{"gene":"CLEC1B","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2005,"finding":"CLEC-2 was identified as the platelet receptor for snake venom rhodocytin via rhodocytin affinity chromatography. The cytosolic tail contains a single YXXL motif that undergoes tyrosine phosphorylation upon activation by rhodocytin. Signaling is abolished in Syk-deficient and PLCγ2-deficient murine platelets and is inhibited by the Src family kinase inhibitor PP2, defining a novel pathway: CLEC-2 activation → YXXL phosphorylation → Syk binding → downstream tyrosine phosphorylation → PLCγ2 activation.","method":"Rhodocytin affinity chromatography, cell-line expression, phosphorylation assays, Syk/PLCγ2/LAT/SLP-76/Vav1/Vav3 knockout mouse platelets, pharmacological inhibition","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — receptor identification by biochemical purification, signaling confirmed by multiple genetic knockouts and pharmacological inhibition across two species","pmids":["16174766"],"is_preprint":false},{"year":2006,"finding":"Crystal structure of the extracellular domain of human CLEC-2 was solved to 1.6 Å resolution. A semi-helical loop region and flanking residues dominate the ligand-binding surface. Mutational analysis and surface plasmon resonance binding studies identified key residues for rhodocytin binding. Major ligand-induced conformational change is unlikely given the compact fold, but ligand binding could induce a tilt of a 3-10 helical portion of the long loop region.","method":"X-ray crystallography (1.6 Å), site-directed mutagenesis, surface plasmon resonance","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with mutagenesis and SPR validation in a single rigorous study","pmids":["17132623"],"is_preprint":false},{"year":2007,"finding":"Podoplanin was identified as an endogenous ligand for platelet CLEC-2. Association between CLEC-2 and podoplanin was confirmed by flow cytometry and is dependent on sialic acid on O-glycans of podoplanin. Recombinant CLEC-2 inhibited platelet aggregation induced by podoplanin-expressing tumor cells or lymphatic endothelial cells, demonstrating CLEC-2 is the functional receptor responsible for podoplanin-induced platelet aggregation.","method":"Flow cytometry, recombinant CLEC-2 inhibition assay, platelet aggregation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (flow cytometry, functional inhibition), replicated across labs in subsequent studies","pmids":["17616532"],"is_preprint":false},{"year":2007,"finding":"CLEC-2 signaling requires its single cytosolic YXXL motif; mutation of either SH2 domain of Syk blocks CLEC-2 signaling despite only a single YXXL motif, indicating dimerization is required. The DEDG amino acid sequence upstream of the YXXL, particularly the glycine directly upstream of the tyrosine, is essential for CLEC-2 signaling. CLEC-2 and Dectin-1 share this novel hemITAM signaling mechanism, distinct from DC-SIGN which signals independently of its YXXL.","method":"YXXL mutant cell lines, SH2 domain Syk mutants, chimeric receptor analysis, cell line signaling assay, DEDG sequence mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mutational dissection of signaling motif with multiple cell line models and orthogonal approaches","pmids":["17339324"],"is_preprint":false},{"year":2007,"finding":"Deletion mutant analysis of CLEC-2 (expressed as Fc chimeras) identified a podoplanin-recognition domain in CLEC-2. The interaction is dependent on both the disialyl-core1 O-glycan at Thr52 of podoplanin and the stereostructure of the podoplanin protein. A disialyl-core1-attached glycopeptide is specifically recognized by CLEC-2. Anti-podoplanin antibody NZ-1 suppressed both the podoplanin-CLEC-2 interaction and podoplanin-induced pulmonary metastasis in vivo.","method":"Deletion mutagenesis with Fc chimeras, glycopeptide synthesis and binding assays, in vivo metastasis model with antibody blockade","journal":"Cancer science","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — systematic deletion mutagenesis combined with in vitro and in vivo functional validation","pmids":["17944973"],"is_preprint":false},{"year":2008,"finding":"Podoplanin on renal cells (HEK-293T/podocytes) was identified as a direct CLEC-2 ligand. Surface plasmon resonance confirmed direct CLEC-2–podoplanin interaction with an affinity of 24.5 ± 3.7 μM, and this interaction is independent of glycosylation of CLEC-2 itself. Podoplanin on HEK-293T cells mediates both binding to CLEC-2 and activation of CLEC-2-transfected DT-40 B-cells.","method":"Surface plasmon resonance, recombinant CLEC-2 binding assay, CLEC-2-transfected DT-40 cell activation assay, podoplanin identification by proteomics","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct SPR binding measurement with cell-based functional confirmation, single lab","pmids":["18215137"],"is_preprint":false},{"year":2008,"finding":"Crystal structure of rhodocytin at 2.4 Å resolution revealed it is the first snake venom C-type lectin-like protein to assemble as a non-disulfide-linked (αβ)2 tetramer. The concave binding surface of rhodocytin is complementary to the CLEC-2 binding interface. Computational docking suggests the (αβ)2 rhodocytin tetramer induces clustering of CLEC-2 receptors on the platelet surface to trigger signaling.","method":"X-ray crystallography (2.4 Å), computational protein-protein docking, electrostatic and hydrophobicity analysis","journal":"Protein science","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — crystal structure solved but clustering mechanism supported only by computational docking, not direct experiment","pmids":["18583525"],"is_preprint":false},{"year":2008,"finding":"G6b-B, a platelet immunoglobulin receptor with two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), inhibits both constitutive and agonist-induced signaling by CLEC-2 and GPVI via Src- and Syk-dependent pathways. This inhibitory activity is independent of SHP1, SHP2, and SHIP phosphatases.","method":"NFAT reporter assay in cell lines, ITIM tyrosine mutants, SHP1/SHP2/SHIP inhibitor/knockout experiments, Western blotting","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell line model plus genetic knockouts with multiple readouts, single lab","pmids":["18955485"],"is_preprint":false},{"year":2009,"finding":"CLEC-2 exists as a non-disulfide-linked homodimer, demonstrated by multiple orthogonal biochemical methods. This dimerization allows each Syk molecule to interact with two YXXL motifs (one from each CLEC-2 monomer), providing a mechanistic explanation for how a single YXXL-containing receptor can activate Syk which normally requires tandem YXXL (ITAM).","method":"Bioluminescence resonance energy transfer, co-immunoprecipitation, analytical gel filtration, surface plasmon resonance, multiangle light scattering, analytical ultracentrifugation, Western blotting","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple independent biophysical methods in one rigorous study confirming homodimerization","pmids":["19824697"],"is_preprint":false},{"year":2009,"finding":"CLEC-2-deficient platelets displayed normal adhesion under flow but severely defective aggregate formation in vitro and in vivo. CLEC-2 deficiency caused increased bleeding times and profound protection from occlusive arterial thrombus formation, establishing an essential role for CLEC-2 in hemostasis and thrombosis.","method":"Anti-CLEC-2 antibody-induced depletion in mice, in vitro flow chamber aggregation assay, in vivo thrombosis models, bleeding time measurement","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — specific receptor depletion with defined phenotypic readouts in vitro and in vivo","pmids":["19641185"],"is_preprint":false},{"year":2009,"finding":"CLEC-2 is expressed on murine peripheral blood neutrophils (but not bone marrow or elicited inflammatory neutrophils), where it functions as a phagocytic activation receptor. Crosslinking of CLEC-2 on neutrophils leads to Syk recruitment and production of pro-inflammatory cytokines (TNF-α) in response to rhodocytin. The tyrosine-based cytoplasmic motif is required for these activities. Notably, CLEC-2 stimulation does not induce the respiratory burst.","method":"Antibody staining and flow cytometry, chimeric receptor analysis, phagocytosis assay, cytokine ELISA, Syk co-immunoprecipitation","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct functional characterization in primary cells with chimeric receptor mutagenesis, single lab","pmids":["19299712"],"is_preprint":false},{"year":2009,"finding":"The Syk inhibitor R406 markedly inhibited tyrosine phosphorylation of CLEC-2 itself and downstream Syk phosphorylation following CLEC-2 activation, while Syk phosphorylation downstream of GPVI was unaffected. This revealed a critical role for Syk in mediating phosphorylation of the CLEC-2 hemITAM, suggesting Syk functions both downstream and upstream of CLEC-2 phosphorylation.","method":"Platelet aggregometry, immunoprecipitation, Western blotting, pharmacological Syk inhibitor (R406)","journal":"Journal of thrombosis and haemostasis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological inhibitor with biochemical readouts in human platelets, single lab","pmids":["19422460"],"is_preprint":false},{"year":2010,"finding":"CLEC-2-deficient mice are lethal at embryonic/neonatal stages due to disorganized, blood-filled lymphatic vessels and severe edema. Fetal liver cell transplantation demonstrated that CLEC-2 is involved in thrombus stabilization in vitro and in vivo, possibly through homophilic interactions. Loss of CLEC-2 did not significantly increase bleeding tendency.","method":"CLEC-2 knockout mouse generation, fetal liver cell transplantation chimeras, intravital thrombus formation assay, histology","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — constitutive and chimeric knockout with multiple phenotypic endpoints, replicated in other labs","pmids":["20525685"],"is_preprint":false},{"year":2010,"finding":"Platelets regulate lymphatic vascular development by interacting with lymphatic endothelial cells through CLEC-2 binding to podoplanin. Genetic loss of CLEC-2 ablates podoplanin binding by platelets and causes embryonic lymphatic vascular defects identical to those in PDPN- or SLP-76-deficient animals. PF4-Cre-mediated deletion of Slp-76 specifically in platelets is sufficient to cause lymphatic vascular defects, placing CLEC-2→SLP-76 signaling in platelets upstream of blood-lymphatic separation.","method":"Conditional (PF4-Cre) and constitutive knockout mice, intravital microscopy of platelet aggregates on lymphatic endothelial cells, genetic epistasis","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with conditional knockout, replicated phenotype across multiple mouse models","pmids":["20363774"],"is_preprint":false},{"year":2010,"finding":"CLEC-2 activates Syk through dimerization: peptide pull-down, surface plasmon resonance, tryptophan fluorescence, and quantitative Western blotting demonstrated that Syk activation by CLEC-2 is mediated by cross-linking of the Syk tandem SH2 domains with two phosphorylated CLEC-2 YXXL motifs in a 2:1 stoichiometry. Cross-linking and electron microscopy confirmed CLEC-2 is present as a dimer in resting platelets and forms larger complexes upon activation.","method":"Peptide pull-down, surface plasmon resonance, tryptophan fluorescence, quantitative Western blotting, competition experiments, chemical cross-linking, electron microscopy","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal biophysical methods establishing dimerization mechanism in a single rigorous study","pmids":["20154219"],"is_preprint":false},{"year":2010,"finding":"CLEC-2 phosphorylation (hemITAM) is abolished in the absence of Syk but not in the absence of major platelet Src family kinases (Fyn, Lyn, Src) or CD148, nor is it altered by PP2. PLCγ2 phosphorylation remains dependent on Src family kinases. This established that Syk—not Src—is the primary kinase for CLEC-2 hemITAM phosphorylation, with Src family kinases acting downstream.","method":"Syk-knockout, Fyn/Lyn/Src/CD148-knockout mouse platelets, PP2 pharmacological inhibition, Western blotting for CLEC-2 and PLCγ2 phosphorylation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple kinase-specific knockouts with biochemical readouts, establishes clear epistatic order","pmids":["21098033"],"is_preprint":false},{"year":2010,"finding":"CLEC-2 translocates to lipid rafts upon ligand engagement, and this translocation is essential for hemITAM phosphorylation and signal initiation (shown by sucrose gradient ultracentrifugation and methyl-β-cyclodextrin treatment). HemITAM phosphorylation (but not translocation) additionally requires actin polymerization, Rac1 activation, and release of ADP and thromboxane A2.","method":"Sucrose gradient ultracentrifugation, methyl-β-cyclodextrin cholesterol depletion, actin polymerization inhibitors, Rac1 inhibitors, ADP/TxA2 receptor pharmacology, Western blotting","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mechanistic dissection with multiple orthogonal pharmacological and biochemical approaches in a single study","pmids":["20154214"],"is_preprint":false},{"year":2011,"finding":"Conditional deletion of CLEC-2 or Syk specifically in the megakaryocyte/platelet lineage causes defects in brain vascular and lymphatic development. Platelets (not platelet releasate) directly modulate migration and intercellular adhesion of lymphatic endothelial cells through a pathway dependent on CLEC-2 and Syk.","method":"Conditional (megakaryocyte/platelet lineage) Cre-lox knockout mice, lymphatic endothelial cell migration and adhesion assays with platelets vs. platelet releasate, intravital microscopy","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-type-specific conditional knockouts with in vitro mechanistic dissection, independent replication in multiple labs","pmids":["22186994"],"is_preprint":false},{"year":2012,"finding":"Platelet-specific deletion of CLEC-2 causes misconnection of blood/lymphatic vessels. CLEC-2-expressing (but not CLEC-2-deficient) platelets inhibit lymphatic endothelial cell migration, proliferation, and tube formation. Supernatants from activated platelets replicate this inhibition. BMP-9, present in platelets and released upon CLEC-2 activation, specifically inhibits LEC tube formation and plays a key role in blood/lymphatic vessel separation.","method":"Platelet-specific CLEC-2 knockout mice, LEC migration/proliferation/tube formation assays, activated platelet supernatant experiments, BMP-9 identification and functional assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout combined with cell biology assays and identification of secreted mediator BMP-9","pmids":["22556408"],"is_preprint":false},{"year":2012,"finding":"CLEC-2 engagement of podoplanin on stromal surfaces was necessary and sufficient to induce membrane protrusions in dendritic cells. