{"gene":"CLEC1B","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":2005,"finding":"CLEC-2 (CLEC1B) was identified as the receptor for snake venom rhodocytin on platelets via rhodocytin affinity chromatography. Ligand binding causes tyrosine phosphorylation of a single YXXL motif in the CLEC-2 cytosolic tail, which is dependent on Src family kinases, and activates Syk and PLCgamma2. Activation is abolished in Syk-/- and PLCgamma2-/- murine platelets and partially reduced in LAT, SLP-76, and Vav1/Vav3-deficient platelets.","method":"Rhodocytin affinity chromatography, expression in cell lines, genetic knockout mice, phosphorylation assays, kinase inhibitors","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1-2 — receptor identification by affinity chromatography, functional reconstitution in cell lines, and multiple genetic knockouts","pmids":["16174766"],"is_preprint":false},{"year":2006,"finding":"Crystal structure of the extracellular domain of human CLEC-2 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 confirmed the key residues involved in rhodocytin binding.","method":"X-ray crystallography, site-directed mutagenesis, surface plasmon resonance","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with mutational and SPR validation","pmids":["17132623"],"is_preprint":false},{"year":2007,"finding":"Podoplanin was identified as an endogenous ligand for CLEC-2 on platelets. The CLEC-2-podoplanin interaction 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.","method":"Flow cytometry binding assays, recombinant protein inhibition, deletion mutant analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — direct binding confirmation by flow cytometry and functional inhibition, replicated across labs","pmids":["17616532"],"is_preprint":false},{"year":2007,"finding":"CLEC-2 and Dectin-1 signal through a novel YXXL-dependent pathway requiring both SH2 domains of Syk (despite having only a single YXXL motif), Src and Tec family kinases, and PLCgamma, with only partial dependence on SLP-76/BLNK adapters. The glycine residue directly upstream of the YXXL tyrosine in the DEDG sequence is critical for CLEC-2 signaling.","method":"Mutant cell line signaling assays, site-directed mutagenesis of YXXL and DEDG motifs, kinase inhibitors","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — reconstitution in mutant cell lines with mutagenesis of critical motifs","pmids":["17339324"],"is_preprint":false},{"year":2007,"finding":"Molecular characterization of the podoplanin-CLEC-2 interaction: CLEC-2 deletion mutants (expressed as Fc chimeras) identified the podoplanin-recognition domain; a disialyl-core1-attached glycopeptide at Thr52 of podoplanin is specifically recognized by CLEC-2; the protein stereostructure of podoplanin is also critical for binding.","method":"Deletion mutant Fc chimeras, synthesized glycopeptides, in vitro binding assays, in vivo metastasis model","journal":"Cancer science","confidence":"High","confidence_rationale":"Tier 1 — domain mapping with deletion mutants, glycopeptide synthesis, in vitro and in vivo validation","pmids":["17944973"],"is_preprint":false},{"year":2008,"finding":"Podoplanin on renal cells (HEK-293T and podocytes) was confirmed as a CLEC-2 ligand. Direct CLEC-2-podoplanin interaction measured by surface plasmon resonance showed an affinity of ~24.5 µM and was shown to be independent of glycosylation of CLEC-2.","method":"Surface plasmon resonance, recombinant protein binding, CLEC-2-transfected DT-40 B-cell activation assay","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 — SPR affinity measurement and functional reconstitution in cell lines","pmids":["18215137"],"is_preprint":false},{"year":2008,"finding":"Crystal structure of rhodocytin (CLEC-2 ligand) solved at 2.4 Å; rhodocytin forms a non-disulfide-linked (αβ)2 tetramer with a concave binding surface complementary to the CLEC-2 binding interface, suggesting it clusters CLEC-2 receptors on the platelet surface to trigger signaling.","method":"X-ray crystallography, computational docking, surface electrostatic analysis","journal":"Protein science","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with computational docking supporting mechanism of receptor clustering","pmids":["18583525"],"is_preprint":false},{"year":2008,"finding":"G6b-B, a platelet immunoglobulin receptor with two ITIM motifs, inhibits constitutive and agonist-induced signaling by CLEC-2 (and GPVI). Inhibition by G6b-B requires its conserved ITIM tyrosines and is independent of SHP1, SHP2, and SHIP phosphatases.","method":"NFAT reporter assay, mutagenesis, cell line signaling studies","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — functional inhibition in cell line model with mutagenesis, single lab","pmids":["18955485"],"is_preprint":false},{"year":2009,"finding":"CLEC-2 is present as a non-disulfide-linked homodimer in resting platelets. This dimerization enables each Syk molecule to interact with two YXXL motifs (one from each CLEC-2 monomer), explaining how a hemITAM activates Syk normally requiring tandem YXXL.","method":"BRET, co-immunoprecipitation, analytical gel filtration, surface plasmon resonance, MALS, analytical ultracentrifugation","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1-2 — multiple biophysical methods confirming dimerization with functional interpretation","pmids":["19824697"],"is_preprint":false},{"year":2009,"finding":"CLEC-2-deficient platelets (via anti-CLEC-2 antibody depletion) show normal adhesion under flow but severely defective aggregate formation in vitro and in vivo, resulting in increased bleeding times and protection from occlusive arterial thrombus formation, establishing CLEC-2 as essential for hemostasis and thrombosis.","method":"Antibody-mediated CLEC-2 depletion, intravital microscopy, in vitro flow chamber assays, bleeding time measurements","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — clean receptor depletion with defined in vivo and in vitro phenotypic readouts","pmids":["19641185"],"is_preprint":false},{"year":2009,"finding":"CLEC-2 is expressed on murine peripheral blood neutrophils (not bone marrow or elicited neutrophils). On neutrophils, CLEC-2 mediates phagocytosis of antibody-coated beads and TNF-alpha production upon rhodocytin stimulation through its tyrosine-based cytoplasmic motif, recruiting Syk.","method":"Monoclonal antibody staining, chimeric receptor analysis, phagocytosis assay, cytokine ELISA, Syk recruitment assay","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — direct functional assays in primary cells with chimeric receptor analysis confirming YXXL requirement","pmids":["19299712"],"is_preprint":false},{"year":2010,"finding":"CLEC-2-deficient mice (Clec-2-/- generated by gene targeting) are lethal at embryonic/neonatal stages with disorganized, blood-filled lymphatic vessels and severe edema. Fetal liver transplantation demonstrated that CLEC-2 is involved in thrombus stabilization in vitro and in vivo, potentially through homophilic interactions, without apparent increase in bleeding tendency.","method":"Gene knockout, fetal liver transplantation, intravital microscopy, laser-induced thrombosis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — constitutive knockout with rescue by transplantation, replicated phenotype","pmids":["20525685"],"is_preprint":false},{"year":2010,"finding":"Platelet CLEC-2 directly binds lymphatic endothelial cell podoplanin (PDPN), activating SLP-76 signaling. Genetic loss of CLEC-2 abolishes PDPN binding by platelets and causes embryonic lymphatic vascular defects; platelet-specific deletion of Slp-76 is sufficient to recapitulate the defect, establishing the platelet CLEC-2/SLP-76 pathway as essential for blood-lymphatic separation.","method":"Genetic knockout (CLEC-2-/-, platelet-specific PF4-Cre Slp-76 deletion), intravital and ex vivo imaging, flow cytometry","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — multiple genetic models with epistasis placing CLEC-2 upstream of SLP-76 in platelet-dependent lymphatic development","pmids":["20363774"],"is_preprint":false},{"year":2010,"finding":"Syk, but not the major platelet Src family kinases (Fyn, Lyn, Src) or CD148, is required for phosphorylation of the CLEC-2 hemITAM by rhodocytin. Src family kinases play critical roles downstream of Syk, regulating Syk activity and other effector proteins rather than initiating CLEC-2 phosphorylation directly.","method":"Syk-/- and Src kinase-deficient mouse platelets, kinase inhibitor PP2, western blotting of phosphorylation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple genetic knockouts establishing Syk's unique role in hemITAM phosphorylation","pmids":["21098033"],"is_preprint":false},{"year":2010,"finding":"CLEC-2 activates Syk through dimerization: phosphorylated CLEC-2 hemITAM recruits Syk in a 2:1 stoichiometry via its tandem SH2 domains. CLEC-2 exists as a dimer in resting platelets and forms larger complexes upon activation, as confirmed by cross-linking and electron microscopy.","method":"Peptide pulldown, surface plasmon resonance, quantitative western blotting, tryptophan fluorescence, cross-linking, electron microscopy","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1 — multiple orthogonal biophysical methods establishing 2:1 Syk:CLEC-2 stoichiometry and dimerization-dependent activation","pmids":["20154219"],"is_preprint":false},{"year":2010,"finding":"CLEC-2 hemITAM phosphorylation requires translocation to lipid rafts upon ligand engagement; this translocation and the subsequent phosphorylation are also critically dependent on actin polymerization, Rac1 activation, and release of ADP and TxA2 as secondary mediators. In contrast, GPVI-ITAM phosphorylation is independent of these events.","method":"Sucrose gradient ultracentrifugation, methyl-beta-cyclodextrin treatment, actin polymerization inhibitors, Rac1 inhibition, ADP/TxA2 pathway inhibitors","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — multiple pharmacological and biochemical approaches with clear mechanistic distinction from GPVI","pmids":["20154214"],"is_preprint":false},{"year":2011,"finding":"Conditional deletion of CLEC-2 or Syk in the megakaryocyte/platelet lineage causes defects in brain vascular and lymphatic development. Platelets (but not platelet releasate) directly modulate migration and intercellular adhesion of lymphatic endothelial cells through a CLEC-2- and Syk-dependent pathway.","method":"Conditional knockout (Cre-lox), in vitro lymphatic endothelial cell migration/adhesion assays, releasate vs. cell contact experiments","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — cell-type-specific conditional knockouts with defined cellular phenotype and mechanistic dissection","pmids":["22186994"],"is_preprint":false},{"year":2012,"finding":"Platelet CLEC-2 regulates blood/lymphatic vessel separation by inhibiting proliferation, migration, and tube formation of lymphatic endothelial cells (LECs). Activated platelets release BMP-9 (present in platelet granules), which plays a key role in inhibiting LEC tube formation; TGF-beta and PF4 also inhibit proliferation/migration.","method":"Platelet-specific CLEC-2 knockout (Cre-lox), in vitro LEC migration/proliferation/tube formation assays, BMP-9 identification in platelets","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — platelet-specific KO with defined cellular assays and identification of secreted mediator","pmids":["22556408"],"is_preprint":false},{"year":2012,"finding":"CLEC-2 engagement by podoplanin in dendritic cells (DCs) rearranges the actin cytoskeleton to promote DC motility. CLEC-2 activation triggers cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation, and F-actin-rich protrusions via Vav signaling and Rac1 activation, enabling DC migration along stromal surfaces and entry into lymphatics.","method":"CLEC-2-deficient DCs, DC migration assays, RhoA/Rac1 activity assays, actin dynamics imaging","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — CLEC-2 KO DCs with defined molecular signaling (RhoA/Rac1) and cellular migratory phenotype","pmids":["22884313"],"is_preprint":false},{"year":2012,"finding":"The triacidic amino acid sequence (DEDG) upstream of the YXXL hemITAM in CLEC-2 is required for hemITAM phosphorylation; mutation of these acidic residues abolishes Syk-dependent signaling. Syk-mediated signaling is restricted to Syk (not Zap-70) in hemITAM receptors.","method":"Site-directed mutagenesis, Syk/Zap-70-deficient cell lines, SPR, phosphorylation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis with SPR and cell line reconstitution establishing functional requirement of acidic motif","pmids":["23264619"],"is_preprint":false},{"year":2013,"finding":"Podoplanin expressed on fibroblastic reticular cells (FRCs) surrounding high endothelial venules (HEVs) activates platelet CLEC-2. CLEC-2-mediated platelet activation induces release of sphingosine-1-phosphate (S1P) from platelets, which promotes VE-cadherin expression on HEVs, maintaining vascular integrity. Infusion of wild-type platelets restored HEV integrity in Clec-2-deficient mice.","method":"Conditional knockout mice, platelet infusion rescue, S1P pathway analysis, VE-cadherin staining, ex vivo HEV treatment","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with platelet rescue and