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Abstract Background Podoplanin (PDPN) expressed on tumour cells interacts with platelet C-type lectin-like receptor 2 (CLEC-2). This study aimed to investigate the role of the PDPN-platelet CLEC-2 interaction in melanoma pulmonary metastasis. Methods Murine melanoma B16-F0 cells, which have two populations that express podoplanin, were sorted by FACS with anti-podoplanin staining to obtain purified PDPN + and PDPN- B16-F0 cells. C57BL/6J mice transplanted with CLEC-2-deficient bone marrow cells were used for in vivo experiments. Results The in vivo data showed that the number of metastatic lung nodules in WT mice injected with PDPN + cells was significantly higher than that in WT mice injected with PDPN- cells and in WT or CLEC-2 KO mice injected with PDPN- cells. In addition, our results revealed that the platelet Syk-dependent signalling pathway contributed to platelet aggregation and melanoma metastasis. Conclusions Our study indicates that the PDPN-CLEC-2 interaction promotes experimental pulmonary metastasis in a mouse melanoma model. Tumour cell-induced platelet aggregation mediated by the interaction between PDPN and CLEC-2 is a key factor in melanoma pulmonary metastasis.

Background: Podoplanin (PDPN) expressed on tumour cells interacts with platelet C-type lectin-like receptor 2 (CLEC-2). This study aimed to investigate the role of the PDPN-platelet CLEC-2 interaction in melanoma pulmonary metastasis. Methods: Murine melanoma B16-F0 cells, which have two populations that express podoplanin, were sorted by FACS with anti-podoplanin staining to obtain purified PDPN + and PDPN- B16-F0 cells. C57BL/6J mice transplanted with CLEC-2-deficient bone marrow cells were used for in vivo experiments. Results: The in vivo data showed that the number of metastatic lung nodules in WT mice injected with PDPN + cells was significantly higher than that in WT mice injected with PDPN- cells and in WT or CLEC-2 KO mice injected with PDPN- cells. In addition, our results revealed that the platelet Syk-dependent signalling pathway contributed to platelet aggregation and melanoma metastasis. Conclusions: Our study indicates that the PDPN-CLEC-2 interaction promotes experimental pulmonary metastasis in a mouse melanoma model. Tumour cell-induced platelet aggregation mediated by the interaction between PDPN and CLEC-2 is a key factor in melanoma pulmonary metastasis. [ABSTRACT FROM AUTHOR]

Cancer cells circulating in the blood attach to platelets by direct cell–cell interactions via several receptor‐counterreceptor contacts and indirectly by fibrin bridges which connect the two cell types by distinct integrin receptors. In the microenvironment of these tumor cell platelet aggregates (TCPAs), the tumor cells are shielded from the shear stress of the blood flow and from attack by the immune system. This supports hematogenous metastasis and tumor cell induced thrombosis. Platelet activation is triggered by binding of podoplanin on cancer cells to the platelet receptor Clec‐2. Therefore, we hypothesize that targeting this initial step will prevent the entire cascade leading to the formation of TCPAs. Rhodocytin, a heterodimeric (αβ)2 C‐type lectin from the Malayan pit viper Calloselasma rhodostoma, binds to Clec‐2 and thereby induces TCPA formation. Remarkably, mutations in rhodocytin that disturbed formation of oligomers, blocked the podoplanin‐Clec‐2 axis and prevented platelet activation. Therefore, we used lysine reactive chemicals to modify rhodocytin isolated from the crude snake venom. Blue native gel electrophoresis and far western blotting showed a change of rhodocytin's suprastructure triggered by acetylation and PEGylation. Mass spectrometry analysis of altered lysines suggested that their modifications interfered with the formation of rhodocytin tetramers. When tested in assays for tumor cell induced platelet aggregation, we found that derivatization turned rhodocytin from an agonist into an antagonist. This observation indicates that Clec‐2 is a valid target receptor molecule to curb TCPA formation and to prevent hematogenous metastasis and tumor cell induced thrombosis in cancer patients. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Src tyrosine kinases and spleen tyrosine kinase (Syk) have recently been shown to contribute to sustained platelet aggregation on collagen under arterial shear. In the present study, we have investigated whether Src and Syk are required for aggregation under minimal shear following activation of glycoprotein VI (GPVI) and have extended this to C-type lectin-like receptor-2 (CLEC-2) which signals through the same pathway. Aggregation was induced by the GPVI ligand collagen-related peptide (CRP) and the CLEC-2 ligand rhodocytin and monitored by light transmission aggregometry (LTA). Aggregation and tyrosine phosphorylation by both receptors were sustained for up to 50 min. The addition of inhibitors of Src, Syk or Bruton’s tyrosine kinase (Btk) at 150 sec, by which time aggregation was maximal, induced rapid loss of tyrosine phosphorylation of their downstream proteins, but only Src kinase inhibition caused a weak (~10%) reversal in light transmission. A similar effect was observed when the inhibitors were combined with apyrase and indomethacin or glycoprotein IIb-IIIa (GPIIb-IIIa) antagonist, eptifibatide. On the other hand, activation of GPIIb-IIIa by GPVI in a diluted platelet suspension, as measured by binding of fluorescein isothiocyanate-labeled antibody specific for the activated GPIIb-IIIa (FITC-PAC1), was reversed on the addition of Src and Syk inhibitors showing that integrin activation is rapidly reversible in the absence of outside-in signals. The results demonstrate that Src but not Syk and Btk contribute to sustained aggregation as monitored by LTA, possibly as a result of inhibition of outside-in signaling from GPIIb-IIIa to the cytoskeleton through a Syk-independent pathway. This is in contrast to the role of Syk in supporting sustained aggregation on collagen under arterial shear.

