tumor cell invasive ability as well as EMT induction, especially by platelets, are still not fully understood.MethodsSubclonal TE11A cells were isolated from the human esophageal squamous carcinoma cell line TE11 and the effects of anti-PDPN neutralizing antibody as well as PDPN gene knockout on platelet-induced EMT-related gene expression were measured. Also, the effects of PDPN deficiency on cellular invasive ability and motility were assessed.ResultsPDPN-null cells were able to provoke platelet aggregation, suggesting that PDPN contribution to platelet activation in these cells is marginal. Nevertheless, expression of platelet-induced EMT-related genes, including vimentin, was impaired by PDPN-neutralizing antibody as well as PDPN deficiency, while their effects on TGF-β-induced gene expression were marginal. Unexpectedly, PDPN gene ablation, at least in either allele, engendered spontaneous N-cadherin upregulation and claudin-1 downregulation. Despite these seemingly EMT-like alterations, PDPN deficiency impaired cellular motility and invasive ability even after TGF-β-induced EMT induction.ConclusionsThese results suggested that, while PDPN seems to function in favor of maintaining the epithelial state of this cell line, it is indispensable for platelet-mediated induction of particular mesenchymal marker genes as well as the potentiation of motility and invasion capacity.

Epitope Analysis of an Antihorse Podoplanin Monoclonal Antibody PMab-219.

Podoplanin (PDPN), which is a mucin-type membrane glycoprotein, is expressed on lymphatic endothelial cells and epithelial cells of many organs. PDPN is also overexpressed in several malignant cancers, and its expression is associated with cancer progression and poor prognosis. Human PDPN possesses three platelet aggregation-stimulating (PLAG) domains and the PLAG-like domain (PLD), which binds to C-type lectin-like receptor-2 (CLEC-2). Previously, we reported a novel antihorse PDPN (horPDPN) monoclonal antibody (mAb), PMab-219, using Cell-Based Immunization and Screening (CBIS) method. PMab-219 specifically detected horPDPN-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/horPDPN) cells and FHK-TcL3.1, a horse kidney cell line, using flow cytometry. In addition, PMab-219 strongly stained horse tissues such as renal podocytes or lymphatic endothelial cells by immunohistochemistry. However, the specific binding epitope of PMab-219 for horPDPN remains to be clarified. In this study, a series of deletion mutants or point mutants of horPDPN were produced for analyzing the PMab-219 epitope using flow cytometry. The analysis of deletion mutants showed that N-terminus of PMab-219 epitope exists between 55th amino acid (aa) and 60th aa of horPDPN. Furthermore, the analysis of point mutants demonstrated that the critical epitope of PMab-219, which was developed by CBIS method, could include Val59, Arg61, Ser62, and Thr63 of horPDPN, indicating that PMab-219 epitope is independent of PLAG domain or PLD of horPDPN.

Epitope Mapping of Monoclonal Antibody PMab-52 Against Cat Podoplanin.

The mucin-type membrane glycoprotein podoplanin (PDPN) is frequently overexpressed in numerous malignant cancers, including squamous cell carcinoma, germinal neoplasia, mesothelioma, lung cancer, oral cancer, and brain tumor. PDPN expression is strongly associated with cancer progression and poor prognosis. Furthermore, PDPN binds to C-type lectin-like receptor 2 (CLEC-2) on platelets, followed by PDPN-mediated platelet aggregation to facilitate tumor metastasis. We have previously reported a novel anti-cat PDPN (cPDPN) monoclonal antibody (mAb), PMab-52, which specifically detects cPDPN using flow cytometry analysis and successfully identifies cPDPN in feline squamous cell carcinomas. However, the specific binding epitope of cPDPN for PMab-52 remains unelucidated. In this study, a series of deletion or point mutants of cPDPN were utilized for investigating the binding epitopes of PMab-52 using flow cytometry and Western blotting. The findings of this study revealed that the critical epitopes of platelet aggregation-stimulating domain 4 (PLAG4) of cPDPN are responsible for the binding of PMab-52 to cPDPN.

Characterization of the Anti-Bovine Podoplanin Monoclonal Antibody PMab-44.

