A role of platelet C-type lectin-like receptor-2 and its ligand podoplanin in vascular biology.

PURPOSE OF REVIEW: Platelets are essential for hemostasis and are also vital in lymphatic and lung development and the maintenance of vascular integrity. Platelet activation receptor C-type lectin-like receptor 2 (CLEC-2) and its endogenous ligand podoplanin (PDPN) in lymphatic endothelial cells (LECs) and other cells regulate these processes. This review aims to comprehensively summarize the roles of platelet CLEC-2 and PDPN. This review also focuses on discussing the underlying mechanisms by which platelet CLEC-2 and PDPN mediate blood/lymphatic separation. FINDINGS: CLEC-2/PDPN-induced platelet activation in the primary lymph sacs, developmental lymphovenous junctions, neonatal mesentery, and the site of tumor lymphangiogenesis prevents blood/lymphatic vessel misconnection. Further, CLEC-2/PDPN-induced platelet activation is essential for lung development. Mice deficient in CLEC-2 or PDPN show blood-filled lymphatics, lung malformations, and cerebrovascular abnormalities. CLEC-2 deletion in steady-state adult mice did not result in blood/lymphatic vessel mixing. In adulthood, CLEC-2 maintains vascular integrity and that of high endothelial venules in lymph nodes. CLEC-2 deletion in adulthood results in hemorrhage under inflammatory conditions, and hemolymph nodes. SUMMARY: The platelet CLEC-2/LEC PDPN interaction prevents blood/lymphatic vessel mixing at active remodeling sites of the blood/lymphatic system, but not in steady-state adult mice. This interaction also regulates vascular integrity when vascular permeability increases before and after birth.

Impact of Hemostasis on the Lymphatic System in Development and Disease.

Lymphatic vessels form a systemic network that maintains interstitial fluid homeostasis and regulates immune responses and is strictly separated from the circulatory system. During embryonic development, lymphatic endothelial cells originate from blood vascular endothelial cells in the cardinal veins and form lymph sacs. Platelets are critical for separating lymph sacs from the cardinal veins through interactions between CLEC-2 (C-type lectin-like receptor-2) and PDPN (podoplanin) in lymphatic endothelial cells. Therefore, deficiencies of these genes cause blood-filled lymphatic vessels, leading to abnormal lymphatic vessel maturation. The junction between the thoracic duct and the subclavian vein has valves and forms physiological thrombi dependent on CLEC-2/PDPN signaling to prevent blood backflow into the thoracic duct. In addition, platelets regulate lymphangiogenesis and maintain blood/lymphatic separation in pathological conditions, such as wound healing and inflammatory diseases. More recently, it was reported that the entire hemostatic system is involved in lymphangiogenesis. Thus, the hemostatic system plays a crucial role in the establishment, maintenance, and rearrangement of lymphatic networks and contributes to body fluid homeostasis, which suggests that the hemostatic system is a potential target for treating lymphatic disorders. This review comprehensively summarizes the role of the hemostatic system in lymphangiogenesis and lymphatic vessel function and discusses challenges and future perspectives.

Periosteum-derived podoplanin-expressing stromal cells regulate nascent vascularization during epiphyseal marrow development.

Bone marrow development and endochondral bone formation occur simultaneously. During endochondral ossification, periosteal vasculatures and stromal progenitors invade the primary avascular cartilaginous anlage, which induces primitive marrow development. We previously determined that bone marrow podoplanin (PDPN)-expressing stromal cells exist in the perivascular microenvironment and promote megakaryopoiesis and erythropoiesis. In this study, we aimed to examine the involvement of PDPN-expressing stromal cells in postnatal bone marrow generation. Using histological analysis, we observed that periosteum-derived PDPN-expressing stromal cells infiltrated the cartilaginous anlage of the postnatal epiphysis and populated on the primitive vasculature of secondary ossification center. Furthermore, immunophenotyping and cellular characteristic analyses indicated that the PDPN-expressing stromal cells constituted a subpopulation of the skeletal stem cell lineage. In vitro xenovascular model cocultured with human umbilical vein endothelial cells and PDPN-expressing skeletal stem cell progenies showed that PDPN-expressing stromal cells maintained vascular integrity via the release of angiogenic factors and vascular basement membrane-related extracellular matrices. We show that in this process, Notch signal activation committed the PDPN-expressing stromal cells into a dominant state with basement membrane-related extracellular matrices, especially type IV collagens. Our findings suggest that the PDPN-expressing stromal cells regulate the integrity of the primitive vasculatures in the epiphyseal nascent marrow. To the best of our knowledge, this is the first study to comprehensively examine how PDPN-expressing stromal cells contribute to marrow development and homeostasis.