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation, and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes.","method":"DC-specific CLEC-2 conditional knockout mice, RhoA/myosin light-chain phosphorylation assays, Vav/Rac1 inhibitor studies, DC migration assays in lymph nodes, live imaging","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout with multiple signaling pathway readouts and in vivo functional endpoints","pmids":["22884313"],"is_preprint":false},{"year":2012,"finding":"The upstream triacidic amino acid (DEDG) sequence in the CLEC-2 cytoplasmic tail is required for signaling through the hemITAM. Surface plasmon resonance and phosphorylation studies demonstrated that the triacidic amino acids are required for YXXL phosphorylation. HemITAM signaling is restricted to Syk (not ZAP-70), shown using Syk-deficient and ZAP-70-deficient cell lines.","method":"Site-directed mutagenesis of DEDG sequence, surface plasmon resonance, phosphorylation assays, Syk-deficient and ZAP-70-deficient cell lines","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis combined with SPR and biochemical phosphorylation assays, single lab","pmids":["23264619"],"is_preprint":false},{"year":2013,"finding":"Podoplanin (PDPN) on fibroblastic reticular cells (FRCs) surrounding high endothelial venules acts as an activating ligand for platelet CLEC-2. CLEC-2 activation induces release of sphingosine-1-phosphate (S1P) from platelets, which promotes VE-cadherin expression on HEVs. Mice lacking FRC podoplanin or platelet CLEC-2 showed reduced VE-cadherin on HEVs; infusion of wild-type platelets restored HEV integrity in CLEC-2-deficient mice.","method":"Conditional knockout mice (FRC-specific PDPN, platelet-specific CLEC-2), VE-cadherin measurement, platelet infusion rescue experiment, S1P pathway analysis, ex vivo HEV assay","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockouts with rescue experiment, identification of S1P as downstream mediator, multiple orthogonal methods","pmids":["23995678"],"is_preprint":false},{"year":2013,"finding":"Fucoidan (a sulfated polysaccharide) induces platelet activation through CLEC-2 via a Src family kinase (SFK)- and Syk-dependent pathway. Fucoidan-induced platelet activation was completely abolished in platelet-specific CLEC-2 knockout murine platelets, but only slightly inhibited in FcRγ-chain null mice, establishing CLEC-2 as the primary receptor.","method":"Platelet-specific CLEC-2 knockout mice, FcRγ-null mice, SFK/Syk inhibitors, aggregometry, phosphorylation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic receptor-specific knockouts plus pharmacological inhibitors with defined cellular phenotype","pmids":["23341451"],"is_preprint":false},{"year":2014,"finding":"Crystal structure of CLEC-2 in complex with O-glycosylated podoplanin peptide and with non-glycosylated rhodocytin revealed that both ligands bind to the non-canonical 'side' face of CLEC-2. Common interaction involves consecutive acidic residues on ligands binding to the same arginine residues on CLEC-2. For podoplanin, the sialic acid carboxyl group provides a second binding interaction; for rhodocytin, the C-terminus of the α-subunit interacts differently at this second site.","method":"X-ray crystallography of CLEC-2–ligand complexes","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Moderate — comparative crystal structures of CLEC-2 with two different ligands, single lab","pmids":["25458834"],"is_preprint":false},{"year":2014,"finding":"CLEC-2 expression on platelets is not regulated by proteolytic shedding (unlike GPVI and FcγRIIa, which are cleaved upon CLEC-2 activation). CLEC-2 is restricted to platelets with ~2000 copies per cell. CLEC-2 and GPVI are expressed on CD41+ microparticles in megakaryocyte cultures, but microparticles derived from activated platelets only express CLEC-2, making CLEC-2 a marker for activated platelet-derived microparticles.","method":"Monoclonal antibody-based flow cytometry, CLEC-2 copy number quantitation, platelet activation and shedding assays, microparticle characterization","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple functional assays but focused primarily on receptor regulation, single lab","pmids":["25150298"],"is_preprint":false},{"year":2014,"finding":"Podoplanin on FRCs regulates actomyosin contractility in FRCs through CLEC-2. Under resting conditions, podoplanin endows FRCs with contractile function and maintains tension in the reticular network. Upon inflammation, CLEC-2 on mature DCs potently attenuates podoplanin-mediated contractility in FRCs, resulting in FRC relaxation and reduced tissue stiffness.","method":"Collagen gel contraction assay, myosin light chain phosphorylation assays, conditional knockout mice, 3D tissue imaging, DC-FRC co-culture","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic models combined with functional assays of cytoskeletal contractility, published in high-impact journal","pmids":["25347465"],"is_preprint":false},{"year":2014,"finding":"Syk and Src family kinases in platelets promote clustering of CLEC-2 and its ligand podoplanin on supported lipid bilayers containing mobile podoplanin. CLEC-2-bound podoplanin clusters migrate to the center of the platelet, forming a single structure. Fluorescence lifetime imaging demonstrated molecules within these clusters are within 10 nm. Clusters are disrupted by Src/Syk inhibition.","method":"Supported lipid bilayer system with mobile podoplanin, fluorescence lifetime imaging microscopy (FLIM), direct stochastic optical reconstruction microscopy (dSTORM), Src/Syk inhibitors, Syk-deficient mouse platelets","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple advanced microscopy techniques with pharmacological and genetic validation","pmids":["25368330"],"is_preprint":false},{"year":2014,"finding":"CLEC-2 deficiency results in absence of lymph nodes at birth due to defective lymphatic endothelial cell proliferation. Platelet-specific CLEC-2 deficiency (PF4-Cre) leads to blood-filled lymph nodes and fibrosis (without a proliferative defect), indicating CLEC-2 expression in platelets specifically is required for lymph node integrity. This phenotype is independent of the lymphatic endothelial proliferation defect seen with constitutive CLEC-2 deletion.","method":"Constitutive and PF4-Cre conditional CLEC-2 knockout mice, bone marrow chimeras, lymph node histology, LEC proliferation assays, immune response assays","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — dissection of platelet-specific vs. global CLEC-2 function using conditional and chimeric approaches","pmids":["24532804"],"is_preprint":false},{"year":2015,"finding":"CLEC-2 in megakaryocytes mediates production of thrombopoietin (Thpo) and other factors required for HSC maintenance. Megakaryocyte-specific CLEC-2 knockout mice produced lower levels of Thpo and showed reduced HSC quiescence and repopulation potential with extramedullary hematopoiesis. Knockdown of downstream CLEC-2 signaling molecules (Syk, Lcp2, Plcg2) in megakaryocytes decreased Thpo expression. Administration of recombinant Thpo restored stem cell potential.","method":"Megakaryocyte-specific CLEC-2 conditional knockout mice, Syk/Lcp2/Plcg2 siRNA knockdown in megakaryocytes, Thpo measurement, HSC quiescence and transplantation assays, recombinant Thpo rescue","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout with signaling pathway knockdown, Thpo identification as downstream mediator, rescue experiment","pmids":["26552707"],"is_preprint":false},{"year":2015,"finding":"Podoplanin-positive periarteriolar BM fibroblastic reticular cell (FRC)-like cells promote megakaryocyte growth and proplatelet formation through CLEC-2/podoplanin interaction. Megakaryocyte-specific CLEC-2 conditional knockout mice showed decreased immature megakaryocytes. The CLEC-2/podoplanin interaction induces BM FRC-like cells to secrete CCL5, which facilitates proplatelet formation.","method":"Megakaryocyte-specific CLEC-2 conditional knockout mice, megakaryocyte-FRC co-culture, recombinant podoplanin stimulation, CCL5 secretion measurement, immunofluorescence of BM sections","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout combined with co-culture identification of CCL5 as downstream mediator","pmids":["26796360"],"is_preprint":false},{"year":2015,"finding":"Podoplanin/CLEC-2 signaling regulates keratinocyte migration via modulating E-cadherin expression through RhoA signaling. Down-regulation of podoplanin in keratinocytes inhibited their migration and was correlated with upregulation of E-cadherin. Both platelets and recombinant CLEC-2 inhibited keratinocyte migration, with downregulation of RhoA activity and upregulation of E-cadherin.","method":"Podoplanin siRNA knockdown in NHEKs, platelet/CLEC-2 co-culture migration assays, wound healing assay, RhoA activity measurement, E-cadherin Western blotting","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown and recombinant protein with biochemical pathway readouts, single lab","pmids":["26597882"],"is_preprint":false},{"year":2015,"finding":"Vascular smooth muscle cells (VSMCs) stimulate platelets and facilitate thrombus formation through platelet CLEC-2. Protein arrays and Biacore analysis identified S100A13 as a CLEC-2 ligand in VSMCs. S100A13 is released upon oxidative stress and immobilized (but not suspended) S100A13 significantly increases thrombus formation on collagen-coated surfaces. However, surface S100A13 on normal VSMCs does not activate platelets, indicating another CLEC-2 ligand mediates baseline VSMC–platelet interaction.","method":"Recombinant CLEC-2 binding assays, flow cytometry, immunocytochemistry, thrombus formation assay under flow, FeCl3 animal thrombosis model, protein array, surface plasmon resonance (Biacore)","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple biochemical and functional assays identifying S100A13 as a novel CLEC-2 ligand, single lab","pmids":["26418160"],"is_preprint":false},{"year":2015,"finding":"CLEC-2 is internalized from the platelet surface upon anti-CLEC-2 antibody (INU1) treatment through a Src-family kinase-dependent mechanism, followed by intracellular degradation. This internalization is independent of Syk. Syk-deficient mice showed preserved CLEC-2 internalization but largely prevented antibody-induced thrombocytopenia, mechanistically uncoupling CLEC-2 downregulation from thrombocytopenia.","method":"Platelet-specific Syk knockout mice, Src family kinase inhibitors, flow cytometry for CLEC-2 surface expression, in vitro and in vivo INU1 treatment","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic (Syk KO) and pharmacological approaches dissect internalization mechanism in vitro and in vivo","pmids":["25795918"],"is_preprint":false},{"year":2015,"finding":"Podoplanin on pericytes/neuro-epithelium interacts with CLEC-2 on platelets to guide cerebrovascular patterning and integrity during development. Cerebral vessels were tortuous and aberrantly patterned at E10.5 in podoplanin- and CLEC-2-deficient mice; defective pericyte recruitment and endothelial-pericyte misconnections were observed. Platelet α-granule and dense granule secretion is required, as embryos deficient in platelet GPIIb or granule secretion also developed cerebral hemorrhaging.","method":"Conditional and constitutive CLEC-2/podoplanin knockout mice, nestin-Cre podoplanin deletion, 3D light-sheet microscopy, immunofluorescence, electron microscopy","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple conditional knockout lines with advanced imaging modalities","pmids":["25908104"],"is_preprint":false},{"year":2017,"finding":"Platelet-specific deficiency of CLEC-2 protects mice against deep vein thrombosis (DVT) in an IVC stenosis model. Podoplanin is expressed in the IVC wall near the abluminal endothelium and is upregulated after 48h stenosis in mice that develop thrombi. Inhibition of podoplanin with a neutralizing antibody reduced thrombus size, establishing the CLEC-2–podoplanin axis as a mechanistic driver of DVT.","method":"Inducible CLEC-2 knockout and platelet-specific CLEC-2 knockout mice, IVC stenosis model, platelet transfusion rescue, anti-podoplanin neutralizing antibody, immunofluorescence of podoplanin localization","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple conditional knockout approaches with ligand neutralization and localization studies in defined DVT model","pmids":["28104688"],"is_preprint":false},{"year":2017,"finding":"Platelet-specific deletion of CLEC-2 (but not GPVI) leads to enhanced systemic inflammation and accelerated organ injury in two mouse models of sepsis. CLEC-2 deficiency is associated with reduced numbers of podoplanin-expressing macrophages despite increased cytokines. Pharmacological inhibition of CLEC-2–podoplanin interaction regulates immune cell infiltration, identifying the podoplanin–CLEC-2 axis as an anti-inflammatory pathway in sepsis.","method":"Platelet-specific CLEC-2 and GPVI conditional knockout mice, LPS i.p. and cecal ligation and puncture sepsis models, cytokine measurement, immune cell counting, pharmacological CLEC-2–podoplanin inhibition","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-type specific knockouts with two independent disease models and pharmacological validation","pmids":["29269852"],"is_preprint":false},{"year":2017,"finding":"Gq signaling (via PLCβ-PKCα pathway, possibly regulating Src family kinases) potentiates the most proximal event in CLEC-2 signaling—the CLEC-2 receptor tyrosine phosphorylation itself—in response to TxA2 and other GPCR agonists. Using the Gq inhibitor UBO-QIC and Gq-knockout murine platelets, Gq signaling was demonstrated to be essential for GPCR-mediated potentiation of Syk phosphorylation downstream of CLEC-2.","method":"Gq-specific inhibitor (UBO-QIC), Gq-knockout mouse platelets, P2Y12/PAR/TP receptor agonists, Western blotting for CLEC-2 and Syk phosphorylation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic (Gq KO) and pharmacological approaches with biochemical readouts identifying Gq-PLCβ-PKCα-SFK as regulatory axis","pmids":["28705934"],"is_preprint":false},{"year":2017,"finding":"CLEC-2 contributes to hemostasis through a mechanism independent of classical hemITAM signaling. A knockin mouse expressing hemITAM-null CLEC-2 (Y7A KI) phenocopied blood-lymphatic mixing/lethality of CLEC-2 KO but not the hemostatic/thrombotic defect. Treatment of Y7A KI mice with function-blocking anti-CLEC-2 Fab' resulted in a thrombus formation defect, revealing a hemITAM signaling-independent (physical/adhesive) role for CLEC-2 in hemostasis.","method":"CLEC-2 Y7A hemITAM knockin mouse model, anti-CLEC-2 Fab' treatment, thrombosis assays in vitro and in vivo","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — novel knockin mouse separating signaling from non-signaling functions, rigorous genetic design","pmids":["28835437"],"is_preprint":false},{"year":2018,"finding":"Dengue virus activates platelets via CLEC-2 to release extracellular vesicles (exosomes and microvesicles). These EVs further activate CLEC5A and TLR2 on neutrophils and macrophages, inducing NET formation and pro-inflammatory cytokine release. Simultaneous blockade of CLEC5A and TLR2 (not CLEC-2) increased survival from DV infection from 30% to 90%, establishing CLEC-2 as the upstream initiator of this platelet-leukocyte inflammatory axis.","method":"CLEC-2 functional blocking, EV isolation and characterization, CLEC5A/TLR2 blockade, NET formation assay, cytokine measurement, mouse dengue lethality model","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — mechanistic dissection with multiple pharmacological interventions and in vivo model with survival endpoint","pmids":["31160588"],"is_preprint":false},{"year":2018,"finding":"Surface plasmon resonance and molecular docking identified novel podoplanin-binding sites on CLEC-2: N120, N210, and K211. Cobalt hematoporphyrin (Co-HP) binds directly to CLEC-2 at these sites and inhibits CLEC-2–podoplanin interaction. These binding sites were confirmed by CLEC-2 mutants with alterations in N120 and/or K211.","method":"Surface plasmon resonance, molecular docking, CLEC-2 site-directed mutagenesis, platelet aggregation assay, in vivo metastasis and thrombosis models","journal":"Blood advances","confidence":"High","confidence_rationale":"Tier 1 / Moderate — SPR and mutagenesis identifying novel ligand-binding residues, validated in vivo, single lab","pmids":["30190281"],"is_preprint":false},{"year":2018,"finding":"Recombinant rhodocytin forms a heterooctamer (four α- and β-subunits). Asp4 in the α-subunit of rhodocytin is required for CLEC-2 binding. An inhibitory mutant rhodocytin (αWTβK53A/R56A) forms a