identification of downstream mediator (S1P), multiple orthogonal methods","pmids":["23995678"],"is_preprint":false},{"year":2013,"finding":"Fucoidan (a sulfated polysaccharide) is a novel CLEC-2 agonist; fucoidan-induced platelet activation is abolished in platelet-specific CLEC-2 knockout platelets, occurs via Src and Syk kinase-dependent signaling, and is largely independent of the GPVI/FcRgamma pathway.","method":"CLEC-2 platelet-specific KO, aggregometry, tyrosine phosphorylation assays, FcRgamma-/- mice","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — platelet-specific KO with direct comparison to GPVI pathway","pmids":["23341451"],"is_preprint":false},{"year":2014,"finding":"Crystal structures of CLEC-2 in complex with podoplanin glycopeptide and rhodocytin reveal that both ligands bind to the non-canonical 'side' face of CLEC-2. A common interaction mode involves consecutive acidic residues on ligands interacting with arginine residues on CLEC-2; the second binding site differs: sialic acid carboxyl from podoplanin vs. rhodocytin C-terminus interact differently.","method":"X-ray crystallography of CLEC-2 complexes with podoplanin peptide and rhodocytin","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 — comparative crystal structures of two CLEC-2 ligand complexes revealing mechanism of versatile binding","pmids":["25458834"],"is_preprint":false},{"year":2014,"finding":"Podoplanin regulates actomyosin contractility in fibroblastic reticular cells (FRCs) through the CLEC-2/podoplanin axis. Under resting conditions, PDPN endows FRCs with contractile function. Upon inflammation, CLEC-2 on mature DCs attenuates PDPN-mediated contractility, resulting in FRC relaxation, reduced tissue stiffness, and an expanded reticular network with enhanced immunity.","method":"Collagen gel contraction assay, FRC co-culture with CLEC-2-expressing DCs, stiffness measurements, podoplanin loss-of-function","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 — mechanistic dissection of CLEC-2/PDPN axis in FRC contractility with multiple functional readouts","pmids":["25347465"],"is_preprint":false},{"year":2014,"finding":"CLEC-2 signaling via Src and Syk kinases promotes platelet adhesion to lymphatic endothelial cells and clustering of CLEC-2 with podoplanin on supported lipid bilayers. CLEC-2/podoplanin clusters migrate to the center of the platelet and are within 10 nm of one another; disrupted by Src/Syk inhibition. CLEC-2 clusters also observed by dSTORM on immobilized podoplanin.","method":"Supported lipid bilayer with mobile podoplanin, FLIM-FRET, dSTORM, Src/Syk kinase inhibitors, Syk-/- mouse platelets","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — super-resolution imaging plus biophysical assays with genetic/pharmacological controls","pmids":["25368330"],"is_preprint":false},{"year":2014,"finding":"CLEC-2 is required for development and maintenance of lymph nodes. Constitutive CLEC-2 deletion impairs lymphatic endothelial cell proliferation resulting in absent LNs; platelet-specific CLEC-2 deletion leads to blood-filled LNs and fibrosis. Platelet CLEC-2 is required for LN integrity and immune cell recirculation.","method":"Constitutive and platelet-specific (PF4-Cre) CLEC-2 knockout, bone marrow chimeras, immunization experiments","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — multiple conditional and constitutive KO models distinguishing LEC proliferative vs. integrity functions","pmids":["24532804"],"is_preprint":false},{"year":2015,"finding":"CLEC-2 in megakaryocytes mediates production of thrombopoietin (Thpo) and other factors via the Syk/Lcp2/Plcg2 signaling axis. Megakaryocyte-specific CLEC-2 deletion (Clec2MkΔ/Δ) reduces Thpo in megakaryocytes, decreases HSC quiescence and repopulation potential, and causes extramedullary hematopoiesis; recombinant Thpo administration restores HSC potential.","method":"Megakaryocyte-specific conditional KO, knockdown of signaling molecules in cultured Mks, recombinant Thpo rescue, HSC transplantation assays","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — conditional KO with rescue experiment and downstream signaling pathway defined","pmids":["26552707"],"is_preprint":false},{"year":2015,"finding":"CLEC-2 drives venous thrombosis through podoplanin interaction; platelet-specific or inducible CLEC-2 deletion protects mice from DVT in IVC stenosis model. Podoplanin is expressed in the IVC wall near the abluminal endothelium and is upregulated in thrombus-bearing vessels. Anti-podoplanin neutralizing antibody reduces thrombus size.","method":"Inducible and platelet-specific CLEC-2 KO, IVC stenosis DVT model, platelet transfusion, anti-podoplanin antibody treatment, intravital imaging","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — multiple genetic and antibody-based approaches with defined in vivo thrombosis phenotype","pmids":["28104688"],"is_preprint":false},{"year":2015,"finding":"Podoplanin-positive bone marrow periarteriolar stromal cells (BM FRC-like cells) promote megakaryocyte growth and proplatelet formation through CLEC-2/podoplanin interaction. This interaction induces BM FRC-like cells to secrete CCL5, which facilitates proplatelet formation.","method":"Platelet/Mk-specific CLEC-2 conditional KO, in vitro megakaryocyte-BM FRC co-culture, recombinant PDPN stimulation, CCL5 ELISA, immunohistochemistry","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — conditional KO plus in vitro co-culture identifying CCL5 as secreted mediator","pmids":["26796360"],"is_preprint":false},{"year":2015,"finding":"Podoplanin and CLEC-2 control cerebrovascular patterning during development. Constitutive or platelet-specific deletion of CLEC-2 causes tortuous, aberrantly patterned cerebral vessels at E10.5 with large hemorrhages and defective pericyte recruitment. Nestin-Cre-driven deletion of podoplanin on neural progenitors also causes cerebral hemorrhaging.","method":"Constitutive and conditional (platelet-specific, neural progenitor-specific Nestin-Cre) knockout mice, 3D light-sheet microscopy, immunofluorescence, electron microscopy","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — multiple conditional KOs with cell-type-specific resolution and 3D imaging","pmids":["25908104"],"is_preprint":false},{"year":2015,"finding":"S100A13 was identified as a CLEC-2 ligand in vascular smooth muscle cells (VSMCs) using protein array and Biacore analysis. VSMCs stimulate platelet granule release and support thrombus formation under flow in a CLEC-2-dependent manner. S100A13 is released under oxidative stress and expressed in atherosclerotic lesions.","method":"Recombinant CLEC-2 binding studies, protein array, surface plasmon resonance (Biacore), CLEC-2-deficient mice, flow chamber thrombosis, FeCl3 thrombosis model","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — SPR identification of novel ligand with in vitro and in vivo functional validation, single lab","pmids":["26418160"],"is_preprint":false},{"year":2015,"finding":"During bacterial infection (Salmonella), inflammation triggers upregulation of podoplanin on podoplanin-expressing monocytes and Kupffer cells at perivascular sites via IFN-gamma, which then activates platelet CLEC-2 to drive thrombus formation independently of classical GPVI-mediated activation. TLR4 and IFN-gamma are required upstream of this process.","method":"Salmonella infection mouse model, intravital liver imaging, cell depletion, TLR4/IFN-gamma/CLEC-2 genetic/antibody-mediated deletion, podoplanin immunostaining","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 — multiple genetic and depletion approaches establishing pathway position in infection-driven thrombosis","pmids":["26571395"],"is_preprint":false},{"year":2017,"finding":"A CLEC-2 knockin mouse expressing CLEC-2 with a non-signaling hemITAM (Y7A KI) recapitulates 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' fragments revealed a hemITAM signaling-independent role for CLEC-2 in hemostasis and thrombosis, suggesting a structural/adhesive contribution.","method":"Knockin mouse (Y7A hemITAM mutation), anti-CLEC-2 Fab' functional blockade, in vivo thrombosis models, tail bleeding time","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — novel knockin model separating signaling-dependent from signaling-independent CLEC-2 functions","pmids":["28835437"],"is_preprint":false},{"year":2017,"finding":"Platelet-specific CLEC-2 deletion leads to enhanced systemic inflammation and accelerated organ injury in sepsis models (LPS and cecal ligation/puncture). 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, suggesting podoplanin activation on macrophages drives the anti-inflammatory action.","method":"Platelet-specific CLEC-2 KO, LPS and CLP sepsis models, immune cell counts, cytokine measurement, pharmacological inhibition","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — platelet-specific KO with defined sepsis phenotype and pharmacological confirmation","pmids":["29269852"],"is_preprint":false},{"year":2017,"finding":"Gq signaling potentiates CLEC-2 signaling via the PLCβ-PKCα pathway, which regulates Src family kinase activity upstream of CLEC-2 hemITAM phosphorylation. TxA2 (via Gq) specifically potentiates CLEC-2 receptor tyrosine phosphorylation and downstream Syk/PLCgamma2 phosphorylation.","method":"Gq inhibitor (UBO-QIC), Gq-/- mouse platelets, pharmacological pathway dissection, western blotting of phosphorylation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — genetic and pharmacological Gq ablation defining the positive feedback mechanism in CLEC-2 signaling","pmids":["28705934"],"is_preprint":false},{"year":2018,"finding":"Cobalt hematoporphyrin (Co-HP) directly binds to CLEC-2 at residues N120, N210, and K211 (confirmed by CLEC-2 mutant analysis), which are distinct from the previously known podoplanin-binding sites. Co-HP inhibits CLEC-2-mediated platelet aggregation and suppresses tumor metastasis and arterial/venous thrombosis in vivo without prolonging bleeding time.","method":"SPR, molecular docking, CLEC-2 mutant analysis, in vitro platelet aggregation, in vivo thrombosis and metastasis models","journal":"Blood advances","confidence":"High","confidence_rationale":"Tier 1 — SPR binding site identification validated by mutagenesis with in vitro and in vivo functional confirmation","pmids":["30190281"],"is_preprint":false},{"year":2018,"finding":"Platelets play an essential role in lung development through CLEC-2/podoplanin interaction. Platelet-specific CLEC-2 deletion in mice causes lung malformation (absence of alveolar duct myofibroblasts) and neonatal lethality. LEC-specific podoplanin deletion phenocopies this defect; CLEC-2/podoplanin interaction on LECs stimulates adMYF differentiation of lung mesothelial cells via TGF-beta signaling.","method":"Platelet-specific and LEC-specific conditional knockout mice, histology, immunostaining, TGF-beta pathway analysis, thrombocytopenia model","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — cell-type-specific KOs with rescue experiments and identification of TGF-beta as mediator","pmids":["29853539"],"is_preprint":false},{"year":2018,"finding":"Recombinant rhodocytin forms a heterooctamer (four α- and β-subunits); Asp4 in the α-subunit is required for CLEC-2 binding. An inhibitory mutant rhodocytin (αWTβK53A/R56A forming a heterotetramer) binds CLEC-2 without inducing platelet aggregation and blocks CLEC-2-podoplanin-dependent platelet aggregation and experimental lung metastasis.","method":"Recombinant protein expression (CHO cells), blue native PAGE, flow cytometry, light transmission aggregometry, in vivo lung metastasis model","journal":"Journal of thrombosis and haemostasis","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis with structural (native PAGE) and functional (platelet aggregation, in vivo metastasis) validation","pmids":["29488681"],"is_preprint":false},{"year":2018,"finding":"Tetraspanin CD37 controls CLEC-2 membrane organization in dendritic cells. A specific CLEC-2-CD37 interaction was identified; CD37-/- myeloid cells have reduced surface CLEC-2. Loss of CD37 impairs CLEC-2 recruitment to podoplanin, DC adhesion, migration velocity, actin protrusion formation in 3D collagen, and CLEC-2-dependent inhibition of FRC actomyosin contractility.","method":"Co-immunoprecipitation, CD37-/- DCs, DC migration assays, microcontact printing, 3D collagen matrix assays, FRC contraction assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — Co-IP identifying interaction plus multiple functional assays in CD37-/- DCs","pmids":["30185523"],"is_preprint":false},{"year":2019,"finding":"Dengue virus activates platelets via CLEC-2 to release extracellular vesicles (exosomes and microvesicles). These CLEC-2-induced EVs activate CLEC5A and TLR2 on neutrophils and macrophages, triggering NET formation and proinflammatory cytokine release. Simultaneous blockade of CLEC5A and TLR2 increased survival from 30 to 90% in dengue-infected mice.","method":"In vitro platelet activation, EV isolation, neutrophil/macrophage activation assays, in vivo dengue mouse model, CLEC5A/TLR2 blockade","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — mechanistic pathway established from CLEC-2 activation to EV release to downstream innate immune activation with in vivo