C-type lectin-like receptor-2 in platelets mediates ferric chloride-induced platelet activation and attenuates ferroptosis of endothelial cells.

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.

Abnormalities in C-type lectin-like receptor 2 in a patient with Gorham-Stout disease: the first case report.

Background: Gorham-Stout disease (GSD) is a form of lymphangiomatosis of unknown etiology, characterized by abnormal distribution of lymphatic vessels. Platelets and lymphangiogenesis are closely related via C-type lectin-like receptor 2 (CLEC-2)/podoplanin. Key Clinical Question: Despite similarities between abnormal lymphatic vessels in CLEC-2-deficient mice and patients with GSD, whether CLEC-2 on platelets is involved in GSD pathogenesis is unknown. Clinical Approach: We examined CLEC-2 expression in platelets of a patient with lethal GSD. Most of the patient's platelets expressed aberrant CLEC-2 that was not detectable by certain monoclonal antibodies for human CLEC-2. Further, this population was not activated by a CLEC-2-activating snake venom, rhodocytin. Possible causes of abnormal CLEC-2 including anti-CLEC-2 autoantibodies, podoplanin binding to CLEC-2, and pathogenic CLEC1B gene alteration were excluded. Conclusions: We believe that this is the first report of a patient with structurally and functionally abnormal CLEC-2. CLEC-2 abnormality may be associated with dysregulated lymphangiogenesis in GSD.

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.

Cancer-associated fibroblasts promote venous thrombosis through podoplanin/CLEC-2 interaction in podoplanin-negative lung cancer mouse model.

BACKGROUND: Cancer-associated thrombosis (CAT) is the leading cause of morbidity and mortality. Cancer-associated fibroblasts (CAFs) are a prominent component of the tumor microenvironment that contributes to cancer progression through direct cell-cell interactions and the release of extracellular vesicles (EVs). However, the role of CAFs in CAT remains unclear. OBJECTIVE: This study aims to investigate whether CAFs aggravate CAT and the underlying molecular mechanism using a preclinical mouse lung cancer model. METHODS: We designed a Lewis lung carcinoma (LLC) tumor-bearing mouse model. CAFs were characterized using fluorescence immunohistostaining. The presence of podoplanin, a platelet-activating membrane protein through C-type lectin-like receptor 2 (CLEC-2), in EVs isolated from primary CAFs or LLC tumor tissues was assessed by immunoblotting. The platelet activation and aggregation abilities of the EVs were quantified using flow cytometry. Podoplanin plasma levels were measured by enzyme-linked immunosorbent assay. Venous thrombosis was induced in the femoral vein using 2.5% ferric chloride. The anti-CLEC-2 monoclonal antibody 2A2B10 was used to deplete CLEC-2 on the surface of the platelets. RESULTS: CAFs expressing CD90, PDGFRß, HSP47, CD34, and vimentin, co-expressed podoplanin and induced platelet activation and aggregation in a CLEC-2-dependent manner. Tumor-bearing mice showed elevated podoplanin plasma levels. CAF-EV injection and tumor-bearing mice showed shorter occlusion time in the venous thrombosis model. Although tumor growth was not altered, antibody-induced CLEC-2 depletion suppressed venous thrombosis in the tumor-bearing state but not in the healthy condition. CONCLUSION: CAFs and CAF-derived EVs induce CLEC-2-dependent platelet aggregation and aggravate venous thrombosis.

A Case of Congenital Leukemia With MYB-GATA1 Fusion Gene in a Female Patient.

We report a female newborn with acute myelogenous leukemia (AML) associated with a MYB-GATA1 fusion gene. Morphologic findings of myeloid lineage were obtained using light microscopy. Cytogenetic analysis of peripheral blood showed a complex karyotype: 46,X,-X,add(3)(q21),der(6)add(6)(q21)del(6)(q?), +mar1[5]/46,XX[15]. Targeted RNA sequencing revealed a MYB-GATA1 fusion gene. Reduced-dose AML-type chemotherapy resulted in remission and survival for >3 years without relapse. The present case demonstrated the feasibility of carrying out targeted RNA sequencing for identifying MYB-GATA1 and supports the notion that neonatal AML with MYB-GATA1 with reduced chemotherapy may show better prognosis than other highly toxic therapies.

Detection of SARS-CoV-2 RNA Using RT-qPCR in Saliva Samples and Nasopharyngeal, Lingual, and Buccal Mucosal Swabs.