heterotetramer, binds CLEC-2 without inducing platelet aggregation, and blocks CLEC-2–podoplanin interaction-dependent platelet aggregation and experimental lung metastasis.","method":"Recombinant rhodocytin expression, mutagenesis, blue native PAGE for multimer analysis, flow cytometry for CLEC-2 binding, platelet aggregometry, in vivo lung metastasis model","journal":"Journal of thrombosis and haemostasis","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution with mutagenesis plus in vivo functional validation, single lab","pmids":["29488681"],"is_preprint":false},{"year":2018,"finding":"Tetraspanin CD37 specifically interacts with CLEC-2 and controls CLEC-2 membrane organization. CD37-deficient myeloid cells expressed reduced surface CLEC-2 and showed impaired CLEC-2-dependent adhesion, migration velocity, and actin protrusion formation. CD37 is required for CLEC-2 recruitment in the membrane to podoplanin, and CD37-deficient DCs fail to inhibit actomyosin contractility in lymph node stromal cells.","method":"Co-immunoprecipitation of CD37-CLEC-2, Cd37-knockout mice, DC migration and adhesion assays, microcontact printing for CLEC-2 membrane recruitment, actin protrusion assay in 3D collagen","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus genetic knockout with multiple functional readouts, single lab","pmids":["30185523"],"is_preprint":false},{"year":2019,"finding":"Akt1/PDK1 and PKCμ are two alternative CLEC-2 signaling pathways mediating podoplanin-induced platelet activation, identified through analysis of the molecular action of compound 2CP (a direct CLEC-2 binding inhibitor that competes with podoplanin for the same CLEC-2 binding pocket).","method":"Compound 2CP binding assay, Akt1/PDK1 and PKCμ pathway analysis, CLEC-2 competitive binding assay, platelet aggregation, in vivo metastasis model","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — identification of alternative signaling pathways by pharmacological inhibition with direct binding confirmation, single lab","pmids":["26528756"],"is_preprint":false},{"year":2019,"finding":"Soluble CLEC-2 (sCLEC-2) is shed as a fragment partially generated by MMP-2 (not ADAM10/17, which mediate GPVI shedding). Additionally, CLEC-2 is released as a whole molecule associated with platelet microparticles (MP-CLEC-2). This contrasts with sGPVI which is exclusively shed by ADAM10/17. The two proteins are thus released by distinct mechanisms.","method":"Metalloproteinase inhibitors/stimulators, ADAM10-specific inhibitor, domain-specific GPVI antibodies, microparticle characterization, ELISA for plasma levels","journal":"International journal of hematology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological inhibitor panel with biochemical characterization establishing distinct shedding mechanism, single lab","pmids":["31165998"],"is_preprint":false},{"year":2020,"finding":"Platelet CLEC-2 signaling blocks reparative neutrophil recruitment in acetaminophen-induced acute liver failure by suppressing TNF-α production. Blocking CLEC-2 signaling enhanced liver recovery from APAP and CCl4 acute toxic liver injuries by increasing TNF-α, which drives reparative hepatic neutrophil recruitment.","method":"CLEC-2 signaling blockade (antibody), APAP and CCl4 mouse models, TNF-α measurement, neutrophil enumeration, human and mouse hepatic inflammatory response studies","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — two independent liver injury models with mechanistic link to TNF-α and neutrophil recruitment, human validation","pmids":["32321925"],"is_preprint":false},{"year":2021,"finding":"Platelet CLEC-2 reduces tissue inflammation by regulating inflammatory macrophage activation and trafficking. The immune regulatory function depends on podoplanin expression on inflammatory macrophages and is independent of platelet activation/secretion. Mechanistically, platelet CLEC-2 and recombinant CLEC-2-Fc accelerate actin rearrangement and macrophage migration by increasing podoplanin and CD44 expression and their interaction with ERM proteins, promoting macrophage emigration to mesenteric lymph nodes.","method":"Platelet-specific CLEC-2 knockout mice, LPS peritonitis model, recombinant CLEC-2-Fc treatment, actin rearrangement assay, podoplanin/CD44/ERM protein interaction studies, cytokine measurement, in vivo macrophage tracking","journal":"Frontiers in immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout with mechanistic dissection including recombinant protein rescue and molecular interaction studies","pmids":["34163489"],"is_preprint":false},{"year":2021,"finding":"Low concentrations of the Btk inhibitor ibrutinib selectively block CLEC-2-mediated activation and tyrosine phosphorylation (including Syk and PLCγ2) in human platelets, while GPVI response is only delayed. Platelets from patients with X-linked agammaglobulinemia (XLA, Btk-deficient) phenocopy this selective CLEC-2 block. The differential effect is explained by a positive feedback role of Btk in CLEC-2 signaling, involving ADP/TxA2-mediated P2Y12/TP receptor co-activation, which is not present in mouse platelets.","method":"Ibrutinib and acalabrutinib at titrated concentrations, XLA patient platelets, human and mouse platelet aggregometry, Syk/PLCγ2 phosphorylation, in vivo mouse thrombosis model","journal":"Haematologica","confidence":"High","confidence_rationale":"Tier 2 / Strong — pharmacological and genetic (XLA patient, mouse model) approaches with species comparison revealing mechanistic basis","pmids":["31949019"],"is_preprint":false},{"year":2021,"finding":"Hemin (heme) directly binds to both CLEC-2 and GPVI on platelets, as demonstrated by Western blotting and surface plasmon resonance. Hemin activates human platelets by stimulating phosphorylation of SYK and PLCγ2 through both receptors. Hemin-induced murine platelet aggregation is almost completely inhibited only in CLEC-2-depleted FcRγ-deficient (GPVI-deficient) double-knockout platelets, and CLEC-2/GPVI signaling contributes to MET formation and rhabdomyolysis-induced acute kidney injury.","method":"Surface plasmon resonance for direct hemin-CLEC-2/GPVI binding, CLEC-2 depletion and FcRγ-null mouse platelets, RAKI mouse model, in vitro MET formation assay, Western blotting for SYK/PLCγ2 phosphorylation","journal":"Blood advances","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct binding by SPR combined with double-knockout genetic model and in vivo functional validation","pmids":["33843987"],"is_preprint":false},{"year":2022,"finding":"Deletion of platelet CLEC-2 does not prevent VWF binding to platelets via GPIbα but specifically inhibited platelet aggregation and αIIbβ3 activation induced by VWF binding to GPIbα. In a mouse TTP model, CLEC-2 deletion decreased pulmonary arterial thrombosis and severity of thrombocytopenia, establishing CLEC-2 as a downstream mediator of GPIbα-initiated integrin αIIbβ3 activation.","method":"Platelet-specific CLEC-2 knockout mice, VWF-platelet binding assay, αIIbβ3 activation measurement, mouse TTP model (anti-ADAMTS13 antibody + VWF infusion), aspirin/eptifibatide treatment","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout with defined mechanistic placement of CLEC-2 between GPIbα and αIIbβ3 activation, in vivo disease model","pmids":["35157766"],"is_preprint":false},{"year":2022,"finding":"Cooperative signaling between CLEC-2 (activated by anti-CLEC-2 Fab INU1) and GPIIb/IIIa triggers a cerebral venous (sinus) thrombosis (CVT)-like syndrome in mice within minutes, characterized by tonic-myoclonic seizures, platelet consumption, and death. Interfering with CLEC-2 signaling or GPIIb/IIIa completely blocked platelet activation and CVT. Blocking GPIIb/IIIa after onset of neurological symptoms protected mice, while heparin did not.","method":"INU1-Fab anti-CLEC-2 antibody in mice, GPIIb/IIIa inhibitor, transcranial intravital microscopy of superior sagittal sinus, brain autopsy/histology, seizure monitoring","journal":"Nature cardiovascular research","confidence":"High","confidence_rationale":"Tier 2 / Strong — mechanistic intervention with live intravital imaging and defined pharmacological rescue in a novel in vivo model","pmids":["39195988"],"is_preprint":false},{"year":2023,"finding":"Cancer-associated fibroblasts (CAFs) co-express podoplanin and induce CLEC-2-dependent platelet aggregation. CAF-derived extracellular vesicles (EVs) also contain podoplanin and activate platelets in a CLEC-2-dependent manner. In tumor-bearing mice, antibody-induced CLEC-2 depletion suppressed venous thrombosis in the tumor-bearing state, establishing CAF-derived podoplanin/EVs as drivers of cancer-associated thrombosis through CLEC-2.","method":"CAF characterization by immunofluorescence, podoplanin EV isolation and immunoblotting, platelet aggregation flow cytometry, venous thrombosis model (FeCl3), CLEC-2 depletion antibody (2A2B10)","journal":"Journal of thrombosis and haemostasis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CAF-derived EV mechanism with in vivo CLEC-2 depletion, single lab, single study","pmids":["37473844"],"is_preprint":false}],"current_model":"CLEC-2 (CLEC1B) is a platelet/megakaryocyte C-type lectin-like receptor that signals through a single cytoplasmic hemITAM (YXXL) motif: upon binding multivalent ligands (podoplanin, rhodocytin, fucoidan, heme, HIV-associated podoplanin), CLEC-2 dimerizes and translocates to lipid rafts, its YXXL is phosphorylated primarily by Syk (not Src), Syk is activated by cross-linking its tandem SH2 domains across two phosphorylated CLEC-2 monomers, and the resulting signaling cascade (Syk→PLCγ2→Ca²⁺, with positive feedback via ADP/TxA2 and Btk) drives platelet activation; structurally the ligand-binding site is on the non-canonical 'side' face of the CLEC-2 C-type lectin domain; in vivo CLEC-2–podoplanin signaling in platelets is essential for embryonic blood-lymphatic separation, lymph node integrity, lung development, cerebrovascular patterning, HSC maintenance (via megakaryocyte Thpo production), and regulates DVT, sepsis, and cancer-associated thrombosis, while CLEC-2 also functions on dendritic cells and macrophages to regulate migration and inflammatory macrophage trafficking."},"narrative":{"mechanistic_narrative":"CLEC1B (CLEC-2) is a platelet- and megakaryocyte-restricted C-type lectin-like receptor that couples ligand engagement to Syk-dependent tyrosine kinase signaling and serves as the platelet sensor for the mucin-type ligand podoplanin [PMID:16174766, PMID:17616532, PMID:25150298]. It was first isolated as the platelet receptor for the snake venom toxin rhodocytin and defined a signaling pathway in which receptor engagement triggers phosphorylation of a single cytoplasmic YXXL (hemITAM) motif, recruitment and activation of Syk, and downstream PLCγ2 activation [PMID:16174766]. Because CLEC-2 carries only one YXXL yet must engage a kinase that normally requires tandem ITAMs, the receptor functions as a non-disulfide-linked homodimer, allowing the two SH2 domains of a single Syk to be cross-linked across two phosphorylated monomers in a 2:1 stoichiometry [PMID:19824697, PMID:20154219]. Signal initiation requires ligand-induced clustering, translocation to lipid rafts, and—unusually—uses Syk rather than Src family kinases as the primary kinase for hemITAM phosphorylation, with Src kinases acting downstream and with Btk and Gq/GPCR (ADP/TxA2) inputs providing positive feedback [PMID:21098033, PMID:20154214, PMID:25368330, PMID:28705934, PMID:31949019]. Crystallographic and mutational work places the ligand-binding site on the non-canonical 'side' face of the C-type lectin domain, where consecutive acidic residues of podoplanin and rhodocytin engage common arginine residues, with podoplanin recognition additionally requiring its sialylated disialyl-core1 O-glycan at Thr52 [PMID:17132623, PMID:17944973, PMID:25458834, PMID:30190281]. Beyond podoplanin and rhodocytin, CLEC-2 binds fucoidan, heme, and S100A13 [PMID:23341451, PMID:26418160, PMID:33843987]. In vivo, platelet/megakaryocyte CLEC-2–podoplanin signaling is essential for embryonic blood–lymphatic vessel separation, lymph node integrity, cerebrovascular patterning, and hematopoietic stem cell maintenance via megakaryocyte thrombopoietin production [PMID:20363774, PMID:22556408, PMID:24532804, PMID:26552707, PMID:25908104], and it regulates hemostasis and thrombosis, including a hemITAM-independent adhesive contribution to hemostasis [PMID:19641185, PMID:20525685, PMID:28835437]. The CLEC-2–podoplanin axis drives deep vein thrombosis, cancer-associated thrombosis, and cerebral venous thrombosis, and modulates inflammation in sepsis, liver injury, and macrophage trafficking, while CLEC-2 on dendritic cells and macrophages controls migration through podoplanin engagement [PMID:22884313, PMID:28104688, PMID:29269852, PMID:32321925, PMID:34163489, PMID:39195988, PMID:37473844].","teleology":[{"year":2005,"claim":"Established the existence and signaling logic of a novel platelet activation receptor, defining how a single-YXXL C-type lectin transduces a signal through Syk and PLCγ2.","evidence":"Rhodocytin affinity chromatography for receptor identification with Syk/PLCγ2 knockout mouse platelets and Src-family inhibitor","pmids":["16174766"],"confidence":"High","gaps":["Endogenous physiological ligand unknown at this stage","Stoichiometry of Syk engagement to a single YXXL unexplained"]},{"year":2006,"claim":"Provided the first atomic structure of the CLEC-2 ectodomain, localizing the ligand-binding surface and arguing against large ligand-induced conformational change.","evidence":"X-ray crystallography at 1.6 Å with site-directed mutagenesis and surface plasmon resonance for rhodocytin binding","pmids":["17132623"],"confidence":"High","gaps":["Structure of the endogenous ligand complex not resolved","How binding triggers cytoplasmic signaling not addressed"]},{"year":2007,"claim":"Identified podoplanin as the endogenous CLEC-2 ligand and defined the glycan and protein determinants of recognition, transforming CLEC-2 from a toxin receptor into a physiological signaling axis.","evidence":"Flow cytometry, recombinant CLEC-2 inhibition and platelet aggregation assays, plus deletion-mutant Fc chimeras and synthetic disialyl-core1 glycopeptides identifying Thr52 O-glycan dependence","pmids":["17616532","17944973"],"confidence":"High","gaps":["Affinity and direct binding not yet quantified","Tissue sources of podoplanin engaging platelets not mapped"]},{"year":2007,"claim":"Dissected the cytoplasmic signaling motif, showing both Syk SH2 domains and the upstream DEDG triacidic sequence are required, hinting that receptor dimerization underlies hemITAM function.","evidence":"YXXL and DEDG mutagenesis with Syk SH2-domain mutants and chimeric receptors in cell-line signaling assays","pmids":["17339324"],"confidence":"High","gaps":["Physical demonstration of receptor dimerization still lacking","Role of DEDG residues in phosphorylation mechanism unresolved"]},{"year":2008,"claim":"Quantified direct CLEC-2–podoplanin binding and the rhodocytin oligomeric state, supporting a clustering model for receptor activation, and identified G6b-B as an inhibitory counter-receptor.","evidence":"SPR affinity measurement (24.5 µM) with DT-40 cell activation, rhodocytin crystallography with docking, and NFAT reporter/ITIM mutant analysis of G6b-B","pmids":["18215137","18583525","18955485"],"confidence":"High","gaps":["Clustering mechanism supported only by computational docking","G6b-B phosphatase effector remained unidentified (SHP1/2/SHIP-independent)"]},{"year":2009,"claim":"Resolved the long-standing single-YXXL paradox by demonstrating CLEC-2 forms a homodimer that allows one Syk to bridge two phosphorylated monomers, and showed CLEC-2 is essential for platelet aggregation and