validation","pmids":["31160588"],"is_preprint":false},{"year":2019,"finding":"Shed CLEC-2 is generated independently of ADAM10/17 (which shed GPVI) and is partially generated by MMP-2. CLEC-2 is released as a shed fragment or as a whole molecule associated with platelet microparticles (MP-CLEC-2), unlike GPVI which is not associated with microparticles under normal conditions.","method":"Metalloproteinase inhibitors/stimulators, anti-domain-specific antibodies, microparticle isolation, immunoblotting","journal":"International journal of hematology","confidence":"Medium","confidence_rationale":"Tier 2 — pharmacological dissection of shedding mechanism, single lab","pmids":["31165998"],"is_preprint":false},{"year":2020,"finding":"Platelet CLEC-2 signaling impairs hepatic recovery after acetaminophen-induced acute liver failure by suppressing TNF-alpha production and reducing reparative hepatic neutrophil recruitment. Blocking platelet CLEC-2 signaling enhances liver recovery by increasing TNF-alpha levels which drive beneficial neutrophil responses.","method":"CLEC-2-deficient mice, APAP and CCl4 liver injury models, neutrophil depletion, cytokine measurement, flow cytometry, human liver samples","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — CLEC-2 KO with defined mechanistic pathway (TNF-alpha/neutrophil recruitment) validated in human and mouse tissue","pmids":["32321925"],"is_preprint":false},{"year":2021,"finding":"CLEC-2 exists as a monomer and homo-dimer in resting platelets and forms higher-order oligomers following ligand activation. Syk binding to phosphorylated hemITAM is mediated by tandem SH2 domains binding to PIP2/PIP3 at the membrane. Low-level Syk activity in resting platelets (due to Src family kinase phosphorylation) is amplified by receptor clustering, which disturbs the kinase-phosphatase equilibrium to trigger hemITAM phosphorylation.","method":"Structural analysis, membrane lipid binding studies, review of published data integrating biophysical and biochemical evidence","journal":"Platelets","confidence":"Medium","confidence_rationale":"Tier 2 — mechanistic model integrating published biophysical data, single synthesis paper","pmids":["33819136"],"is_preprint":false},{"year":2021,"finding":"Low concentrations of the Btk inhibitor ibrutinib (and acalabrutinib) selectively block CLEC-2-mediated platelet activation and tyrosine phosphorylation including Syk and PLCgamma2 in human platelets, while only delaying GPVI responses. This differential effect is explained by Btk's positive feedback role in CLEC-2 signaling (via ADP/TxA2/P2Y12/TP receptors), which is not present in mouse platelets.","method":"Human platelets with ibrutinib/acalabrutinib, XLA patient platelets (Btk-deficient), aggregometry, phosphorylation assays, mouse in vivo thrombosis","journal":"Haematologica","confidence":"High","confidence_rationale":"Tier 2 — genetic (XLA) and pharmacological Btk ablation distinguishing CLEC-2 from GPVI pathway, human and mouse data","pmids":["31949019"],"is_preprint":false},{"year":2021,"finding":"Platelet CLEC-2 reduces inflammatory macrophage accumulation during peritonitis. CLEC-2 engagement of podoplanin on macrophages (upregulated during inflammation) accelerates actin rearrangement and macrophage migration by increasing podoplanin and CD44 expression and their interaction with ERM proteins, promoting macrophage emigration to draining lymph nodes.","method":"Platelet-specific CLEC-2 KO, recombinant CLEC-2-Fc, LPS peritonitis model, macrophage migration assays, ERM protein interaction studies, flow cytometry","journal":"Frontiers in immunology","confidence":"High","confidence_rationale":"Tier 2 — KO model plus recombinant protein with defined molecular mechanism (podoplanin/CD44/ERM) and in vivo validation","pmids":["34163489"],"is_preprint":false},{"year":2021,"finding":"Hemin (free heme) directly binds to both CLEC-2 and GPVI on platelets (confirmed by western blotting and SPR), activating platelet aggregation via SYK and PLCgamma2 phosphorylation. Double knockout of CLEC-2 and FcRgamma (GPVI-equivalent) nearly abolishes hemin-induced platelet aggregation and attenuates rhabdomyolysis-induced acute kidney injury.","method":"SPR binding, western blotting, CLEC-2-depleted and FcRgamma-/- mice (single and double KO), RAKI mouse model, in vitro MET formation","journal":"Blood advances","confidence":"High","confidence_rationale":"Tier 1-2 — SPR direct binding plus double-KO mouse model establishing cooperative roles","pmids":["33843987"],"is_preprint":false},{"year":2022,"finding":"Cooperative signaling between CLEC-2 and GPIIb/IIIa triggers cerebral venous sinus thrombosis (CVT)-like syndrome in mice. INU1-Fab-induced CLEC-2 signaling combined with GPIIb/IIIa activation causes rapid CVT. Interfering with CLEC-2 signaling or inhibiting GPIIb/IIIa completely blocked platelet activation and CVT; blocking GPIIb/IIIa after symptom onset protected against death.","method":"Antibody-induced CLEC-2 signaling model, intravital transcranial microscopy, CLEC-2 signaling inhibition, GPIIb/IIIa antagonist treatment","journal":"Nature cardiovascular research","confidence":"High","confidence_rationale":"Tier 2 — novel mouse model establishing cooperative receptor mechanism with pharmacological interventions","pmids":["39195988"],"is_preprint":false},{"year":2015,"finding":"Targeted downregulation of platelet CLEC-2 by antibody INU1 occurs through Src family kinase-dependent receptor internalization (not Syk-dependent), presumably followed by intracellular degradation. In platelet-specific Syk-deficient mice, INU1-induced CLEC-2 internalization was fully preserved while the associated thrombocytopenia was largely prevented, mechanistically uncoupling the two processes.","method":"Anti-CLEC-2 antibody (INU1) treatment, Src family kinase inhibitors, platelet-specific Syk-deficient mice, receptor internalization assays in vitro and in vivo","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — genetic and pharmacological dissection separating internalization from thrombocytopenia mechanisms","pmids":["25795918"],"is_preprint":false},{"year":2006,"finding":"Platelets express CLEC-2, which binds HIV-1 independently of the viral envelope protein, facilitating HIV-1 capture by platelets. CLEC-2 and DC-SIGN together are required for efficient HIV-1 binding; captured HIV-1 remains infectious for several days, suggesting CLEC-2 may facilitate HIV-1 dissemination.","method":"Flow cytometry binding assays, DC-SIGN and CLEC-2 inhibitors, platelet HIV-1 capture assays, infectivity assays","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 — direct binding and inhibitor studies, single lab","pmids":["16940507"],"is_preprint":false},{"year":2015,"finding":"Podoplanin/CLEC-2 signaling regulates keratinocyte migration during wound healing. CLEC-2 addition and podoplanin knockdown both inhibit keratinocyte migration. CLEC-2 downregulates RhoA activity and upregulates E-cadherin in keratinocytes, providing a molecular mechanism for the regulation of migration.","method":"podoplanin siRNA in NHEK cells, CLEC-2 protein addition, wound healing and transwell migration assays, RhoA activity assay, E-cadherin immunostaining","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 — siRNA and recombinant protein in primary human cells with defined downstream signaling, single lab","pmids":["26597882"],"is_preprint":false},{"year":2020,"finding":"Phosphoproteomic analysis of rhodocytin-activated platelets identified 363 differentially phosphorylated tyrosine residues. Syk phosphorylation at Tyr525+526 occurs even with ADP and TxA2 pathway inhibitors, while Src-pTyr419 and PLCgamma2-pTyr759 require ADP/TxA2 feedback. Novel components of the CLEC-2 signalosome identified include adaptors DAPP1, Dok1/3, CASS4, Nck1/2, kinases FAK1, FES, FGR, JAK2, SHIP2, and membrane proteins G6F, JAM-A, PECAM-1, TLT-1.","method":"Mass spectrometry-based phosphotyrosine proteomics, 2D-DIGE, kinase inhibitors, Ca2+ mobilization assays","journal":"Thrombosis and haemostasis","confidence":"Medium","confidence_rationale":"Tier 2 — comprehensive phosphoproteomics with pharmacological dissection, single lab","pmids":["31901221"],"is_preprint":false}],"current_model":"CLEC-2 (CLEC1B) is a platelet and megakaryocyte C-type lectin receptor that binds its primary endogenous ligand podoplanin (and also rhodocytin, fucoidan, hemin, and S100A13) on its non-canonical 'side' face as revealed by crystal structures; ligand engagement induces CLEC-2 dimerization and higher-order clustering, recruits Syk (2:1 stoichiometry via tandem SH2 domains binding two phosphorylated hemITAM YXXL motifs from a dimer), requires lipid raft translocation, actin polymerization, and ADP/TxA2 positive feedback (via Gq/PLCβ/PKCα/Src) to amplify hemITAM phosphorylation, and activates a Syk→SLP-76→PLCgamma2→Ca2+ signaling cascade that triggers platelet aggregation, granule secretion (releasing S1P, TGF-beta, BMP-9, CCL5), and EV release, with physiological roles in blood-lymphatic separation during embryogenesis, HEV integrity, megakaryopoiesis, lymph node maintenance, lung development, dendritic cell motility, and pathological roles in thrombosis, tumor metastasis, and thromboinflammation."},"narrative":{"teleology":[{"year":2005,"claim":"Identifying CLEC-2 as the platelet receptor for rhodocytin established that a single hemITAM YXXL motif could activate a Syk→PLCγ2 signaling cascade, defining a new receptor class distinct from classical dual-ITAM receptors.","evidence":"Rhodocytin affinity chromatography, Syk−/− and PLCγ2−/− mouse platelets, kinase inhibitors","pmids":["16174766"],"confidence":"High","gaps":["Endogenous ligand unknown","Mechanism by which single YXXL engages tandem SH2 domains of Syk unresolved","In vivo physiological role not established"]},{"year":2006,"claim":"The 1.6 Å crystal structure of CLEC-2 revealed the ligand-binding surface centered on a semi-helical loop, providing the first structural framework for understanding receptor–ligand specificity.","evidence":"X-ray crystallography, site-directed mutagenesis, SPR","pmids":["17132623"],"confidence":"High","gaps":["No co-crystal with ligand yet","Dimerization interface not resolved"]},{"year":2007,"claim":"Identification of podoplanin as the endogenous CLEC-2 ligand answered what physiological signal activates this receptor and revealed dependence on sialylated O-glycans, while parallel signaling studies showed the DEDG acidic motif upstream of YXXL is critical for hemITAM function.","evidence":"Flow cytometry binding, recombinant protein inhibition, glycopeptide mapping, mutagenesis in mutant cell lines","pmids":["17616532","17944973","17339324"],"confidence":"High","gaps":["Affinity of CLEC-2/podoplanin interaction not quantified","Structural basis of glycan recognition unknown","In vivo developmental role not yet demonstrated"]},{"year":2009,"claim":"Demonstration that CLEC-2 exists as a pre-formed homodimer resolved how a single hemITAM receptor engages Syk's tandem SH2 domains — each SH2 domain binds one phospho-YXXL from a different monomer — and antibody-mediated CLEC-2 depletion established its requirement for thrombus stability in vivo.","evidence":"BRET, co-IP, analytical ultracentrifugation, MALS, intravital microscopy, in vivo bleeding/thrombosis models","pmids":["19824697","19641185"],"confidence":"High","gaps":["Stoichiometry of Syk binding not yet directly measured","Developmental role not yet tested genetically"]},{"year":2010,"claim":"CLEC-2 knockout mice revealed that hemITAM signaling via Syk/SLP-76 on platelets is essential for embryonic blood–lymphatic separation, while biophysical studies confirmed 2:1 Syk:CLEC-2 stoichiometry and showed that hemITAM phosphorylation requires lipid raft translocation, actin polymerization, and ADP/TxA2 positive feedback.","evidence":"Constitutive and platelet-specific conditional KOs, fetal liver transplant, quantitative SPR/cross-linking/EM, lipid raft fractionation, pharmacological inhibitors","pmids":["20525685","20363774","20154219","20154214","21098033"],"confidence":"High","gaps":["Relative contributions of Syk versus Src in initiating versus amplifying signaling debated","How lipid raft translocation is triggered mechanistically unclear","Whether CLEC-2 has signaling-independent roles unknown"]},{"year":2012,"claim":"Beyond platelets, CLEC-2 was shown to regulate dendritic cell motility through RhoA inhibition and Rac1/Vav-dependent actin protrusions upon podoplanin engagement, while in platelets, CLEC-2 activation releases BMP-9 and TGF-β to inhibit lymphatic endothelial cell proliferation and tube formation.","evidence":"CLEC-2-deficient DCs, migration/actin dynamics assays, RhoA/Rac1 activity assays, platelet-specific KO, LEC functional assays","pmids":["22884313","22556408"],"confidence":"High","gaps":["Whether DC and platelet CLEC-2 signaling pathways diverge at the level of Syk unclear","How BMP-9 is stored and released from platelet granules not defined"]},{"year":2013,"claim":"The physiological function of platelet CLEC-2 was extended to maintaining high endothelial venule integrity through podoplanin-dependent S1P release promoting VE-cadherin expression, and fucoidan was identified as an additional CLEC-2 agonist.","evidence":"Conditional KO with platelet rescue, S1P pathway analysis, VE-cadherin staining, fucoidan