A type I transmembrane sialoglycoprotein podoplanin (PDPN) is expressed in several normal cells, including podocytes of the kidney, type I alveolar cells of the lung, and lymphatic endothelial cells. We recently produced an anti-bovine PDPN (bovPDPN) monoclonal antibody (mAb), PMab-44, by immunizing mice with recombinant proteins of bovPDPN. In this study, we determined the critical epitope of PMab-44 for the recognition of bovPDPN using many deletion mutants and point mutants of bovPDPN. Flow cytometric analyses revealed that the epitope of PMab-44 was Glu46-Thr50, which corresponds to platelet aggregation-stimulating (PLAG) domain-3. The important amino acids in the PMab-44 epitope were determined to be Glu46, Tyr48, and Thr50. Western blot analysis also confirmed these results, indicating that the PLAG domain of bovPDPN is also important in immunogenicity for producing useful anti-PDPN mAbs.

MAP Tag: A Novel Tagging System for Protein Purification and Detection

Protein purification is an essential procedure in fields such as biochemistry, molecular biology, and biophysics. Acquiring target proteins with high quality and purity is still difficult, although several tag systems have been established for protein purification. Affinity tag systems are excellent because they possess high affinity and specificity for acquiring the target proteins. Nevertheless, further affinity tag systems are needed to compensate for several disadvantages of the presently available affinity tag systems. Herein, we developed a novel affinity tag system designated as the MAP tag system. This system is composed of a rat anti-mouse podoplanin monoclonal antibody (clone PMab-1) and MAP tag (GDGMVPPGIEDK) derived from the platelet aggregation-stimulating domain of mouse podoplanin. PMab-1 possesses high affinity and specificity for the MAP tag, and the PMab-1/MAP tag complex dissociates in the presence of the epitope peptide, indicating that the MAP tag system is suitable for protein purification. We successfully purified several proteins, including a nuclear protein, soluble proteins, and a membrane protein using the MAP tag system. The MAP tag system is very useful not only for protein purification but also in protein detection systems such as western blot and flow cytometric analyses. Taken together, these findings indicate that the MAP tag system could be a powerful tool for protein purification and detection.

PMab-38 Recognizes Canine Podoplanin of Squamous Cell Carcinomas.

Podoplanin, a type I transmembrane protein, is expressed in lymphatic endothelial cells. Although we previously developed an anticanine podoplanin monoclonal antibody (mAb), PMab-38, immunohistochemistry (IHC) showed that it did not react with canine lymphatic endothelial cells. Here, we determined whether PMab-38 recognizes canine podoplanin of squamous cell carcinomas (SCCs) and clarified its epitope. In IHC, PMab-38 reacted with 83% of SCCs (15/18 cases). Flow cytometry showed that the epitope of PMab-38 was different from that of the platelet aggregation-stimulating domain of the N-terminus, which was detected by almost all antipodoplanin mAbs such as D2-40 or NZ-1. PMab-38 is expected to be useful for investigating the function of podoplanin in canine tumors.

Abstract 1275: Suppression of platelet aggregation and tumor metastasis by anti-podoplanin mAbs recognizing a novel CLEC-2 binding domain

Abstract Tumor-induced platelet aggregation promotes tumor growth and hematogenous metastasis by secreting growth factors from platelets, by avoiding immune system and shear stress, and by stimulating the formation of tumor emboli in microvasculature. Thus, tumor-platelet interaction could be a promising target for cancer therapy. We previously identified a sialoglycoprotein podoplanin, also known as Aggrus, as a key factor in tumor-induced platelet aggregation enhancing tumor metastasis. Podoplanin is expressed in various cancers and reported to be a candidate for cancer stem cell marker. Podoplanin-induced platelet aggregation depends on binding to its platelet receptor CLEC-2. Here, we report the discovery of a novel platelet aggregation-stimulating domain PLAG4 in human podoplanin critical for the binding to CLEC-2. Mutant analyses indicated that PLAG4 exhibits a predominant CLEC-2 binding ability and platelet-aggregating function when compared to the previously reported PLAG3. Moreover, mutation of both PLAG3 and PLAG4 domains could almost completely attenuate its CLEC-2 binding ability and its platelet-aggregation activity. To validate the inhibitory effect against PLAG4 domain, we established anti-PLAG4-neutralizing monoclonal antibodies (mAbs), PG4D1 and PG4D2. Both mAbs recognized PLAG4 domain and exhibited a high affinity to podoplanin. We confirmed those neutralizing ability against PLAG4-CLEC-2 interaction and platelet aggregation. Remarkably, the administration of each mAbs significantly suppressed pulmonary metastasis of podoplanin expressing tumor. Taken together, we suggest that PLAG4 is the novel CLEC-2 binding and platelet aggregation-stimulating domain on podoplanin and has a role in podoplanin-mediated metastasis formation. Blocking the podoplanin-CLEC-2 interaction targeting PLAG3 or PLAG4 would be a promising strategy for suppressing hematogenous metastasis. Citation Format: Takaya Sekiguchi, Ai Takemoto, Naoya Fujita. Suppression of platelet aggregation and tumor metastasis by anti-podoplanin mAbs recognizing a novel CLEC-2 binding domain. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1275.