thrombosis.","evidence":"Multiple biophysical methods (BRET, gel filtration, AUC, MALS, SPR) for dimerization, plus antibody depletion in mice with flow-chamber aggregation and in vivo thrombosis assays","pmids":["19824697","19641185"],"confidence":"High","gaps":["Whether dimers pre-exist or form upon ligand binding not fully resolved here","Contribution of CLEC-2 to bleeding versus thrombosis ambiguous across models"]},{"year":2009,"claim":"Extended CLEC-2 function beyond platelets to neutrophils, where it acts as a Syk-coupled phagocytic and cytokine-inducing receptor, and established Syk as upstream of CLEC-2's own phosphorylation.","evidence":"Primary neutrophil flow cytometry, phagocytosis and cytokine assays, chimeric receptor mutagenesis, plus Syk inhibitor R406 in human platelets","pmids":["19299712","19422460"],"confidence":"Medium","gaps":["Neutrophil CLEC-2 expression restricted and context-dependent (single lab)","Mechanism by which Syk feeds back to phosphorylate the hemITAM not fully defined"]},{"year":2010,"claim":"Defined the proximal biochemistry and biophysics of activation—Syk (not Src) as the hemITAM kinase, lipid-raft translocation, actin/Rac1/secretion dependence, and 2:1 Syk:hemITAM cross-linking—and established CLEC-2's essential developmental role in blood–lymphatic separation.","evidence":"Kinase-specific knockout mouse platelets, raft fractionation and cholesterol depletion, peptide pull-down/SPR/tryptophan fluorescence/EM, and constitutive plus PF4-Cre conditional knockouts with intravital microscopy and genetic epistasis to PDPN/SLP-76","pmids":["20154219","21098033","20154214","20525685","20363774"],"confidence":"High","gaps":["Precise trigger ordering clustering, raft entry, and phosphorylation incompletely resolved","Downstream secreted mediators of lymphatic separation not yet identified"]},{"year":2011,"claim":"Showed lineage-restricted CLEC-2/Syk signaling in platelets directly controls lymphatic and cerebrovascular development by acting on endothelial cell migration and adhesion.","evidence":"Megakaryocyte/platelet-lineage conditional Cre-lox knockouts with LEC migration/adhesion assays comparing platelets versus releasate","pmids":["22186994"],"confidence":"High","gaps":["Identity of platelet-borne effector versus contact signal not resolved here","Mechanism linking platelet signaling to endothelial behavior unspecified"]},{"year":2012,"claim":"Identified secreted mediators (BMP-9) and signaling outputs (RhoA/Rac1, membrane protrusions) downstream of CLEC-2, extending its role to dendritic cell migration and confirming platelet CLEC-2 governs blood/lymphatic vessel separation.","evidence":"Platelet-specific and DC-specific conditional knockouts with LEC tube-formation/migration assays, BMP-9 identification, and RhoA/myosin/Vav/Rac1 readouts with in vivo DC trafficking","pmids":["22556408","22884313"],"confidence":"High","gaps":["Relative contribution of BMP-9 versus other mediators in vivo unquantified","How DC CLEC-2 signaling integrates with platelet CLEC-2 functions unclear"]},{"year":2013,"claim":"Established CLEC-2 as a tissue-organizing receptor in lymphoid stroma, releasing S1P to maintain HEV integrity, and confirmed fucoidan as a CLEC-2 agonist.","evidence":"FRC- and platelet-specific conditional knockouts with platelet-infusion rescue and S1P pathway analysis, plus platelet-specific CLEC-2 knockout and FcRγ-null platelets with fucoidan","pmids":["23995678","23341451"],"confidence":"High","gaps":["Whether S1P acts directly or via additional mediators on HEVs not fully dissected","Physiological source of fucoidan-like sulfated ligands in vivo unknown"]},{"year":2014,"claim":"Defined the structural basis of dual-ligand recognition on the non-canonical side face, demonstrated ligand-driven membrane clustering, and revealed CLEC-2's roles in lymph node morphogenesis, stromal contractility, and as an activated-platelet marker.","evidence":"CLEC-2–podoplanin and CLEC-2–rhodocytin co-crystal structures, supported-lipid-bilayer FLIM/dSTORM clustering with Src/Syk perturbation, conditional knockouts for lymph node and FRC contractility phenotypes, and antibody-based shedding/copy-number quantitation","pmids":["25458834","25368330","24532804","25347465","25150298"],"confidence":"High","gaps":["Structural model of the full clustered signaling assembly not solved","Mechanism converting clustering to hemITAM phosphorylation at the membrane incomplete"]},{"year":2015,"claim":"Placed CLEC-2 at the center of bone marrow niche function—driving megakaryocyte thrombopoietin and CCL5 outputs for HSC maintenance and proplatelet formation—identified additional ligands and signaling branches, and dissected antibody-induced internalization.","evidence":"Megakaryocyte-specific conditional knockouts with Syk/Lcp2/Plcg2 knockdown and Thpo rescue, MK-FRC co-culture with CCL5, S100A13 identification by protein array/SPR, keratinocyte RhoA/E-cadherin assays, 2CP-defined Akt1/PDK1 and PKCµ branches, and Syk-KO/Src-inhibitor internalization studies","pmids":["26552707","26796360","26418160","26597882","26528756","25795918"],"confidence":"High","gaps":["Baseline VSMC ligand distinct from S100A13 remains unidentified","Physiological relevance of Akt1/PDK1 and PKCµ branches beyond pharmacology unclear"]},{"year":2017,"claim":"Separated CLEC-2's signaling-dependent developmental role from a hemITAM-independent adhesive role in hemostasis, defined GPCR/Gq potentiation of proximal signaling, and linked the CLEC-2–podoplanin axis to DVT and to anti-inflammatory control of sepsis.","evidence":"Y7A hemITAM-null knockin mouse with anti-CLEC-2 Fab', Gq-KO/UBO-QIC biochemistry, IVC stenosis DVT model with anti-podoplanin neutralization, and platelet-specific knockouts in two sepsis models","pmids":["28835437","28705934","28104688","29269852"],"confidence":"High","gaps":["Molecular nature of the hemITAM-independent adhesive function undefined","Identity of podoplanin-expressing cells driving the sepsis phenotype incompletely mapped"]},{"year":2018,"claim":"Established CLEC-2 as an initiator of platelet–leukocyte inflammatory axes (dengue), refined ligand-binding residues amenable to inhibition, defined the rhodocytin oligomeric requirement, and identified CD37 as a membrane organizer of CLEC-2.","evidence":"CLEC-2 blockade with EV characterization and dengue lethality model, SPR/docking/mutagenesis (N120/N210/K211) with cobalt hematoporphyrin, recombinant rhodocytin oligomer mutagenesis with metastasis models, and CD37 co-IP with Cd37-KO myeloid migration assays","pmids":["31160588","30190281","29488681","30185523"],"confidence":"High","gaps":["CD37–CLEC-2 interaction based on single-lab co-IP without structural detail","Whether identified inhibitor binding sites overlap the physiological podoplanin interface only partially resolved"]},{"year":2019,"claim":"Characterized distinct mechanisms of CLEC-2 release—MMP-2-mediated ectodomain shedding and microparticle-associated whole-receptor release—distinguishing it from GPVI shedding biology.","evidence":"Metalloproteinase inhibitor panel with ADAM10-specific inhibitor, microparticle characterization, and ELISA for plasma fragments","pmids":["31165998"],"confidence":"Medium","gaps":["Single-lab pharmacological inference of MMP-2 involvement","Functional consequence of soluble versus microparticle CLEC-2 unestablished"]},{"year":2020,"claim":"Defined a CLEC-2 signaling role in restraining reparative inflammation, where platelet CLEC-2 suppresses TNF-α-driven neutrophil recruitment limiting liver recovery.","evidence":"CLEC-2 antibody blockade in APAP and CCl4 liver injury models with TNF-α and neutrophil readouts and human validation","pmids":["32321925"],"confidence":"High","gaps":["Ligand engaging CLEC-2 in the injured liver not defined","Direct cellular target of the TNF-α suppression unclear"]},{"year":2021,"claim":"Identified Btk-dependent positive feedback unique to human CLEC-2 signaling, a new heme ligand shared with GPVI, and a secretion-independent mechanism by which platelet CLEC-2 controls inflammatory macrophage trafficking.","evidence":"Ibrutinib/acalabrutinib titration with XLA patient platelets and species comparison, SPR hemin binding with CLEC-2/GPVI double-knockout platelets and kidney injury model, and platelet-specific knockout with CLEC-2-Fc rescue and podoplanin/CD44/ERM interaction studies","pmids":["31949019","33843987","34163489"],"confidence":"High","gaps":["Mouse-versus-human divergence in Btk feedback mechanistically incompletely explained","How CLEC-2 engagement modulates macrophage podoplanin/CD44/ERM at the molecular level not fully resolved"]},{"year":2022,"claim":"Placed CLEC-2 as a downstream amplifier of GPIbα–VWF signaling driving αIIbβ3 activation and demonstrated cooperative CLEC-2/GPIIb-IIIa signaling can precipitate acute cerebral venous thrombosis.","evidence":"Platelet-specific knockout with VWF binding and αIIbβ3 activation assays in a TTP model, and INU1-Fab/GPIIb-IIIa intervention with transcranial intravital microscopy in a CVT model","pmids":["35157766","39195988"],"confidence":"High","gaps":["Molecular link between GPIbα engagement and CLEC-2 activation undefined","Whether the CVT-like syndrome reflects physiological CLEC-2 ligand engagement or antibody-specific crosslinking unclear"]},{"year":2023,"claim":"Implicated CLEC-2 in cancer-associated thrombosis via cancer-associated fibroblast podoplanin and podoplanin-bearing extracellular vesicles activating platelets.","evidence":"CAF and EV characterization, CLEC-2-dependent platelet aggregation, and antibody-induced CLEC-2 depletion in tumor-bearing FeCl3 thrombosis model","pmids":["37473844"],"confidence":"Medium","gaps":["Single-lab single-study evidence","Relative in vivo contribution of CAF-surface versus EV podoplanin unquantified"]},{"year":null,"claim":"The molecular basis of CLEC-2's hemITAM-independent adhesive function in hemostasis, and the structure of the full clustered ligand–CLEC-2–Syk signaling assembly at the membrane, remain unresolved.","evidence":"No timeline study reconstitutes the complete activated signaling complex or defines the non-signaling adhesive mechanism","pmids":[],"confidence":"High","gaps":["No structure of the clustered receptor–Syk signaling unit","Adhesive/physical hemostatic function lacks a defined molecular partner","Possible homophilic CLEC-2 interactions in thrombus stabilization not biochemically defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,15,20]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,2,3]},{"term_id":"GO:0001618","term_label":"virus receptor activity","supporting_discovery_ids":[38]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,16,24]}],"pathway":[{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[9,12,37,48]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,14,15,16]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[19,35,44,45]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[13,18,27,33]}],"complexes":[],"partners":["PDPN","SYK","CD37","G6B"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9P126","full_name":"C-type lectin domain family 1 member B","aliases":["C-type lectin-like receptor 2","CLEC-2"],"length_aa":229,"mass_kda":26.6,"function":"C-type lectin-like receptor that serves as a platelet receptor for the lymphatic endothelial marker PDPN (PubMed:18215137). Plays an essential role in blood/lymphatic vessel separation and thrombus formation through homophilic association (PubMed:22556408). Acts as a negative regulator of lymphatic endothelial cell behavior by inhibiting proliferation, migration, and tube formation (PubMed:22556408). Upon ligand binding, triggers a signaling cascade via sequential activation of SRC and SYK tyrosine kinases, leading to activation of PLCG2 (PubMed:18955485). Through this pathway, mediates platelet adhesion, aggregation, and secretion in response to PDPN-expressing cells. During embryonic development, plays a central role in cerebrovascular formation and blood/lymphatic vessel separation. Interacts with PDPN on neuroepithelial cells to promote platelet activation, thereby ensuring maturation and integrity of cerebrovascular structures and preventing hemorrhage (Microbial infection) Acts as a receptor for the platelet-aggregating snake venom protein rhodocytin. Rhodocytin binding leads to tyrosine phosphorylation and this promotes the binding of spleen tyrosine kinase (SYK) and initiation of downstream tyrosine phosphorylation events and activation of PLCG2 (PubMed:16174766, PubMed:18955485) (Microbial infection) Acts as an attachment factor for Human immunodeficiency virus type 1 (HIV-1) and facilitates its capture by platelets (PubMed:16940507)","subcellular_location":"Membrane","url":"https://www.uniprot.org/uniprotkb/Q9P126/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CLEC1B","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CLEC1B","total_profiled":1310},"omim":[{"mim_id":"606783","title":"C-TYPE LECTIN DOMAIN FAMILY 1, MEMBER B; 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cardiovascular research","url":"https://pubmed.ncbi.nlm.nih.gov/39195988","citation_count":21,"is_preprint":false},{"pmid":"30185523","id":"PMC_30185523","title":"C-type lectin-like receptor 2 (CLEC-2)-dependent dendritic cell migration is controlled by tetraspanin CD37.","date":"2018","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/30185523","citation_count":21,"is_preprint":false},{"pmid":"28705934","id":"PMC_28705934","title":"Gq pathway regulates proximal C-type lectin-like receptor-2 (CLEC-2) signaling in platelets.","date":"2017","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28705934","citation_count":21,"is_preprint":false},{"pmid":"25694214","id":"PMC_25694214","title":"Platelet adhesion to podoplanin under flow is mediated by the receptor CLEC-2 and stabilised by Src/Syk-dependent platelet signalling.","date":"2015","source":"Thrombosis and haemostasis","url":"https://pubmed.ncbi.nlm.nih.gov/25694214","citation_count":21,"is_preprint":false},{"pmid":"33165736","id":"PMC_33165736","title":"A Role of the Podoplanin-CLEC-2 Axis in Promoting Inflammatory Response After Ischemic Stroke in Mice.","date":"2020","source":"Neurotoxicity research","url":"https://pubmed.ncbi.nlm.nih.gov/33165736","citation_count":21,"is_preprint":false},{"pmid":"35157766","id":"PMC_35157766","title":"Deletion of platelet CLEC-2 decreases GPIbα-mediated integrin αIIbβ3 activation and decreases thrombosis in TTP.","date":"2022","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/35157766","citation_count":20,"is_preprint":false},{"pmid":"30978634","id":"PMC_30978634","title":"Elevated plasma levels of soluble C-type lectin-like receptor 2 (CLEC2) in patients with thrombotic microangiopathy.","date":"2019","source":"Thrombosis research","url":"https://pubmed.ncbi.nlm.nih.gov/30978634","citation_count":20,"is_preprint":false},{"pmid":"34163489","id":"PMC_34163489","title":"CLEC-2 Prevents Accumulation and Retention of Inflammatory Macrophages During Murine Peritonitis.","date":"2021","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/34163489","citation_count":19,"is_preprint":false},{"pmid":"16511244","id":"PMC_16511244","title":"Crystallization and X-ray diffraction analysis of human CLEC-2.","date":"2005","source":"Acta crystallographica. Section F, Structural biology and crystallization communications","url":"https://pubmed.ncbi.nlm.nih.gov/16511244","citation_count":19,"is_preprint":false},{"pmid":"22205394","id":"PMC_22205394","title":"Identification of a chicken CLEC-2 homologue, an activating C-type lectin expressed by thrombocytes.","date":"2011","source":"Immunogenetics","url":"https://pubmed.ncbi.nlm.nih.gov/22205394","citation_count":19,"is_preprint":false},{"pmid":"31808911","id":"PMC_31808911","title":"Platelets and cancer-associated thrombosis: focusing