Ligand Podoplanin Research Articles

Diphenyl-tetrazol-propanamide Derivatives Act as Dual-Specific Antagonists of Platelet CLEC-2 and Glycoprotein VI.

Platelet C-type lectin-like receptor 2 (CLEC-2) induces platelet activation and aggregation after clustering by its ligand podoplanin (PDPN). PDPN, which is not normally expressed in cells in contact with blood flow, is induced in inflammatory immune cells and some malignant tumor cells, thereby increasing the risk of venous thromboembolism (VTE) and tumor metastasis. Therefore, small-molecule compounds that can interfere with the PDPN-CLEC-2 axis have the potential to become selective antiplatelet agents. Using molecular docking analysis of CLEC-2 and a PDPN-CLEC-2 binding-inhibition assay, we identified a group of diphenyl-tetrazol-propanamide derivatives as novel CLEC-2 inhibitors. A total of 12 hit compounds also inhibited PDPN-induced platelet aggregation in humans and mice. Unexpectedly, these compounds also fit the collagen-binding pocket of the glycoprotein VI molecule, thereby inhibiting collagen interaction. These compounds also inhibited collagen-induced platelet aggregation, and one compound ameliorated collagen-induced thrombocytopenia in mice. For clinical use, these compounds will require a degree of chemical modification to decrease albumin binding. Nonetheless, as dual activation of platelets by collagen and PDPN-positive cells is expected to occur after the rupture of atherosclerotic plaques, these dual antagonists could represent a promising pharmacophore, particularly for arterial thrombosis, in addition to VTE and metastasis.

Stimulation of platelet aggregation by affinity captured rhodocytin from the Malayan pit viper Calloselasma rhodostoma

The receptor protein CLEC-2 on platelet membranes is the target of the endogenous ligand podoplanin found on cancer cells and of rhodocytin, a snake venom component of the Malayan pit viper Calloselasma rhodostoma. Ligand binding results in platelet activation, increased blood coagulation and thrombosis. In an effort to isolate rhodocytin, we have purified CLEC-2 as bait from E. coli. Affinity captured rhodocytin interacted with mammalian CLEC-2 and stimulated platelet aggregation in a dose dependent manner.

Monocyte upregulation of podoplanin during early sepsis induces complement inhibitor release to protect liver function.

Multiple organ failure in sepsis is a progressive failure of several interdependent organ systems. Liver dysfunction occurs early during sepsis and is directly associated with patient death; however, the underlying mechanism of liver dysfunction is unclear. Platelet transfusion benefits patients with sepsis, and inhibition of complement activation protects liver function in septic animals. Herein, we explored the potential link between platelets, complement activation, and liver dysfunction in sepsis. We found that deletion of platelet C-type lectin-like receptor 2 (CLEC-2) exacerbated liver dysfunction in early sepsis. Platelet CLEC-2-deficient mice exhibited higher complement activation, more severe complement attack in the liver, and lower plasma levels of complement inhibitors at early time points after E. coli infection. Circulating monocytes expressed the CLEC-2 ligand podoplanin in early sepsis, and podoplanin binding induced release of complement inhibitors from platelets. Injection of complement inhibitors released from platelets reduced complement attack and attenuated liver dysfunction in septic mice. These findings indicate a new function of platelets in the regulation of complement activation during sepsis.

Lymphatic blood filling in CLEC-2-deficient mouse models

C-type lectin-like receptor 2 (CLEC-2) is considered as a potential drug target in settings of wound healing, inflammation, and infection. A potential barrier to this is evidence that CLEC-2 and its ligand podoplanin play a critical role in preventing lymphatic vessel blood filling in mice throughout life. In this study, this aspect of CLEC-2/podoplanin function is investigated in more detail using new and established mouse models of CLEC-2 and podoplanin deficiency, and models of acute and chronic vascular remodeling. We report that CLEC-2 expression on platelets is not required to maintain a barrier between the blood and lymphatic systems in unchallenged mice, post-development. However, under certain conditions of chronic vascular remodeling, such as during tumorigenesis, deficiency in CLEC-2 can lead to lymphatic vessel blood filling. These data provide a new understanding of the function of CLEC-2 in adult mice and confirm the essential nature of CLEC-2-driven platelet activation in vascular developmental programs. This work expands our understanding of how lymphatic blood filling is prevented by CLEC-2-dependent platelet function and provides a context for the development of safe targeting strategies for CLEC-2 and podoplanin.

Platelets and cancer-associated thrombosis: focusing on the platelet activation receptor CLEC-2 and podoplanin.