ABSTRACT To initiate an antileishmanial adaptive immune response, dendritic cells (DCs) must carry Leishmania antigens from peripheral tissues to local draining lymph nodes. However, the migratory capacity of DCs is largely compromised during Leishmania donovani infection. The molecular mechanism underlying this defective DC migration is not yet fully understood. Here, we demonstrate that L. donovani infection impaired the lymph node homing ability of DCs by decreasing C-type lectin receptor 2 (CLEC-2) expression. L. donovani exerted this inhibitory effect by inducing transforming growth factor-β (TGF-β) secretion from DCs. Indeed, TGF-β produced in this manner inhibited nuclear factor-κB (NF-κB)-mediated CLEC-2 expression on DCs by activating c-Src. Notably, suppression of c-Src expression significantly improved the arrival of DCs in draining lymph nodes by preventing L. donovani-induced CLEC-2 downregulation on DCs. These findings reveal a unique mechanism by which L. donovani inhibits DC migration to lymph nodes and suggest a key role for TGF-β, c-Src, and CLEC-2 in regulating this process. IMPORTANCE Dendritic cells (DCs) play a key role in initiating T cell-mediated protective immunity against visceral leishmaniasis (VL), the second most lethal parasitic disease in the world. However, the T cell-inducing ability of DCs critically depends on the extent of DC migration to regional lymph nodes. Notably, the migration of DCs is reported to be impaired during VL. The cause of this impaired DC migration, however, remains ill-defined. Here, we provide the first evidence that L. donovani, the causative agent of VL, attenuates the lymph node homing capacity of DCs by decreasing C-type lectin receptor 2 (CLEC-2) expression on DCs. Additionally, we have demonstrated how L. donovani mediates this inhibitory effect. Overall, our work has revealed a unique mechanism underlying L. donovani-induced impairment of DC migration and suggests a potential strategy to improve antileishmanial T cell activity by increasing DC arrival in lymph nodes.

Upon initial immune challenge, dendritic cells (DCs) migrate to lymph nodes and interact with fibroblastic reticular cells (FRCs) via C-type lectin-like receptor 2 (CLEC-2). CLEC-2 binds to the membrane glycoprotein podoplanin (PDPN) on FRCs, inhibiting actomyosin contractility through the FRC network and permitting lymph node expansion. The hyaluronic acid receptor CD44 is known to be required for FRCs to respond to DCs but the mechanism of action is not fully elucidated. Here, we use DNA-PAINT, a quantitative single molecule super-resolution technique, to visualize and quantify how PDPN clustering is regulated in the plasma membrane of FRCs. Our results indicate that CLEC-2 interaction leads to the formation of large PDPN clusters (i.e. more than 12 proteins per cluster) in a CD44-dependent manner. These results suggest that CD44 expression is required to stabilize large pools of PDPN at the membrane of FRCs upon CLEC-2 interaction, revealing the molecular mechanism through which CD44 facilitates cellular crosstalk between FRCs and DCs.

As a specific lymphatic marker and a key ligand of C-type lectin-like receptor 2 (CLEC-2), podoplanin (Pdpn) is involved in various physiological and pathological processes such as growth and development, respiration, blood coagulation, lymphangiogenesis, angiogenesis, and inflammation. Thrombotic diseases constitute a major cause of disability and mortality in adults, in which thrombosis and inflammation play a crucial role. Recently, increasing evidence demonstrates the distribution and function of this glycoprotein in thrombotic diseases such as atherosclerosis, ischemic stroke, venous thrombosis, ischemic-reperfusion injury (IRI) of kidney and liver, and myocardial infarction. Evidence showed that after ischemia, Pdpn can be acquired over time by a heterogeneous cell population, whichmay not express Pdpn in normal conditions. In this review, the research progresses in understanding the roles andmechanisms of podoplanin in thromobotic diseases are summarized. The challenges of podoplanin-targeted approaches for disease prognosis and preventions are also discussed. [ABSTRACT FROM AUTHOR]

Podoplanin (PDPN) is known to induce platelet aggregation via interacting with the C-type lectin-like receptor-2 on platelets and is involved in postoperative venous thromboembolism (VTE) formation. In this study, we investigate the correlation between soluble C-type lectin-like receptor (sCLEC-2) levels and PDPN expression in patients with high grade gliomas and the relationship between sCLEC-2 levels and the occurrence of VTE. Forty-four patients harboring high grade gliomas, treated surgically at the Department of Neurosurgery, Niigata University from April 2018 to August 2020, were included. Patients with high grade gliomas were divided into isocitrate dehydrogenase (IDH)- wildtype and mutant groups, and the presence or absence of VTE and the intensity of PDPN by immunohistochemistry were confirmed. Platelet counts, as well as plasma sCLEC-2 and PDPN were measured in these patients. Furthermore, the levels of sCLEC-2 concentration were divided by the platelet count (C2PAC index) for comparison. IDH-wildtype glioma patients highly expressed PDPN (P < 0.001) compared to IDH-mutant glioma patients. In total, 9 (20.5 %) patients were diagnosed with VTE during the follow-up period, of which 8 patients harbored IDH-wildtype gliomas, and one patient an IDH-mutant glioma. Mean sCLEC-2 levels and C2PAC index in patients with IDH-wildtype gliomas were significantly higher than that of low or no PDPN expression group, which included patients with IDH-mutant gliomas (P = 0.0004, P = 0.0002). In patients with IDH-wildtype gliomas, the C2PAC index in patients with VTE was significantly higher than in patients without VTE (P = 0.0492). The optimal cutoff point of C2PAC for predicting VTE in IDH-wildtype glioma patients was 3.7 with a sensitivity of 87.5 % and specificity of 51.9 %. Platelet activation is strongly involved in the development of VTE in patients with IDH-wildtype high grade gliomas, and C2PAC index is a potential marker to detect VTE formation after surgery. [Display omitted] • Soluble C-type lectin-like receptor (sCLEC-2) levels were correlate with podoplanin (PDPN) expression in patients with high grade gliomas. • IDH-wildtype glioma patients highly expressed PDPN compared to IDH-mutant glioma patients. • Platelet activation is strongly involved in the development of venous thromboembolism (VTE) in patients with IDH-wildtype high grade gliomas. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Src tyrosine kinases and spleen tyrosine kinase (Syk) have recently been shown to contribute to sustained platelet aggregation on collagen under arterial shear. In the present study, we have investigated whether Src and Syk are required for aggregation under minimal shear following activation of glycoprotein VI (GPVI) and have extended this to C-type lectin-like receptor-2 (CLEC-2) which signals through the same pathway. Aggregation was induced by the GPVI ligand collagen-related peptide (CRP) and the CLEC-2 ligand rhodocytin and monitored by light transmission aggregometry (LTA). Aggregation and tyrosine phosphorylation by both receptors were sustained for up to 50 min. The addition of inhibitors of Src, Syk or Bruton's tyrosine kinase (Btk) at 150 sec, by which time aggregation was maximal, induced rapid loss of tyrosine phosphorylation of their downstream proteins, but only Src kinase inhibition caused a weak (~10%) reversal in light transmission. A similar effect was observed when the inhibitors were combined with apyrase and indomethacin or glycoprotein IIb-IIIa (GPIIb-IIIa) antagonist, eptifibatide. On the other hand, activation of GPIIb-IIIa by GPVI in a diluted platelet suspension, as measured by binding of fluorescein isothiocyanate-labeled antibody specific for the activated GPIIb-IIIa (FITC-PAC1), was reversed on the addition of Src and Syk inhibitors showing that integrin activation is rapidly reversible in the absence of outside-in signals. The results demonstrate that Src but not Syk and Btk contribute to sustained aggregation as monitored by LTA, possibly as a result of inhibition of outside-in signaling from GPIIb-IIIa to the cytoskeleton through a Syk-independent pathway. This is in contrast to the role of Syk in supporting sustained aggregation on collagen under arterial shear. [ABSTRACT FROM AUTHOR]