activation in CLEC-2 KO platelets","pmids":["23995678","23341451"],"confidence":"High","gaps":["Whether S1P release is the sole downstream mediator of HEV maintenance not excluded","Fucoidan binding site on CLEC-2 unknown"]},{"year":2014,"claim":"Co-crystal structures of CLEC-2 with both podoplanin and rhodocytin revealed a shared non-canonical 'side' binding face utilizing arginine–acidic residue interactions, providing a structural explanation for ligand promiscuity, while super-resolution imaging showed Src/Syk-dependent CLEC-2/podoplanin clustering within 10 nm on platelet membranes.","evidence":"X-ray crystallography of two ligand complexes, dSTORM/FLIM-FRET, supported lipid bilayer with Syk−/− platelets","pmids":["25458834","25368330"],"confidence":"High","gaps":["Full-length receptor structure including transmembrane and cytoplasmic domains not solved","Structural basis of higher-order clustering beyond dimers unknown"]},{"year":2015,"claim":"Multiple in vivo studies established CLEC-2/podoplanin as critical for cerebrovascular patterning, lymph node development, megakaryopoiesis (via Thpo production), venous thrombosis, and infection-driven thromboinflammation, while S100A13 was identified as an additional CLEC-2 ligand on vascular smooth muscle cells.","evidence":"Constitutive/conditional KOs (platelet-specific, Mk-specific, Nestin-Cre), IVC stenosis DVT model, anti-PDPN antibody, Salmonella infection model, SPR/protein array for S100A13, HSC transplant","pmids":["25908104","24532804","26552707","28104688","26571395","26418160"],"confidence":"High","gaps":["Relative quantitative contributions of multiple CLEC-2 ligands in different vascular beds undefined","Mechanistic link between Mk CLEC-2 and Thpo transcription not fully resolved","S100A13 binding site not mapped"]},{"year":2017,"claim":"A hemITAM-dead knockin (Y7A) distinguished signaling-dependent from signaling-independent CLEC-2 functions: blood–lymphatic separation requires hemITAM signaling, whereas hemostasis/thrombosis also involves a structural/adhesive contribution independent of YXXL phosphorylation. Gq/PLCβ/PKCα/Src was defined as the positive feedback loop potentiating hemITAM phosphorylation.","evidence":"Y7A knockin mouse with anti-CLEC-2 Fab blockade, in vivo thrombosis models, Gq−/− platelets, pharmacological pathway dissection","pmids":["28835437","28705934"],"confidence":"High","gaps":["Nature of signaling-independent adhesive mechanism unknown","Whether Gq feedback loop operates in non-platelet CLEC-2-expressing cells not tested"]},{"year":2018,"claim":"CLEC-2/podoplanin was shown to be essential for lung alveolar development via TGF-β-dependent myofibroblast differentiation, CD37 was identified as a tetraspanin partner controlling CLEC-2 membrane organization in DCs, and a small-molecule antagonist (Co-HP) binding a site distinct from podoplanin inhibited thrombosis and metastasis without bleeding.","evidence":"Platelet-specific and LEC-specific conditional KOs, CD37−/− DC functional assays and co-IP, Co-HP SPR/mutagenesis with in vivo thrombosis/metastasis models","pmids":["29853539","30185523","30190281"],"confidence":"High","gaps":["How Co-HP binding inhibits signaling allosterically not structurally resolved","CD37 interaction interface not mapped","Lung phenotype in humans with CLEC-2 loss unknown"]},{"year":2019,"claim":"CLEC-2 activation by dengue virus triggers platelet extracellular vesicle release that activates innate immune receptors CLEC5A and TLR2 on neutrophils/macrophages, establishing a platelet-to-innate-immune amplification loop in viral thromboinflammation.","evidence":"In vitro platelet EV isolation, neutrophil/macrophage activation assays, dengue mouse model with CLEC5A/TLR2 blockade","pmids":["31160588"],"confidence":"High","gaps":["Whether EV-mediated amplification applies to other viral infections not tested","Specific viral component engaging CLEC-2 not identified"]},{"year":2020,"claim":"Phosphoproteomic profiling of CLEC-2-activated platelets revealed 363 differentially phosphorylated tyrosine sites and identified novel signalosome components (DAPP1, Dok1/3, CASS4, FAK1, FES, JAK2), while in vivo studies showed platelet CLEC-2 impairs hepatic recovery after acute injury by suppressing TNF-α-driven neutrophil recruitment.","evidence":"MS-based phosphotyrosine proteomics with kinase inhibitors; CLEC-2 KO mice in APAP/CCl4 liver injury models with cytokine/neutrophil analysis","pmids":["31901221","32321925"],"confidence":"High","gaps":["Functional validation of most novel signalosome components pending","Whether CLEC-2's liver injury role is podoplanin-dependent not directly tested"]},{"year":2021,"claim":"Btk was positioned as a critical node in CLEC-2 positive feedback in human (but not mouse) platelets, hemin was confirmed as a direct CLEC-2 and GPVI co-ligand relevant to rhabdomyolysis, and CLEC-2/podoplanin interaction on macrophages was shown to accelerate their emigration from inflamed peritoneum via ERM-mediated cytoskeletal remodeling.","evidence":"XLA patient platelets and ibrutinib/acalabrutinib treatment; SPR plus double-KO (CLEC-2/FcRγ) in RAKI model; platelet-specific CLEC-2 KO in peritonitis with recombinant CLEC-2-Fc and ERM studies","pmids":["31949019","33843987","34163489"],"confidence":"High","gaps":["Species differences in Btk feedback not structurally explained","Hemin binding site on CLEC-2 not mapped","How CLEC-2 engagement on macrophages upregulates podoplanin/CD44 not mechanistically resolved"]},{"year":2022,"claim":"Cooperative signaling between CLEC-2 and GPIIb/IIIa was shown to trigger cerebral venous sinus thrombosis, demonstrating that CLEC-2 does not act in isolation but synergizes with integrin signaling for pathological thrombus formation in specific vascular beds.","evidence":"Antibody-induced CLEC-2 activation model, intravital transcranial microscopy, GPIIb/IIIa antagonist rescue","pmids":["39195988"],"confidence":"High","gaps":["Molecular mechanism of CLEC-2/GPIIb/IIIa crosstalk not defined","Relevance to human CVT (e.g., vaccine-induced) not established"]},{"year":null,"claim":"Key unresolved questions include the full-length structure of CLEC-2 including transmembrane and cytoplasmic domains, the structural basis of higher-order clustering beyond dimers, functional validation of newly identified signalosome components, the molecular nature of CLEC-2's signaling-independent adhesive role in hemostasis, and whether human CLEC-2 loss-of-function mutations cause developmental vascular disease.","evidence":"","pmids":[],"confidence":"Low","gaps":["No full-length receptor structure","No human genetic disease linked to CLEC1B mutations in the timeline","Signaling-independent adhesive mechanism molecularly undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,2,5,22]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[18,23,44]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,8,14,15,24]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[39,40]}],"pathway":[{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[0,9,14,15,27,46]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,3,13,34,42]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[10,18,31,33,39,44]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[11,12,16,25,29,36]}],"complexes":["CLEC-2 homodimer"],"partners":["PDPN","SYK","LCP2","PLCG2","CD37","ITGA2B"],"other_free_text":[]},"mechanistic_narrative":"CLEC-2 (CLEC1B) is a C-type lectin-like receptor expressed on platelets, megakaryocytes, dendritic cells, and neutrophils that functions as a hemITAM-coupled pattern recognition receptor linking innate immune sensing to hemostasis, vascular development, and inflammation. Its primary endogenous ligand podoplanin and exogenous agonists (rhodocytin, fucoidan, hemin, S100A13) bind a non-canonical 'side' face of the extracellular domain, inducing receptor dimerization and higher-order clustering that enables Syk recruitment in a 2:1 stoichiometry via tandem SH2 domains bridging two phosphorylated hemITAM YXXL motifs; hemITAM phosphorylation itself requires lipid raft translocation, actin polymerization, and positive feedback through ADP/TxA2/Gq/PLCβ/PKCα/Src pathways, driving a Syk→SLP-76→PLCγ2→Ca²⁺ cascade that triggers platelet aggregation, granule secretion (releasing S1P, TGF-β, BMP-9, CCL5), and extracellular vesicle release [PMID:16174766, PMID:25458834, PMID:20154219, PMID:20154214, PMID:28705934, PMID:31160588]. Genetically, platelet CLEC-2/podoplanin signaling is essential for embryonic blood–lymphatic vessel separation, lymph node development and maintenance, cerebrovascular patterning, lung alveolar duct formation, HEV vascular integrity, and megakaryopoiesis, and contributes pathologically to arterial and venous thrombosis, infection-driven thromboinflammation, tumor metastasis, and regulation of macrophage-mediated inflammation [PMID:20525685, PMID:20363774, PMID:25908104, PMID:29853539, PMID:23995678, PMID:24532804, PMID:26552707, PMID:28104688, PMID:26571395, PMID:34163489]."},"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; CLEC1B","url":"https://www.omim.org/entry/606783"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"liver","ntpm":74.8}],"url":"https://www.proteinatlas.org/search/CLEC1B"},"hgnc":{"alias_symbol":["CLEC2","CLEC-2"],"prev_symbol":[]},"alphafold":{"accession":"Q9P126","domains":[{"cath_id":"3.10.100.10","chopping":"107-222","consensus_level":"high","plddt":95.7933,"start":107,"end":222},{"cath_id":"1.20.5","chopping":"33-101","consensus_level":"medium","plddt":90.6823,"start":33,"end":101}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P126","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P126-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P126-F1-predicted_aligned_error_v6.png","plddt_mean":88.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CLEC1B","jax_strain_url":"https://www.jax.org/strain/search?query=CLEC1B"},"sequence":{"accession":"Q9P126","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9P126.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9P126/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P126"}},"corpus_meta":[{"pmid":"17616532","id":"PMC_17616532","title":"Involvement of the snake toxin receptor CLEC-2, in podoplanin-mediated platelet activation, by cancer cells.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17616532","citation_count":397,"is_preprint":false},{"pmid":"16174766","id":"PMC_16174766","title":"A novel Syk-dependent mechanism of platelet activation by the C-type lectin receptor CLEC-2.","date":"2005","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/16174766","citation_count":392,"is_preprint":false},{"pmid":"20363774","id":"PMC_20363774","title":"Platelets regulate lymphatic vascular development through CLEC-2-SLP-76 signaling.","date":"2010","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/20363774","citation_count":332,"is_preprint":false},{"pmid":"23995678","id":"PMC_23995678","title":"Podoplanin maintains high endothelial venule integrity by interacting with platelet CLEC-2.","date":"2013","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/23995678","citation_count":258,"is_preprint":false},{"pmid":"30601137","id":"PMC_30601137","title":"Functional significance of the platelet immune receptors GPVI and CLEC-2.","date":"2019","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/30601137","citation_count":246,"is_preprint":false},{"pmid":"22884313","id":"PMC_22884313","title":"Podoplanin-rich stromal networks induce dendritic cell motility via activation of the C-type lectin receptor CLEC-2.","date":"2012","source":"Immunity","url":"https://pubmed.ncbi.nlm.nih.gov/22884313","citation_count":235,"is_preprint":false},{"pmid":"17944973","id":"PMC_17944973","title":"Molecular analysis of the pathophysiological binding of the platelet aggregation-inducing factor podoplanin to the C-type lectin-like receptor CLEC-2.","date":"2007","source":"Cancer science","url":"https://pubmed.ncbi.nlm.nih.gov/17944973","citation_count":227,"is_preprint":false},{"pmid":"25347465","id":"PMC_25347465","title":"The CLEC-2-podoplanin axis controls the contractility of fibroblastic reticular cells and lymph node microarchitecture.","date":"2014","source":"Nature immunology","url":"https://pubmed.ncbi.nlm.nih.gov/25347465","citation_count":219,"is_preprint":false},{"pmid":"16940507","id":"PMC_16940507","title":"DC-SIGN and CLEC-2 mediate human immunodeficiency virus type 1 capture by platelets.","date":"2006","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/16940507","citation_count":205,"is_preprint":false},{"pmid":"20525685","id":"PMC_20525685","title":"Essential in vivo roles of the C-type lectin receptor CLEC-2: embryonic/neonatal lethality of CLEC-2-deficient mice by blood/lymphatic misconnections and impaired thrombus formation of CLEC-2-deficient platelets.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20525685","citation_count":199,"is_preprint":false},{"pmid":"31160588","id":"PMC_31160588","title":"Extracellular vesicles from CLEC2-activated platelets enhance dengue virus-induced lethality via CLEC5A/TLR2.","date":"2019","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/31160588","citation_count":190,"is_preprint":false},{"pmid":"19641185","id":"PMC_19641185","title":"CLEC-2 is an essential platelet-activating receptor in hemostasis and thrombosis.","date":"2009","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/19641185","citation_count":174,"is_preprint":false},{"pmid":"17339324","id":"PMC_17339324","title":"The C-type lectin receptors CLEC-2 and Dectin-1, but not DC-SIGN, signal via a novel YXXL-dependent signaling cascade.