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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.

Abstract: In humans, platelet count within the normal range is required for physiological hemostasis, but, adversely, platelets also support pathological thrombosis. Moreover, by releasing growth factors, they may enhance neoplastic proliferation. We hypothesize that platelet count correlates with platelet-dependent pathologies, even within the range of hemostatic competence. Because platelet production is promoted by thrombopoietin signaling through the myeloproliferative leukemia virus oncogene (cMPL), a receptor expressed on megakaryocytes, we evaluated the feasibility of selective targeting of hepatic thrombopoietin production to test this hypothesis. We synthesized murine- and primate-specific antisense oligonucleotides (THPO-ASO) that silence hepatic thrombopoietin gene (THPO) expression without blocking extrahepatic THPO. Repeated doses of THPO-ASO were administered to mice and a baboon, causing a sustained 50% decline in plasma thrombopoietin levels and platelet count within 4 weeks in both species. To investigate whether reducing platelet count within the translationally relevant hemostatic range could alter a neoplastic process, we administered THPO-ASO to 6-week-old transgenic MMTV-PyMT mice that develop early ductal atypia that progresses into cMPL-negative fatal metastatic breast cancer within 2 to 3 months. THPO-ASO treatment increased the average time to euthanasia (primary humane endpoint) at 2 cm3 combined palpable tumor volume. Our results show that THPO-ASO reduced blood platelet count, plasma platelet factor 4, vascular endothelial growth factor, thrombopoietin levels, bone marrow megakaryocyte density, tumor growth rate, proliferation index, vascularization, platelet and macrophage content, and pulmonary metastases vs untreated controls. These findings confirm that sustained and moderate pharmacological platelet count reduction is feasible with THPO-ASO administration and can delay progression of certain platelet-dependent pathological processes within a safe hemostatic platelet count range.

Platelet recruitment to sites of vascular injury is mediated by von Willebrand factor (VWF). The shear-induced unraveling of ultra-large VWF multimers causes the formation of a “stringlike” conformation, which rapidly recruits platelets from the bloodstream. A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) regulates this process by cleaving VWF to prevent aberrant platelet adhesion; it is unclear whether the activity of ADAMTS13 itself is regulated. The serine proteases α-thrombin and plasmin have been shown to cleave ADAMTS13. Based on sequence homology, we hypothesized that activated coagulation factor XI (FXIa) would likewise cleave ADAMTS13. Our results show that FXIa cleaves ADAMTS13 at the C-terminal domains, generating a truncated ADAMTS13 with a deletion of part of the thrombospondin type-1 domain and the CUB1-2 domains, while α-thrombin cleaves ADAMTS13 near the CUB1-2 domains and plasmin cleaves ADAMTS13 at the metalloprotease domain and at the C-terminal domain. Using a cell surface immunoassay, we observed that FXIa induced the deletion of the CUB1-2 domains from ADAMTS13 on the surface of endothelial cells. Removal of the C-terminal domain of ADAMTS13 by FXIa or α-thrombin caused an increase in ADAMTS13 activity as measured by a fluorogenic substrate (FRETS) and blocked the ability of ADAMTS13 to cleave VWF on the endothelial cell surface, resulting in persistence of VWF strands and causing an increase in platelet adhesion under flow conditions. We have demonstrated a novel mechanism for coagulation proteinases including FXIa in regulating ADAMTS13 activity and function. This may represent an additional hemostatic function by which FXIa promotes local platelet deposition at sites of vessel injury.

The platelet receptor CLEC-2 is involved in thrombosis/hemostasis, but its ligand, podoplanin, is expressed only in advanced atherosclerotic lesions. We investigated CLEC-2 ligands in vessel walls. Recombinant CLEC-2 bound to early atherosclerotic lesions and normal arterial walls, co-localizing with vascular smooth muscle cells (VSMCs). Flow cytometry and immunocytochemistry showed that recombinant CLEC-2, but not an anti-podoplanin antibody, bound to VSMCs, suggesting that CLEC-2 ligands other than podoplanin are present in VSMCs. VSMCs stimulated platelet granule release and supported thrombus formation under flow, dependent on CLEC-2. The time to occlusion in a FeCl3-induced animal thrombosis model was significantly prolonged in the absence of CLEC-2. Because the internal elastic lamina was lacerated in our FeCl3-induced model, we assume that the interaction between CLEC-2 and its ligands in VSMCs induces thrombus formation. Protein arrays and Biacore analysis were used to identify S100A13 as a CLEC-2 ligand in VSMCs. However, S100A13 is not responsible for the above-described VSMC-induced platelet activation, because S100A13 is not expressed on the surface of normal VSMCs. S100A13 was released upon oxidative stress and expressed in the luminal area of atherosclerotic lesions. Suspended S100A13 did not activate platelets, but immobilized S100A13 significantly increased thrombus formation on collagen-coated surfaces. Taken together, we proposed that VSMCs stimulate platelets through CLEC-2, possibly leading to thrombus formation after plaque erosion and stent implantation, where VSMCs are exposed to blood flow. Furthermore, we identified S100A13 as one of the ligands on VSMCs.