C-type lectin-like receptor-2 (CLEC-2) is a key regulator of kappa-carrageenan-induced tail thrombosis model in mice.

Kappa-carrageenan (KCG), which is used to induce thrombosis in laboratory animals for antithrombotic drug screening, can trigger platelet aggregation. However, the cell-surface receptor and related signaling pathways remain unclear. In this study, we investigated the molecular basis of KCG-induced platelet activation using light-transmittance aggregometry, flow cytometry, western blotting, and surface plasmon resonance assays using platelets from platelet receptor-deficient mice and recombinant proteins. KCG-induced tail thrombosis was also evaluated in mice lacking the platelet receptor. We found that KCG induces platelet aggregation with α-granule secretion, activated integrin αIIbß3, and phosphatidylserine exposure. As this aggregation was significantly inhibited by the Src family kinase inhibitor and spleen tyrosine kinase (Syk) inhibitor, a tyrosine kinase-dependent pathway is required. Platelets exposed to KCG exhibited intracellular tyrosine phosphorylation of Syk, linker activated T cells, and phospholipase C gamma 2. KCG-induced platelet aggregation was abolished in platelets from C-type lectin-like receptor-2 (CLEC-2)-deficient mice, but not in platelets pre-treated with glycoprotein VI-blocking antibody, JAQ1. Surface plasmon resonance assays showed a direct association between murine/human recombinant CLEC-2 and KCG. KCG-induced thrombosis and thrombocytopenia were significantly inhibited in CLEC-2-deficient mice. Our findings show that KCG induces platelet activation via CLEC-2.

Thrombosis is a serious medical condition that occurs when blood clots form in the blood vessels and can lead to heart attacks or strokes. Animal models are important for evaluating the effectiveness of drugs in thrombosis treatment. Kappa-carrageenan (KCG) is a food thickener and a substance used to induce clot formation in laboratory animals. In this study, we investigated the molecular basis of KCG-induced platelet activation, which is an important step in thrombosis development. We found that KCG activates platelets via a receptor called C-type lectin-like receptor 2 (CLEC-2), leading to a prothrombotic state in mice. We also showed that KCG-induced tail thrombosis (CTT) is significantly inhibited in CLEC-2 deficient mice. Our findings suggest that CLEC-2-mediated platelet activation plays a key role in the pathogenesis of thrombosis and CLEC-2 May participate in innate immunity as a receptor for sulfate-polysaccharide.Abbreviation; CLEC-2: C-type lectin-like receptor 2; CRP: collagen-related peptide; CTT: KCGN-induced tail thrombosis; DIC: disseminated intravascular coagulation; EDTA: ethylenediaminetetraacetic acid; GPVI: glycoprotein VI; HRP: horseradish peroxidase; KCG: Κ-Carrageenan; LAT: linker activated T cells; LDS: lithium dodecyl sulfate; LTA: light-transmittance aggregometry; MFI: mean fluorescence intensity; PFA: paraformaldehyde; PLCγ2: phospholipase C gamma 2; PS: phosphatidylserine; Syk: spleen tyrosine kinase; Co-HP: Cobalt-hematoporphyrin.

High plasma soluble CLEC-2 level predicts oxygen therapy requirement in patients with COVID-19.