Establishment of Mouse Monoclonal Antibody LpMab-13 Against Human Podoplanin.

Podoplanin (PDPN)/Aggrus is a type-I transmembrane sialoglycoprotein, which possesses a platelet aggregation-stimulating (PLAG) domain. The O-glycosylation on Thr52 of human PDPN (hPDPN) is critical for the interaction of hPDPN with C-type lectin-like receptor-2 (CLEC-2), resulting in platelet aggregation. Many anti-hPDPN monoclonal antibodies (MAbs) against PLAG domains and non-PLAG domains have been established; however, mouse anti-PLAG2/3 MAb, the epitope of which is consistent with rat anti-PLAG2/3 MAb NZ-1, has not been established. NZ-1 inhibits the hPDPN-CLEC-2 interaction and is also useful for anti-PA tag MAb. We recently established CasMab technology to produce MAbs against membranous proteins. Herein, we produced a novel anti-hPDPN MAb, LpMab-13, which binds to PLAG2/3 domains. LpMab-13 recognized endogenous hPDPN of cancer cells, including glioblastoma, oral cancer, lung cancer, and malignant mesothelioma, and normal cells such as lymphatic endothelial cells and podocytes of kidney in Western blot, flow cytometry, and immunohistochemistry. LpMab-13 recognized glycan-deficient hPDPN in flow cytometry, indicating that the interaction between LpMab-13 and hPDPN is independent of its glycosylation. The minimum epitope of LpMab-13 was identified as Ala42-Asp49 of hPDPN using Western blot and flow cytometry. The combination of different epitope-possessing MAbs could be advantageous for the hPDPN-targeting diagnosis and therapy.

Establishment of Mouse Monoclonal Antibody LpMab-13 Against Human Podoplanin.

Podoplanin (PDPN)/Aggrus is a type-I transmembrane sialoglycoprotein, which possesses a platelet aggregation-stimulating (PLAG) domain. The O-glycosylation on Thr52 of human PDPN (hPDPN) is critical for the interaction of hPDPN with C-type lectin-like receptor-2 (CLEC-2), resulting in platelet aggregation. Many anti-hPDPN monoclonal antibodies (MAbs) against PLAG domains and non-PLAG domains have been established; however, mouse anti-PLAG2/3 MAb, the epitope of which is consistent with rat anti-PLAG2/3 MAb NZ-1, has not been established. NZ-1 inhibits the hPDPN-CLEC-2 interaction and is also useful for anti-PA tag MAb. We recently established CasMab technology to produce MAbs against membranous proteins. Herein, we produced a novel anti-hPDPN MAb, LpMab-13, which binds to PLAG2/3 domains. LpMab-13 recognized endogenous hPDPN of cancer cells, including glioblastoma, oral cancer, lung cancer, and malignant mesothelioma, and normal cells such as lymphatic endothelial cells and podocytes of kidney in Western blot, flow cytometry, and immunohistochemistry. LpMab-13 recognized glycan-deficient hPDPN in flow cytometry, indicating that the interaction between LpMab-13 and hPDPN is independent of its glycosylation. The minimum epitope of LpMab-13 was identified as Ala42-Asp49 of hPDPN using Western blot and flow cytometry. The combination of different epitope-possessing MAbs could be advantageous for the hPDPN-targeting diagnosis and therapy.

LpMab-12 Established by CasMab Technology Specifically Detects Sialylated O-Glycan on Thr52 of Platelet Aggregation-Stimulating Domain of Human Podoplanin.

