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Soluble CLEC-2 is anticipated to have various clinical applications as a novel biomarker for in vivo platelet activation, assessable using plasma obtained through routine sampling procedures. While sCLEC-2 has been measured using ELISA, we have developed a highly sensitive chemiluminescence enzyme immunoassay (CLEIA) reagent that can yield results in approximately 19 minutes. This study aims to assess its fundamental performance and explore the molecular forms of sCLEC-2 measured in patient samples. We examined the sensitivity, precision, linearity, influence of endogenous substances, residual platelets, as well as the correlation with the ELISA method, for the sCLEC-2 CLEIA reagent. The CLEIA method demonstrated sufficient sensitivity for levels observed in healthy donors, and its basic performance was satisfactory. It exhibited a strong correlation with the previously described ELISA method, with reference ranges that did not significantly differ from the ELISA data. The sCLEC-2 reference range for males was significantly higher than that for females. Since it is known that sCLEC-2 exists in shed form and microparticle form, we investigated molecular forms of sCLEC-2 measured by the CLEIA in in vitro-activated platelets and in patients’ plasma using gel filtration. It is considered that the CLEIA method shows significantly stronger reactivity with the shed form compared to the microparticle form. Studies using gel filtration of patient samples also suggest that the shed form is being primarily measured. The sCLEC-2 CLEIA reagent exhibits robust performance and is promising for clinical applications.

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.

AbstractKappa-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 αIIbβ3, 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.

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 a-granule secretion, activated integrin aIIbß3, 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 pretreated with glycoprotein VI-blocking antibody, JAQ1. Surface plasmon resonance assays showed a direct association between murine/human recombinant CLEC-2 and KCG. KCGinduced thrombosis and thrombocytopenia were significantly inhibited in CLEC-2-deficient mice. Our findings show that KCG induces platelet activation via CLEC-2. [ABSTRACT FROM AUTHOR]