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17339324","citation_count":171,"is_preprint":false},{"pmid":"23991201","id":"PMC_23991201","title":"Platelets promote tumor growth and metastasis via direct interaction between Aggrus/podoplanin and CLEC-2.","date":"2013","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23991201","citation_count":165,"is_preprint":false},{"pmid":"31778548","id":"PMC_31778548","title":"Platelets and cancer-associated thrombosis: focusing on the platelet activation receptor CLEC-2 and podoplanin.","date":"2019","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/31778548","citation_count":154,"is_preprint":false},{"pmid":"20345705","id":"PMC_20345705","title":"GPVI and CLEC-2 in hemostasis and vascular integrity.","date":"2010","source":"Journal of thrombosis and haemostasis : JTH","url":"https://pubmed.ncbi.nlm.nih.gov/20345705","citation_count":150,"is_preprint":false},{"pmid":"28104688","id":"PMC_28104688","title":"Mice with a deficiency in CLEC-2 are protected against deep vein thrombosis.","date":"2017","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/28104688","citation_count":143,"is_preprint":false},{"pmid":"26571395","id":"PMC_26571395","title":"Inflammation drives thrombosis after Salmonella infection via CLEC-2 on platelets.","date":"2015","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/26571395","citation_count":134,"is_preprint":false},{"pmid":"22556408","id":"PMC_22556408","title":"Platelet activation receptor CLEC-2 regulates blood/lymphatic vessel separation by inhibiting proliferation, migration, and tube formation of lymphatic endothelial cells.","date":"2012","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22556408","citation_count":129,"is_preprint":false},{"pmid":"29269852","id":"PMC_29269852","title":"The podoplanin-CLEC-2 axis inhibits inflammation in sepsis.","date":"2017","source":"Nature 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Ligand binding causes tyrosine phosphorylation of a single YXXL motif in the CLEC-2 cytosolic tail, which is dependent on Src family kinases, and activates Syk and PLCgamma2. Activation is abolished in Syk-/- and PLCgamma2-/- murine platelets and partially reduced in LAT, SLP-76, and Vav1/Vav3-deficient platelets.\",\n      \"method\": \"Rhodocytin affinity chromatography, expression in cell lines, genetic knockout mice, phosphorylation assays, kinase inhibitors\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — receptor identification by affinity chromatography, functional reconstitution in cell lines, and multiple genetic knockouts\",\n      \"pmids\": [\"16174766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Crystal structure of the extracellular domain of human CLEC-2 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 confirmed the key residues involved in rhodocytin binding.\",\n      \"method\": \"X-ray crystallography, site-directed mutagenesis, surface plasmon resonance\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with mutational and SPR validation\",\n      \"pmids\": [\"17132623\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Podoplanin was identified as an endogenous ligand for CLEC-2 on platelets. The CLEC-2-podoplanin interaction 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.\",\n      \"method\": \"Flow cytometry binding assays, recombinant protein inhibition, deletion mutant analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct binding confirmation by flow cytometry and functional inhibition, replicated across labs\",\n      \"pmids\": [\"17616532\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CLEC-2 and Dectin-1 signal through a novel YXXL-dependent pathway requiring both SH2 domains of Syk (despite having only a single YXXL motif), Src and Tec family kinases, and PLCgamma, with only partial dependence on SLP-76/BLNK adapters. The glycine residue directly upstream of the YXXL tyrosine in the DEDG sequence is critical for CLEC-2 signaling.\",\n      \"method\": \"Mutant cell line signaling assays, site-directed mutagenesis of YXXL and DEDG motifs, kinase inhibitors\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution in mutant cell lines with mutagenesis of critical motifs\",\n      \"pmids\": [\"17339324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Molecular characterization of the podoplanin-CLEC-2 interaction: CLEC-2 deletion mutants (expressed as Fc chimeras) identified the podoplanin-recognition domain; a disialyl-core1-attached glycopeptide at Thr52 of podoplanin is specifically recognized by CLEC-2; the protein stereostructure of podoplanin is also critical for binding.\",\n      \"method\": \"Deletion mutant Fc chimeras, synthesized glycopeptides, in vitro binding assays, in vivo metastasis model\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — domain mapping with deletion mutants, glycopeptide synthesis, in vitro and in vivo validation\",\n      \"pmids\": [\"17944973\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Podoplanin on renal cells (HEK-293T and podocytes) was confirmed as a CLEC-2 ligand. Direct CLEC-2-podoplanin interaction measured by surface plasmon resonance showed an affinity of ~24.5 µM and was shown to be independent of glycosylation of CLEC-2.\",\n      \"method\": \"Surface plasmon resonance, recombinant protein binding, CLEC-2-transfected DT-40 B-cell activation assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — SPR affinity measurement and functional reconstitution in cell lines\",\n      \"pmids\": [\"18215137\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Crystal structure of rhodocytin (CLEC-2 ligand) solved at 2.4 Å; rhodocytin forms a non-disulfide-linked (αβ)2 tetramer with a concave binding surface complementary to the CLEC-2 binding interface, suggesting it clusters CLEC-2 receptors on the platelet surface to trigger signaling.\",\n      \"method\": \"X-ray crystallography, computational docking, surface electrostatic analysis\",\n      \"journal\": \"Protein science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with computational docking supporting mechanism of receptor clustering\",\n      \"pmids\": [\"18583525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"G6b-B, a platelet immunoglobulin receptor with two ITIM motifs, inhibits constitutive and agonist-induced signaling by CLEC-2 (and GPVI). Inhibition by G6b-B requires its conserved ITIM tyrosines and is independent of SHP1, SHP2, and SHIP phosphatases.\",\n      \"method\": \"NFAT reporter assay, mutagenesis, cell line signaling studies\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional inhibition in cell line model with mutagenesis, single lab\",\n      \"pmids\": [\"18955485\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CLEC-2 is present as a non-disulfide-linked homodimer in resting platelets. This dimerization enables each Syk molecule to interact with two YXXL motifs (one from each CLEC-2 monomer), explaining how a hemITAM activates Syk normally requiring tandem YXXL.\",\n      \"method\": \"BRET, co-immunoprecipitation, analytical gel filtration, surface plasmon resonance, MALS, analytical ultracentrifugation\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple biophysical methods confirming dimerization with functional interpretation\",\n      \"pmids\": [\"19824697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CLEC-2-deficient platelets (via anti-CLEC-2 antibody depletion) show normal adhesion under flow but severely defective aggregate formation in vitro and in vivo, resulting in increased bleeding times and protection from occlusive arterial thrombus formation, establishing CLEC-2 as essential for hemostasis and thrombosis.\",\n      \"method\": \"Antibody-mediated CLEC-2 depletion, intravital microscopy, in vitro flow chamber assays, bleeding time measurements\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean receptor depletion with defined in vivo and in vitro phenotypic readouts\",\n      \"pmids\": [\"19641185\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CLEC-2 is expressed on murine peripheral blood neutrophils (not bone marrow or elicited neutrophils). On neutrophils, CLEC-2 mediates phagocytosis of antibody-coated beads and TNF-alpha production upon rhodocytin stimulation through its tyrosine-based cytoplasmic motif, recruiting Syk.\",\n      \"method\": \"Monoclonal antibody staining, chimeric receptor analysis, phagocytosis assay, cytokine ELISA, Syk recruitment assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct functional assays in primary cells with chimeric receptor analysis confirming YXXL requirement\",\n      \"pmids\": [\"19299712\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CLEC-2-deficient mice (Clec-2-/- generated by gene targeting) are lethal at embryonic/neonatal stages with disorganized, blood-filled lymphatic vessels and severe edema. Fetal liver transplantation demonstrated that CLEC-2 is involved in thrombus stabilization in vitro and in vivo, potentially through homophilic interactions, without apparent increase in bleeding tendency.\",\n      \"method\": \"Gene knockout, fetal liver transplantation, intravital microscopy, laser-induced thrombosis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — constitutive knockout with rescue by transplantation, replicated phenotype\",\n      \"pmids\": [\"20525685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Platelet CLEC-2 directly binds lymphatic endothelial cell podoplanin (PDPN), activating SLP-76 signaling. Genetic loss of CLEC-2 abolishes PDPN binding by platelets and causes embryonic lymphatic vascular defects; platelet-specific deletion of Slp-76 is sufficient to recapitulate the defect, establishing the platelet CLEC-2/SLP-76 pathway as essential for blood-lymphatic separation.\",\n      \"method\": \"Genetic knockout (CLEC-2-/-, platelet-specific PF4-Cre Slp-76 deletion), intravital and ex vivo imaging, flow cytometry\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic models with epistasis placing CLEC-2 upstream of SLP-76 in platelet-dependent lymphatic development\",\n      \"pmids\": [\"20363774\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Syk, but not the major platelet Src family kinases (Fyn, Lyn, Src) or CD148, is required for phosphorylation of the CLEC-2 hemITAM by rhodocytin. Src family kinases play critical roles downstream of Syk, regulating Syk activity and other effector proteins rather than initiating CLEC-2 phosphorylation directly.\",\n      \"method\": \"Syk-/- and Src kinase-deficient mouse platelets, kinase inhibitor PP2, western blotting of phosphorylation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic knockouts establishing Syk's unique role in hemITAM phosphorylation\",\n      \"pmids\": [\"21098033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CLEC-2 activates Syk through dimerization: phosphorylated CLEC-2 hemITAM recruits Syk in a 2:1 stoichiometry via its tandem SH2 domains. CLEC-2 exists as a dimer in resting platelets and forms larger complexes upon activation, as confirmed by cross-linking and electron microscopy.\",\n      \"method\": \"Peptide pulldown, surface plasmon resonance, quantitative western blotting, tryptophan fluorescence, cross-linking, electron microscopy\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple orthogonal biophysical methods establishing 2:1 Syk:CLEC-2 stoichiometry and dimerization-dependent activation\",\n      \"pmids\": [\"20154219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CLEC-2 hemITAM phosphorylation requires translocation to lipid rafts upon ligand engagement; this translocation and the subsequent phosphorylation are also critically dependent on actin polymerization, Rac1 activation, and release of ADP and TxA2 as secondary mediators. In contrast, GPVI-ITAM phosphorylation is independent of these events.\",\n      \"method\": \"Sucrose gradient ultracentrifugation, methyl-beta-cyclodextrin treatment, actin polymerization inhibitors, Rac1 inhibition, ADP/TxA2 pathway inhibitors\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple pharmacological and biochemical approaches with clear mechanistic distinction from GPVI\",\n      \"pmids\": [\"20154214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Conditional deletion of CLEC-2 or Syk in the megakaryocyte/platelet lineage causes defects in brain vascular and lymphatic development. Platelets (but not platelet releasate) directly modulate migration and intercellular adhesion of lymphatic endothelial cells through a CLEC-2- and Syk-dependent pathway.\",\n      \"method\": \"Conditional knockout (Cre-lox), in vitro lymphatic endothelial cell migration/adhesion assays, releasate vs. cell contact experiments\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific conditional knockouts with defined cellular phenotype and mechanistic dissection\",\n      \"pmids\": [\"22186994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Platelet CLEC-2 regulates blood/lymphatic vessel separation by inhibiting proliferation, migration, and tube formation of lymphatic endothelial cells (LECs). Activated platelets release BMP-9 (present in platelet granules), which plays a key role in inhibiting LEC tube formation; TGF-beta and PF4 also inhibit proliferation/migration.