Predicting the clinical course and allocating limited medical resources appropriately is crucial during the COVID-19 pandemic. Platelets are involved in microthrombosis, a critical pathogenesis of COVID-19; however, the role of soluble CLEC-2 (sCLEC-2), a novel platelet activation marker, in predicting the prognosis of COVID-19 remains unexplored. We enrolled 108 patients with COVID-19, hospitalized between January 2021 and May 2022, to evaluate the clinical use of sCLEC-2 as a predictive marker. sCLEC-2 levels were measured in plasma sampled on admission, as well as interleukin-6, cell-free DNA, von Willebrand factor, and thrombomodulin. We retrospectively classified the patients into two groups - those who required oxygenation during hospitalization (oxygenated group) and those who did not (unoxygenated group) - and compared their clinical and laboratory characteristics. The correlation between sCLEC-2 and the other parameters was validated. The sCLEC-2 level was significantly higher in the oxygenated group (188.8 pg/mL vs. 296.1 pg/mL). Multivariate analysis identified high sCLEC-2 levels (odds ratio per 10 pg/mL:1.25) as an independent predictor of oxygen therapy requirement. sCLEC-2 was positively correlated with cell-free DNA, supporting the association between platelet activation and neutrophil extracellular traps. In conclusion, sCLEC-2 is a clinically valuable marker in predicting oxygen therapy requirements for patients with COVID-19.

What is the context? During the COVID-19 epidemic with tremendous damage to healthcare systems worldwide, predicting the clinical course of patients and allocating limited medical resources appropriately is crucial.Platelets are involved in microthrombosis - a critical pathogenesis of COVID-19. The role of soluble CLEC-2 (sCLEC-2), a novel in vivo platelet activation marker, in predicting the prognosis of COVID-19 remains unexplored.What is new? sCLEC-2 is an independent predictive marker of oxygen therapy requirement in COVID-19.What is the impact? In most cases, patients requiring oxygen therapy must be hospitalized. The ability to predict such cases during the COVID-19 epidemic, when medical recourses are depleted, may contribute to the appropriate allocation of medical resources.

Thrombotic Mechanism Involving Platelet Activation, Hypercoagulability and Hypofibrinolysis in Coronavirus Disease 2019.

Coronavirus disease 2019 (COVID-19) has spread, with thrombotic complications being increasingly frequently reported. Although thrombosis is frequently complicated in septic patients, there are some differences in the thrombosis noted with COVID-19 and that noted with bacterial infections. The incidence (6-26%) of thrombosis varied among reports in patients with COVID-19; the incidences of venous thromboembolism and acute arterial thrombosis were 4.8-21.0% and 0.7-3.7%, respectively. Although disseminated intravascular coagulation (DIC) is frequently associated with bacterial infections, a few cases of DIC have been reported in association with COVID-19. Fibrin-related markers, such as D-dimer levels, are extremely high in bacterial infections, whereas soluble C-type lectin-like receptor 2 (sCLEC-2) levels are high in COVID-19, suggesting that hypercoagulable and hyperfibrinolytic states are predominant in bacterial infections, whereas hypercoagulable and hypofibrinolytic states with platelet activation are predominant in COVID-19. Marked platelet activation, hypercoagulability and hypofibrinolytic states may cause thrombosis in patients with COVID-19.

Early recognition of sepsis-induced coagulopathy using the C2PAC index: a ratio of soluble type C lectin-like receptor 2 (sCLEC-2) level and platelet count.

C-type lectin-like receptor 2 (CLEC-2) is a platelet-activated receptor expressed on the surface of platelet membranes. Soluble CLEC-2 (sCLEC-2) has been receiving attention as a predictive marker for thrombotic predisposition. The present study examined the relationship between sCLEC-2 level and degree of coagulation disorder in septic patients. Seventy septic patients were divided into the sepsis-induced disseminated intravascular coagulation (DIC) (SID) group (n = 44) and non-SID group (n = 26). The sCLEC-2 levels were compared between the two groups. Because we suspected that the sCLEC-2 level was affected by the platelet count, we calculated the sCLEC-2/platelet count ratio (C2PAC index). We further divided septic patients into four groups using the Japanese Association for Acute Medicine (JAAM) DIC scoring system (DIC scores: 0-1, 2-3, 4-5, and 6-8). The C2PAC index was significantly higher in the SID group (2.6 ± 1.7) compared with the non-SID group (1.2 ± 0.5) (P < .001). The C2PAC indexes in the four JAAM DIC score groups were 0.9 ± 0.3, 1.1 ± 0.3, 1.7 ± 0.7, and 3.6 ± 1.0, respectively, and this index increased significantly as the DIC score increased (P < .001). According to the receiver-operating curve analysis, the area under the curve (AUC) and optimal cutoff value for the diagnosis of SID were 0.8051 and 1.4 (sensitivity, 75.0%; specificity, 76.9%), respectively. When the C2PAC index and D-dimer level, one of the main fibrinolytic markers, were selected as predictive markers for SID diagnosis in stepwise multiple logistic regression analysis, it was possible to diagnose SID with a high probability (AUC, 0.9528; sensitivity, 0.9545; specificity, 0.8846). The C2PAC index is a useful predictor of SID progression and diagnosis in septic patients.