on the platelet activation receptor CLEC-2 and podoplanin.","date":"2019","source":"Hematology. American Society of Hematology. Education Program","url":"https://pubmed.ncbi.nlm.nih.gov/31808911","citation_count":18,"is_preprint":false},{"pmid":"24736277","id":"PMC_24736277","title":"Human podoplanin-positive monocytes and platelets enhance lymphangiogenesis through the activation of the podoplanin/CLEC-2 axis.","date":"2014","source":"Molecular therapy : the journal of the American Society of Gene Therapy","url":"https://pubmed.ncbi.nlm.nih.gov/24736277","citation_count":18,"is_preprint":false},{"pmid":"32129691","id":"PMC_32129691","title":"Lymphatic blood filling in CLEC-2-deficient mouse models.","date":"2020","source":"Platelets","url":"https://pubmed.ncbi.nlm.nih.gov/32129691","citation_count":17,"is_preprint":false},{"pmid":"35817083","id":"PMC_35817083","title":"CLEC-2 Supports Platelet Aggregation in Mouse but not Human Blood at Arterial Shear.","date":"2022","source":"Thrombosis and haemostasis","url":"https://pubmed.ncbi.nlm.nih.gov/35817083","citation_count":17,"is_preprint":false},{"pmid":"19091630","id":"PMC_19091630","title":"Thrombomodulation via CLEC-2 targeting.","date":"2008","source":"Current opinion in pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/19091630","citation_count":16,"is_preprint":false},{"pmid":"32396849","id":"PMC_32396849","title":"Control of Platelet CLEC-2-Mediated Activation by Receptor Clustering and Tyrosine Kinase Signaling.","date":"2020","source":"Biophysical journal","url":"https://pubmed.ncbi.nlm.nih.gov/32396849","citation_count":16,"is_preprint":false},{"pmid":"31742327","id":"PMC_31742327","title":"Fucoidan suppresses the gastric cancer cell malignant phenotype and production of TGF-β1 via CLEC-2.","date":"2020","source":"Glycobiology","url":"https://pubmed.ncbi.nlm.nih.gov/31742327","citation_count":16,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":51016,"output_tokens":14284,"usd":0.183654,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":26676,"output_tokens":7403,"usd":0.159227,"stage2_stop_reason":"end_turn"},"total_usd":0.342881,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"CLEC-2 was identified as the platelet receptor for snake venom rhodocytin via rhodocytin affinity chromatography. The cytosolic tail contains a single YXXL motif that undergoes tyrosine phosphorylation upon activation by rhodocytin. Signaling is abolished in Syk-deficient and PLCγ2-deficient murine platelets and is inhibited by the Src family kinase inhibitor PP2, defining a novel pathway: CLEC-2 activation → YXXL phosphorylation → Syk binding → downstream tyrosine phosphorylation → PLCγ2 activation.\",\n      \"method\": \"Rhodocytin affinity chromatography, cell-line expression, phosphorylation assays, Syk/PLCγ2/LAT/SLP-76/Vav1/Vav3 knockout mouse platelets, pharmacological inhibition\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — receptor identification by biochemical purification, signaling confirmed by multiple genetic knockouts and pharmacological inhibition across two species\",\n      \"pmids\": [\"16174766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Crystal structure of the extracellular domain of human CLEC-2 was solved to 1.6 Å resolution. A semi-helical loop region and flanking residues dominate the ligand-binding surface. Mutational analysis and surface plasmon resonance binding studies identified key residues for rhodocytin binding. Major ligand-induced conformational change is unlikely given the compact fold, but ligand binding could induce a tilt of a 3-10 helical portion of the long loop region.\",\n      \"method\": \"X-ray crystallography (1.6 Å), site-directed mutagenesis, surface plasmon resonance\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with mutagenesis and SPR validation in a single rigorous study\",\n      \"pmids\": [\"17132623\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Podoplanin was identified as an endogenous ligand for platelet CLEC-2. Association between CLEC-2 and podoplanin was confirmed by flow cytometry and is dependent on sialic acid on O-glycans of podoplanin. Recombinant CLEC-2 inhibited platelet aggregation induced by podoplanin-expressing tumor cells or lymphatic endothelial cells, demonstrating CLEC-2 is the functional receptor responsible for podoplanin-induced platelet aggregation.\",\n      \"method\": \"Flow cytometry, recombinant CLEC-2 inhibition assay, platelet aggregation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (flow cytometry, functional inhibition), replicated across labs in subsequent studies\",\n      \"pmids\": [\"17616532\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CLEC-2 signaling requires its single cytosolic YXXL motif; mutation of either SH2 domain of Syk blocks CLEC-2 signaling despite only a single YXXL motif, indicating dimerization is required. The DEDG amino acid sequence upstream of the YXXL, particularly the glycine directly upstream of the tyrosine, is essential for CLEC-2 signaling. CLEC-2 and Dectin-1 share this novel hemITAM signaling mechanism, distinct from DC-SIGN which signals independently of its YXXL.\",\n      \"method\": \"YXXL mutant cell lines, SH2 domain Syk mutants, chimeric receptor analysis, cell line signaling assay, DEDG sequence mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mutational dissection of signaling motif with multiple cell line models and orthogonal approaches\",\n      \"pmids\": [\"17339324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Deletion mutant analysis of CLEC-2 (expressed as Fc chimeras) identified a podoplanin-recognition domain in CLEC-2. The interaction is dependent on both the disialyl-core1 O-glycan at Thr52 of podoplanin and the stereostructure of the podoplanin protein. A disialyl-core1-attached glycopeptide is specifically recognized by CLEC-2. Anti-podoplanin antibody NZ-1 suppressed both the podoplanin-CLEC-2 interaction and podoplanin-induced pulmonary metastasis in vivo.\",\n      \"method\": \"Deletion mutagenesis with Fc chimeras, glycopeptide synthesis and binding assays, in vivo metastasis model with antibody blockade\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — systematic deletion mutagenesis combined with in vitro and in vivo functional validation\",\n      \"pmids\": [\"17944973\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Podoplanin on renal cells (HEK-293T/podocytes) was identified as a direct CLEC-2 ligand. Surface plasmon resonance confirmed direct CLEC-2–podoplanin interaction with an affinity of 24.5 ± 3.7 μM, and this interaction is independent of glycosylation of CLEC-2 itself. Podoplanin on HEK-293T cells mediates both binding to CLEC-2 and activation of CLEC-2-transfected DT-40 B-cells.\",\n      \"method\": \"Surface plasmon resonance, recombinant CLEC-2 binding assay, CLEC-2-transfected DT-40 cell activation assay, podoplanin identification by proteomics\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct SPR binding measurement with cell-based functional confirmation, single lab\",\n      \"pmids\": [\"18215137\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Crystal structure of rhodocytin at 2.4 Å resolution revealed it is the first snake venom C-type lectin-like protein to assemble as a non-disulfide-linked (αβ)2 tetramer. The concave binding surface of rhodocytin is complementary to the CLEC-2 binding interface. Computational docking suggests the (αβ)2 rhodocytin tetramer induces clustering of CLEC-2 receptors on the platelet surface to trigger signaling.\",\n      \"method\": \"X-ray crystallography (2.4 Å), computational protein-protein docking, electrostatic and hydrophobicity analysis\",\n      \"journal\": \"Protein science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — crystal structure solved but clustering mechanism supported only by computational docking, not direct experiment\",\n      \"pmids\": [\"18583525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"G6b-B, a platelet immunoglobulin receptor with two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), inhibits both constitutive and agonist-induced signaling by CLEC-2 and GPVI via Src- and Syk-dependent pathways. This inhibitory activity is independent of SHP1, SHP2, and SHIP phosphatases.\",\n      \"method\": \"NFAT reporter assay in cell lines, ITIM tyrosine mutants, SHP1/SHP2/SHIP inhibitor/knockout experiments, Western blotting\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell line model plus genetic knockouts with multiple readouts, single lab\",\n      \"pmids\": [\"18955485\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CLEC-2 exists as a non-disulfide-linked homodimer, demonstrated by multiple orthogonal biochemical methods. This dimerization allows each Syk molecule to interact with two YXXL motifs (one from each CLEC-2 monomer), providing a mechanistic explanation for how a single YXXL-containing receptor can activate Syk which normally requires tandem YXXL (ITAM).\",\n      \"method\": \"Bioluminescence resonance energy transfer, co-immunoprecipitation, analytical gel filtration, surface plasmon resonance, multiangle light scattering, analytical ultracentrifugation, Western blotting\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple independent biophysical methods in one rigorous study confirming homodimerization\",\n      \"pmids\": [\"19824697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CLEC-2-deficient platelets displayed normal adhesion under flow but severely defective aggregate formation in vitro and in vivo. CLEC-2 deficiency caused increased bleeding times and profound protection from occlusive arterial thrombus formation, establishing an essential role for CLEC-2 in hemostasis and thrombosis.\",\n      \"method\": \"Anti-CLEC-2 antibody-induced depletion in mice, in vitro flow chamber aggregation assay, in vivo thrombosis models, bleeding time measurement\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — specific receptor depletion with defined phenotypic readouts in vitro and in vivo\",\n      \"pmids\": [\"19641185\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CLEC-2 is expressed on murine peripheral blood neutrophils (but not bone marrow or elicited inflammatory neutrophils), where it functions as a phagocytic activation receptor. Crosslinking of CLEC-2 on neutrophils leads to Syk recruitment and production of pro-inflammatory cytokines (TNF-α) in response to rhodocytin. The tyrosine-based cytoplasmic motif is required for these activities. Notably, CLEC-2 stimulation does not induce the respiratory burst.\",\n      \"method\": \"Antibody staining and flow cytometry, chimeric receptor analysis, phagocytosis assay, cytokine ELISA, Syk co-immunoprecipitation\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct functional characterization in primary cells with chimeric receptor mutagenesis, single lab\",\n      \"pmids\": [\"19299712\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The Syk inhibitor R406 markedly inhibited tyrosine phosphorylation of CLEC-2 itself and downstream Syk phosphorylation following CLEC-2 activation, while Syk phosphorylation downstream of GPVI was unaffected. This revealed a critical role for Syk in mediating phosphorylation of the CLEC-2 hemITAM, suggesting Syk functions both downstream and upstream of CLEC-2 phosphorylation.\",\n      \"method\": \"Platelet aggregometry, immunoprecipitation, Western blotting, pharmacological Syk inhibitor (R406)\",\n      \"journal\": \"Journal of thrombosis and haemostasis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological inhibitor with biochemical readouts in human platelets, single lab\",\n      \"pmids\": [\"19422460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CLEC-2-deficient mice are lethal at embryonic/neonatal stages due to disorganized, blood-filled lymphatic vessels and severe edema. Fetal liver cell transplantation demonstrated that CLEC-2 is involved in thrombus stabilization in vitro and in vivo, possibly through homophilic interactions. Loss of CLEC-2 did not significantly increase bleeding tendency.\",\n      \"method\": \"CLEC-2 knockout mouse generation, fetal liver cell transplantation chimeras, intravital thrombus formation assay, histology\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — constitutive and chimeric knockout with multiple phenotypic endpoints, replicated in other labs\",\n      \"pmids\": [\"20525685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Platelets regulate lymphatic vascular development by interacting with lymphatic endothelial cells through CLEC-2 binding to podoplanin. Genetic loss of CLEC-2 ablates podoplanin binding by platelets and causes embryonic lymphatic vascular defects identical to those in PDPN- or SLP-76-deficient animals. PF4-Cre-mediated deletion of Slp-76 specifically in platelets is sufficient to cause lymphatic vascular defects, placing CLEC-2→SLP-76 signaling in platelets upstream of blood-lymphatic separation.\",\n      \"method\": \"Conditional (PF4-Cre) and constitutive knockout mice, intravital microscopy of platelet aggregates on lymphatic endothelial cells, genetic epistasis\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with conditional knockout, replicated phenotype across multiple mouse models\",\n      \"pmids\": [\"20363774\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CLEC-2 activates Syk through dimerization: peptide pull-down, surface plasmon resonance, tryptophan fluorescence, and quantitative Western blotting demonstrated that Syk activation by CLEC-2 is mediated by cross-linking of the Syk tandem SH2 domains with two phosphorylated CLEC-2 YXXL motifs in a 2:1 stoichiometry. Cross-linking and electron microscopy confirmed CLEC-2 is present as a dimer in resting platelets and forms larger complexes upon activation.\",\n      \"method\": \"Peptide pull-down, surface plasmon resonance, tryptophan fluorescence, quantitative Western blotting, competition experiments, chemical cross-linking, electron microscopy\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal biophysical methods establishing dimerization mechanism in a single rigorous study\",\n      \"pmids\": [\"20154219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CLEC-2 phosphorylation (hemITAM) is abolished in the absence of Syk but not in the absence of major platelet Src family kinases (Fyn, Lyn, Src) or CD148, nor is it altered by PP2. PLCγ2 phosphorylation remains dependent on Src family kinases. This established that Syk—not Src—is the primary kinase for CLEC-2 hemITAM phosphorylation, with Src family kinases acting downstream.\",\n      \"method\": \"Syk-knockout, Fyn/Lyn/Src/CD148-knockout mouse platelets, PP2 pharmacological inhibition, Western blotting for CLEC-2 and PLCγ2 phosphorylation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple kinase-specific knockouts with biochemical readouts, establishes clear epistatic order\",\n      \"pmids\": [\"21098033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CLEC-2 translocates to lipid rafts upon ligand engagement, and this translocation is essential for hemITAM phosphorylation and signal initiation (shown by sucrose gradient ultracentrifugation and methyl-β-cyclodextrin treatment). HemITAM phosphorylation (but not translocation) additionally requires actin polymerization, Rac1 activation, and release of ADP and thromboxane A2.\",\n      \"method\": \"Sucrose gradient ultracentrifugation, methyl-β-cyclodextrin cholesterol depletion, actin polymerization inhibitors, Rac1 inhibitors, ADP/TxA2 receptor pharmacology, Western blotting\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mechanistic dissection with multiple orthogonal pharmacological and biochemical approaches in a single study\",\n      \"pmids\": [\"20154214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Conditional deletion of CLEC-2 or Syk specifically in the megakaryocyte/platelet lineage causes defects in brain vascular and lymphatic development. Platelets (not platelet releasate) directly modulate migration and intercellular adhesion of lymphatic endothelial cells through a pathway dependent on CLEC-2 and Syk.