\",\n      \"method\": \"Platelet-specific CLEC-2 knockout (Cre-lox), in vitro LEC migration/proliferation/tube formation assays, BMP-9 identification in platelets\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — platelet-specific KO with defined cellular assays and identification of secreted mediator\",\n      \"pmids\": [\"22556408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"CLEC-2 engagement by podoplanin in dendritic cells (DCs) rearranges the actin cytoskeleton to promote DC motility. CLEC-2 activation triggers cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation, and F-actin-rich protrusions via Vav signaling and Rac1 activation, enabling DC migration along stromal surfaces and entry into lymphatics.\",\n      \"method\": \"CLEC-2-deficient DCs, DC migration assays, RhoA/Rac1 activity assays, actin dynamics imaging\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — CLEC-2 KO DCs with defined molecular signaling (RhoA/Rac1) and cellular migratory phenotype\",\n      \"pmids\": [\"22884313\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The triacidic amino acid sequence (DEDG) upstream of the YXXL hemITAM in CLEC-2 is required for hemITAM phosphorylation; mutation of these acidic residues abolishes Syk-dependent signaling. Syk-mediated signaling is restricted to Syk (not Zap-70) in hemITAM receptors.\",\n      \"method\": \"Site-directed mutagenesis, Syk/Zap-70-deficient cell lines, SPR, phosphorylation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis with SPR and cell line reconstitution establishing functional requirement of acidic motif\",\n      \"pmids\": [\"23264619\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Podoplanin expressed on fibroblastic reticular cells (FRCs) surrounding high endothelial venules (HEVs) activates platelet CLEC-2. CLEC-2-mediated platelet activation induces release of sphingosine-1-phosphate (S1P) from platelets, which promotes VE-cadherin expression on HEVs, maintaining vascular integrity. Infusion of wild-type platelets restored HEV integrity in Clec-2-deficient mice.\",\n      \"method\": \"Conditional knockout mice, platelet infusion rescue, S1P pathway analysis, VE-cadherin staining, ex vivo HEV treatment\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with platelet rescue and identification of downstream mediator (S1P), multiple orthogonal methods\",\n      \"pmids\": [\"23995678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Fucoidan (a sulfated polysaccharide) is a novel CLEC-2 agonist; fucoidan-induced platelet activation is abolished in platelet-specific CLEC-2 knockout platelets, occurs via Src and Syk kinase-dependent signaling, and is largely independent of the GPVI/FcRgamma pathway.\",\n      \"method\": \"CLEC-2 platelet-specific KO, aggregometry, tyrosine phosphorylation assays, FcRgamma-/- mice\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — platelet-specific KO with direct comparison to GPVI pathway\",\n      \"pmids\": [\"23341451\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structures of CLEC-2 in complex with podoplanin glycopeptide and rhodocytin reveal that both ligands bind to the non-canonical 'side' face of CLEC-2. A common interaction mode involves consecutive acidic residues on ligands interacting with arginine residues on CLEC-2; the second binding site differs: sialic acid carboxyl from podoplanin vs. rhodocytin C-terminus interact differently.\",\n      \"method\": \"X-ray crystallography of CLEC-2 complexes with podoplanin peptide and rhodocytin\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — comparative crystal structures of two CLEC-2 ligand complexes revealing mechanism of versatile binding\",\n      \"pmids\": [\"25458834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Podoplanin regulates actomyosin contractility in fibroblastic reticular cells (FRCs) through the CLEC-2/podoplanin axis. Under resting conditions, PDPN endows FRCs with contractile function. Upon inflammation, CLEC-2 on mature DCs attenuates PDPN-mediated contractility, resulting in FRC relaxation, reduced tissue stiffness, and an expanded reticular network with enhanced immunity.\",\n      \"method\": \"Collagen gel contraction assay, FRC co-culture with CLEC-2-expressing DCs, stiffness measurements, podoplanin loss-of-function\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic dissection of CLEC-2/PDPN axis in FRC contractility with multiple functional readouts\",\n      \"pmids\": [\"25347465\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CLEC-2 signaling via Src and Syk kinases promotes platelet adhesion to lymphatic endothelial cells and clustering of CLEC-2 with podoplanin on supported lipid bilayers. CLEC-2/podoplanin clusters migrate to the center of the platelet and are within 10 nm of one another; disrupted by Src/Syk inhibition. CLEC-2 clusters also observed by dSTORM on immobilized podoplanin.\",\n      \"method\": \"Supported lipid bilayer with mobile podoplanin, FLIM-FRET, dSTORM, Src/Syk kinase inhibitors, Syk-/- mouse platelets\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — super-resolution imaging plus biophysical assays with genetic/pharmacological controls\",\n      \"pmids\": [\"25368330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CLEC-2 is required for development and maintenance of lymph nodes. Constitutive CLEC-2 deletion impairs lymphatic endothelial cell proliferation resulting in absent LNs; platelet-specific CLEC-2 deletion leads to blood-filled LNs and fibrosis. Platelet CLEC-2 is required for LN integrity and immune cell recirculation.\",\n      \"method\": \"Constitutive and platelet-specific (PF4-Cre) CLEC-2 knockout, bone marrow chimeras, immunization experiments\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple conditional and constitutive KO models distinguishing LEC proliferative vs. integrity functions\",\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 via the Syk/Lcp2/Plcg2 signaling axis. Megakaryocyte-specific CLEC-2 deletion (Clec2MkΔ/Δ) reduces Thpo in megakaryocytes, decreases HSC quiescence and repopulation potential, and causes extramedullary hematopoiesis; recombinant Thpo administration restores HSC potential.\",\n      \"method\": \"Megakaryocyte-specific conditional KO, knockdown of signaling molecules in cultured Mks, recombinant Thpo rescue, HSC transplantation assays\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with rescue experiment and downstream signaling pathway defined\",\n      \"pmids\": [\"26552707\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CLEC-2 drives venous thrombosis through podoplanin interaction; platelet-specific or inducible CLEC-2 deletion protects mice from DVT in IVC stenosis model. Podoplanin is expressed in the IVC wall near the abluminal endothelium and is upregulated in thrombus-bearing vessels. Anti-podoplanin neutralizing antibody reduces thrombus size.\",\n      \"method\": \"Inducible and platelet-specific CLEC-2 KO, IVC stenosis DVT model, platelet transfusion, anti-podoplanin antibody treatment, intravital imaging\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic and antibody-based approaches with defined in vivo thrombosis phenotype\",\n      \"pmids\": [\"28104688\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Podoplanin-positive bone marrow periarteriolar stromal cells (BM FRC-like cells) promote megakaryocyte growth and proplatelet formation through CLEC-2/podoplanin interaction. This interaction induces BM FRC-like cells to secrete CCL5, which facilitates proplatelet formation.\",\n      \"method\": \"Platelet/Mk-specific CLEC-2 conditional KO, in vitro megakaryocyte-BM FRC co-culture, recombinant PDPN stimulation, CCL5 ELISA, immunohistochemistry\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO plus in vitro co-culture identifying CCL5 as secreted mediator\",\n      \"pmids\": [\"26796360\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Podoplanin and CLEC-2 control cerebrovascular patterning during development. Constitutive or platelet-specific deletion of CLEC-2 causes tortuous, aberrantly patterned cerebral vessels at E10.5 with large hemorrhages and defective pericyte recruitment. Nestin-Cre-driven deletion of podoplanin on neural progenitors also causes cerebral hemorrhaging.\",\n      \"method\": \"Constitutive and conditional (platelet-specific, neural progenitor-specific Nestin-Cre) knockout mice, 3D light-sheet microscopy, immunofluorescence, electron microscopy\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple conditional KOs with cell-type-specific resolution and 3D imaging\",\n      \"pmids\": [\"25908104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"S100A13 was identified as a CLEC-2 ligand in vascular smooth muscle cells (VSMCs) using protein array and Biacore analysis. VSMCs stimulate platelet granule release and support thrombus formation under flow in a CLEC-2-dependent manner. S100A13 is released under oxidative stress and expressed in atherosclerotic lesions.\",\n      \"method\": \"Recombinant CLEC-2 binding studies, protein array, surface plasmon resonance (Biacore), CLEC-2-deficient mice, flow chamber thrombosis, FeCl3 thrombosis model\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — SPR identification of novel ligand with in vitro and in vivo functional validation, single lab\",\n      \"pmids\": [\"26418160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"During bacterial infection (Salmonella), inflammation triggers upregulation of podoplanin on podoplanin-expressing monocytes and Kupffer cells at perivascular sites via IFN-gamma, which then activates platelet CLEC-2 to drive thrombus formation independently of classical GPVI-mediated activation. TLR4 and IFN-gamma are required upstream of this process.\",\n      \"method\": \"Salmonella infection mouse model, intravital liver imaging, cell depletion, TLR4/IFN-gamma/CLEC-2 genetic/antibody-mediated deletion, podoplanin immunostaining\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic and depletion approaches establishing pathway position in infection-driven thrombosis\",\n      \"pmids\": [\"26571395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"A CLEC-2 knockin mouse expressing CLEC-2 with a non-signaling hemITAM (Y7A KI) recapitulates 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' fragments revealed a hemITAM signaling-independent role for CLEC-2 in hemostasis and thrombosis, suggesting a structural/adhesive contribution.\",\n      \"method\": \"Knockin mouse (Y7A hemITAM mutation), anti-CLEC-2 Fab' functional blockade, in vivo thrombosis models, tail bleeding time\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — novel knockin model separating signaling-dependent from signaling-independent CLEC-2 functions\",\n      \"pmids\": [\"28835437\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Platelet-specific CLEC-2 deletion leads to enhanced systemic inflammation and accelerated organ injury in sepsis models (LPS and cecal ligation/puncture). 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, suggesting podoplanin activation on macrophages drives the anti-inflammatory action.\",\n      \"method\": \"Platelet-specific CLEC-2 KO, LPS and CLP sepsis models, immune cell counts, cytokine measurement, pharmacological inhibition\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — platelet-specific KO with defined sepsis phenotype and pharmacological confirmation\",\n      \"pmids\": [\"29269852\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Gq signaling potentiates CLEC-2 signaling via the PLCβ-PKCα pathway, which regulates Src family kinase activity upstream of CLEC-2 hemITAM phosphorylation. TxA2 (via Gq) specifically potentiates CLEC-2 receptor tyrosine phosphorylation and downstream Syk/PLCgamma2 phosphorylation.\",\n      \"method\": \"Gq inhibitor (UBO-QIC), Gq-/- mouse platelets, pharmacological pathway dissection, western blotting of phosphorylation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic and pharmacological Gq ablation defining the positive feedback mechanism in CLEC-2 signaling\",\n      \"pmids\": [\"28705934\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Cobalt hematoporphyrin (Co-HP) directly binds to CLEC-2 at residues N120, N210, and K211 (confirmed by CLEC-2 mutant analysis), which are distinct from the previously known podoplanin-binding sites. Co-HP inhibits CLEC-2-mediated platelet aggregation and suppresses tumor metastasis and arterial/venous thrombosis in vivo without prolonging bleeding time.\",\n      \"method\": \"SPR, molecular docking, CLEC-2 mutant analysis, in vitro platelet aggregation, in vivo thrombosis and metastasis models\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — SPR binding site identification validated by mutagenesis with in vitro and in vivo functional confirmation\",\n      \"pmids\": [\"30190281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Platelets play an essential role in lung development through CLEC-2/podoplanin interaction. Platelet-specific CLEC-2 deletion in mice causes lung malformation (absence of alveolar duct myofibroblasts) and neonatal lethality. LEC-specific podoplanin deletion phenocopies this defect; CLEC-2/podoplanin interaction on LECs stimulates adMYF differentiation of lung mesothelial cells via TGF-beta signaling.