Platelets enhance malignant behaviours of gastric cancer cells via direct contacts.

In this study, we aimed to analyse human cancer cell-platelet interactions in functional cell analyses and explore the molecular mechanisms behind tumour progression. Various functional analyses of gastric cancer (GC) cells were performed after direct/indirect co-incubation with platelets derived from GC patients. Further detailed expression and signalling analyses were performed after co-culture with direct and indirect GC cells-platelet contact. Malignant behaviours of cancer cells, such as proliferation, migration, invasion and adhesion, were significantly enhanced after direct co-incubation with platelets. Microarray analyses demonstrated changes in multiple genes, including epithelial-mesenchymal transition (EMT)-related genes. Among them, matrix metalloproteinase 9 was notably upregulated, which was validated by quantitative reverse transcription-polymerase chain reaction and western blot. Further, this change was only observed after direct co-incubation with platelets. This study demonstrated that platelets from GC patients promote malignant behaviours of GC cells through EMT-related signalling, especially by direct contact with tumour cells.

Platelet C-Type Lectin-Like Receptor 2 Reduces Cholestatic Liver Injury in Mice.

Cholestatic liver injury leads to liver dysfunction. The available evidence suggests that platelets can either promote or reduce liver injury and fibrosis. This study focused on the functions of the C-type lectin-like receptor 2 (CLEC-2), a new special platelet receptor that binds with podoplanin-activating platelets. The role of CLEC-2 and podoplanin in cholestatic liver injury was investigated. Mice were injected intraperitoneally with weekly doses of anti-CLEC-2 antibody (2A2B10) to achieve effective CLEC-2 inhibition in their platelets. Next, left and middle hepatic bile duct ligation (BDL) procedures were performed, and mice were euthanized 1 week later (2A2B10-BDL group). In addition, mice were prepared for control groups, and relevant histological and laboratory variables were compared among these groups. The inhibition of CLEC-2 resulted in increasing hepatocellular necrosis, hepatic inflammation, and liver fibrosis. In addition, podoplanin was strongly expressed in hepatic sinusoidal endothelial cells in BDL-treated mice. Moreover, in 2A2B10-BDL mice, total plasma bile acid levels were significantly increased. In summary, podoplanin is expressed on hepatic sinusoidal endothelial cells upon BDL. Platelets bind with podoplanin via CLEC-2 and become activated. As a result, the total bile acid pool is decreased. Therefore, the CLEC-2-podoplanin interaction promotes liver protection and inhibits liver fibrosis after cholestatic liver injury.

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.

Clinical characteristics and molecular analysis of USA300 and ST 764 methicillin-resistant Staphylococcus aureus isolates from outpatients in Japan by PCR-Based open reading frame typing.

INTRODUCTION: USA300 is the most common community-acquired methicillin-resistant Staphylococcus aureus (MRSA) strain. Sequence type (ST) 764 MRSA is a new local variant of the ST 5 lineage. The objective of this study was to determine the clinical characteristics of USA300 and ST 764 infections among outpatients in Japan. METHODS: We obtained MRSA isolates from 132 outpatients who visited our hospital from January 2016 to December 2017 and compared USA300 infection group to ST 764 infection group. Molecular analysis, including that of various toxins and other virulence factors, of the MRSA isolates were performed. In particular, we investigated the relationships among PCR-based open reading frame typing (POT) scores, MRSA clones, and virulence factors. RESULTS: Twenty-seven USA300 isolates (20.5%) and 16 ST 764 isolates (12.1%) were identified. Although USA300 and ST 764 had lower rates of risk factors, their infection rates were higher. USA300-infected patients had higher rates of deep skin and soft tissue infections compared with the non-USA300 CA-MRSA-infected patients. Notably, the USA300 and ST 764 isolates had unique POT scores. CONCLUSIONS: Our results indicated that USA300 MRSA was spreading in an area 120 km west of Tokyo, Japan. We observed multiple cases of ST 764 MRSA infection, raising concerns about the antimicrobial resistance of ST 764, as it limits the choices of antibiotics to treat infection. The POT score can predict the presence of toxins and virulence factors, as well as the clone identity of MRSA with high accuracy.