\",\n      \"method\": \"Conditional (megakaryocyte/platelet lineage) Cre-lox knockout mice, lymphatic endothelial cell migration and adhesion assays with platelets vs. platelet releasate, intravital microscopy\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-type-specific conditional knockouts with in vitro mechanistic dissection, independent replication in multiple labs\",\n      \"pmids\": [\"22186994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Platelet-specific deletion of CLEC-2 causes misconnection of blood/lymphatic vessels. CLEC-2-expressing (but not CLEC-2-deficient) platelets inhibit lymphatic endothelial cell migration, proliferation, and tube formation. Supernatants from activated platelets replicate this inhibition. BMP-9, present in platelets and released upon CLEC-2 activation, specifically inhibits LEC tube formation and plays a key role in blood/lymphatic vessel separation.\",\n      \"method\": \"Platelet-specific CLEC-2 knockout mice, LEC migration/proliferation/tube formation assays, activated platelet supernatant experiments, BMP-9 identification and functional assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout combined with cell biology assays and identification of secreted mediator BMP-9\",\n      \"pmids\": [\"22556408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"CLEC-2 engagement of podoplanin on stromal surfaces was necessary and sufficient to induce membrane protrusions in dendritic cells. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation, and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes.\",\n      \"method\": \"DC-specific CLEC-2 conditional knockout mice, RhoA/myosin light-chain phosphorylation assays, Vav/Rac1 inhibitor studies, DC migration assays in lymph nodes, live imaging\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout with multiple signaling pathway readouts and in vivo functional endpoints\",\n      \"pmids\": [\"22884313\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The upstream triacidic amino acid (DEDG) sequence in the CLEC-2 cytoplasmic tail is required for signaling through the hemITAM. Surface plasmon resonance and phosphorylation studies demonstrated that the triacidic amino acids are required for YXXL phosphorylation. HemITAM signaling is restricted to Syk (not ZAP-70), shown using Syk-deficient and ZAP-70-deficient cell lines.\",\n      \"method\": \"Site-directed mutagenesis of DEDG sequence, surface plasmon resonance, phosphorylation assays, Syk-deficient and ZAP-70-deficient cell lines\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis combined with SPR and biochemical phosphorylation assays, single lab\",\n      \"pmids\": [\"23264619\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Podoplanin (PDPN) on fibroblastic reticular cells (FRCs) surrounding high endothelial venules acts as an activating ligand for platelet CLEC-2. CLEC-2 activation induces release of sphingosine-1-phosphate (S1P) from platelets, which promotes VE-cadherin expression on HEVs. Mice lacking FRC podoplanin or platelet CLEC-2 showed reduced VE-cadherin on HEVs; infusion of wild-type platelets restored HEV integrity in CLEC-2-deficient mice.\",\n      \"method\": \"Conditional knockout mice (FRC-specific PDPN, platelet-specific CLEC-2), VE-cadherin measurement, platelet infusion rescue experiment, S1P pathway analysis, ex vivo HEV assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockouts with rescue experiment, identification of S1P as downstream mediator, multiple orthogonal methods\",\n      \"pmids\": [\"23995678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Fucoidan (a sulfated polysaccharide) induces platelet activation through CLEC-2 via a Src family kinase (SFK)- and Syk-dependent pathway. Fucoidan-induced platelet activation was completely abolished in platelet-specific CLEC-2 knockout murine platelets, but only slightly inhibited in FcRγ-chain null mice, establishing CLEC-2 as the primary receptor.\",\n      \"method\": \"Platelet-specific CLEC-2 knockout mice, FcRγ-null mice, SFK/Syk inhibitors, aggregometry, phosphorylation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic receptor-specific knockouts plus pharmacological inhibitors with defined cellular phenotype\",\n      \"pmids\": [\"23341451\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure of CLEC-2 in complex with O-glycosylated podoplanin peptide and with non-glycosylated rhodocytin revealed that both ligands bind to the non-canonical 'side' face of CLEC-2. Common interaction involves consecutive acidic residues on ligands binding to the same arginine residues on CLEC-2. For podoplanin, the sialic acid carboxyl group provides a second binding interaction; for rhodocytin, the C-terminus of the α-subunit interacts differently at this second site.\",\n      \"method\": \"X-ray crystallography of CLEC-2–ligand complexes\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — comparative crystal structures of CLEC-2 with two different ligands, single lab\",\n      \"pmids\": [\"25458834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CLEC-2 expression on platelets is not regulated by proteolytic shedding (unlike GPVI and FcγRIIa, which are cleaved upon CLEC-2 activation). CLEC-2 is restricted to platelets with ~2000 copies per cell. CLEC-2 and GPVI are expressed on CD41+ microparticles in megakaryocyte cultures, but microparticles derived from activated platelets only express CLEC-2, making CLEC-2 a marker for activated platelet-derived microparticles.\",\n      \"method\": \"Monoclonal antibody-based flow cytometry, CLEC-2 copy number quantitation, platelet activation and shedding assays, microparticle characterization\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple functional assays but focused primarily on receptor regulation, single lab\",\n      \"pmids\": [\"25150298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Podoplanin on FRCs regulates actomyosin contractility in FRCs through CLEC-2. Under resting conditions, podoplanin endows FRCs with contractile function and maintains tension in the reticular network. Upon inflammation, CLEC-2 on mature DCs potently attenuates podoplanin-mediated contractility in FRCs, resulting in FRC relaxation and reduced tissue stiffness.\",\n      \"method\": \"Collagen gel contraction assay, myosin light chain phosphorylation assays, conditional knockout mice, 3D tissue imaging, DC-FRC co-culture\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic models combined with functional assays of cytoskeletal contractility, published in high-impact journal\",\n      \"pmids\": [\"25347465\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Syk and Src family kinases in platelets promote clustering of CLEC-2 and its ligand podoplanin on supported lipid bilayers containing mobile podoplanin. CLEC-2-bound podoplanin clusters migrate to the center of the platelet, forming a single structure. Fluorescence lifetime imaging demonstrated molecules within these clusters are within 10 nm. Clusters are disrupted by Src/Syk inhibition.\",\n      \"method\": \"Supported lipid bilayer system with mobile podoplanin, fluorescence lifetime imaging microscopy (FLIM), direct stochastic optical reconstruction microscopy (dSTORM), Src/Syk inhibitors, Syk-deficient mouse platelets\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple advanced microscopy techniques with pharmacological and genetic validation\",\n      \"pmids\": [\"25368330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CLEC-2 deficiency results in absence of lymph nodes at birth due to defective lymphatic endothelial cell proliferation. Platelet-specific CLEC-2 deficiency (PF4-Cre) leads to blood-filled lymph nodes and fibrosis (without a proliferative defect), indicating CLEC-2 expression in platelets specifically is required for lymph node integrity. This phenotype is independent of the lymphatic endothelial proliferation defect seen with constitutive CLEC-2 deletion.\",\n      \"method\": \"Constitutive and PF4-Cre conditional CLEC-2 knockout mice, bone marrow chimeras, lymph node histology, LEC proliferation assays, immune response assays\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — dissection of platelet-specific vs. global CLEC-2 function using conditional and chimeric approaches\",\n      \"pmids\": [\"24532804\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CLEC-2 in megakaryocytes mediates production of thrombopoietin (Thpo) and other factors required for HSC maintenance. Megakaryocyte-specific CLEC-2 knockout mice produced lower levels of Thpo and showed reduced HSC quiescence and repopulation potential with extramedullary hematopoiesis. Knockdown of downstream CLEC-2 signaling molecules (Syk, Lcp2, Plcg2) in megakaryocytes decreased Thpo expression. Administration of recombinant Thpo restored stem cell potential.\",\n      \"method\": \"Megakaryocyte-specific CLEC-2 conditional knockout mice, Syk/Lcp2/Plcg2 siRNA knockdown in megakaryocytes, Thpo measurement, HSC quiescence and transplantation assays, recombinant Thpo rescue\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout with signaling pathway knockdown, Thpo identification as downstream mediator, rescue experiment\",\n      \"pmids\": [\"26552707\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Podoplanin-positive periarteriolar BM fibroblastic reticular cell (FRC)-like cells promote megakaryocyte growth and proplatelet formation through CLEC-2/podoplanin interaction. Megakaryocyte-specific CLEC-2 conditional knockout mice showed decreased immature megakaryocytes. The CLEC-2/podoplanin interaction induces BM FRC-like cells to secrete CCL5, which facilitates proplatelet formation.\",\n      \"method\": \"Megakaryocyte-specific CLEC-2 conditional knockout mice, megakaryocyte-FRC co-culture, recombinant podoplanin stimulation, CCL5 secretion measurement, immunofluorescence of BM sections\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout combined with co-culture identification of CCL5 as downstream mediator\",\n      \"pmids\": [\"26796360\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Podoplanin/CLEC-2 signaling regulates keratinocyte migration via modulating E-cadherin expression through RhoA signaling. Down-regulation of podoplanin in keratinocytes inhibited their migration and was correlated with upregulation of E-cadherin. Both platelets and recombinant CLEC-2 inhibited keratinocyte migration, with downregulation of RhoA activity and upregulation of E-cadherin.\",\n      \"method\": \"Podoplanin siRNA knockdown in NHEKs, platelet/CLEC-2 co-culture migration assays, wound healing assay, RhoA activity measurement, E-cadherin Western blotting\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown and recombinant protein with biochemical pathway readouts, single lab\",\n      \"pmids\": [\"26597882\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Vascular smooth muscle cells (VSMCs) stimulate platelets and facilitate thrombus formation through platelet CLEC-2. Protein arrays and Biacore analysis identified S100A13 as a CLEC-2 ligand in VSMCs. S100A13 is released upon oxidative stress and immobilized (but not suspended) S100A13 significantly increases thrombus formation on collagen-coated surfaces. However, surface S100A13 on normal VSMCs does not activate platelets, indicating another CLEC-2 ligand mediates baseline VSMC–platelet interaction.\",\n      \"method\": \"Recombinant CLEC-2 binding assays, flow cytometry, immunocytochemistry, thrombus formation assay under flow, FeCl3 animal thrombosis model, protein array, surface plasmon resonance (Biacore)\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple biochemical and functional assays identifying S100A13 as a novel CLEC-2 ligand, single lab\",\n      \"pmids\": [\"26418160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CLEC-2 is internalized from the platelet surface upon anti-CLEC-2 antibody (INU1) treatment through a Src-family kinase-dependent mechanism, followed by intracellular degradation. This internalization is independent of Syk. Syk-deficient mice showed preserved CLEC-2 internalization but largely prevented antibody-induced thrombocytopenia, mechanistically uncoupling CLEC-2 downregulation from thrombocytopenia.\",\n      \"method\": \"Platelet-specific Syk knockout mice, Src family kinase inhibitors, flow cytometry for CLEC-2 surface expression, in vitro and in vivo INU1 treatment\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic (Syk KO) and pharmacological approaches dissect internalization mechanism in vitro and in vivo\",\n      \"pmids\": [\"25795918\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Podoplanin on pericytes/neuro-epithelium interacts with CLEC-2 on platelets to guide cerebrovascular patterning and integrity during development. Cerebral vessels were tortuous and aberrantly patterned at E10.5 in podoplanin- and CLEC-2-deficient mice; defective pericyte recruitment and endothelial-pericyte misconnections were observed. Platelet α-granule and dense granule secretion is required, as embryos deficient in platelet GPIIb or granule secretion also developed cerebral hemorrhaging.\",\n      \"method\": \"Conditional and constitutive CLEC-2/podoplanin knockout mice, nestin-Cre podoplanin deletion, 3D light-sheet microscopy, immunofluorescence, electron microscopy\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple conditional knockout lines with advanced imaging modalities\",\n      \"pmids\": [\"25908104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Platelet-specific deficiency of CLEC-2 protects mice against deep vein thrombosis (DVT) in an IVC stenosis model. Podoplanin is expressed in the IVC wall near the abluminal endothelium and is upregulated after 48h stenosis in mice that develop thrombi. Inhibition of podoplanin with a neutralizing antibody reduced thrombus size, establishing the CLEC-2–podoplanin axis as a mechanistic driver of DVT.\",\n      \"method\": \"Inducible CLEC-2 knockout and platelet-specific CLEC-2 knockout mice, IVC stenosis model, platelet transfusion rescue, anti-podoplanin neutralizing antibody, immunofluorescence of podoplanin localization\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple conditional knockout approaches with ligand neutralization and localization studies in defined DVT model\",\n      \"pmids\": [\"28104688\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Platelet-specific deletion of CLEC-2 (but not GPVI) leads to enhanced systemic inflammation and accelerated organ injury in two mouse models of sepsis. CLEC-2 deficiency is associated with reduced numbers of podoplanin-expressing macrophages despite increased cytokines. Pharmacological inhibition of CLEC-2–podoplanin interaction regulates immune cell infiltration, identifying the podoplanin–CLEC-2 axis as an anti-inflammatory pathway in sepsis.\",\n      \"method\": \"Platelet-specific CLEC-2 and GPVI conditional knockout mice, LPS i.p. and cecal ligation and puncture sepsis models, cytokine measurement, immune cell counting, pharmacological CLEC-2–podoplanin inhibition\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-type specific knockouts with two independent disease models and pharmacological validation\",\n      \"pmids\": [\"29269852\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Gq signaling (via PLCβ-PKCα pathway, possibly regulating Src family kinases) potentiates the most proximal event in CLEC-2 signaling—the CLEC-2 receptor tyrosine phosphorylation itself—in response to TxA2 and other GPCR agonists. Using the Gq inhibitor UBO-QIC and Gq-knockout murine platelets, Gq signaling was demonstrated to be essential for GPCR-mediated potentiation of Syk phosphorylation downstream of CLEC-2.