\",\n      \"method\": \"Platelet-specific and LEC-specific conditional knockout mice, histology, immunostaining, TGF-beta pathway analysis, thrombocytopenia model\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific KOs with rescue experiments and identification of TGF-beta as mediator\",\n      \"pmids\": [\"29853539\"],\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 is required for CLEC-2 binding. An inhibitory mutant rhodocytin (αWTβK53A/R56A forming a heterotetramer) binds CLEC-2 without inducing platelet aggregation and blocks CLEC-2-podoplanin-dependent platelet aggregation and experimental lung metastasis.\",\n      \"method\": \"Recombinant protein expression (CHO cells), blue native PAGE, flow cytometry, light transmission aggregometry, in vivo lung metastasis model\",\n      \"journal\": \"Journal of thrombosis and haemostasis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis with structural (native PAGE) and functional (platelet aggregation, in vivo metastasis) validation\",\n      \"pmids\": [\"29488681\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Tetraspanin CD37 controls CLEC-2 membrane organization in dendritic cells. A specific CLEC-2-CD37 interaction was identified; CD37-/- myeloid cells have reduced surface CLEC-2. Loss of CD37 impairs CLEC-2 recruitment to podoplanin, DC adhesion, migration velocity, actin protrusion formation in 3D collagen, and CLEC-2-dependent inhibition of FRC actomyosin contractility.\",\n      \"method\": \"Co-immunoprecipitation, CD37-/- DCs, DC migration assays, microcontact printing, 3D collagen matrix assays, FRC contraction assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP identifying interaction plus multiple functional assays in CD37-/- DCs\",\n      \"pmids\": [\"30185523\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Dengue virus activates platelets via CLEC-2 to release extracellular vesicles (exosomes and microvesicles). These CLEC-2-induced EVs activate CLEC5A and TLR2 on neutrophils and macrophages, triggering NET formation and proinflammatory cytokine release. Simultaneous blockade of CLEC5A and TLR2 increased survival from 30 to 90% in dengue-infected mice.\",\n      \"method\": \"In vitro platelet activation, EV isolation, neutrophil/macrophage activation assays, in vivo dengue mouse model, CLEC5A/TLR2 blockade\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic pathway established from CLEC-2 activation to EV release to downstream innate immune activation with in vivo validation\",\n      \"pmids\": [\"31160588\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Shed CLEC-2 is generated independently of ADAM10/17 (which shed GPVI) and is partially generated by MMP-2. CLEC-2 is released as a shed fragment or as a whole molecule associated with platelet microparticles (MP-CLEC-2), unlike GPVI which is not associated with microparticles under normal conditions.\",\n      \"method\": \"Metalloproteinase inhibitors/stimulators, anti-domain-specific antibodies, microparticle isolation, immunoblotting\",\n      \"journal\": \"International journal of hematology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological dissection of shedding mechanism, single lab\",\n      \"pmids\": [\"31165998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Platelet CLEC-2 signaling impairs hepatic recovery after acetaminophen-induced acute liver failure by suppressing TNF-alpha production and reducing reparative hepatic neutrophil recruitment. Blocking platelet CLEC-2 signaling enhances liver recovery by increasing TNF-alpha levels which drive beneficial neutrophil responses.\",\n      \"method\": \"CLEC-2-deficient mice, APAP and CCl4 liver injury models, neutrophil depletion, cytokine measurement, flow cytometry, human liver samples\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — CLEC-2 KO with defined mechanistic pathway (TNF-alpha/neutrophil recruitment) validated in human and mouse tissue\",\n      \"pmids\": [\"32321925\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CLEC-2 exists as a monomer and homo-dimer in resting platelets and forms higher-order oligomers following ligand activation. Syk binding to phosphorylated hemITAM is mediated by tandem SH2 domains binding to PIP2/PIP3 at the membrane. Low-level Syk activity in resting platelets (due to Src family kinase phosphorylation) is amplified by receptor clustering, which disturbs the kinase-phosphatase equilibrium to trigger hemITAM phosphorylation.\",\n      \"method\": \"Structural analysis, membrane lipid binding studies, review of published data integrating biophysical and biochemical evidence\",\n      \"journal\": \"Platelets\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic model integrating published biophysical data, single synthesis paper\",\n      \"pmids\": [\"33819136\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Low concentrations of the Btk inhibitor ibrutinib (and acalabrutinib) selectively block CLEC-2-mediated platelet activation and tyrosine phosphorylation including Syk and PLCgamma2 in human platelets, while only delaying GPVI responses. This differential effect is explained by Btk's positive feedback role in CLEC-2 signaling (via ADP/TxA2/P2Y12/TP receptors), which is not present in mouse platelets.\",\n      \"method\": \"Human platelets with ibrutinib/acalabrutinib, XLA patient platelets (Btk-deficient), aggregometry, phosphorylation assays, mouse in vivo thrombosis\",\n      \"journal\": \"Haematologica\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic (XLA) and pharmacological Btk ablation distinguishing CLEC-2 from GPVI pathway, human and mouse data\",\n      \"pmids\": [\"31949019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Platelet CLEC-2 reduces inflammatory macrophage accumulation during peritonitis. CLEC-2 engagement of podoplanin on macrophages (upregulated during inflammation) accelerates actin rearrangement and macrophage migration by increasing podoplanin and CD44 expression and their interaction with ERM proteins, promoting macrophage emigration to draining lymph nodes.\",\n      \"method\": \"Platelet-specific CLEC-2 KO, recombinant CLEC-2-Fc, LPS peritonitis model, macrophage migration assays, ERM protein interaction studies, flow cytometry\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO model plus recombinant protein with defined molecular mechanism (podoplanin/CD44/ERM) and in vivo validation\",\n      \"pmids\": [\"34163489\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Hemin (free heme) directly binds to both CLEC-2 and GPVI on platelets (confirmed by western blotting and SPR), activating platelet aggregation via SYK and PLCgamma2 phosphorylation. Double knockout of CLEC-2 and FcRgamma (GPVI-equivalent) nearly abolishes hemin-induced platelet aggregation and attenuates rhabdomyolysis-induced acute kidney injury.\",\n      \"method\": \"SPR binding, western blotting, CLEC-2-depleted and FcRgamma-/- mice (single and double KO), RAKI mouse model, in vitro MET formation\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — SPR direct binding plus double-KO mouse model establishing cooperative roles\",\n      \"pmids\": [\"33843987\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cooperative signaling between CLEC-2 and GPIIb/IIIa triggers cerebral venous sinus thrombosis (CVT)-like syndrome in mice. INU1-Fab-induced CLEC-2 signaling combined with GPIIb/IIIa activation causes rapid CVT. Interfering with CLEC-2 signaling or inhibiting GPIIb/IIIa completely blocked platelet activation and CVT; blocking GPIIb/IIIa after symptom onset protected against death.\",\n      \"method\": \"Antibody-induced CLEC-2 signaling model, intravital transcranial microscopy, CLEC-2 signaling inhibition, GPIIb/IIIa antagonist treatment\",\n      \"journal\": \"Nature cardiovascular research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — novel mouse model establishing cooperative receptor mechanism with pharmacological interventions\",\n      \"pmids\": [\"39195988\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Targeted downregulation of platelet CLEC-2 by antibody INU1 occurs through Src family kinase-dependent receptor internalization (not Syk-dependent), presumably followed by intracellular degradation. In platelet-specific Syk-deficient mice, INU1-induced CLEC-2 internalization was fully preserved while the associated thrombocytopenia was largely prevented, mechanistically uncoupling the two processes.\",\n      \"method\": \"Anti-CLEC-2 antibody (INU1) treatment, Src family kinase inhibitors, platelet-specific Syk-deficient mice, receptor internalization assays in vitro and in vivo\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic and pharmacological dissection separating internalization from thrombocytopenia mechanisms\",\n      \"pmids\": [\"25795918\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Platelets express CLEC-2, which binds HIV-1 independently of the viral envelope protein, facilitating HIV-1 capture by platelets. CLEC-2 and DC-SIGN together are required for efficient HIV-1 binding; captured HIV-1 remains infectious for several days, suggesting CLEC-2 may facilitate HIV-1 dissemination.\",\n      \"method\": \"Flow cytometry binding assays, DC-SIGN and CLEC-2 inhibitors, platelet HIV-1 capture assays, infectivity assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct binding and inhibitor studies, single lab\",\n      \"pmids\": [\"16940507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Podoplanin/CLEC-2 signaling regulates keratinocyte migration during wound healing. CLEC-2 addition and podoplanin knockdown both inhibit keratinocyte migration. CLEC-2 downregulates RhoA activity and upregulates E-cadherin in keratinocytes, providing a molecular mechanism for the regulation of migration.\",\n      \"method\": \"podoplanin siRNA in NHEK cells, CLEC-2 protein addition, wound healing and transwell migration assays, RhoA activity assay, E-cadherin immunostaining\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — siRNA and recombinant protein in primary human cells with defined downstream signaling, single lab\",\n      \"pmids\": [\"26597882\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Phosphoproteomic analysis of rhodocytin-activated platelets identified 363 differentially phosphorylated tyrosine residues. Syk phosphorylation at Tyr525+526 occurs even with ADP and TxA2 pathway inhibitors, while Src-pTyr419 and PLCgamma2-pTyr759 require ADP/TxA2 feedback. Novel components of the CLEC-2 signalosome identified include adaptors DAPP1, Dok1/3, CASS4, Nck1/2, kinases FAK1, FES, FGR, JAK2, SHIP2, and membrane proteins G6F, JAM-A, PECAM-1, TLT-1.\",\n      \"method\": \"Mass spectrometry-based phosphotyrosine proteomics, 2D-DIGE, kinase inhibitors, Ca2+ mobilization assays\",\n      \"journal\": \"Thrombosis and haemostasis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — comprehensive phosphoproteomics with pharmacological dissection, single lab\",\n      \"pmids\": [\"31901221\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CLEC-2 (CLEC1B) is a platelet and megakaryocyte C-type lectin receptor that binds its primary endogenous ligand podoplanin (and also rhodocytin, fucoidan, hemin, and S100A13) on its non-canonical 'side' face as revealed by crystal structures; ligand engagement induces CLEC-2 dimerization and higher-order clustering, recruits Syk (2:1 stoichiometry via tandem SH2 domains binding two phosphorylated hemITAM YXXL motifs from a dimer), requires lipid raft translocation, actin polymerization, and ADP/TxA2 positive feedback (via Gq/PLCβ/PKCα/Src) to amplify hemITAM phosphorylation, and activates a Syk→SLP-76→PLCgamma2→Ca2+ signaling cascade that triggers platelet aggregation, granule secretion (releasing S1P, TGF-beta, BMP-9, CCL5), and EV release, with physiological roles in blood-lymphatic separation during embryogenesis, HEV integrity, megakaryopoiesis, lymph node maintenance, lung development, dendritic cell motility, and pathological roles in thrombosis, tumor metastasis, and thromboinflammation.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CLEC-2 (CLEC1B) is a C-type lectin-like receptor expressed on platelets, megakaryocytes, dendritic cells, and neutrophils that functions as a hemITAM-coupled pattern recognition receptor linking innate immune sensing to hemostasis, vascular development, and inflammation. Its primary endogenous ligand podoplanin and exogenous agonists (rhodocytin, fucoidan, hemin, S100A13) bind a non-canonical 'side' face of the extracellular domain, inducing receptor dimerization and higher-order clustering that enables Syk recruitment in a 2:1 stoichiometry via tandem SH2 domains bridging two phosphorylated hemITAM YXXL motifs; hemITAM phosphorylation itself requires lipid raft translocation, actin polymerization, and positive feedback through ADP/TxA2/Gq/PLCβ/PKCα/Src pathways, driving a Syk→SLP-76→PLCγ2→Ca²⁺ cascade that triggers platelet aggregation, granule secretion (releasing S1P, TGF-β, BMP-9, CCL5), and extracellular vesicle release [PMID:16174766, PMID:25458834, PMID:20154219, PMID:20154214, PMID:28705934, PMID:31160588]. Genetically, platelet CLEC-2/podoplanin signaling is essential for embryonic blood–lymphatic vessel separation, lymph node development and maintenance, cerebrovascular patterning, lung alveolar duct formation, HEV vascular integrity, and megakaryopoiesis, and contributes pathologically to arterial and venous thrombosis, infection-driven thromboinflammation, tumor metastasis, and regulation of macrophage-mediated inflammation [PMID:20525685, PMID:20363774, PMID:25908104, PMID:29853539, PMID:23995678, PMID:24532804, PMID:26552707, PMID:28104688, PMID:26571395, PMID:34163489].