Coronavirus disease 2019 is diagnosed based on the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in nasopharyngeal swabs or saliva samples using reverse-transcription quantitative polymerase chain reaction. Nasopharyngeal swabs should be collected by medical professionals who are covered with full personal protective equipment (PPE), while saliva samples can be collected by patients themselves without any PPE. However, collecting saliva is difficult for people who are unable to follow instructions, including infants or unconscious patients. Owing to the high viscosity of saliva, special attention is required to handle saliva samples in laboratories. To solve these problems, we compared lingual and buccal mucosal swabs (oral swabs) with nasopharyngeal swabs and saliva samples. Among 13 patients who had a positive result for SARS-CoV-2 RNA in their nasopharyngeal swabs, 8 and 10 patients had a positive result for SARS-CoV-2 RNA in their saliva (concordance rate, 61.5%) and oral swabs (76.9%), respectively. Among the eight patients with a positive result for SARS-CoV-2 RNA in saliva, seven (87.5%) had SARS-CoV-2 detected in their oral swabs. We could not obtain saliva samples from four patients, but we found perfect concordance of SARS-CoV-2 positivity between the nasopharyngeal and oral swabs. Therefore, oral swabs can be used for SARS-CoV-2 RNA detection.

Heme activates platelets and exacerbates rhabdomyolysis-induced acute kidney injury via CLEC-2 and GPVI/FcRγ.

There is increasing evidence that platelets participate in multiple pathophysiological processes other than thrombosis and hemostasis, such as immunity, inflammation, embryonic development, and cancer progression. A recent study revealed that heme (hemin)-activated platelets induce macrophage extracellular traps (METs) and exacerbate rhabdomyolysis-induced acute kidney injury (RAKI); however, how hemin activates platelets remains unclear. Here, we report that both C-type lectin-like receptor-2 (CLEC-2) and glycoprotein VI (GPVI) are platelet hemin receptors and are involved in the exacerbation of RAKI. We investigated hemin-induced platelet aggregation in humans and mice, binding of hemin to CLEC-2 and GPVI, the RAKI-associated phenotype in a mouse model, and in vitro MET formation. Using western blotting and surface plasmon resonance, we showed that hemin activates human platelets by stimulating the phosphorylation of SYK and PLCγ2 and directly binding to both CLEC-2 and GPVI. Furthermore, hemin-induced murine platelet aggregation was partially reduced in CLEC-2-depleted and FcRγ-deficient (equivalent to GPVI-deficient) platelets and almost completely inhibited in CLEC-2-depleted FcRγ-deficient (double-knockout) platelets. In addition, hemin-induced murine platelet aggregation was inhibited by the CLEC-2 inhibitor cobalt hematoporphyrin or GPVI antibody (JAQ-1). Renal dysfunction, tubular injury, and MET formation were attenuated in double-knockout RAKI mice. Furthermore, in vitro MET formation assay showed that the downstream signaling pathway of CLEC-2 and GPVI is involved in MET formation. We propose that both CLEC-2 and GPVI in platelets play an important role in RAKI development.

Diversity unearthed by the estimated molecular phylogeny and ecologically quantitative characteristics of uncultured Ehrlichia bacteria in Haemaphysalis ticks, Japan.

Ehrlichia species are obligatory intracellular bacteria transmitted by arthropods, and some of these species cause febrile diseases in humans and livestock. Genome sequencing has only been performed with cultured Ehrlichia species, and the taxonomic status of such ehrlichiae has been estimated by core genome-based phylogenetic analysis. However, many uncultured ehrlichiae exist in nature throughout the world, including Japan. This study aimed to conduct a molecular-based taxonomic and ecological characterization of uncultured Ehrlichia species or genotypes from ticks in Japan. We first surveyed 616 Haemaphysalis ticks by p28-PCR screening and analyzed five additional housekeeping genes (16S rRNA, groEL, gltA, ftsZ, and rpoB) from 11 p28-PCR-positive ticks. Phylogenetic analyses of the respective genes showed similar trees but with some differences. Furthermore, we found that V1 in the V1-V9 regions of Ehrlichia 16S rRNA exhibited the greatest variability. From an ecological viewpoint, the amounts of ehrlichiae in a single tick were found to equal approx. 6.3E+3 to 2.0E+6. Subsequently, core-partial-RGGFR-based phylogenetic analysis based on the concatenated sequences of the five housekeeping loci revealed six Ehrlichia genotypes, which included potentially new Ehrlichia species. Thus, our approach contributes to the taxonomic profiling and ecological quantitative analysis of uncultured or unidentified Ehrlichia species or genotypes worldwide.

Growth Characteristics of Rickettsia Species LON Strains Closely Related to Rickettsia japonica Isolated from Haemaphysalis longicornis Ticks in Mouse Derived L929 and Human-Derived THP-1 Host Cell Lines.