Podoplanin (PDPN), also known as Aggrus, possesses three tandem repeat of platelet aggregation-stimulating (PLAG) domains in its N-terminus. Among the PLAG domains, sialylated O-glycan on Thr52 of PLAG3 is essential for the binding to C-type lectin-like receptor-2 (CLEC-2) and the platelet-aggregating activity of human PDPN (hPDPN). Although various anti-hPDPN monoclonal antibodies (mAbs) have been generated, no specific mAb has been reported to target the epitope containing glycosylated Thr52. We recently established CasMab technology to develop mAbs against glycosylated membrane proteins. Herein, we report the development of a novel anti-glycopeptide mAb (GpMab), LpMab-12. LpMab-12 detected endogenous hPDPN by flow cytometry. Immunohistochemical analyses also showed that hPDPN-expressing lymphatic endothelial and cancer cells were clearly labeled by LpMab-12. The minimal epitope of LpMab-12 was identified as Asp49–Pro53 of hPDPN. Furthermore, LpMab-12 reacted with the synthetic glycopeptide of hPDPN, corresponding to 38–54 amino acids (hpp3854: 38-EGGVAMPGAEDDVVTPG-54), which carries α2–6 sialylated N-acetyl-D-galactosamine (GalNAc) on Thr52. LpMab-12 did not recognize non-sialylated GalNAc-attached glycopeptide, indicating that sialylated GalNAc on Thr52 is necessary for the binding of LpMab-12 to hPDPN. Thus, LpMab-12 could serve as a new diagnostic tool for determining whether hPDPN possesses the sialylation on Thr52, a site-specific post-translational modification critical for the hPDPN association with CLEC-2.

Critical Epitope of Anti-Rabbit Podoplanin Monoclonal Antibodies for Immunohistochemical Analysis.

Podoplanin (PDPN) is a type I transmembrane sialoglycoprotein, which is expressed in several normal cells, including lymphatic endothelial cells throughout the body, podocytes of the kidney, and lung type I alveolar cells of the lung. We have established many monoclonal antibodies (mAbs) against human PDPN, mouse PDPN, and rat PDPN. In addition, we recently produced an anti-rabbit PDPN mAb, PMab-32, which was established by immunizing mice with recombinant proteins of rabbit PDPN. Herein, we compared the reactivity of PMab-32 with that of newly established anti-rabbit PDPN mAbs, PMab-33 and PMab-21, against normal tissues in immunohistochemistry. PMab-32 reacted with podocytes, type I alveolar cells, and lymphatic endothelial cells, whereas PMab-33 detected only podocytes and type I alveolar cells but not lymphatic endothelial cells. PMab-21 was not useful in immunohistochemistry. We identified the epitope of PMab-32 and PMab-33 as Ser61-Ala68 of rabbit PDPN using western blot and flow cytometric analyses. In contrast, the epitope of PMab-21 was identified as Leu44-Glu48, which is corresponding to platelet aggregation-stimulating (PLAG) domain, indicating that Ser61-Ala68 of rabbit PDPN is a more appropriate epitope for immunohistochemistry compared with PLAG domain. PMab-32 could be useful for uncovering the function of rabbit PDPN.

Novel Monoclonal Antibody LpMab-17 Developed by CasMab Technology Distinguishes Human Podoplanin from Monkey Podoplanin.

Podoplanin (PDPN) is a type-I transmembrane sialoglycoprotein, which possesses a platelet aggregation-stimulating (PLAG) domain in its N-terminus. Among the three PLAG domains, O-glycan on Thr52 of PLAG3 is critical for the binding with C-type lectin-like receptor-2 (CLEC-2) and is essential for platelet-aggregating activity of PDPN. Although many anti-PDPN monoclonal antibodies (mAbs) have been established, almost all mAbs bind to PLAG domains. We recently established CasMab technology to produce mAbs against membranous proteins. Using CasMab technology, we produced a novel anti-PDPN mAb, LpMab-17, which binds to non-PLAG domains. LpMab-17 clearly detected endogenous PDPN of cancer cells and normal cells in Western-blot, flow cytometry, and immunohistochemistry. LpMab-17 recognized glycan-deficient PDPN in flow cytometry, indicating that the interaction between LpMab-17 and PDPN is independent of its glycosylation. The minimum epitope of LpMab-17 was identified as Gly77-Asp82 of PDPN using enzyme-linked immunosorbent assay. Of interest, LpMab-17 did not bind to monkey PDPN, whereas the homology is 94% between human PDPN and monkey PDPN, indicating that the epitope of LpMab-17 is unique compared with the other anti-PDPN mAbs. The combination of different epitope-possessing mAbs could be advantageous for the PDPN-targeting diagnosis or therapy.

Development of Monoclonal Antibody LpMab-10 Recognizing Non-glycosylated PLAG1/2 Domain Including Thr34 of Human Podoplanin.