Patients with cancer have an increased risk of thromboembolism, which is the second leading cause of death in these patients. Several mechanisms of the prothrombotic state in these patients have been proposed. Among them are a platelet activation receptor, C-type lectin-like receptor 2 (CLEC-2), and its endogenous ligand podoplanin, which are the focus of this review. CLEC-2 is almost specifically expressed in platelets/megakaryocytes in humans. A membrane protein, podoplanin is expressed in certain types of cancer cells, including squamous cell carcinoma, brain tumor, and osteosarcoma, in addition to several normal tissues, including kidney podocytes and lymphatic endothelial cells but not vascular endothelial cells. In the bloodstream, podoplanin induces platelet activation by binding to CLEC-2 and facilitates hematogenous cancer metastasis and cancer-associated thrombosis. In an experimental lung metastasis model, the pharmacological depletion of CLEC-2 from platelets in mice resulted in a marked reduction of lung metastasis of podoplanin-expressing B16F10 cells. Control mice with B16F10 orthotopically inoculated in the back skin showed massive thrombus formation in the lungs, but the cancer-associated thrombus formation in CLEC-2-depleted mice was significantly inhibited, suggesting that CLEC-2-podoplanin interaction stimulates cancer-associated thrombosis. Thromboinflammation induced ectopic podoplanin expression in vascular endothelial cells or macrophages, which may also contribute to cancer-associated thrombosis. CLEC-2 depletion in cancer-bearing mice resulted in not only reduced cancer-associated thrombosis but also reduced levels of plasma inflammatory cytokines, anemia, and sarcopenia, suggesting that cancer-associated thrombosis may cause thromboinflammation and cancer cachexia. Blocking CLEC-2-podoplanin interaction may be a novel therapeutic strategy in patients with podoplanin-expressing cancer.

CLEC2 and CLEC5A: Pathogenic Host Factors in Acute Viral Infections.

The protective roles of endosomal toll-like receptors (TLRs) and cytosolic nucleic acid sensors are well elucidated, but the pathogenic host factors during viral infections remain unclear. Spleen tyrosine kinase (Syk)-coupled C-type lectins (CLECs) CLEC2 and CLEC5A are highly expressed on platelets and myeloid cells, respectively. CLEC2 has been shown to recognize snake venom aggretin and the endogenous ligand podoplanin and acts as a critical regulator in the development and immunothrombosis. Although CLEC2 has been reported to interact with type I immunodeficiency virus (HIV-1), its role in viral infections is still unclear. CLEC5A binds to fucose and mannose moieties of dengue virus membrane glycans, as well as to N-acetylglucosamine (GlcNAc)/N-acetylmuramic acid (MurNAc) disaccharides that form the backbone of L. monocytogenes peptidoglycans. Recently, we demonstrated that both CLEC2 and CLEC5A are critical in microbe-induced “neutrophil extracellular trap” (NET) formation and proinflammatory cytokine production. Moreover, activation of CLEC2 by dengue virus (DV) and H5N1 influenza virus (IAV) induces the release of extracellular vesicles (EVs), which further enhance NETosis and proinflammatory cytokine production via CLEC5A and Toll-like receptor 2 (TLR2). These findings not only illustrate the immunomodulatory effects of EVs during platelet-leukocyte interactions, but also demonstrate the critical roles of CLEC2 and CLEC5A in acute viral infections.

Platelet glycoprotein VI aids in local immunity during pneumonia-derived sepsis caused by gram-negative bacteria

AbstractPlatelet collagen receptor glycoprotein VI (GPVI) and podoplanin receptor C-type lectin-like receptor 2 (CLEC2) are receptors implicated in platelet activation that both signal via an immunoreceptor tyrosine–based activation motif. Platelets are necessary for host defense and prevention of hemorrhage during sepsis, but the role of platelet GPVI and CLEC2 herein is unknown. To investigate this, we infected mice depleted of platelet GPVI or CLEC2 by antibody treatment or GPVI−/− mice with the common human sepsis pathogen Klebsiella pneumoniae via the airways to induce pneumonia-derived sepsis. The GPVI ligand collagen and the CLEC2 ligand podoplanin were constitutively present in the lung, whereas the GPVI ligands fibrin and histone were induced during pneumonia. During late-stage infection, both mice depleted of GPVI and GPVI−/− mice showed increased bacterial growth in lungs, and GPVI−/− mice also showed increased bacterial growth in distant body sites. Despite higher bacterial loads, GPVI-depleted mice showed reduced platelet numbers, platelet activation, and platelet–leukocyte complex formation in the bronchoalveolar space. Consistently, in human whole blood, GPVI stimulation of platelets increased platelet–leukocyte complex formation and leukocyte activation, which was accompanied by enhanced phagocytosis of Klebsiella. GPVI-depleted mice showed increased lung hemorrhage during infection, but not to the extent observed in platelet-depleted mice, and lung bleeding was not significantly different between GPVI−/− and wild-type mice. CLEC2 depletion did not affect any of the responses during pneumonia. These results suggest that platelet GPVI, but not CLEC2, contributes to local host defense during pneumonia-derived sepsis by enhancing leukocyte function.