Abstract All irreversible Bruton tyrosine kinase (Btk) inhibitors including ibrutinib and acalabrutinib induce platelet dysfunction and increased bleeding risk. New reversible Btk inhibitors were developed, like MK‐1026. The mechanism underlying increased bleeding tendency with Btk inhibitors remains unclear. We investigated the effects of ibrutinib, acalabrutinib and MK‐1026 on platelet function in healthy volunteers, patients and Btk‐deficient mice, together with off‐target effects on tyrosine kinase phosphorylation. All inhibitors suppressed GPVI‐ and CLEC‐2‐mediated platelet aggregation, activation and secretion in a dose‐dependent manner. Only ibrutinib inhibited thrombus formation on vWF‐co‐coated surfaces, while on collagen this was not affected. In blood from Btk‐deficient mice, collagen‐induced thrombus formation under flow was reduced, but preincubation with either inhibitor was without additional effects. MK‐1026 showed less off‐target effects upon GPVI‐induced TK phosphorylation as compared to ibrutinib and acalabrutinib. In ibrutinib‐treated patients, GPVI‐stimulated platelet activation, and adhesion on vWF‐co‐coated surfaces were inhibited, while CLEC‐2 stimulation induced variable responses. The dual inhibition of GPVI and CLEC‐2 signalling by Btk inhibitors might account for the increased bleeding tendency, with ibrutinib causing more high‐grade bleedings due to additional inhibition of platelet‐vWF interaction. As MK‐1026 showed less off‐target effects and only affected activation of isolated platelets, it might be promising for future treatment.

Charge interactions play a critical role in the activation of the innate immune system by damage- and pathogen-associated molecular pattern receptors. The ability of these receptors to recognize a wide spectrum of ligands through a common mechanism is critical in host defense. In this article, we argue that platelet glycoprotein receptors that signal through conserved tyrosine-based motifs function as pattern recognition receptors (PRRs) for charged endogenous and exogenous ligands, including sulfated polysaccharides, charged proteins and nanoparticles. This is exemplified by GPVI, CLEC-2 and PEAR1 which are activated by a wide spectrum of endogenous and exogenous ligands, including diesel exhaust particles, sulfated polysaccharides and charged surfaces. We propose that this mechanism has evolved to drive rapid activation of platelets at sites of injury, but that under some conditions it can drive occlusive thrombosis, for example, when blood comes into contact with infectious agents or toxins. In this Opinion Article, we discuss mechanisms behind charge-mediated platelet activation and opportunities for designing nanoparticles and related agents such as dendrimers as novel antithrombotics.

GPVI and CLEC-2 have emerged as promising targets for long-term prevention of both arterial thrombosis and thrombo-inflammation with a decreased bleeding risk relative to current drugs. However, while there are potent blocking antibodies of both receptors, their protein nature comes with decreased bioavailability, making formulation for oral medication challenging. Small molecules are able to overcome these limitations, but there are many challenges in developing antagonists of nanomolar potency, which is necessary when considering the structural features that underlie the interaction of CLEC-2 and GPVI with their protein ligands. In this review, we describe current small-molecule inhibitors for both receptors and strategies to overcome such limitations, including considerations when it comes to in silico drug design and the importance of complex compound library selection.

The platelet C-type lectin-like receptor CLEC-2 drives inflammation-driven venous thrombosis in mouse models of thrombo-inflammatory disease with a minimal effect on hemostasis identifying it as a target for a new class of antiplatelet agent. Here, we discuss how the protein structure and dynamic arrangement of CLEC-2 on the platelet membrane helps the receptor, which has a single YxxL motif (known as a hemITAM), to trigger intracellular signaling. CLEC-2 exists as a monomer and homo-dimer within resting platelets and forms higher-order oligomers following ligand activation, a process that is mediated by the multivalent nature of its ligands and the binding of the tandem SH2 domains of Syk to the phosphorylated hemITAM and concomitantly to PIP2 or PIP3 to localize it to the membrane. We propose that a low level of active Syk is present at the membrane in resting platelets due to phosphorylation by Src family kinases and that clustering of receptors disturbs the equilibrium between kinases and phosphatases, triggering phosphorylation of the CLEC-2 hemITAM and recruitment of Syk. Knowledge of the structure of CLEC-2 and the mechanism of platelet activation has important implications for development of therapeutics.

Podoplanin is a small mucin-like glycoprotein involved in several physiological and pathological processes in the brain including development, angiogenesis, tumors, ischemic stroke and other neurological disorders. Podoplanin expression is upregulated in different cell types including choroid plexus epithelial cells, glial cells, as well as periphery infiltrated immune cells during brain development and neurological disorders. As a transmembrane protein, podoplanin interacts with other molecules in the same or neighboring cells. In the past, a lot of studies reported a pleiotropic role of podoplanin in the modulation of thrombosis, inflammation, lymphangiogenesis, angiogenesis, immune surveillance, epithelial mesenchymal transition, as well as extracellular matrix remodeling in periphery, which have been well summarized and discussed. Recently, mounting evidence demonstrates the distribution and function of this molecule in brain development and neurological disorders. In this review, we summarize the research progresses in understanding the roles and mechanisms of podoplanin in the development and disorders of the nervous system. The challenges of podoplanin-targeted approaches for disease prognosis and preventions are also discussed. [ABSTRACT FROM AUTHOR]

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.