Podoplanin (PDPN) is a type-I transmembrane sialoglycoprotein that possesses a platelet aggregation-stimulating (PLAG) domain in the N-terminus. PLAG domain includes three tandem repeats of eight amino acids: PLAG1, PLAG2, and PLAG3. Among the three PLAG domains, O-glycan on Thr52 of PLAG3 is critical for binding with C-type lectin-like receptor-2 (CLEC-2) and is essential for platelet-aggregating activity of PDPN. In contrast, the glycosylation of Thr34 of PLAG1 of human PDPN remains to be clarified. Herein, we developed and characterized a novel anti-PDPN monoclonal antibody, LpMab-10, which targets PLAG1/2 domain. LpMab-10 detects endogenous PDPN of cancer cells and normal cells independently of glycosylation. The minimum epitope of LpMab-10 was identified as Glu33-Gly45 of PDPN using Western blot and flow cytometry. The Thr34 of PLAG1 is critical for LpMab-10 recognition, and O-glycan is not included in LpMab-10 epitope, indicating that Thr34 of PLAG1 is not O-glycosylated. In immunocytochemical and immunohistochemical analyses, LpMab-10 strongly detected PDPN-expressing tumor cells. By using monoclonal antibodies against different Ser/Thr, including epitopes of PDPN, it becomes possible to determine whether Ser/Thr residues of PDPN are O-glycosylated.

The chimeric antibody chLpMab-7 targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC rather than via its neutralizing activity.

Podoplanin (PDPN/Aggrus/T1α) binds to C-type lectin-like receptor-2 (CLEC-2) and induces platelet aggregation. PDPN is associated with malignant progression, tumor metastasis, and poor prognosis in several types of cancer. Although many anti-human PDPN (hPDPN) monoclonal antibodies (mAbs), such as D2-40 and NZ-1, have been established, these epitopes are limited to the platelet aggregation-stimulating (PLAG) domain (amino acids 29-54) of hPDPN. Recently, we developed a novel mouse anti-hPDPN mAb, LpMab-7, which is more sensitive than D2-40 and NZ-1, using the Cancer-specific mAb (CasMab) method. The epitope of LpMab-7 was shown to be entirely different from that of NZ-1, a neutralizing mAb against the PLAG domain according to an inhibition assay and lectin microarray analysis. In the present study, we produced a mouse-human chimeric anti-hPDPN mAb, chLpMab-7. ChLpMab-7 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Furthermore, chLpMab-7 inhibited the growth of hPDPN-expressing tumors in vivo. Although chLpMab-7 recognizes a non-PLAG domain of hPDPN, it suppressed the hematogenous metastasis of hPDPN-expressing tumors. These results indicated that chLpMab-7 suppressed tumor development and hematogenous metastasis in a neutralization-independent manner. In conclusion, hPDPN shows promise as a target in the development of a novel antibody-based therapy.

Characterization of Monoclonal Antibody LpMab-7 Recognizing Non-PLAG Domain of Podoplanin.

Podoplanin (PDPN/Aggrus/T1α), a platelet aggregation-inducing type I transmembrane sialoglycoprotein, is involved in tumor invasion and metastasis. Furthermore, podoplanin expression was reported to be involved in poor prognosis of several cancers. Although many anti-podoplanin monoclonal antibodies (MAbs), such as NZ-1 and D2-40, have been established, those epitopes are limited to platelet aggregation-stimulating (PLAG) domain of podoplanin. In this study, we developed and characterized a novel anti-podoplanin MAb, LpMab-7, that is more sensitive than NZ-1 in immunohistochemistry. We identified the minimum epitope of LpMab-7 as Arg79-Leu83 of human podoplanin, which is different from PLAG domain, using ELISA, Western blot analysis, and flow cytometry. Because LpMab-7 has high sensitivity against podoplanin, LpMab-7 is expected to be useful for molecular targeting therapy for podoplanin-expressing cancers.

The impact of Aggrus/podoplanin on platelet aggregation and tumour metastasis

Platelets are small blood components that play indispensable roles in the initial stages of coagulation. In addition to their role in haemostasis, platelets participate in inflammation and tissue regeneration under physiological conditions. Recent studies also revealed the role of platelets under pathological conditions, including the oncogenic process. Platelets enhance tumour growth and metastasis by secreting many growth factors and angiogenic factors or by forming a coat around tumour cells in the blood stream. We previously discovered Aggrus (also known as podoplanin, gp36, gp38P, T1alpha and OTS-8) expressed on tumour cell surfaces as a key molecule for tumour-induced platelet aggregation. Aggrus expression is increased in various malignant tumours such as squamous cell carcinomas, mesotheliomas, glioblastomas and osteosarcomas. Detailed analysis revealed that Aggrus contains three tandem repeats of platelet aggregation-stimulating (PLAG) domains that are associated with its platelet aggregation-inducing ability. PLAG domains of Aggrus are involved in binding to its platelet receptor, C-type lectin-like receptor 2 (CLEC-2). Neutralizing monoclonal antibodies that interfere with Aggrus-CLEC-2 binding attenuate Aggrus-induced platelet aggregation, tumour cell growth and metastasis formation. Aggrus is also expressed in advanced atherosclerotic lesions, suggesting that Aggrus is associated with thrombus formation on disrupted atherosclerotic lesions. These data suggest that Aggrus is a promising cell surface target for developing new therapies against cancer and thrombosis.