C-type lectin-like receptor 2 (CLEC-2)-dependent dendritic cell migration is controlled by tetraspanin CD37.

Cell migration is central to evoking a potent immune response. Dendritic cell (DC) migration to lymph nodes is dependent on the interaction of C-type lectin-like receptor 2 (CLEC-2; encoded by the gene Clec1b), expressed by DCs, with podoplanin, expressed by lymph node stromal cells, although the underlying molecular mechanisms remain elusive. Here, we show that CLEC-2-dependent DC migration is controlled by tetraspanin CD37, a membrane-organizing protein. We identified a specific interaction between CLEC-2 and CD37, and myeloid cells lacking CD37 (Cd37-/-) expressed reduced surface CLEC-2. CLEC-2-expressing Cd37-/- DCs showed impaired adhesion, migration velocity and displacement on lymph node stromal cells. Moreover, Cd37-/- DCs failed to form actin protrusions in a 3D collagen matrix upon podoplanin-induced CLEC-2 stimulation, phenocopying CLEC-2-deficient DCs. Microcontact printing experiments revealed that CD37 is required for CLEC-2 recruitment in the membrane to its ligand podoplanin. Finally, Cd37-/- DCs failed to inhibit actomyosin contractility in lymph node stromal cells, thus phenocopying CLEC-2-deficient DCs. This study demonstrates that tetraspanin CD37 controls CLEC-2 membrane organization and provides new molecular insights into the mechanisms underlying CLEC-2-dependent DC migration.This article has an associated First Person interview with the first author of the paper.

Platelet CLEC-2 protects against lung injury via effects of its ligand podoplanin on inflammatory alveolar macrophages in the mouse.

There is no therapeutic intervention proven to prevent acute respiratory distress syndrome (ARDS). Novel mechanistic insights into the pathophysiology of ARDS are therefore required. Platelets are implicated in regulating many of the pathogenic processes that occur during ARDS; however, the mechanisms remain elusive. The platelet receptor CLEC-2 has been shown to regulate vascular integrity at sites of acute inflammation. Therefore the purpose of this study was to establish the role of CLEC-2 and its ligand podoplanin in a mouse model of ARDS. Platelet-specific CLEC-2-deficient, as well as alveolar epithelial type I cell (AECI)-specific or hematopoietic-specific podoplanin deficient, mice were established using cre-loxP strategies. Combining these with intratracheal (IT) instillations of lipopolysaccharide (LPS), we demonstrate that arterial oxygen saturation decline in response to IT-LPS in platelet-specific CLEC-2-deficient mice is significantly augmented. An increase in bronchoalveolar lavage (BAL) neutrophils and protein was also observed 48 h post-IT-LPS, with significant increases in pro-inflammatory chemokines detected in BAL of platelet-specific CLEC-2-deficient animals. Deletion of podoplanin from hematopoietic cells but not AECIs also reduces lung function and increases pro-inflammatory chemokine expression following IT-LPS. Furthermore, we demonstrate that following IT-LPS, platelets are present in BAL in aggregates with neutrophils, which allows for CLEC-2 interaction with podoplanin expressed on BAL inflammatory alveolar macrophages. Taken together, these data suggest that the platelet CLEC-2-podoplanin signaling axis regulates the severity of lung inflammation in mice and is a possible novel target for therapeutic intervention in patients at risk of developing ARDS.