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Identifying CLEC-2 as the platelet receptor for rhodocytin established that a single hemITAM YXXL motif could activate a Syk→PLCγ2 signaling cascade, defining a new receptor class distinct from classical dual-ITAM receptors.\",\n      \"evidence\": \"Rhodocytin affinity chromatography, Syk−/− and PLCγ2−/− mouse platelets, kinase inhibitors\",\n      \"pmids\": [\"16174766\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous ligand unknown\", \"Mechanism by which single YXXL engages tandem SH2 domains of Syk unresolved\", \"In vivo physiological role not established\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"The 1.6 Å crystal structure of CLEC-2 revealed the ligand-binding surface centered on a semi-helical loop, providing the first structural framework for understanding receptor–ligand specificity.\",\n      \"evidence\": \"X-ray crystallography, site-directed mutagenesis, SPR\",\n      \"pmids\": [\"17132623\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No co-crystal with ligand yet\", \"Dimerization interface not resolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identification of podoplanin as the endogenous CLEC-2 ligand answered what physiological signal activates this receptor and revealed dependence on sialylated O-glycans, while parallel signaling studies showed the DEDG acidic motif upstream of YXXL is critical for hemITAM function.\",\n      \"evidence\": \"Flow cytometry binding, recombinant protein inhibition, glycopeptide mapping, mutagenesis in mutant cell lines\",\n      \"pmids\": [\"17616532\", \"17944973\", \"17339324\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Affinity of CLEC-2/podoplanin interaction not quantified\", \"Structural basis of glycan recognition unknown\", \"In vivo developmental role not yet demonstrated\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstration that CLEC-2 exists as a pre-formed homodimer resolved how a single hemITAM receptor engages Syk's tandem SH2 domains — each SH2 domain binds one phospho-YXXL from a different monomer — and antibody-mediated CLEC-2 depletion established its requirement for thrombus stability in vivo.\",\n      \"evidence\": \"BRET, co-IP, analytical ultracentrifugation, MALS, intravital microscopy, in vivo bleeding/thrombosis models\",\n      \"pmids\": [\"19824697\", \"19641185\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of Syk binding not yet directly measured\", \"Developmental role not yet tested genetically\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"CLEC-2 knockout mice revealed that hemITAM signaling via Syk/SLP-76 on platelets is essential for embryonic blood–lymphatic separation, while biophysical studies confirmed 2:1 Syk:CLEC-2 stoichiometry and showed that hemITAM phosphorylation requires lipid raft translocation, actin polymerization, and ADP/TxA2 positive feedback.\",\n      \"evidence\": \"Constitutive and platelet-specific conditional KOs, fetal liver transplant, quantitative SPR/cross-linking/EM, lipid raft fractionation, pharmacological inhibitors\",\n      \"pmids\": [\"20525685\", \"20363774\", \"20154219\", \"20154214\", \"21098033\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contributions of Syk versus Src in initiating versus amplifying signaling debated\", \"How lipid raft translocation is triggered mechanistically unclear\", \"Whether CLEC-2 has signaling-independent roles unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Beyond platelets, CLEC-2 was shown to regulate dendritic cell motility through RhoA inhibition and Rac1/Vav-dependent actin protrusions upon podoplanin engagement, while in platelets, CLEC-2 activation releases BMP-9 and TGF-β to inhibit lymphatic endothelial cell proliferation and tube formation.\",\n      \"evidence\": \"CLEC-2-deficient DCs, migration/actin dynamics assays, RhoA/Rac1 activity assays, platelet-specific KO, LEC functional assays\",\n      \"pmids\": [\"22884313\", \"22556408\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether DC and platelet CLEC-2 signaling pathways diverge at the level of Syk unclear\", \"How BMP-9 is stored and released from platelet granules not defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"The physiological function of platelet CLEC-2 was extended to maintaining high endothelial venule integrity through podoplanin-dependent S1P release promoting VE-cadherin expression, and fucoidan was identified as an additional CLEC-2 agonist.\",\n      \"evidence\": \"Conditional KO with platelet rescue, S1P pathway analysis, VE-cadherin staining, fucoidan activation in CLEC-2 KO platelets\",\n      \"pmids\": [\"23995678\", \"23341451\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether S1P release is the sole downstream mediator of HEV maintenance not excluded\", \"Fucoidan binding site on CLEC-2 unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Co-crystal structures of CLEC-2 with both podoplanin and rhodocytin revealed a shared non-canonical 'side' binding face utilizing arginine–acidic residue interactions, providing a structural explanation for ligand promiscuity, while super-resolution imaging showed Src/Syk-dependent CLEC-2/podoplanin clustering within 10 nm on platelet membranes.\",\n      \"evidence\": \"X-ray crystallography of two ligand complexes, dSTORM/FLIM-FRET, supported lipid bilayer with Syk−/− platelets\",\n      \"pmids\": [\"25458834\", \"25368330\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length receptor structure including transmembrane and cytoplasmic domains not solved\", \"Structural basis of higher-order clustering beyond dimers unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Multiple in vivo studies established CLEC-2/podoplanin as critical for cerebrovascular patterning, lymph node development, megakaryopoiesis (via Thpo production), venous thrombosis, and infection-driven thromboinflammation, while S100A13 was identified as an additional CLEC-2 ligand on vascular smooth muscle cells.\",\n      \"evidence\": \"Constitutive/conditional KOs (platelet-specific, Mk-specific, Nestin-Cre), IVC stenosis DVT model, anti-PDPN antibody, Salmonella infection model, SPR/protein array for S100A13, HSC transplant\",\n      \"pmids\": [\"25908104\", \"24532804\", \"26552707\", \"28104688\", \"26571395\", \"26418160\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative quantitative contributions of multiple CLEC-2 ligands in different vascular beds undefined\", \"Mechanistic link between Mk CLEC-2 and Thpo transcription not fully resolved\", \"S100A13 binding site not mapped\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"A hemITAM-dead knockin (Y7A) distinguished signaling-dependent from signaling-independent CLEC-2 functions: blood–lymphatic separation requires hemITAM signaling, whereas hemostasis/thrombosis also involves a structural/adhesive contribution independent of YXXL phosphorylation. Gq/PLCβ/PKCα/Src was defined as the positive feedback loop potentiating hemITAM phosphorylation.\",\n      \"evidence\": \"Y7A knockin mouse with anti-CLEC-2 Fab blockade, in vivo thrombosis models, Gq−/− platelets, pharmacological pathway dissection\",\n      \"pmids\": [\"28835437\", \"28705934\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nature of signaling-independent adhesive mechanism unknown\", \"Whether Gq feedback loop operates in non-platelet CLEC-2-expressing cells not tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"CLEC-2/podoplanin was shown to be essential for lung alveolar development via TGF-β-dependent myofibroblast differentiation, CD37 was identified as a tetraspanin partner controlling CLEC-2 membrane organization in DCs, and a small-molecule antagonist (Co-HP) binding a site distinct from podoplanin inhibited thrombosis and metastasis without bleeding.\",\n      \"evidence\": \"Platelet-specific and LEC-specific conditional KOs, CD37−/− DC functional assays and co-IP, Co-HP SPR/mutagenesis with in vivo thrombosis/metastasis models\",\n      \"pmids\": [\"29853539\", \"30185523\", \"30190281\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Co-HP binding inhibits signaling allosterically not structurally resolved\", \"CD37 interaction interface not mapped\", \"Lung phenotype in humans with CLEC-2 loss unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"CLEC-2 activation by dengue virus triggers platelet extracellular vesicle release that activates innate immune receptors CLEC5A and TLR2 on neutrophils/macrophages, establishing a platelet-to-innate-immune amplification loop in viral thromboinflammation.\",\n      \"evidence\": \"In vitro platelet EV isolation, neutrophil/macrophage activation assays, dengue mouse model with CLEC5A/TLR2 blockade\",\n      \"pmids\": [\"31160588\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether EV-mediated amplification applies to other viral infections not tested\", \"Specific viral component engaging CLEC-2 not identified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Phosphoproteomic profiling of CLEC-2-activated platelets revealed 363 differentially phosphorylated tyrosine sites and identified novel signalosome components (DAPP1, Dok1/3, CASS4, FAK1, FES, JAK2), while in vivo studies showed platelet CLEC-2 impairs hepatic recovery after acute injury by suppressing TNF-α-driven neutrophil recruitment.\",\n      \"evidence\": \"MS-based phosphotyrosine proteomics with kinase inhibitors; CLEC-2 KO mice in APAP/CCl4 liver injury models with cytokine/neutrophil analysis\",\n      \"pmids\": [\"31901221\", \"32321925\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional validation of most novel signalosome components pending\", \"Whether CLEC-2's liver injury role is podoplanin-dependent not directly tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Btk was positioned as a critical node in CLEC-2 positive feedback in human (but not mouse) platelets, hemin was confirmed as a direct CLEC-2 and GPVI co-ligand relevant to rhabdomyolysis, and CLEC-2/podoplanin interaction on macrophages was shown to accelerate their emigration from inflamed peritoneum via ERM-mediated cytoskeletal remodeling.\",\n      \"evidence\": \"XLA patient platelets and ibrutinib/acalabrutinib treatment; SPR plus double-KO (CLEC-2/FcRγ) in RAKI model; platelet-specific CLEC-2 KO in peritonitis with recombinant CLEC-2-Fc and ERM studies\",\n      \"pmids\": [\"31949019\", \"33843987\", \"34163489\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Species differences in Btk feedback not structurally explained\", \"Hemin binding site on CLEC-2 not mapped\", \"How CLEC-2 engagement on macrophages upregulates podoplanin/CD44 not mechanistically resolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Cooperative signaling between CLEC-2 and GPIIb/IIIa was shown to trigger cerebral venous sinus thrombosis, demonstrating that CLEC-2 does not act in isolation but synergizes with integrin signaling for pathological thrombus formation in specific vascular beds.\",\n      \"evidence\": \"Antibody-induced CLEC-2 activation model, intravital transcranial microscopy, GPIIb/IIIa antagonist rescue\",\n      \"pmids\": [\"39195988\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism of CLEC-2/GPIIb/IIIa crosstalk not defined\", \"Relevance to human CVT (e.g., vaccine-induced) not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the full-length structure of CLEC-2 including transmembrane and cytoplasmic domains, the structural basis of higher-order clustering beyond dimers, functional validation of newly identified signalosome components, the molecular nature of CLEC-2's signaling-independent adhesive role in hemostasis, and whether human CLEC-2 loss-of-function mutations cause developmental vascular disease.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No full-length receptor structure\", \"No human genetic disease linked to CLEC1B mutations in the timeline\", \"Signaling-independent adhesive mechanism molecularly undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 2, 5, 22]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [18, 23, 44]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 8, 14, 15, 24]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [39, 40]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [0, 9, 14, 15, 27, 46]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 3, 13, 34, 42]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [10, 18, 31, 33, 39, 44]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [11, 12, 16, 25, 29, 36]}\n    ],\n    \"complexes\": [\n      \"CLEC-2 homodimer\"\n    ],\n    \"partners\": [\n      \"PDPN\",\n      \"SYK\",\n      \"LCP2\",\n      \"PLCG2\",\n      \"CD37\",\n      \"ITGA2B\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}