Antiplatelet medications comprise the cornerstone of treatment for diseases that involve arterial thrombosis, including acute coronary syndromes (ACS), stroke and peripheral arterial disease. However, antiplatelet medications may cause bleeding and, furthermore, thrombotic events may still recur despite treatment. The interaction of collagen with GPVI receptors on the surface of platelets has been identified as one of the major players in the pathophysiology of arterial thrombosis that occurs following atherosclerotic plaque rupture. Promisingly, GPVI deficiency in humans appears to have a minimal impact on bleeding. These findings together suggest that targeting platelet GPVI may provide a novel treatment strategy that provides additional antithrombotic efficacy with minimal disruption of normal hemostasis compared to conventional antiplatelet medications. CLEC-2 is gaining interest as a therapeutic target for a variety of thrombo-inflammatory disorders including deep vein thrombosis (DVT) with treatment also predicted to cause minimal disruption to hemostasis. GPVI and CLEC-2 signal through Src, Syk and Tec family tyrosine kinases, providing additional strategies for inhibiting both receptors. In this review, we summarize the evidence regarding GPVI and CLEC-2 and strategies for inhibiting these receptors to inhibit platelet recruitment and activation in thrombotic diseases.

Abstract In this article, the State of the Art lecture “Platelet CLEC‐2 and Lung Development” presented at the ISTH congress 2019 is reviewed. During embryonic development, blood cells are often considered as porters of nutrition and oxygen but not as active influencers of cell differentiation. However, recent studies revealed that platelets actively facilitate cell differentiation by releasing biological substances during development. C‐type lectin‐like receptor 2 (CLEC‐2) has been identified as a receptor for the platelet‐activating snake venom rhodocytin. An endogenous ligand of CLEC‐2 is the membrane protein podoplanin (PDPN), which is expressed on the surface of certain types of tumor cells and lymphatic endothelial cells (LECs). Deletion of CLEC‐2 from platelets in mice results in death just after birth due to lung malformation and blood/lymphatic vessel separation. During development, lymphatic vessels are derived from cardinal veins. At this stage, platelets are activated by binding of CLEC‐2 to LEC PDPN and release trandforming growth factor‐β (TGF‐β). This cytokine inhibits LEC migration and proliferation, facilitating blood/lymphatic vessel separation. TGF‐β released upon platelet‐expressed CLEC‐2/LEC PDPN also facilitates differentiation of lung mesothelial cells into alveolar duct myofibroblasts (adMYFs) in the developing lung. AdMYFs generate elastic fibers inside the lung, so that the lung can be properly inflated. Thus, platelets act as an ultimate natural drug delivery system that enables biological substances to be specifically delivered to the target at high concentrations by receptor/ligand interactions during development.

C-type lectin-like receptor 2 (CLEC-2) expressed on megakaryocytes plays important roles in megakaryopoiesis. We found that CLEC-2 was expressed in about 20% of phenotypical long-term hematopoietic stem cells (LT-HSCs), which expressed lower levels of HSC-specific genes and produced larger amounts of megakaryocyte-related molecules than CLEC-2low LT-HSCs. Although CLEC-2high LT-HSCs had immature clonogenic activity, cultured CLEC-2high LT-HSCs preferentially differentiated into megakaryocytes. CLEC-2high HSCs yielded 6.8 times more megakaryocyte progenitors (MkPs) and 6.0 times more platelets 2 weeks and 1 week after transplantation compared with CLEC-2low LT-HSCs. However, platelet yield from CLEC-2high HSCs gradually declined with the loss of MkPs, while CLEC-2low HSCs self-renewed long-term, indicating that CLEC-2high LT-HSCs mainly contribute to early megakaryopoiesis. Treatment with pI:C and LPS increased the proportion of CLEC-2high LT-HSCs within LT-HSCs. Almost all CLEC-2low LT-HSCs were in the G0 phase and barely responded to pI:C. In contrast, 54% of CLEC-2high LT-HSCs were in G0, and pI:C treatment obliged CLEC-2high LT-HSCs to enter the cell cycle and differentiate into megakaryocytes, indicating that CLEC-2high LT-HSCs are primed for cell cycle entry and rapidly yield platelets in response to inflammatory stress. In conclusion, CLEC-2high LT-HSCs appear to act as a reserve for emergent platelet production under stress conditions. [ABSTRACT FROM AUTHOR]

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.

Losartan and honokiol are small molecules which have been described to inhibit aggregation of platelets by collagen. Losartan has been proposed to block clustering of GPVI but not to affect binding of collagen. Honokiol has been reported to bind directly to GPVI but only at a concentration that is three orders of magnitude higher than that needed for inhibition of aggregation. The mechanism of action of both inhibitors is so far unclear. In the present study, we confirm the inhibitory effects of both agents on platelet aggregation by collagen and show that both also block the aggregation induced by the activation of CLEC-2 or the low affinity immune receptor FcγRIIa at similar concentrations. For GPVI and CLEC-2, this inhibition is associated with a reduction in protein tyrosine phosphorylation of multiple proteins including Syk. In contrast, on a collagen surface, spreading of platelets and clustering of GPVI (measured by single molecule localisation microscopy) was not altered by losartan or honokiol. Furthermore, in flow whole-blood, both inhibitors suppressed the formation of multi-layered platelet thrombi at arteriolar shear rates at concentrations that hardly affect collagen-induced platelet aggregation in platelet rich plasma. Together, these results demonstrate that losartan and honokiol have multiple effects on platelets which should be considered in the use of these compounds as anti-platelet agents.

