Katsue Suzuki-Inoue

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.

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. (Cancer Sci 2008; 99: 54–61)

DC-SIGN and CLEC-2 Mediate Human Immunodeficiency Virus Type 1 Capture by Platelets

ABSTRACT Platelets can engulf human immunodeficiency virus type 1 (HIV-1), and a significant amount of HIV-1 in the blood of infected individuals is associated with these cells. However, it is unclear how platelets capture HIV-1 and whether platelet-associated virus remains infectious. DC-SIGN and other lectins contribute to capture of HIV-1 by dendritic cells (DCs) and facilitate HIV-1 spread in DC/T-cell cocultures. Here, we show that platelets express both the C-type lectin-like receptor 2 (CLEC-2) and low levels of DC-SIGN. CLEC-2 bound to HIV-1, irrespective of the presence of the viral envelope protein, and facilitated HIV-1 capture by platelets. However, a substantial fraction of the HIV-1 binding activity of platelets was dependent on DC-SIGN. A combination of DC-SIGN and CLEC-2 inhibitors strongly reduced HIV-1 association with platelets, indicating that these lectins are required for efficient HIV-1 binding to platelets. Captured HIV-1 was maintained in an infectious state over several days, suggesting that HIV-1 can escape degradation by platelets and might use these cells to promote its spread. Our results identify CLEC-2 as a novel HIV-1 attachment factor and provide evidence that platelets capture and transfer infectious HIV-1 via DC-SIGN and CLEC-2, thereby possibly facilitating HIV-1 dissemination in infected patients.

Platelet GPIb‐IX‐V‐dependent signaling

Summary.  Although the signaling pathways related to GPIb‐IX‐V have not been fully elucidated, an accumulating body of evidence suggests that phospholipase C (PLC)γ2 activation, subsequent Ca++ release and oscillations constitute an essential signal transduction pathway related to GPIb‐IX‐V. Src family kinases are required for PLCγ2 activation, while FcRγ‐chain/FcγRIIA may be dispensable for PLCγ2 activation. Although PI‐3K serves to potentiate various signaling events culminating in αIIbβ3 activation, PI‐3K activity may be dispensable for Src‐PLCγ2 activation in GPIb‐IX‐V‐mediated signaling. Glycosphingolipid‐enriched microdomains (GEMs) appear to provide platforms for the signal transduction pathway related to GIb‐IX‐V, as the interaction between GPIb‐IX‐V and Src or PLCγ2 tyrosine phosphorylation occurs exclusively in GEMs.

Platelet Activation Receptor CLEC-2 Regulates Blood/Lymphatic Vessel Separation by Inhibiting Proliferation, Migration, and Tube Formation of Lymphatic Endothelial Cells

Background: Mice deficient in the platelet receptor CLEC-2 for podoplanin showed impaired blood/lymphatic vessel separation. Results: Functions of lymphatic endothelial cells are inhibited by platelet releasates and BMP-9, which we identified as a novel releasate. Conclusion: Granule contents including BMP-9 released upon platelet activation by CLEC-2-podoplanin interaction may contribute to the separation in vivo. Significance: We proposed a novel mechanism of platelet-mediated blood/lymphatic vessel separation. The platelet activation receptor CLEC-2 plays crucial roles in thrombosis/hemostasis, tumor metastasis, and lymphangiogenesis, although its role in thrombosis/hemostasis remains controversial. An endogenous ligand for CLEC-2, podoplanin, is expressed in lymphatic endothelial cells (LECs). We and others have reported that CLEC-2-deficiency is lethal at mouse embryonic/neonatal stages associated with blood-filled lymphatics, indicating that CLEC-2 is essential for blood/lymphatic vessel separation. However, its mechanism, and whether CLEC-2 in platelets is necessary for this separation, remains unknown. We found that specific deletion of CLEC-2 from platelets leads to the misconnection of blood/lymphatic vessels. CLEC-2+/+ platelets, but not by CLEC-2−/− platelets, inhibited LEC migration, proliferation, and tube formation but had no effect on human umbilical vein endothelial cells. Additionally, supernatants from activated platelets significantly inhibited these three functions in LECs, suggesting that released granule contents regulate blood/lymphatic vessel separation. Bone morphologic protein-9 (BMP-9), which we found to be present in platelets and released upon activation, appears to play a key role in regulating LEC functions. Only BMP-9 inhibited tube formation, although other releasates including transforming growth factor-β and platelet factor 4 inhibited proliferation and/or migration. We propose that platelets regulate blood/lymphatic vessel separation by inhibiting the proliferation, migration, and tube formation of LECs, mainly because of the release of BMP-9 upon activation by CLEC-2/podoplanin interaction.

Novel platelet activation receptor CLEC-2: from discovery to prospects.

