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

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 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. 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</p

Otx2 Is Involved in the Regional Specification of the Developing Retinal Pigment Epithelium by Preventing the Expression of Sox2 and <em>Fgf8</em>, Factors That Induce Neural Retina Differentiation

<div><p>The retinal pigment epithelium (RPE) shares its developmental origin with the neural retina (NR). When RPE development is disrupted, cells in the presumptive RPE region abnormally differentiate into NR-like cells. Therefore, the prevention of NR differentiation in the presumptive RPE area seems to be essential for regionalizing the RPE during eye development. However, its molecular mechanisms are not fully understood. In this study, we conducted a functional inhibition of a transcription factor Otx2, which is required for RPE development, using early chick embryos. The functional inhibition of <em>Otx2</em> in chick eyes, using a recombinant gene encoding a dominant negative form of Otx2, caused the outer layer of the optic cup (the region forming the RPE, when embryos normally develop) to abnormally form an ectopic NR. In that ectopic NR, the characteristics of the RPE did not appear and NR markers were ectopically expressed. Intriguingly, the repression of <em>Otx2</em> function also caused the ectopic expression of <em>Fgf8</em> and Sox2 in the outer layer of the optic cup (the presumptive RPE region of normally developing eyes). These two factors are known to be capable of inducing NR cell differentiation in the presumptive RPE region, and are not expressed in the normally developing RPE region. Here, we suggest that <em>Otx2</em> prevents the presumptive RPE region from forming the NR by repressing the expression of both <em>Fgf8</em> and Sox2 which induce the NR cell fate.</p> </div

Schematic representation of how Otx2 functions in the regional specification of the RPE and NR in the OC.

<p>In developing chick eyes, Sox2 and <i>Fgf8</i> are expressed in the OC and induce NR differentiation. However, the expression domains of Sox2 and <i>Fgf8</i> are restricted to the inner layer of the OC, since Otx2 is expressed in the outer layer of the OC and represses the expression of Sox2 and <i>Fgf8</i>. As a result, the Sox2 and <i>Fgf8</i>-positive inner layer of the OC is induced to form the NR, whereas the Sox2 and <i>Fgf8</i>-negative outer layer is prevented from forming the NR and instead differentiates into the RPE.</p

Vascular Smooth Muscle Cells Stimulate Platelets and Facilitate Thrombus Formation through Platelet CLEC-2: Implications in Atherothrombosis

<div><p>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.</p></div

Alterations of expression patterns of transcription factors following <i>EnR-Otx2</i> transfection.

<p>Immunohistological staining of Sox2, Pax6 and Pax2 in sections of eyes transfected with empty vector (A–I) or <i>EnR -Otx2</i> (J–R). A–C and J–L indicate the expression of Sox2 (magenta). A and J are merged images with GFP (green). DAPI (blue) in C is used to ease observation of tissue structures of the RPE and NR. D–F and M–O indicate the expression of Pax6 (magenta). D and M are merged images with GFP (green). G–I and P–R indicate the expression of Pax2 (magenta). G and P are merged images with GFP (green). C, F, I, L, O and R are magnified images of the boxes in B, E, H, K, N and Q, respectively. Dashed lines highlight the RPE of control eyes (B–I) or the thickened outer layer of <i>EnR -Otx2</i>-transfected eyes (K–R). Arrows and asterisks in A, J, K, M and N indicate the peripheral and proximal areas of the outer layer of the OC, respectively. The central area of the outer layer of the OC corresponds to the area between the arrow and the asterisk. Arrowheads in B highlight the Sox2-positive small area of the RPE. The upper and lower sides of each image correspond to the dorsal and ventral sides of the specimen, respectively. In J–R, ‘RPE’ refers to the abnormally thickened outer layer, apparently ‘ectopic NR’. RPE, retinal pigment epithelium. NR, neural retina. Le, lens. Scale bars: 100 µm.</p

Expression patterns of <i>Six3</i> and <i>Lhx2</i> in <i>EnR-Otx2</i>-transfected eyes.

<p><i>In situ</i> hybridization analyses of the expression of <i>Six3</i> (B and E) and <i>Lhx2</i> (C and F) in sections of eyes transfected with an empty vector (A–C) or <i>EnR-Otx2</i> (D–F). A and D indicate GFP signals (green). A–C and D–F are serial sections. Dashed lines in A–C indicate boundaries between the RPE and NR. Dashed lines in D–F highlight the thickened outer layer of <i>EnR-Otx2</i>-transfected eyes. The upper and lower sides of each image correspond to the dorsal and ventral sides of the specimen, respectively. In D-F,‘RPE’ refers to the abnormally thickened outer layer, apparently ‘ectopic NR’. RPE, retinal pigment epithelium. NR, neural retina. Le, lens. Scale bars: 100 µm.</p

Recombinant CLEC-2 bound to early and advanced atherosclerotic lesions.

<p>A) Advanced lesions (fibrofatty plaques) of the human abdominal aorta were stained by (i) human CLEC-2-rabbit Fc2, (ii) rabbit Fc2, (iii) anti-CD68 (clone PGM-1), and iv) anti-smooth muscle actin. Magnified Fig 1A(i) was inserted into (ii). B) Early lesions (diffuse intimal thickening) were stained by (i) human CLEC-2-rabbit Fc2, (ii) rabbit Fc2, (iii) anti-CD68 (clone PGM-1), and iv) anti-smooth muscle actin. Magnified Fig 1Bi was inserted into (ii).</p

Expression pattern of <i>Otx2</i> and the dominant negative activity of <i>EnR-Otx2.</i>

<p>(A–F) <i>In situ</i> hybridization analyses of <i>Otx2</i> expression and immunohistological analyses of Mitf expression. A and B are serial sections of an HH10 embryo, as well as C and D of an HH12 embryo, and E and F of an HH17 embryo. A, C and E show expression of <i>Otx2</i>, and B, D and F show Mitf staining. Asterisks in A and B indicate the OV. Arrows in A indicate the ventral area of the OV where <i>Otx2</i> is weakly expressed. Arrowheads in C and D highlight the sites where <i>Otx2</i> and Mitf are strongly expressed. Upper and lower sides of panels A–F correspond to dorsal and ventral sides of the embryos, respectively. (G) <i>Dct</i> promoter activity in D407 cells. The <i>Dct</i> promoter fused to <i>luciferase</i> was co-transfected with the vectors, as follows. Co-transfection with: empty vector (Lane 1), <i>Otx2</i> (Lane 2), <i>Otx2</i> and <i>EnR-Otx2</i> (Lane 3), <i>Otx</i>2 and <i>EnRΔC-Otx2</i> (Lane 4), <i>EnR-Otx2</i> (Lane 5) and <i>EnR-Otx2</i> and <i>EnRΔC-Otx2</i> (Lane 6). The histogram presents means ± SD. RPE, retinal pigment epithelium. NR, neural retina. Le, lens. Scale bars: 100 µm.</p

Recombinant S100A13 protein associated with CLEC-2.

<p>A) Different concentrations of His-S100A13 were flowed over an immobilized hCLEC-2-rFc2 or a control rFc2-coated surface. The arrows indicate the beginning and the end of perfusion of S100A13. The results from one experiment are shown that is representative of the other three. RU indicates resonance units. B) (i) Platelets spreading on the surface of BSA, collagen, or S100A13 were investigated. (ii) Magnified images of adhered WT platelets on the surfaces coated with BSA or recombinant S100A13. (iii) Quantification of adherent platelets in the images in (i). Adherent platelets were counted. C) Western blotting with anti-S100A13 or anti-β3 integrin antibody. Plt represents platelets. The data are representative of three experiments.</p