Abstract 350: Activation of Platelet CLEC-2 in Hematoma Expansion After Intracerebral Hemorrhage: Finding an Effective Dose

Intracerebral hemorrhage (ICH) is the least treatable and one of the most disabling stroke subtypes, with both the primary and secondary brain injuries mainly due to hematoma expansion (HE) within the first few hours, followed by an inflammatory and oxidative injury. Almost all ICH patients experience some degree of expansion, and ICH volume can be an independent determinant of mortality and functional outcome. Previous studies found that reduced platelet activity is associated with early ICH growth and worse functional outcomes. The platelet C-type lectin-like receptor 2 (CLEC-2) elicits robust platelet activation upon stimulation. Thus, we hypothesized that activation of CLEC-2 would result in activation and aggregation of platelets, reducing HE, and improving functional outcomes after ICH. We administered two CLEC-2 agonists (Fucoidan and the endogenous ligand Podoplanin) at varying doses after murine ICH and measured hemorrhage volume, brain water content, and behavior. We also collected blood from podoplanin-treated mice and conducted hematologic tests. We further tested Rhodocytin, a known CLEC-2 activator, for whole blood clotting times, platelet functions, and soluble fibrin in human blood samples. We found that treatment with Fucoidan did not reduce hemorrhage volume or brain water content and had no effect on neurological function, compared to vehicle. Further, Podoplanin treatment showed a tendency to reduce brain water content but did not affect hematoma volume or behavior. Our in vitro studies showed lower fibrinogen levels with podoplanin treatment, compared to sham and vehicle, but no effect on partial thromboplastin time, prothrombin time, or INR. At higher doses, Rhodocytin resulted in immediate clotting, while at lower doses, it significantly increased soluble fibrin, consistent with initiation of clotting seen in the higher doses. In conclusion, our findings indicate that activation of CLEC-2 at the doses tested does not reduce ICH volume or improve outcomes, but did affect hemostatic parameters. Thus, the agonists may produce favorable outcomes with a different dosing regimen. Further studies are needed to confirm the mechanism by which CLEC-2 is acting in ICH, effective doses, and the effect of altered bleeding parameters.

Role of CLEC-2-driven platelet activation in the pathogenesis of toxic liver damage

BackgroundToxic liver injury from drugs including paracetamol is the main cause of acute liver failure in developed countries. The mechanisms that drive irreversible liver failure are poorly understood; platelets could have an important role in this process given their roles beyond haemostasis, including liver regeneration. Ligation of the platelet receptor CLEC-2 with its cognate ligand podoplanin (PDPN) powerfully activates platelets; we sought to investigate the role of CLEC-2 in the pathogenesis of acute liver failure. MethodsParacetamol or carbon tetrachloride (CCl4) were used to induce acute liver damage in mice. The role of CLEC-2-mediated platelet activation was investigated in mice with conditional deletions for either the platelet CLEC-2 receptor (PF4creCLEC1bfl/fl) or PDPN (Vav1-iCre+PDPNfl/fl), or with specific function blocking antibodies. Liver necrosis, and the subsequent inflammatory response, was gauged by assessment of hepatic leucocyte infiltration and measurement of liver histological and serum markers. FindingsInitial liver injury after CCl4 and paracetamol administration was similar in both wild-type (WT) and CLEC-2-deficient mice. Abrogating CLEC-2-driven platelet activation accelerated liver healing from both toxic insults: mean serum alanine aminotransferase [ALT] after paracetamol administration was 1264 IU/L (SE 296·5) in WT mice versus 52·00 (5·00) in CLEC-2-deficient mice (n=5–8, p=0·0078); and after CCl4 4451 (886·3) versus 367 (99·35) (n=4–8, p=0·0015). Targeting this pathway therapeutically with a specific PDPN function blocking antibody in WT mice also enhanced liver healing: after CCl4 administration mean ALT in control antibody treated mice was 5482 (SE 785·4) versus 598·8 (102·4) in anti-PDPN antibody treated mice (n=6, p=0·0001), and after paracetamol 2850 (1128) versus 194·5 (61·26) (p=0·0176). In-vitro experiments showed that CLEC-2-deficient platelets interacted with Kupffer cells to enhance production of tumour necrosis factor α (TNFα) and increase accumulation of hepatic neutrophils. Healing was prevented by either blocking TNFα or depleting neutrophils in mice. Upregulation of PDPN on Kupffer cells in human acute liver failure suggests that this pathway is also activated in human beings. InterpretationPlatelets are involved in determining the outcome of the sterile inflammatory response to toxic liver injury. Platelet activation via CLEC-2 in the context of an acute liver injury inhibits TNFα-driven reparative inflammation mediated by neutrophils. The fact that blocking CLEC-2-mediated platelet activation enhances neutrophil-driven liver repair without causing bleeding, suggests that this could be a completely novel treatment for human acute liver failure. FundingWellcome Trust.