Non-pathogenic Rickettsia species LON strains closely related to an agent of Japanese spotted fever (JSF), R. japonica, were isolated in Japan from Haemaphysalis longicornis ticks in 2001. However, the biological properties of LONs in mammalian host cells are poorly understood. In this study, microscopic analysis showed that LONs in a mouse-derived L929 host cell line were rod shaped with sizes of 0.3-0.5 × 0.5-2.0 µm. Molecular analysis revealed the existence of a LON-specific disrupted open reading frame in R. japonica-related group-specific DNA regions. Growth kinetics of LON-2 and LON-13 strains analyzed by a quantitative real-time PCR showed 100-fold or more increment of LONs cultured in L929 host cells at 30°C and slightly less increment at 33°C, and 25-fold increment in human-derived THP-1 host cells at 35°C on day 7 (168 h) post infection. The generation times of the two LON strains cultured in L929 and THP-1 were estimated to be 9.4-12.9 h and 9.6-10.9 h, respectively. To our knowledge, this is the first report on the biological characteristics of Rickettsia sp. LON strains in mammalian cells, which may provide significant information for the experimental approaches for other rickettsiae.

Insight of diagnostic performance using B-cell epitope antigens derived from triple P44-related proteins of Anaplasma phagocytophilum.

Human granulocytic anaplasmosis (HGA) is caused by Anaplasma phagocytophilum. Indirect immunofluorescence assay (IFA) is generally used for HGA serodiagnosis. A. phagocytophilum immunodominant P44 major outer membrane proteins are encoded by p44/msp2 multigene family, responsible for IFA reactivity. However, because multiple P44-related proteins may involve immunoreactivity in IFA, the available diagnostic antigens remain obscure. In this study, we identified 12 B-cell epitopes on triple P44-related proteins using peptide array that reacted with 4 HGA patients' sera. Then, peptide spot immunoassay using 14 synthetic peptides derived from those 12 epitopes as antigens was applied for the detection of antibody to A. phagocytophilum from patients with fever of unknown origin. The sensitivities and diagnostic efficiencies of this immunoassay were higher than those of Western blot analysis using 3 recombinant proteins previously developed. Thus, the immunoassay using our epitope-derived antigens, which has higher diagnostic performances, may have significant benefit for HGA serodiagnosis.

Molecular Detection and Characterization of p44/msp2 Multigene Family of Anaplasma phagocytophilum from Haemaphysalis longicornis in Mie Prefecture, Japan.

Anaplasma phagocytophilum is an obligate intracellular bacterium that causes human granulocytic anaplasmosis (HGA), an emerging tick-borne infectious disease. This bacterium expresses various 44-kDa major outer membrane proteins encoded by the p44/msp2 multigene family to avoid the host immune system. We previously detected A. phagocytophilum p44/msp2 from the tick Haemaphysalis longicornis in Mie Prefecture, Japan in 2008. In this study, we further investigated a total of 483 H. longicornis ticks (220 adults and 263 nymphs) collected from the Mie Prefecture by PCR targeting p44/msp2 to characterize the p44/msp2 multigene family of A. phagocytophilum. Six of the 483 ticks tested were PCR-positive for A. phagocytophilum p44/msp2, and these positive individuals were at the nymph stage of the tick life cycle. Cloning, sequencing, and phylogenetic analyses of the amplicons revealed that the 11 p44/msp2 clones obtained from the positive ticks shared a 54.9%-99.3% amino acid sequence similarity with the 27 previously identified clones from HGA patients in Japan. In particular, 6 p44/msp2 clones displayed the highest similarities (97.2%-99.3%) with 3 previously identified clones (FJ417343, FJ417345, FJ417357). Thus, the data from this study provide important public health information regarding A. phagocytophilum infection transmitted by H. longicornis ticks, especially at the nymph stage.

Combined Coagulopathy Can Induce Both Hemorrhagic and Thrombotic Complications in Multiple Myeloma.

Coagulation abnormalities are a rare but critical complication associated with plasma cell diseases. We herein present a case of multiple myeloma (MM) with complicated coagulopathy. Initially, the patient showed severe bleeding tendency due to concomitant acquired hemophilia A and acquired von Willebrand syndrome. Interestingly, the patient also exhibited hyperactivation of factor IX. During treatment for MM, the bleeding complications were ameliorated; however, the patient had central retinal vein occlusion. All of the coagulation abnormalities were completely resolved after the complete remission of MM. This case suggests that MM patients may have concomitant risks for both bleeding and thromboembolic complications.