The platelet C-type lectin-like receptor CLEC-2 drives inflammation-driven venous thrombosis in mouse models of thrombo-inflammatory disease with a minimal effect on hemostasis identifying it as a target for a new class of antiplatelet agent. Here, we discuss how the protein structure and dynamic arrangement of CLEC-2 on the platelet membrane helps the receptor, which has a single YxxL motif (known as a hemITAM), to trigger intracellular signaling. CLEC-2 exists as a monomer and homo-dimer within resting platelets and forms higher-order oligomers following ligand activation, a process that is mediated by the multivalent nature of its ligands and the binding of the tandem SH2 domains of Syk to the phosphorylated hemITAM and concomitantly to PIP2 or PIP3 to localize it to the membrane. We propose that a low level of active Syk is present at the membrane in resting platelets due to phosphorylation by Src family kinases and that clustering of receptors disturbs the equilibrium between kinases and phosphatases, triggering phosphorylation of the CLEC-2 hemITAM and recruitment of Syk. Knowledge of the structure of CLEC-2 and the mechanism of platelet activation has important implications for development of therapeutics. [ABSTRACT FROM AUTHOR]

GPVI and CLEC-2 have emerged as promising targets for long-term prevention of both arterial thrombosis and thrombo-inflammation with a decreased bleeding risk relative to current drugs. However, while there are potent blocking antibodies of both receptors, their protein nature comes with decreased bioavailability, making formulation for oral medication challenging. Small molecules are able to overcome these limitations, but there are many challenges in developing antagonists of nanomolar potency, which is necessary when considering the structural features that underlie the interaction of CLEC-2 and GPVI with their protein ligands. In this review, we describe current small-molecule inhibitors for both receptors and strategies to overcome such limitations, including considerations when it comes to in silico drug design and the importance of complex compound library selection. [ABSTRACT FROM AUTHOR]

Platelets play a key role in the development, progression and resolution of the inflammatory response during sterile inflammation and infection, although the mechanism is not well understood. Here we show that platelet CLEC-2 reduces tissue inflammation by regulating inflammatory macrophage activation and trafficking from the inflamed tissues. The immune regulatory function of CLEC-2 depends on the expression of its ligand, podoplanin, upregulated on inflammatory macrophages and is independent of platelet activation and secretion. Mechanistically, platelet CLEC-2 and also recombinant CLEC-2-Fc accelerates actin rearrangement and macrophage migration by increasing the expression of podoplanin and CD44, and their interaction with the ERM proteins. During ongoing inflammation, induced by lipopolysaccharide, treatment with rCLEC-2-Fc induces the rapid emigration of peritoneal inflammatory macrophages to mesenteric lymph nodes, thus reducing the accumulation of inflammatory macrophages in the inflamed peritoneum. This is associated with a significant decrease in pro-inflammatory cytokine, TNF-α and an increase in levels of immunosuppressive, IL-10 in the peritoneum. Increased podoplanin expression and actin remodelling favour macrophage migration towards CCL21, a soluble ligand for podoplanin and chemoattractant secreted by lymph node lymphatic endothelial cells. Macrophage efflux to draining lymph nodes induces T cell priming. In conclusion, we show that platelet CLEC-2 reduces the inflammatory phenotype of macrophages and their accumulation, leading to diminished tissue inflammation. These immunomodulatory functions of CLEC-2 are a novel strategy to reduce tissue inflammation and could be therapeutically exploited through rCLEC-2-Fc, to limit the progression to chronic inflammation.

C-type lectin-like receptor 2 (CLEC-2, also known as CLEC-1b) is expressed on platelets, Kupffer cells and other immune cells, and binds to various ligands including the mucin-like protein podoplanin (PDPN). The role of CLEC-2 in infection and immunity has become increasingly evident in recent years. CLEC-2 is involved in platelet activation, tumor cell metastasis, separation of blood/lymphatic vessels, and cerebrovascular patterning during embryonic development. In this review, we have discussed the role of CLEC-2 in thromboinflammation, and focused on the recent research.

C-type lectin-like receptor 2 (CLEC-2, also known as CLEC-1b) is expressed on platelets, Kupffer cells and other immune cells, and binds to various ligands including the mucin-like protein podoplanin (PDPN). The role of CLEC-2 in infection and immunity has become increasingly evident in recent years. CLEC-2 is involved in platelet activation, tumor cell metastasis, separation of blood/lymphatic vessels, and cerebrovascular patterning during embryonic development. In this review, we have discussed the role of CLEC-2 in thromboinflammation, and focused on the recent research. [ABSTRACT FROM AUTHOR]

Platelets play a key role in the development, progression and resolution of the inflammatory response during sterile inflammation and infection, although the mechanism is not well understood. Here we show that platelet CLEC-2 reduces tissue inflammation by regulating inflammatory macrophage activation and trafficking from the inflamed tissues. The immune regulatory function of CLEC-2 depends on the expression of its ligand, podoplanin, upregulated on inflammatory macrophages and is independent of platelet activation and secretion. Mechanistically, platelet CLEC-2 and also recombinant CLEC-2-Fc accelerates actin rearrangement and macrophage migration by increasing the expression of podoplanin and CD44, and their interaction with the ERM proteins. During ongoing inflammation, induced by lipopolysaccharide, treatment with rCLEC-2-Fc induces the rapid emigration of peritoneal inflammatory macrophages to mesenteric lymph nodes, thus reducing the accumulation of inflammatory macrophages in the inflamed peritoneum. This is associated with a significant decrease in pro-inflammatory cytokine, TNF-α and an increase in levels of immunosuppressive, IL-10 in the peritoneum. Increased podoplanin expression and actin remodelling favour macrophage migration towards CCL21, a soluble ligand for podoplanin and chemoattractant secreted by lymph node lymphatic endothelial cells. Macrophage efflux to draining lymph nodes induces T cell priming. In conclusion, we show that platelet CLEC-2 reduces the inflammatory phenotype of macrophages and their accumulation, leading to diminished tissue inflammation. These immunomodulatory functions of CLEC-2 are a novel strategy to reduce tissue inflammation and could be therapeutically exploited through rCLEC-2-Fc, to limit the progression to chronic inflammation. [ABSTRACT FROM AUTHOR]