Platelets play an essential role in murine lung development through Clec-2/podoplanin interaction.

Platelets participate in not only thrombosis and hemostasis but also other pathophysiological processes, including tumor metastasis and inflammation. However, the putative role of platelets in the development of solid organs has not yet been described. Here, we report that platelets regulate lung development through the interaction between the platelet-activation receptor, C-type lectin-like receptor-2 (Clec-2; encoded by Clec1b), and its ligand, podoplanin, a membrane protein. Clec-2 deletion in mouse platelets led to lung malformation, which caused respiratory failure and neonatal lethality. In these embryos, α-smooth muscle actin-positive alveolar duct myofibroblasts (adMYFs) were almost absent in the primary alveolar septa, which resulted in loss of alveolar elastic fibers and lung malformation. Our data suggest that the lack of adMYFs is caused by abnormal differentiation of lung mesothelial cells (luMCs), the major progenitor of adMYFs. In the developing lung, podoplanin expression is detected in alveolar epithelial cells (AECs), luMCs, and lymphatic endothelial cells (LECs). LEC-specific podoplanin knockout mice showed neonatal lethality and Clec1b-/--like lung developmental abnormalities. Notably, these Clec1b-/--like lung abnormalities were also observed after thrombocytopenia or transforming growth factor-ß depletion in fetuses. We propose that the interaction between Clec-2 on platelets and podoplanin on LECs stimulates adMYF differentiation of luMCs through transforming growth factor-ß signaling, thus regulating normal lung development.

Potential therapeutics for antiphospholipid antibody associated thrombocytopenia: A systematic review and meta-analysis.

Objectives: Thrombocytopenia is frequently observed in antiphospholipid antibody (aPL) carriers. Due to the paradoxical risks of thrombosis and hemorrhage, the management of aPL-associated thrombocytopenia (APAT) is often deductive. We aimed to investigate the efficacy and safety of therapeutic approaches for APAT through a systematic review.Methods: Four therapeutic approaches for APAT, including antiplatelet agents, glucocorticoids, splenectomy and thrombopoietin receptor agonists, were selected. Clinical trials evaluating therapeutic outcomes including the remission, complications, mortality and relapse, were searched in MEDLINE, EMBASE and CENTRAL from the inception dates to 28 November 2016. A meta-analysis was performed to calculate risk ratios (RRs) and 95% confidence intervals (CIs) using random-effects models.Results: Out of 1407 papers, eight controlled clinical trials were included. In patients with APAT, the remission rates were higher in patients on glucocorticoids (RR 8.33 [95% CI 3.07-22.6]) or splenectomy (RR 8.37 [95% CI 1.61-43.7]) than in patients without those treatments. There was no significant association between glucocorticoids and thrombosis (RR 1.57 [95% CI, 0.17-14.9]) or between splenectomy and hemorrhage (RR 0.17 [95% CI 0.02-1.28]). The extracted data of mortality and relapse rate were not available for synthesis.Conclusion: Glucocorticoids or splenectomy seemed suitable therapeutic approaches for APAT.

[A role of platelets beyond hemostasis].

It is well known that platelets play a crucial role in hemostasis, but it has recently been revealed that platelets are also necessary for organ development. The platelet activation receptor CLEC-2 activates platelets by binding to the membrane protein, podoplanin, on the surface of lymphatic endothelial cells. This results in the release of TGF-ß family from activated platelets to facilitate blood/lymphatic vessel separation. TGF-ß also acts on lung mesothelial cells, which leads to their differentiation into alveolar duct myofibroblasts (adMYFs) and their migration into the inside of the lung. adMYFs generate elastin, which gives elasticity to the lung. Therefore, mice deficient in either CLEC-2 or podoplanin exhibit blood/lymphatic vessel misconnection and die just after birth due to respiratory failure. It had been previously surmised that biologically active substances from cells act on neighboring cells, leading to organ development, and the role of blood cells in organ development had not been elucidated. However, it has recently been demonstrated that blood platelets contain biological active substances in their granules, which are released when and where necessary by specific interactions between platelet receptors and their ligands. Now platelets are recognized as a "biological package" that actively facilitates organ development.