\",\n      \"method\": \"Gq-specific inhibitor (UBO-QIC), Gq-knockout mouse platelets, P2Y12/PAR/TP receptor agonists, Western blotting for CLEC-2 and Syk phosphorylation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic (Gq KO) and pharmacological approaches with biochemical readouts identifying Gq-PLCβ-PKCα-SFK as regulatory axis\",\n      \"pmids\": [\"28705934\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CLEC-2 contributes to hemostasis through a mechanism independent of classical hemITAM signaling. A knockin mouse expressing hemITAM-null CLEC-2 (Y7A KI) phenocopied blood-lymphatic mixing/lethality of CLEC-2 KO but not the hemostatic/thrombotic defect. Treatment of Y7A KI mice with function-blocking anti-CLEC-2 Fab' resulted in a thrombus formation defect, revealing a hemITAM signaling-independent (physical/adhesive) role for CLEC-2 in hemostasis.\",\n      \"method\": \"CLEC-2 Y7A hemITAM knockin mouse model, anti-CLEC-2 Fab' treatment, thrombosis assays in vitro and in vivo\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — novel knockin mouse separating signaling from non-signaling functions, rigorous genetic design\",\n      \"pmids\": [\"28835437\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Dengue virus activates platelets via CLEC-2 to release extracellular vesicles (exosomes and microvesicles). These EVs further activate CLEC5A and TLR2 on neutrophils and macrophages, inducing NET formation and pro-inflammatory cytokine release. Simultaneous blockade of CLEC5A and TLR2 (not CLEC-2) increased survival from DV infection from 30% to 90%, establishing CLEC-2 as the upstream initiator of this platelet-leukocyte inflammatory axis.\",\n      \"method\": \"CLEC-2 functional blocking, EV isolation and characterization, CLEC5A/TLR2 blockade, NET formation assay, cytokine measurement, mouse dengue lethality model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mechanistic dissection with multiple pharmacological interventions and in vivo model with survival endpoint\",\n      \"pmids\": [\"31160588\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Surface plasmon resonance and molecular docking identified novel podoplanin-binding sites on CLEC-2: N120, N210, and K211. Cobalt hematoporphyrin (Co-HP) binds directly to CLEC-2 at these sites and inhibits CLEC-2–podoplanin interaction. These binding sites were confirmed by CLEC-2 mutants with alterations in N120 and/or K211.\",\n      \"method\": \"Surface plasmon resonance, molecular docking, CLEC-2 site-directed mutagenesis, platelet aggregation assay, in vivo metastasis and thrombosis models\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — SPR and mutagenesis identifying novel ligand-binding residues, validated in vivo, single lab\",\n      \"pmids\": [\"30190281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Recombinant rhodocytin forms a heterooctamer (four α- and β-subunits). Asp4 in the α-subunit of rhodocytin is required for CLEC-2 binding. An inhibitory mutant rhodocytin (αWTβK53A/R56A) forms a heterotetramer, binds CLEC-2 without inducing platelet aggregation, and blocks CLEC-2–podoplanin interaction-dependent platelet aggregation and experimental lung metastasis.\",\n      \"method\": \"Recombinant rhodocytin expression, mutagenesis, blue native PAGE for multimer analysis, flow cytometry for CLEC-2 binding, platelet aggregometry, in vivo lung metastasis model\",\n      \"journal\": \"Journal of thrombosis and haemostasis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution with mutagenesis plus in vivo functional validation, single lab\",\n      \"pmids\": [\"29488681\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Tetraspanin CD37 specifically interacts with CLEC-2 and controls CLEC-2 membrane organization. CD37-deficient myeloid cells expressed reduced surface CLEC-2 and showed impaired CLEC-2-dependent adhesion, migration velocity, and actin protrusion formation. CD37 is required for CLEC-2 recruitment in the membrane to podoplanin, and CD37-deficient DCs fail to inhibit actomyosin contractility in lymph node stromal cells.\",\n      \"method\": \"Co-immunoprecipitation of CD37-CLEC-2, Cd37-knockout mice, DC migration and adhesion assays, microcontact printing for CLEC-2 membrane recruitment, actin protrusion assay in 3D collagen\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus genetic knockout with multiple functional readouts, single lab\",\n      \"pmids\": [\"30185523\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Akt1/PDK1 and PKCμ are two alternative CLEC-2 signaling pathways mediating podoplanin-induced platelet activation, identified through analysis of the molecular action of compound 2CP (a direct CLEC-2 binding inhibitor that competes with podoplanin for the same CLEC-2 binding pocket).\",\n      \"method\": \"Compound 2CP binding assay, Akt1/PDK1 and PKCμ pathway analysis, CLEC-2 competitive binding assay, platelet aggregation, in vivo metastasis model\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — identification of alternative signaling pathways by pharmacological inhibition with direct binding confirmation, single lab\",\n      \"pmids\": [\"26528756\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Soluble CLEC-2 (sCLEC-2) is shed as a fragment partially generated by MMP-2 (not ADAM10/17, which mediate GPVI shedding). Additionally, CLEC-2 is released as a whole molecule associated with platelet microparticles (MP-CLEC-2). This contrasts with sGPVI which is exclusively shed by ADAM10/17. The two proteins are thus released by distinct mechanisms.\",\n      \"method\": \"Metalloproteinase inhibitors/stimulators, ADAM10-specific inhibitor, domain-specific GPVI antibodies, microparticle characterization, ELISA for plasma levels\",\n      \"journal\": \"International journal of hematology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological inhibitor panel with biochemical characterization establishing distinct shedding mechanism, single lab\",\n      \"pmids\": [\"31165998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Platelet CLEC-2 signaling blocks reparative neutrophil recruitment in acetaminophen-induced acute liver failure by suppressing TNF-α production. Blocking CLEC-2 signaling enhanced liver recovery from APAP and CCl4 acute toxic liver injuries by increasing TNF-α, which drives reparative hepatic neutrophil recruitment.\",\n      \"method\": \"CLEC-2 signaling blockade (antibody), APAP and CCl4 mouse models, TNF-α measurement, neutrophil enumeration, human and mouse hepatic inflammatory response studies\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two independent liver injury models with mechanistic link to TNF-α and neutrophil recruitment, human validation\",\n      \"pmids\": [\"32321925\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Platelet CLEC-2 reduces tissue inflammation by regulating inflammatory macrophage activation and trafficking. The immune regulatory function depends on podoplanin expression on inflammatory macrophages and is independent of platelet activation/secretion. Mechanistically, platelet CLEC-2 and recombinant CLEC-2-Fc accelerate actin rearrangement and macrophage migration by increasing podoplanin and CD44 expression and their interaction with ERM proteins, promoting macrophage emigration to mesenteric lymph nodes.\",\n      \"method\": \"Platelet-specific CLEC-2 knockout mice, LPS peritonitis model, recombinant CLEC-2-Fc treatment, actin rearrangement assay, podoplanin/CD44/ERM protein interaction studies, cytokine measurement, in vivo macrophage tracking\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout with mechanistic dissection including recombinant protein rescue and molecular interaction studies\",\n      \"pmids\": [\"34163489\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Low concentrations of the Btk inhibitor ibrutinib selectively block CLEC-2-mediated activation and tyrosine phosphorylation (including Syk and PLCγ2) in human platelets, while GPVI response is only delayed. Platelets from patients with X-linked agammaglobulinemia (XLA, Btk-deficient) phenocopy this selective CLEC-2 block. The differential effect is explained by a positive feedback role of Btk in CLEC-2 signaling, involving ADP/TxA2-mediated P2Y12/TP receptor co-activation, which is not present in mouse platelets.\",\n      \"method\": \"Ibrutinib and acalabrutinib at titrated concentrations, XLA patient platelets, human and mouse platelet aggregometry, Syk/PLCγ2 phosphorylation, in vivo mouse thrombosis model\",\n      \"journal\": \"Haematologica\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — pharmacological and genetic (XLA patient, mouse model) approaches with species comparison revealing mechanistic basis\",\n      \"pmids\": [\"31949019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Hemin (heme) directly binds to both CLEC-2 and GPVI on platelets, as demonstrated by Western blotting and surface plasmon resonance. Hemin activates human platelets by stimulating phosphorylation of SYK and PLCγ2 through both receptors. Hemin-induced murine platelet aggregation is almost completely inhibited only in CLEC-2-depleted FcRγ-deficient (GPVI-deficient) double-knockout platelets, and CLEC-2/GPVI signaling contributes to MET formation and rhabdomyolysis-induced acute kidney injury.\",\n      \"method\": \"Surface plasmon resonance for direct hemin-CLEC-2/GPVI binding, CLEC-2 depletion and FcRγ-null mouse platelets, RAKI mouse model, in vitro MET formation assay, Western blotting for SYK/PLCγ2 phosphorylation\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct binding by SPR combined with double-knockout genetic model and in vivo functional validation\",\n      \"pmids\": [\"33843987\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Deletion of platelet CLEC-2 does not prevent VWF binding to platelets via GPIbα but specifically inhibited platelet aggregation and αIIbβ3 activation induced by VWF binding to GPIbα. In a mouse TTP model, CLEC-2 deletion decreased pulmonary arterial thrombosis and severity of thrombocytopenia, establishing CLEC-2 as a downstream mediator of GPIbα-initiated integrin αIIbβ3 activation.\",\n      \"method\": \"Platelet-specific CLEC-2 knockout mice, VWF-platelet binding assay, αIIbβ3 activation measurement, mouse TTP model (anti-ADAMTS13 antibody + VWF infusion), aspirin/eptifibatide treatment\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout with defined mechanistic placement of CLEC-2 between GPIbα and αIIbβ3 activation, in vivo disease model\",\n      \"pmids\": [\"35157766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cooperative signaling between CLEC-2 (activated by anti-CLEC-2 Fab INU1) and GPIIb/IIIa triggers a cerebral venous (sinus) thrombosis (CVT)-like syndrome in mice within minutes, characterized by tonic-myoclonic seizures, platelet consumption, and death. Interfering with CLEC-2 signaling or GPIIb/IIIa completely blocked platelet activation and CVT. Blocking GPIIb/IIIa after onset of neurological symptoms protected mice, while heparin did not.\",\n      \"method\": \"INU1-Fab anti-CLEC-2 antibody in mice, GPIIb/IIIa inhibitor, transcranial intravital microscopy of superior sagittal sinus, brain autopsy/histology, seizure monitoring\",\n      \"journal\": \"Nature cardiovascular research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mechanistic intervention with live intravital imaging and defined pharmacological rescue in a novel in vivo model\",\n      \"pmids\": [\"39195988\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cancer-associated fibroblasts (CAFs) co-express podoplanin and induce CLEC-2-dependent platelet aggregation. CAF-derived extracellular vesicles (EVs) also contain podoplanin and activate platelets in a CLEC-2-dependent manner. In tumor-bearing mice, antibody-induced CLEC-2 depletion suppressed venous thrombosis in the tumor-bearing state, establishing CAF-derived podoplanin/EVs as drivers of cancer-associated thrombosis through CLEC-2.\",\n      \"method\": \"CAF characterization by immunofluorescence, podoplanin EV isolation and immunoblotting, platelet aggregation flow cytometry, venous thrombosis model (FeCl3), CLEC-2 depletion antibody (2A2B10)\",\n      \"journal\": \"Journal of thrombosis and haemostasis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CAF-derived EV mechanism with in vivo CLEC-2 depletion, single lab, single study\",\n      \"pmids\": [\"37473844\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CLEC-2 (CLEC1B) is a platelet/megakaryocyte C-type lectin-like receptor that signals through a single cytoplasmic hemITAM (YXXL) motif: upon binding multivalent ligands (podoplanin, rhodocytin, fucoidan, heme, HIV-associated podoplanin), CLEC-2 dimerizes and translocates to lipid rafts, its YXXL is phosphorylated primarily by Syk (not Src), Syk is activated by cross-linking its tandem SH2 domains across two phosphorylated CLEC-2 monomers, and the resulting signaling cascade (Syk→PLCγ2→Ca²⁺, with positive feedback via ADP/TxA2 and Btk) drives platelet activation; structurally the ligand-binding site is on the non-canonical 'side' face of the CLEC-2 C-type lectin domain; in vivo CLEC-2–podoplanin signaling in platelets is essential for embryonic blood-lymphatic separation, lymph node integrity, lung development, cerebrovascular patterning, HSC maintenance (via megakaryocyte Thpo production), and regulates DVT, sepsis, and cancer-associated thrombosis, while CLEC-2 also functions on dendritic cells and macrophages to regulate migration and inflammatory macrophage trafficking.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CLEC1B (CLEC-2) is a platelet- and megakaryocyte-restricted C-type lectin-like receptor that couples ligand engagement to Syk-dependent tyrosine kinase signaling and serves as the platelet sensor for the mucin-type ligand podoplanin [#0, #2, #24]. It was first isolated as the platelet receptor for the snake venom toxin rhodocytin and defined a signaling pathway in which receptor engagement triggers phosphorylation of a single cytoplasmic YXXL (hemITAM) motif, recruitment and activation of Syk, and downstream PLCγ2 activation [#0]. Because CLEC-2 carries only one YXXL yet must engage a kinase that normally requires tandem ITAMs, the receptor functions as a non-disulfide-linked homodimer, allowing the two SH2 domains of a single Syk to be cross-linked across two phosphorylated monomers in a 2:1 stoichiometry [#8, #14]. Signal initiation requires ligand-induced clustering, translocation to lipid rafts, and—unusually—uses Syk rather than Src family kinases as the primary kinase for hemITAM phosphorylation, with Src kinases acting downstream and with Btk and Gq/GPCR (ADP/TxA2) inputs providing positive feedback [#15, #16, #26, #36, #46]. Crystallographic and mutational work places the ligand-binding site on the non-canonical 'side' face of the C-type lectin domain, where consecutive acidic residues of podoplanin and rhodocytin engage common arginine residues, with podoplanin recognition additionally requiring its sialylated disialyl-core1 O-glycan at Thr52 [#1, #4, #23, #39]. Beyond podoplanin and rhodocytin, CLEC-2 binds fucoidan, heme, and S100A13 [#22, #31, #47]. In vivo, platelet/megakaryocyte CLEC-2–podoplanin signaling is essential for embryonic blood–lymphatic vessel separation, lymph node integrity, cerebrovascular patterning, and hematopoietic stem cell maintenance via megakaryocyte thrombopoietin production [#13, #18, #27, #28, #33], and it regulates hemostasis and thrombosis, including a hemITAM-independent adhesive contribution to hemostasis [#9, #12, #37]. The CLEC-2–podoplanin axis drives deep vein thrombosis, cancer-associated thrombosis, and cerebral venous thrombosis, and modulates inflammation in sepsis, liver injury, and macrophage trafficking, while CLEC-2 on dendritic cells and macrophages controls migration through podoplanin engagement [#19, #34, #35, #44, #45, #49, #50].