Podoplanin is a key molecule for enhancing tumor‐induced platelet aggregation. Podoplanin interacts with CLEC‐2 on platelets via PLatelet Aggregation–inducing domains (PLAGs). Among our generated antibodies, those targeting the fourth PLAG domain (PLAG4) strongly suppress podoplanin–CLEC‐2 binding and podoplanin‐expressing tumor growth and metastasis. We previously performed a single‐dose toxicity study of PLAG4‐targeting anti‐podoplanin–neutralizing antibodies and found no acute toxicity in cynomolgus monkeys. To confirm the therapeutic efficacy and toxicity of podoplanin‐targeting antibodies, a syngeneic mouse model that enables repeated dose toxicity tests is needed. Replacement of mouse PLAG1‐PLAG4 domains with human homologous domains drastically decreased the platelet‐aggregating activity. Therefore, we searched the critical domain of the platelet‐aggregating activity in mouse podoplanin and found that the mouse PLAG4 domain played a critical role in platelet aggregation, similar to the human PLAG4 domain. Human/mouse chimeric podoplanin, in which a limited region containing mouse PLAG4 was replaced with human homologous region, exhibited a similar platelet‐aggregating activity to wild‐type mouse podoplanin. Thus, we generated knock‐in mice with human/mouse chimeric podoplanin expression (PdpnKI/KI mice). Our previously established PLAG4‐targeting antibodies could suppress human/mouse chimeric podoplanin–mediated platelet aggregation and tumor growth in PdpnKI/KI mice. Repeated treatment of PdpnKI/KI mice with antibody‐dependent cell‐mediated cytotoxicity activity–possessing PG4D2 antibody did not result in toxicity or changes in hematological and biochemical parameters. Our results suggest that anti‐podoplanin–neutralizing antibodies could be used safely as novel anti‐tumor agents. Our generated PdpnKI/KI mice are useful for investigating the efficacy and toxicity of human podoplanin–targeting drugs. [ABSTRACT FROM AUTHOR]

Patients with cancer are at a high risk of venous thromboembolism (VTE), studies have shown that high expression of podoplanin (PDPN) in tumors is associated with increased risk of VTE. Two human malignant cell lines (NCI-H226 and C8161) expressing high levels of PDPN were selected to explore the role of platelet in cancer-associated venous thrombosis in vitro and in vivo. Immunohistochemical staining using anti-PDPN antibody was performed in the pulmonary carcinoma patients. Both NCI-H226 and C8161 cells expressing high PDPN triggered platelet activation via CLEC-2 in vitro , which was abrogated by an anti-PDPN antibody SZ-168. Furthermore, the in vivo study revealed that injection of CHO-PDPN or C8161 in two mouse model of venous thrombosis activated platelets, increased platelet counts and enhanced thrombosis. More importantly, PDPN-enhanced thrombosis was reduced in mice treated with SZ168. A total of 63.3% tumor specimens stained positive for PDPN. High PDPN expression was associated with an increased risk of VTE and poor prognosis. PDPN expression in tumors induced platelet activation and was related to a high risk of VTE via platelet activation. SZ168 inhibited PDPN-induced platelet activation in vitro and decreased the incidence of VTE in mice. • PDPN expression in tumors induces VTE via platelet activation. • The PDPN antibody SZ168 inhibits PDPN-induced platelet activation in vitro. • SZ168 decreases the incidence of VTE in mice with PDPN-expressing tumors. • High PDPN level is associated with a high risk of VTE in cancer patients. • Targeting hemITAM signaling may serve as a potential therapeutic strategy for cancer-associated venous thrombosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

Abstract The interaction of tumor cells with platelets is a prerequisite for successful hematogenous metastatic dissemination. Upon tumor cell arrival in the blood, tumor cells immediately activate platelets to form a permissive microenvironment. Platelets protect tumor cells from shear forces and assault of NK cells, recruit myeloid cells by secretion of chemokines, and mediate an arrest of the tumor cell platelet embolus at the vascular wall. Subsequently, platelet-derived growth factors confer a mesenchymal-like phenotype to tumor cells and open the capillary endothelium to expedite extravasation in distant organs. Finally, platelet-secreted growth factors stimulate tumor cell proliferation to micrometastatic foci. This review provides a synopsis on the current literature on platelet-mediated effects in cancer metastasis and particularly focuses on platelet adhesion receptors and their role in metastasis. Immunoreceptor tyrosine-based activation motif (ITAM) and hemi ITAM (hemITAM) comprising receptors, especially, glycoprotein VI (GPVI), FcγRIIa, and C-type lectin-like-2 receptor (CLEC-2) are turned in the spotlight since several new mechanisms and contributions to metastasis have been attributed to this family of platelet receptors in the last years.

The podoplanin-CLEC-2 axis is critical in mice for prevention of hemorrhage in the cerebral vasculature during mid-gestation. This raises the question as to how platelets are captured by podoplanin on neuroepithelial cells in a high shear environment. In this study, we demonstrate that mouse platelets form stable aggregates on mouse podoplanin at arterial shear through a CLEC-2 and Src kinase-dependent pathway. Adhesion and aggregation are also dependent on the platelet glycoprotein (GP) receptors, integrin αIIbβ3 and GPIb, and the feedback agonists ADP and thromboxane A2 (TxA2). CLEC-2 does not bind to von Willebrand factor (VWF) suggesting that the interaction with podoplanin is sufficient to both tether and activate platelets. Consistent with this, the surface plasmon resonance measurements reveal that mouse CLEC-2 binds to mouse podoplanin with nanomolar affinity. The present findings demonstrate a novel pathway of hemostasis in which podoplanin supports platelet capture and activation at arteriolar rates of shear.

Platelet activating receptor CLEC-2 has been identified on platelet surface a decade ago. The only confirmed endogenous CLEC-2 agonist is podoplanin. Podoplanin is a transmembrane protein expressed by lymphatic endothelial cells, reticular fibroblastic cells in lymph nodes, kidney podocytes and by cells of certain tumors. Association of CLEC-2 with podoplanin is involved in processes of embryonic development (blood-lymph vessel separation and angiogenesis), maintaining of vascular integrity of small vessels during inflammation and prevention of blood-lymphatic mixing in high endothelial venules. However, CLEC-2 and podoplanin are contributing to tumor metastasis progression, Salmonella sepsis and deep-vein thrombosis. This makes CLEC-2 and podoplanin a perspective target for pharmacological treatment. Aspirin and Ibrutinib are considered to be perspective for abrogation of podoplanin-induced platelet activation via CLEC-2. The present review discusses already known pathological and physiological roles of CLEC-2 and possibilities of a targeted therapy for CLEC-2 associated diseases.