[Peri-arteriolar megakaryopoietic microenvironment via reciprocal CLEC-2/PDPN axis in bone marrow].

Bone marrow (BM), the tissue specializing in the production of hematopoietic cells, consists of multiple components (e.g., extracellular matrixes, vasculatures, and stromal cells) that generate a complex three-dimensional network and several localized microenvironment. These microenvironments regulate hematopoietic stem and progenitor cells, including megakaryocyte lineage cells. In this review, we first provide an overview of the microenvironment for hematopoietic stem cells as an introduction to bone marrow microenvironment and subsequently summarize the microenvironment for megakaryocyte differentiation and maturation (megakaryopoiesis). In the last portion, we describe megakaryocyte regulation by podoplanin-positive peri-arteriolar stromal cells in the mouse bone marrow.

Platelet CLEC-2: Roles Beyond Hemostasis.

C-type lectin-like receptor 2 (CLEC-2) has been identified on the surface of platelets as a receptor for a platelet activating snake venom, rhodocytin/aggretin. CLEC-2 belongs to a C-type lectin superfamily and binds to a sialoglycoprotein, podoplanin, in vivo. Platelets play a crucial role in hemostasis and thrombosis, but recent studies have uncovered multiple roles of platelets beyond hemostasis in physiology and pathology. The interaction between platelet CLEC-2 and podoplanin is the key to several roles of platelets beyond hemostasis. The spatial and temporal expression patterns of podoplanin regulate vascular/lymphatic development, maintenance of vascular integrity, tissue regeneration, and some pathological processes including tumor metastasis and thromboinflammation. CLEC-2 facilitates blood/lymphatic vessel separation during embryonic development by binding to podoplanin on lymphatic endothelial cells. The leakage of platelets from hyperpermeable vessels for maintaining vascular integrity during inflammation depends on CLEC-2. During wound healing, the expression of podoplanin in keratinocytes is upregulated, which helps in the process. Podoplanin is expressed on the surface of tumor cells and facilitates hematogenous metastasis by inducing platelet aggregation through CLEC-2. During thrombotic processes, such as development of deep vein thrombosis, podoplanin is upregulated on unknown cells in the vessel wall in the area of inflammation, facilitates thrombus formation, and promotes further inflammation by binding to CLEC-2. In this article, the roles of platelets beyond hemostasis are comprehensively reviewed.

Podoplanin-positive periarteriolar stromal cells promote megakaryocyte growth and proplatelet formation in mice by CLEC-2.

Megakaryopoiesis is the hierarchical differentiation of hematopoietic stem cells into megakaryocytes. Differentiating megakaryocytes undergo maturation characterized by endomitosis and produce numerous platelets through proplatelet formation. C-type lectin-like receptor 2 (CLEC-2) is a podoplanin (PDPN) receptor mainly expressed on platelets and megakaryocytes. Deletion of platelet/megakaryocyte CLEC-2 causes thrombocytopenia in mice; however, its contribution to megakaryopoiesis remains unknown. Here, we show that megakaryopoiesis is promoted through the CLEC-2/PDPN interaction in the vicinity of arterioles in the bone marrow (BM). We have also identified PDPN-expressing BM arteriolar stromal cells, tentatively termed as BM fibroblastic reticular cell (FRC)-like cells. Platelet/megakaryocyte-specific CLEC-2 conditional knockout (cKO) mice showed a decrease in the number of immature megakaryocytes. CLEC-2 wild-type megakaryocyte expansion was augmented in vitro by the addition of recombinant PDPN, but not cKO megakaryocytes. Moreover, megakaryocyte colonies were colocalized with periarteriolar BM FRC-like cells in the BM. Coculture of megakaryocytes with BM FRC-like cells augmented megakaryocyte expansion, which was dependent upon the CLEC-2/PDPN interaction. Furthermore, we found that the CLEC-2/PDPN interaction induces BM FRC-like cells to secrete chemokine (C-C motif) ligand 5 (CCL5) to facilitate proplatelet formation. These observations indicate that a reciprocal interaction between CLEC-2 on megakaryocytes and PDPN on BM FRC-like cells contributes to the periarteriolar megakaryopoietic microenvironment in mouse BM.