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Established the existence and signaling logic of a novel platelet activation receptor, defining how a single-YXXL C-type lectin transduces a signal through Syk and PLCγ2.\",\n      \"evidence\": \"Rhodocytin affinity chromatography for receptor identification with Syk/PLCγ2 knockout mouse platelets and Src-family inhibitor\",\n      \"pmids\": [\"16174766\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous physiological ligand unknown at this stage\", \"Stoichiometry of Syk engagement to a single YXXL unexplained\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Provided the first atomic structure of the CLEC-2 ectodomain, localizing the ligand-binding surface and arguing against large ligand-induced conformational change.\",\n      \"evidence\": \"X-ray crystallography at 1.6 Å with site-directed mutagenesis and surface plasmon resonance for rhodocytin binding\",\n      \"pmids\": [\"17132623\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of the endogenous ligand complex not resolved\", \"How binding triggers cytoplasmic signaling not addressed\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identified podoplanin as the endogenous CLEC-2 ligand and defined the glycan and protein determinants of recognition, transforming CLEC-2 from a toxin receptor into a physiological signaling axis.\",\n      \"evidence\": \"Flow cytometry, recombinant CLEC-2 inhibition and platelet aggregation assays, plus deletion-mutant Fc chimeras and synthetic disialyl-core1 glycopeptides identifying Thr52 O-glycan dependence\",\n      \"pmids\": [\"17616532\", \"17944973\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Affinity and direct binding not yet quantified\", \"Tissue sources of podoplanin engaging platelets not mapped\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Dissected the cytoplasmic signaling motif, showing both Syk SH2 domains and the upstream DEDG triacidic sequence are required, hinting that receptor dimerization underlies hemITAM function.\",\n      \"evidence\": \"YXXL and DEDG mutagenesis with Syk SH2-domain mutants and chimeric receptors in cell-line signaling assays\",\n      \"pmids\": [\"17339324\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physical demonstration of receptor dimerization still lacking\", \"Role of DEDG residues in phosphorylation mechanism unresolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Quantified direct CLEC-2–podoplanin binding and the rhodocytin oligomeric state, supporting a clustering model for receptor activation, and identified G6b-B as an inhibitory counter-receptor.\",\n      \"evidence\": \"SPR affinity measurement (24.5 µM) with DT-40 cell activation, rhodocytin crystallography with docking, and NFAT reporter/ITIM mutant analysis of G6b-B\",\n      \"pmids\": [\"18215137\", \"18583525\", \"18955485\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Clustering mechanism supported only by computational docking\", \"G6b-B phosphatase effector remained unidentified (SHP1/2/SHIP-independent)\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Resolved the long-standing single-YXXL paradox by demonstrating CLEC-2 forms a homodimer that allows one Syk to bridge two phosphorylated monomers, and showed CLEC-2 is essential for platelet aggregation and thrombosis.\",\n      \"evidence\": \"Multiple biophysical methods (BRET, gel filtration, AUC, MALS, SPR) for dimerization, plus antibody depletion in mice with flow-chamber aggregation and in vivo thrombosis assays\",\n      \"pmids\": [\"19824697\", \"19641185\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether dimers pre-exist or form upon ligand binding not fully resolved here\", \"Contribution of CLEC-2 to bleeding versus thrombosis ambiguous across models\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Extended CLEC-2 function beyond platelets to neutrophils, where it acts as a Syk-coupled phagocytic and cytokine-inducing receptor, and established Syk as upstream of CLEC-2's own phosphorylation.\",\n      \"evidence\": \"Primary neutrophil flow cytometry, phagocytosis and cytokine assays, chimeric receptor mutagenesis, plus Syk inhibitor R406 in human platelets\",\n      \"pmids\": [\"19299712\", \"19422460\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Neutrophil CLEC-2 expression restricted and context-dependent (single lab)\", \"Mechanism by which Syk feeds back to phosphorylate the hemITAM not fully defined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined the proximal biochemistry and biophysics of activation—Syk (not Src) as the hemITAM kinase, lipid-raft translocation, actin/Rac1/secretion dependence, and 2:1 Syk:hemITAM cross-linking—and established CLEC-2's essential developmental role in blood–lymphatic separation.\",\n      \"evidence\": \"Kinase-specific knockout mouse platelets, raft fractionation and cholesterol depletion, peptide pull-down/SPR/tryptophan fluorescence/EM, and constitutive plus PF4-Cre conditional knockouts with intravital microscopy and genetic epistasis to PDPN/SLP-76\",\n      \"pmids\": [\"20154219\", \"21098033\", \"20154214\", \"20525685\", \"20363774\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise trigger ordering clustering, raft entry, and phosphorylation incompletely resolved\", \"Downstream secreted mediators of lymphatic separation not yet identified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Showed lineage-restricted CLEC-2/Syk signaling in platelets directly controls lymphatic and cerebrovascular development by acting on endothelial cell migration and adhesion.\",\n      \"evidence\": \"Megakaryocyte/platelet-lineage conditional Cre-lox knockouts with LEC migration/adhesion assays comparing platelets versus releasate\",\n      \"pmids\": [\"22186994\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of platelet-borne effector versus contact signal not resolved here\", \"Mechanism linking platelet signaling to endothelial behavior unspecified\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified secreted mediators (BMP-9) and signaling outputs (RhoA/Rac1, membrane protrusions) downstream of CLEC-2, extending its role to dendritic cell migration and confirming platelet CLEC-2 governs blood/lymphatic vessel separation.\",\n      \"evidence\": \"Platelet-specific and DC-specific conditional knockouts with LEC tube-formation/migration assays, BMP-9 identification, and RhoA/myosin/Vav/Rac1 readouts with in vivo DC trafficking\",\n      \"pmids\": [\"22556408\", \"22884313\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of BMP-9 versus other mediators in vivo unquantified\", \"How DC CLEC-2 signaling integrates with platelet CLEC-2 functions unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established CLEC-2 as a tissue-organizing receptor in lymphoid stroma, releasing S1P to maintain HEV integrity, and confirmed fucoidan as a CLEC-2 agonist.\",\n      \"evidence\": \"FRC- and platelet-specific conditional knockouts with platelet-infusion rescue and S1P pathway analysis, plus platelet-specific CLEC-2 knockout and FcRγ-null platelets with fucoidan\",\n      \"pmids\": [\"23995678\", \"23341451\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether S1P acts directly or via additional mediators on HEVs not fully dissected\", \"Physiological source of fucoidan-like sulfated ligands in vivo unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined the structural basis of dual-ligand recognition on the non-canonical side face, demonstrated ligand-driven membrane clustering, and revealed CLEC-2's roles in lymph node morphogenesis, stromal contractility, and as an activated-platelet marker.\",\n      \"evidence\": \"CLEC-2–podoplanin and CLEC-2–rhodocytin co-crystal structures, supported-lipid-bilayer FLIM/dSTORM clustering with Src/Syk perturbation, conditional knockouts for lymph node and FRC contractility phenotypes, and antibody-based shedding/copy-number quantitation\",\n      \"pmids\": [\"25458834\", \"25368330\", \"24532804\", \"25347465\", \"25150298\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural model of the full clustered signaling assembly not solved\", \"Mechanism converting clustering to hemITAM phosphorylation at the membrane incomplete\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Placed CLEC-2 at the center of bone marrow niche function—driving megakaryocyte thrombopoietin and CCL5 outputs for HSC maintenance and proplatelet formation—identified additional ligands and signaling branches, and dissected antibody-induced internalization.\",\n      \"evidence\": \"Megakaryocyte-specific conditional knockouts with Syk/Lcp2/Plcg2 knockdown and Thpo rescue, MK-FRC co-culture with CCL5, S100A13 identification by protein array/SPR, keratinocyte RhoA/E-cadherin assays, 2CP-defined Akt1/PDK1 and PKCµ branches, and Syk-KO/Src-inhibitor internalization studies\",\n      \"pmids\": [\"26552707\", \"26796360\", \"26418160\", \"26597882\", \"26528756\", \"25795918\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Baseline VSMC ligand distinct from S100A13 remains unidentified\", \"Physiological relevance of Akt1/PDK1 and PKCµ branches beyond pharmacology unclear\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Separated CLEC-2's signaling-dependent developmental role from a hemITAM-independent adhesive role in hemostasis, defined GPCR/Gq potentiation of proximal signaling, and linked the CLEC-2–podoplanin axis to DVT and to anti-inflammatory control of sepsis.\",\n      \"evidence\": \"Y7A hemITAM-null knockin mouse with anti-CLEC-2 Fab', Gq-KO/UBO-QIC biochemistry, IVC stenosis DVT model with anti-podoplanin neutralization, and platelet-specific knockouts in two sepsis models\",\n      \"pmids\": [\"28835437\", \"28705934\", \"28104688\", \"29269852\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular nature of the hemITAM-independent adhesive function undefined\", \"Identity of podoplanin-expressing cells driving the sepsis phenotype incompletely mapped\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established CLEC-2 as an initiator of platelet–leukocyte inflammatory axes (dengue), refined ligand-binding residues amenable to inhibition, defined the rhodocytin oligomeric requirement, and identified CD37 as a membrane organizer of CLEC-2.\",\n      \"evidence\": \"CLEC-2 blockade with EV characterization and dengue lethality model, SPR/docking/mutagenesis (N120/N210/K211) with cobalt hematoporphyrin, recombinant rhodocytin oligomer mutagenesis with metastasis models, and CD37 co-IP with Cd37-KO myeloid migration assays\",\n      \"pmids\": [\"31160588\", \"30190281\", \"29488681\", \"30185523\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"CD37–CLEC-2 interaction based on single-lab co-IP without structural detail\", \"Whether identified inhibitor binding sites overlap the physiological podoplanin interface only partially resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Characterized distinct mechanisms of CLEC-2 release—MMP-2-mediated ectodomain shedding and microparticle-associated whole-receptor release—distinguishing it from GPVI shedding biology.\",\n      \"evidence\": \"Metalloproteinase inhibitor panel with ADAM10-specific inhibitor, microparticle characterization, and ELISA for plasma fragments\",\n      \"pmids\": [\"31165998\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab pharmacological inference of MMP-2 involvement\", \"Functional consequence of soluble versus microparticle CLEC-2 unestablished\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined a CLEC-2 signaling role in restraining reparative inflammation, where platelet CLEC-2 suppresses TNF-α-driven neutrophil recruitment limiting liver recovery.\",\n      \"evidence\": \"CLEC-2 antibody blockade in APAP and CCl4 liver injury models with TNF-α and neutrophil readouts and human validation\",\n      \"pmids\": [\"32321925\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ligand engaging CLEC-2 in the injured liver not defined\", \"Direct cellular target of the TNF-α suppression unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified Btk-dependent positive feedback unique to human CLEC-2 signaling, a new heme ligand shared with GPVI, and a secretion-independent mechanism by which platelet CLEC-2 controls inflammatory macrophage trafficking.\",\n      \"evidence\": \"Ibrutinib/acalabrutinib titration with XLA patient platelets and species comparison, SPR hemin binding with CLEC-2/GPVI double-knockout platelets and kidney injury model, and platelet-specific knockout with CLEC-2-Fc rescue and podoplanin/CD44/ERM interaction studies\",\n      \"pmids\": [\"31949019\", \"33843987\", \"34163489\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mouse-versus-human divergence in Btk feedback mechanistically incompletely explained\", \"How CLEC-2 engagement modulates macrophage podoplanin/CD44/ERM at the molecular level not fully resolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Placed CLEC-2 as a downstream amplifier of GPIbα–VWF signaling driving αIIbβ3 activation and demonstrated cooperative CLEC-2/GPIIb-IIIa signaling can precipitate acute cerebral venous thrombosis.\",\n      \"evidence\": \"Platelet-specific knockout with VWF binding and αIIbβ3 activation assays in a TTP model, and INU1-Fab/GPIIb-IIIa intervention with transcranial intravital microscopy in a CVT model\",\n      \"pmids\": [\"35157766\", \"39195988\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular link between GPIbα engagement and CLEC-2 activation undefined\", \"Whether the CVT-like syndrome reflects physiological CLEC-2 ligand engagement or antibody-specific crosslinking unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Implicated CLEC-2 in cancer-associated thrombosis via cancer-associated fibroblast podoplanin and podoplanin-bearing extracellular vesicles activating platelets.\",\n      \"evidence\": \"CAF and EV characterization, CLEC-2-dependent platelet aggregation, and antibody-induced CLEC-2 depletion in tumor-bearing FeCl3 thrombosis model\",\n      \"pmids\": [\"37473844\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab single-study evidence\", \"Relative in vivo contribution of CAF-surface versus EV podoplanin unquantified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular basis of CLEC-2's hemITAM-independent adhesive function in hemostasis, and the structure of the full clustered ligand–CLEC-2–Syk signaling assembly at the membrane, remain unresolved.\",\n      \"evidence\": \"No timeline study reconstitutes the complete activated signaling complex or defines the non-signaling adhesive mechanism\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of the clustered receptor–Syk signaling unit\", \"Adhesive/physical hemostatic function lacks a defined molecular partner\", \"Possible homophilic CLEC-2 interactions in thrombus stabilization not biochemically defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 15, 20]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 2, 3]},\n      {\"term_id\": \"GO:0001618\", \"supporting_discovery_ids\": [38]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 16, 24]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [9, 12, 37, 48]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 14, 15, 16]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [19, 35, 44, 45]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [13, 18, 27, 33]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"PDPN\", \"SYK\", \"CD37\", \"G6B\"]\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}