Lymphatic endothelial cells (LEC) express the transmembrane receptor podoplanin whose only known endogenous ligand CLEC-2 is found on platelets. Both podoplanin and CLEC-2 are required for normal lymphangiogenesis as mice lacking either protein develop a blood-lymphatic mixing phenotype. We investigated the roles of podoplanin and its interaction with platelets in migration and tube formation by LEC. Addition of platelets or antibody-mediated crosslinking of podoplanin inhibited LEC migration induced by vascular endothelial growth factors (VEGF-A or VEGF-C), but did not modify basal migration or the response to basic fibroblast growth factor or epidermal growth factor. In addition, platelets and podoplanin crosslinking disrupted networks of LEC formed in co-culture with fibroblasts. Depletion of podoplanin in LEC using siRNA negated the pro-migratory effect of VEGF-A and VEGF-C. Inhibition of RhoA or Rho-kinase reduced LEC migration induced by VEGF-C, but had no further effect after crosslinking of podoplanin, suggesting that podoplanin is required for signaling downstream of VEGF-receptors but upstream of RhoA. Together, these data reveal for the first time that podoplanin is an intrinsic specific regulator of VEGF-mediated migration and network formation in LEC and identify crosslinking of podoplanin by platelets or antibodies as mechanisms to modulate this pathway.

Antiplatelet medications comprise the cornerstone of treatment for diseases that involve arterial thrombosis, including acute coronary syndromes (ACS), stroke and peripheral arterial disease. However, antiplatelet medications may cause bleeding and, furthermore, thrombotic events may still recur despite treatment. The interaction of collagen with GPVI receptors on the surface of platelets has been identified as one of the major players in the pathophysiology of arterial thrombosis that occurs following atherosclerotic plaque rupture. Promisingly, GPVI deficiency in humans appears to have a minimal impact on bleeding. These findings together suggest that targeting platelet GPVI may provide a novel treatment strategy that provides additional antithrombotic efficacy with minimal disruption of normal hemostasis compared to conventional antiplatelet medications. CLEC-2 is gaining interest as a therapeutic target for a variety of thrombo-inflammatory disorders including deep vein thrombosis (DVT) with treatment also predicted to cause minimal disruption to hemostasis. GPVI and CLEC-2 signal through Src, Syk and Tec family tyrosine kinases, providing additional strategies for inhibiting both receptors. In this review, we summarize the evidence regarding GPVI and CLEC-2 and strategies for inhibiting these receptors to inhibit platelet recruitment and activation in thrombotic diseases. [ABSTRACT FROM AUTHOR]

Background: An iron overload status induces ferroptosis, an iron-dependent nonapoptotic cell death, in various pathological conditions. We previously reported that hemin (heme), protoporphyrin-IX with ferric iron, activates platelets via C-type lectin-like receptor-2 (CLEC-2) and glycoprotein VI/FcRγ, but protoporphyrin-IX alone blocks CLEC-2-dependent platelet activation. Therefore, we hypothesized that free iron has the ability to activate platelets., Objectives: This study aimed to elucidate platelet activation mechanisms of iron (ferric chloride), including the identification of signaling pathways and receptors, and to examine whether platelets regulate ferroptosis., Methods: Platelet aggregometry, platelet activation marker expression, and protein phosphorylation were examined in ferric chloride-stimulated human and murine platelets. Inhibitors of platelet activation signaling pathways and receptor-deleted platelets were utilized to identify the responsible signaling pathway and receptor. The effect of platelets on ferroptosis of endothelial cells was investigated in vitro., Results: Ferric chloride induced platelet activation dependent on Src family kinase pathways in humans and mice. Ferric chloride-induced platelet aggregation was almost lost in CLEC-2-depleted murine platelets and wild-type platelets preincubated with recombinant CLEC-2 proteins. Furthermore, coculture of wild-type platelets, but not CLEC-2-deficient platelets, attenuated ferroptosis of endothelial cells in vitro., Conclusion: Ferric chloride activates platelets via CLEC-2 and Src family kinase pathways, and platelets have a protective role in the ferroptosis of endothelial cells dependent on CLEC-2., Competing Interests: Declaration of competing interests The authors declare no competing financial interests., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Summary: Acute respiratory distress syndrome (ARDS) is characterized by a rapid‐onset respiratory failure with a mortality rate of approximately 40%. This physiologic inflammatory process is mediated by disruption of the alveolar‐vascular interface, leading to pulmonary oedema and impaired oxygen exchange, which often warrants mechanical ventilation to increase survival in the acute setting. One of the least understood aspects of ARDS is the role of the platelets in this process. Platelets, which protect vascular integrity, play a pivotal role in the progression and resolution of ARDS. The recent substantiation of the age‐old theory that megakaryocytes are found in the lungs has rejuvenated interest in and raised new questions about the importance of platelets for pulmonary function. In addition to primary haemostasis, platelets provide a myriad of inflammatory functions that are poised to aid the innate immune system. This review focuses on the evidence for regulatory roles of platelets in pulmonary inflammation, with an emphasis on two receptors, CLEC‐2 and TLT‐1. Studies of these receptors identify novel pathways through which platelets may regulate vascular integrity and inflammation in the lungs, thereby influencing the development of ARDS. [ABSTRACT FROM AUTHOR]

Losartan and honokiol are small molecules which have been described to inhibit aggregation of platelets by collagen. Losartan has been proposed to block clustering of GPVI but not to affect binding of collagen. Honokiol has been reported to bind directly to GPVI but only at a concentration that is three orders of magnitude higher than that needed for inhibition of aggregation. The mechanism of action of both inhibitors is so far unclear. In the present study, we confirm the inhibitory effects of both agents on platelet aggregation by collagen and show that both also block the aggregation induced by the activation of CLEC-2 or the low affinity immune receptor FcγRIIa at similar concentrations. For GPVI and CLEC-2, this inhibition is associated with a reduction in protein tyrosine phosphorylation of multiple proteins including Syk. In contrast, on a collagen surface, spreading of platelets and clustering of GPVI (measured by single molecule localisation microscopy) was not altered by losartan or honokiol. Furthermore, in flow whole-blood, both inhibitors suppressed the formation of multi-layered platelet thrombi at arteriolar shear rates at concentrations that hardly affect collagen-induced platelet aggregation in platelet rich plasma. Together, these results demonstrate that losartan and honokiol have multiple effects on platelets which should be considered in the use of these compounds as anti-platelet agents. [ABSTRACT FROM AUTHOR]