Summary.  C‐type lectin‐like receptor 2 (CLEC‐2) has been identified as a receptor for the platelet activating snake venom rhodocytin. CLEC‐2 elicits powerful platelet activation signals in conjunction with Src, Syk kinases, and phospholipase Cγ2, similar to the collagen receptor glycoprotein (GP) VI/FcRγ‐chain complex. In contrast to GPVI/FcRγ, which initiates platelet activation through the tandem YxxL motif immunoreceptor tyrosine‐based activation motif (ITAM), CLEC‐2 signals via the single YxxL motif hemi‐ITAM. The endogenous ligand of CLEC‐2 has been identified as podoplanin, which is expressed on the surface of tumour cells and facilitates tumour metastasis by inducing platelet activation. Studies of CLEC‐2‐deficient mice have revealed several physiological roles of CLEC‐2. Podoplanin is also expressed in lymphatic endothelial cells as well as several other cells, including type I alveolar cells and kidney podocytes, but is absent from vascular endothelial cells. In the developmental stages, when the primary lymph sac is derived from the cardinal vein, podoplanin activates platelets in lymphatic endothelial cells by binding to CLEC‐2, which facilitates blood/lymphatic vessel separation. Moreover, CLEC‐2 is involved in thrombus stabilisation under flow conditions in part through homophilic interactions. However, the absence of CLEC‐2 does not significantly increase bleeding tendency. CLEC‐2 may be a good target protein for novel anti‐platelet drugs or anti‐metastatic drugs having therapeutic and preventive effects on arterial thrombosis and cancer, the primary causes of mortality in developed countries. In this article, we review the mechanisms of signal transduction, structure, expression, and function of CLEC‐2.

CLEC-2 in megakaryocytes is critical for maintenance of hematopoietic stem cells in the bone marrow

Nakamura-Ishizu et al. report that megakaryocytes function as a niche to maintain HSC quiescence through CLEC-2–mediated production of Thpo and other key regulators of HSC function. These findings could enable manipulation of HSCs for clinical application.

Essential in vivo roles of the platelet activation receptor CLEC-2 in tumour metastasis, lymphangiogenesis and thrombus formation

We have recently identified C-type lectin-like receptor 2 (CLEC-2) as a receptor for the platelet activating snake venom rhodocytin. CLEC-2 elicits powerful platelet activation signals in conjunction with single YxxL motif in its cytoplasmic tail, Src, Syk kinases, and phospholipase Cγ2. An endogenous ligand of CLEC-2 has been identified as podoplanin, which is a membrane protein of tumour cells and facilitates tumour metastasis by inducing platelet activation. Studies of CLEC-2-deficient mice have revealed several physiological roles of CLEC-2. Podoplanin is also expressed in lymphatic endothelial cells. In the developmental stages, when the primary lymph sac is derived from the cardinal vein, podoplanin activates platelets in lymphatic endothelial cells, which facilitates blood/lymphatic vessel separation. Moreover, CLEC-2 is involved in thrombus stabilization under flow conditions in part through homophilic interactions. The absence of CLEC-2 does not significantly increase bleeding tendency, implying that CLEC-2 may be a good target protein for anti-platelet drugs in addition to anti-metastatic drugs.

Redundant Mechanism of Platelet Adhesion to Laminin and Collagen under Flow INVOLVEMENT OF VON WILLEBRAND FACTOR AND GLYCOPROTEIN Ib-IX-V

Although the role of collagen in thrombosis has been extensively investigated, the contribution of other extracellular matrices is still unclear. We have recently reported that laminin stimulates platelet spreading through integrin α6β1-dependent activation of the collagen receptor glycoprotein (GP) VI under static condition. Under physiological high and low shear conditions, platelets adhered to laminin, and this was strongly inhibited by an antibody that blocks association between GPIb-IX-V and von Willebrand factor (VWF). Moreover, platelets of type III von Willebrand disease or Bernard-Soulier syndrome adhered to laminin at a low shear condition but not at a high shear condition. The specific binding of laminin to VWF was confirmed by surface plasmin resonance spectroscopy (BIAcore). These findings suggest that laminin supports platelet adhesion depending on the interaction of VWF and GPIb-IX-V under pathophysiological high shear flow. This mechanism is similar to that of collagen. We propose that integrins, GPVI, GPIb-IX-V, and VWF represent a general paradigm for the interaction between platelets and subendothelial matrices.

Platelet receptors activated via mulitmerization: glycoprotein VI, GPIb-IX-V, and CLEC-2.

While very different in structure, GPVI – the major collagen receptor on platelet membranes, the GPIb‐IX‐V complex – the receptor for von Willebrand factor, and CLEC‐2, a novel platelet activation receptor for podoplanin, share several common features in terms of function and platelet activation signal transduction pathways. All employ Src family kinases (SFK), Syk, and other signaling molecules involving tyrosine phosphorylation, similar to those of immunoreceptors for T and B cells. There appear to be overlapping functional roles for these glycoproteins, and in some cases, they can compensate for each other, suggesting a degree of redundancy. New ligands for these receptors are being identified, which broadens their functional relevancy. This is particularly true for CLEC‐2, whose functions beyond hemostasis are being explored. The common mode of signaling, clustering, and localization to glycosphingolipid‐enriched microdomains (GEMs) suggest that GEMs are central to signaling function by ligand‐dependent association of these receptors, SFK, Syk, phosphotyrosine phosphatases, and other signaling molecules.