血小板活性化受容体CLEC-2 とその内因性リガンドがポドプラニンであることの同定と，両者の結合の病態生理学的役割の解明

要約：私達は，血小板活性化蛇毒ロドサイチンの受容体として，新規血小板活性化受容体C-type lectin-like receptor 2（CLEC-2）を血小板上に同定した．またCLEC-2 の生体内リガンドが，ポドプラニンと呼ばれる膜蛋白であることを発見した．ポドプラニンはある種の腫瘍細胞や，リンパ節の細網細胞，腎臓のポドサイト，リンパ管内皮に発現しているが，血管内皮には発現していない．血小板のCLEC-2 は通常の状態ではポドプラニンと接触できないが，発生の途中や，病的状態では接触しうる．私達はCLEC-2 とポドプラニンの結合が様々な病態生理学的役割を持つことを解明した．血小板CLEC-2 は腫瘍細胞に発現するポドプラニンと結合して，腫瘍の血行性転移を促進する．また，血小板CLEC-2 は胎生期に血管から分化して分離するリンパ管内皮細胞のポドプラニンと結合することで，リンパ管と血管の分離を促進する．CLEC-2 はヒトではほぼ血小板・巨核球特異的に発現しているが，流血中で血小板活性化依存性にホモフィリックに結合することで血栓を安定化している．私達は骨髄中にポドプラニンを発現する骨髄細網細胞様細胞を同定した．巨核球のCLEC-2 と骨髄細網細胞様細胞のポドプラニンとの結合で，巨核球の増殖や血小板産生が促進されるという新しい骨髄微小環境を提唱した．CLEC-2 の臨床応用として，血小板活性化に伴って遊離するsoluble CLEC-2 を開発し，生体内血小板活性化のマーカーとすることを試みた．以上，私達はCLEC-2 とそのリガンドの同定を通じて，血小板が様々な血栓止血以外の役割を持つことを示し，臨床応用に向けた取り組みを展開している．

PDGF upregulates CLEC-2 to induce T regulatory cells.

The effect of platelet derived growth factor (PDGF) on immune cells is not elucidated. Here, we demonstrate PDGF inhibited the maturation of human DCs and induced IL-10 secretion. Culture of PDGF-DCs with T cells induced the polarization of T cells towards FoxP3 expressing T regulatory cells that secreted IL-10. Gene expression studies revealed that PDGF induced the expression of C-type lectin like receptor member 2, (CLEC-2) receptor on DCs. Furthermore, DCs transfected with CLEC-2 induced T regulatory cells in DC-T cell co-culture. CLEC-2 is naturally expressed on platelets. Therefore, to confirm whether CLEC-2 is responsible for inducing the T regulatory cells, T cells were cultured with either CLEC-2 expressing platelets or soluble CLEC-2. Both conditions resulted in the induction of regulatory T cells. The generation of T regulatory cells was probably due to the binding of CLEC-2 with its ligand podoplanin on T cells, since crosslinking of podoplanin on the T cells also resulted in the induction of T regulatory cells. These data demonstrate that PDGF upregulates the expression of CLEC-2 on cells to induce T regulatory cells.

Platelet adhesion to podoplanin under flow is mediated by the receptor CLEC-2 and stabilised by Src/Syk-dependent platelet signalling.

SummaryPlatelet-specific deletion of CLEC-2, which signals through Src and Syk kinases, or global deletion of its ligand podoplanin results in blood-filled lymphatics during mouse development. Platelet-specific Syk deficiency phenocopies this defect, indicating that platelet activation is required for lymphatic development. In the present study, we investigated whether CLEC-2-podoplanin interactions could support platelet arrest from blood flow and whether platelet signalling is required for stable platelet adhesion to lymphatic endothelial cells (LECs) and recombinant podoplanin under flow. Perfusion of human or mouse blood over human LEC monolayers led to platelet adhesion and aggregation. Following αIIbβ3 blockade, individual platelets still adhered. Platelet binding occurred at venous but not arterial shear rates. There was no adhesion using CLEC-2-deficient blood or to vascular endothelial cells (which lack podoplanin). Perfusion of human blood over human Fc-podoplanin (hFcPDPN) in the presence of monoclonal antibody IV.3 to block FcγRIIA receptors led to platelet arrest at similar shear rates to those used on LECs. Src and Syk inhibitors significantly reduced global adhesion of human or mouse platelets to LECs and hFcPDPN. A similar result was seen using Syk-deficient mouse platelets. Reduced platelet adhesion was due to a decrease in the stability of binding. In conclusion, our data reveal that CLEC-2 is an adhesive receptor that supports platelet arrest to podoplanin under venous shear. Src/Syk-dependent signalling stabilises platelet adhesion to podoplanin, providing a possible molecular mechanism contributing to the lymphatic defects of Syk-deficient mice.