Molecular analysis of the pathophysiological binding of the platelet aggregation‐inducing factor podoplanin to the C‐type lectin‐like receptor CLEC‐2

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

The Platelet Aggregation-Inducing Factor Aggrus/Podoplanin Promotes Pulmonary Metastasis

Tumor cell-induced platelet aggregation has been reported to facilitate hematogenous metastasis. Aggrus/podoplanin is a platelet aggregation-inducing factor that is up-regulated in a number of human cancers and has been implicated in tumor progression. We studied herein the role of Aggrus in tumor growth, metastasis , and survival in vivo. Aggrus expression in Chinese hamster ovary cells promoted pulmonary metastasis in both an experimental and a spontaneous mouse model. No differences in the size of metastatic foci or in primary tumor growth were found in either set of mice. Aggrus expressing cells , which were covered with platelets, arrested in the lung microvasculature 30 minutes after injection. In addition, lung metastasis resulting from Aggrus expression decreased the survival of the mice. By generating several Aggrus point mutants, we revealed that point mutation at the platelet aggregation-stimulating domain of Aggrus(Thr34 and Thr52) obliterated both platelet aggregation and metastasis. Furthermore, administration of aspirin to mice reduced the number of metastatic foci. These results indicate that Aggrus contributes to the establishment of metastasis by promoting platelet aggregation without affecting subsequent growth. Thus, Aggrus could serve as an ideal therapeutic target for drug development to block metastasis.

Functional glycosylation of human podoplanin: Glycan structure of platelet aggregation-inducing factor

Podoplanin (Aggrus) is a mucin-type sialoglycoprotein that plays a key role in tumor cell-induced platelet aggregation. Podoplanin possesses a platelet aggregation-stimulating (PLAG) domain, and Thr52 in the PLAG domain of human podoplanin is important for its activity. Endogenous or recombinant human podoplanin were purified, and total glycosylation profiles were surveyed by lectin microarray. Analyses of glycopeptides produced by Edman degradation and mass spectrometry revealed that the disialyl-corel (NeuAcα2-3Galβl-3(NeuAcα2-6)GalNAcαl- O-Thr) structure was primarily attached to a glycosylation site at residue Thr52. Sialic acid-deficient podoplanin recovered its activity after additional sialylation. These results indicated that the sialylated Corel at Thr52 is critical for podoplanin-induced platelet aggregation.

Conservation of a platelet activating domain of Aggrus/podoplanin as a platelet aggregation-inducing factor

Human Aggrus/podoplanin is an identified platelet aggregation-inducing factor of cancer cells, which is also known as a specific marker of lymphatic endothelium. Human Aggrus was known to be expressed in seminoma, squamous cell carcinoma, malignant mesothelioma, sarcomas and several brain tumors. In our previous studies, the sialylated O-glycan of human and mouse Aggrus were shown to be critical for its platelet aggregation-inducing activity in the experiments using the glycosylation-deficient Chinese hamster ovary (CHO) cell lines. We newly cloned Aggrus homologues from rat, hamster, dog and bovine cDNAs, in addition to the human and mouse cDNAs, and confirmed there are three tandem repeats of the platelet aggregation-stimulating (PLAG) domain in Aggrus, which were conserved in all homologues. We found that bovine Aggrus has a sporadic deletion mutation in the first PLAG domain, and lacks platelet aggregation-inducing activity. We introduced point mutation in the PLAG domain of Aggrus and showed that either the first or last PLAG domain is critical for activity, but not the middle domain. In addition, we studied the molecular evolutionary process of the PLAG domain of Aggrus. The PLAG domain and its activity appeared after the divergence of avians and mammals. In conclusion, we provide evidence that Aggrus homologues conserved the segment of EDxxVTPG in their extracellular domain which are critical for their platelet aggregation-inducing activities.