Tyro3, Axl, and Mertk receptors differentially participate in platelet activation and thrombus formation.

BACKGROUND: Previously, several studies have shown that Tyro3, Axl, and Mertk (TAM) receptors participate in platelet activation and thrombosis. However, the role of individual receptors is not fully understood. METHODS: Using single receptor-deficient platelets from TAM knockout mice in the C57BL/6 J strain, we performed a knockout study using single TAM-deficient mice. We treated platelets isolated from TAM knockout mice with the Glycoprotein VI (GPVI) agonists convulxin, poly(PHG), and collagen-related triple-helical peptide (CRP), as well as thrombin for in-vitro experiments. We used a laser-induced cremaster arterial injury model for thrombosis experiments in vivo. RESULTS: Deficiency of the tyrosine kinase receptors, Axl or Tyro3, but not Mertk, inhibited aggregation, spreading, JON/A binding, and P-selectin expression of platelets in vitro. In vivo, platelet thrombus formation was significantly decreased in Axl-/- and Tyro3-/- mice, but not in Mertk-/- mice. Upon stimulation with glycoprotein VI (GPVI) agonists, tyrosine phosphorylation of signaling molecules, including spleen tyrosine kinase (Syk) and phospholipase C-γ2 (PLCγ2), was decreased in Axl-/- and Tyro3-/- platelets, but not in Mertk-/- platelets. While platelet aggregation induced by agonists did not differ in the presence or absence of the Gas6 neutralizing antibody, the platelet aggregation was inhibited by anti-Axl or anti-Tyro3 neutralizing antibodies antibody, but not the anti-Mertk antibody. Additionally, the recombinant extracellular domain of Axl or Tyro3, but not that of Mertk, also inhibited platelet aggregation. CONCLUSIONS: These data suggest that Axl and Tyro3, but not Mertk, have an important role in platelet activation and thrombus formation, and mechanistically may do so by a pathway that regulates inside to outside signaling and heterotypic interactions via the extracellular domains of TAMs.

Roles of the CLEC-2-podoplanin interaction in tumor progression.

Podoplanin is a type-I transmembrane sialomucin-like glycoprotein expressed on the surface of several kinds of tumor cells. The podoplanin receptor is a platelet activation receptor known as C-type lectin-like receptor 2 (CLEC-2), which has been identified as a receptor for the platelet-activating snake venom protein rhodocytin. CLEC-2 is highly expressed in platelets and megakaryocytes and expressed at lower levels in liver Kupffer cells. Podoplanin is expressed in certain types of tumor cells, including squamous cell carcinomas, seminomas, and brain tumors. Podoplanin is also expressed in a wide range of normal cells, including fibroblastic reticular cells in lymph nodes, kidney podocytes, and lymphatic endothelial cells, but not vascular endothelial cells. Metastasis of podoplanin-positive lung tumors injected from the tail vein is greatly inhibited in CLEC-2-depleted mice or in anti-podoplanin antibody-treated mice. These findings suggest that the CLEC-2-podoplanin interaction facilitates hematogenous tumor metastasis. Platelets may increase the survival of tumor cells by covering tumor cells and physically protecting them from shear stress or immune cells in the bloodstream. Alternatively, platelets may stimulate the epithelial-mesenchymal transition of tumor cells to facilitate their extravasation from blood vessels. Cell proliferation is stimulated in podoplanin-expressing tumor cells by the coculture with platelets, but the effects of the CLEC-2-podoplanin interaction on tumor growth in vivo are not yet resolved. It is possible that the CLEC-2-podoplanin interaction facilitates tumor-related thrombosis, subsequent inflammation, inflammation-induced cachexia, and reduced survival. Considering these findings, anti-podoplanin and anti-CLEC-2 drugs are promising therapies for the prevention of tumor metastasis, progression, and tumor-related symptoms, which may result in longer survival in cancer patients. There are advantages and disadvantages of anti-podoplanin vs. anti-CLEC-2 therapy. Side effects in podoplanin-expressing normal tissues due to treatment with anti-podoplanin and temporal thrombocytopenia due to treatment with anti-CLEC2 are potential problems, although solutions to these problems have been reported.