• Podoplanin overexpression promoted thrombus formation in rat carotid artery. • Endothelial cells expressed podoplanin involved in the carotid thrombus. • VEGF-A enhanced podoplanin expression and platelet aggregation in endothelial cells. • Anti-podoplanin antibody inhibited platelet aggregation with VEGF-A treated cells. • The results provide novel insight into the mechanisms of plaque erosion. [ABSTRACT FROM AUTHOR]

Abstract Lipid rafts are membrane microdomains that have been proposed to play an important role in several platelet-signalling cascades, including those mediated by the receptors Glycoprotein VI (GPVI), and C-type lectin domain family 1 member B (CLEC-2), both involved in thrombus formation. We have performed a LC-MS/MS proteomic analysis of lipid rafts isolated from platelets activated through GPVI and CLEC-2 as well as from resting platelets. Our aim was to determine the magnitude of changes in lipid rafts protein composition and to elucidate the relevance of these alterations in platelet function. A number of relevant signalling proteins were found enriched in lipid rafts following platelet activation (such as the tyrosine protein kinases Fyn, Lyn and Yes; the G proteins G(i) and G(z); and cAMP protein kinase). Interestingly, our results indicate that the relative enrichment of lipid rafts in these signalling proteins may not be a consequence of protein translocation to these domains upon platelet stimulation, but the result of a massive loss in cytoskeletal proteins after platelet activation. Thus, this study may help to better understand the effects of platelet activation in the reorganization of lipid rafts and set the basis for further proteomic studies of these membrane microdomains in platelets. Significance We performed the first proteomic comparative analysis of lipid rafts- protein composition in platelets activated through GPVI and CLEC-2 receptors and in resting state. We identified a number of signalling proteins essential for platelet activation relatively enriched in platelets activated through both receptors, and we show that lipid rafts reorganization upon platelet activation leads to a loss in cytoskeletal proteins, highly associated to these domains in resting platelets. Graphical abstract Unlabelled Image Highlights • FASP digestion was the most appropriate method for LC-MS/MS analysis of lipid rafts. • Lipid rafts associated to signalling proteins in basal and activated states. • Lipid rafts became relatively enriched in signalling proteins upon platelet activation. • Cytoskeletal proteins were highly associated to lipid rafts in resting platelets. • Upon GPVI and CLEC-2 activation a massive loss in cytoskeletal proteins occurred. [ABSTRACT FROM AUTHOR]

Abstract Victims of Bothrops jararaca snakebites manifest bleedings, blood incoagulability, platelet dysfunction, and thrombocytopenia, and the latter has been directly implicated in the genesis of hemorrhagic diathesis. We addressed herein the direct effects of B. jararaca venom (BjV) on ex vivo platelet aggregation and granule secretion in washed human and mouse platelets. BjV directly aggregated platelets, but the extent of platelet aggregation was lower in human than mouse platelets. On the other hand, BjV (24.4 μg/mL) and thrombin (0.1 U/mL) induced a similar extent of ATP and platelet factor 4 (PF4) secretion in both species. BjV-induced platelet aggregation was independent of the platelet dense body content, as in pearl mouse (Ap3b1 −/−) platelets, whose dense bodies are deficient in adenine nucleotides and serotonin, the extent of platelet aggregation was superior to that induced in BALB/c or C57BL/6 mice. BjV-induced β-hexosaminidase secretion in human platelets was less intense than that evoked by thrombin, and the contrary was observed in mouse platelets. Irreversible inactivation of platelet cyclooxygenase 1 by acetylsalicylic acid did not reduce BjV-induced platelet aggregation. BjV exerted no cytotoxic activity in human and mouse platelets, as evaluated by lactate dehydrogenase loss. Eptifibatide, which inhibits the binding of fibrinogen to platelet glycoprotein complex GPIIb-IIIa, differently blocked BjV-induced platelet aggregation in mice and humans. BjV-induced platelet aggregation did not depend on snake venom serine proteinases nor metalloproteinases in mice, whilst serine proteinases were rather important for platelet aggregation in humans. Our results show that BjV induces direct activation, aggregation, and secretion in human and mouse platelets, but it exerts diverse responses in them, which should be considered in comparative studies to understand pathophysiological events during Bothrops envenomation. Highlights • Bothrops jararaca venom (BjV) induces platelet aggregation and secretion. • BjV-induced platelet aggregation is independent of dense body secretion in mice. • Inhibition of thromboxane A 2 synthesis does not block BjV-induced platelet aggregation in mice nor humans. • GPIIb/IIIa inhibition does not completely abolish BjV-induced platelet aggregation in mice, but it does in humans. • BjV exerts no cytotoxic activity in human or mouse platelets. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The podoplanin-CLEC-2 axis is critical in mice for prevention of hemorrhage in the cerebral vasculature during mid-gestation. This raises the question as to how platelets are captured by podoplanin on neuroepithelial cells in a high shear environment. In this study, we demonstrate that mouse platelets form stable aggregates on mouse podoplanin at arterial shear through a CLEC-2 and Src kinase-dependent pathway. Adhesion and aggregation are also dependent on the platelet glycoprotein (GP) receptors, integrin αIIbβ3 and GPIb, and the feedback agonists ADP and thromboxane A2 (TxA2). CLEC-2 does not bind to von Willebrand factor (VWF) suggesting that the interaction with podoplanin is sufficient to both tether and activate platelets. Consistent with this, the surface plasmon resonance measurements reveal that mouse CLEC-2 binds to mouse podoplanin with nanomolar affinity. The present findings demonstrate a novel pathway of hemostasis in which podoplanin supports platelet capture and activation at arteriolar rates of shear. [ABSTRACT FROM AUTHOR]

Cellmigration 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 formactin protrusions in a 3Dcollagenmatrix upon podoplanininduced 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. [ABSTRACT FROM AUTHOR]