CLE2 Antibody

What is CLEC2 and why are antibodies against it important in research?

CLEC2 (C-type lectin-like receptor 2) is a unique platelet activation receptor that signals through a single YXXL sequence, representing half of an immunoreceptor tyrosine-based activation motif (hemITAM) . The receptor is primarily expressed on platelets with approximately 2000 copies per cell, although murine CLEC2 has also been detected on peripheral blood neutrophils . CLEC2 antibodies are essential research tools because they allow for:

• Identification and quantification of CLEC2 expression on platelets and other cell types

• Investigation of CLEC2-mediated signaling pathways

• Exploration of CLEC2's roles in thrombosis, tumor metastasis, and development

• Development of potential antithrombotic therapeutics targeting CLEC2

The significance of these antibodies is highlighted by their use in demonstrating that CLEC2 and its endogenous ligand podoplanin are crucial for normal development, with mice deficient in either showing defective blood-lymphatic vessel separation .

How do CLEC2 antibodies differ from other platelet receptor antibodies?

CLEC2 antibodies target a receptor with unique regulatory characteristics that distinguish it from other platelet receptor antibodies:

• Unlike antibodies against GPVI and FcγRIIa (which target receptors that undergo proteolytic regulation), CLEC2 antibodies recognize a receptor that is not regulated by proteolysis upon activation .

• CLEC2 antibodies can both activate the receptor (when cross-linked) and block its function (as Fab fragments), allowing for versatile experimental approaches .

• Some CLEC2 antibodies (such as AYP1) recognize conformational epitopes that are lost during the denaturing conditions of SDS-PAGE, making them suitable for specific applications like flow cytometry but not western blotting .

• CLEC2 antibodies can induce proteolysis of other platelet receptors (GPVI and FcγRIIa) without affecting CLEC2 itself, revealing unique cross-regulatory mechanisms between platelet receptors .

This distinct profile makes CLEC2 antibodies particularly valuable for studying the integration of signaling pathways in platelets.

What are the key variants and nomenclature considerations for CLEC2 antibodies?

When working with CLEC2 antibodies, researchers should be aware of several nomenclature and variant considerations:

• CLEC2 is also known as CLEC1B in humans, and antibodies may be labeled with either designation .

• Related family members include CLEC2B (also known as AICL, Clrb, and CLECSF2), which represents a distinct target with different expression patterns and functions .

• Species-specific variants exist with important differences:

  • Human CLEC2 antibodies (e.g., AYP1) are not cross-reactive with murine CLEC2

  • Murine-specific antibodies (e.g., INU1) are used for studies in mice

  • Humanized CLEC2 mouse models (hCLEC-2 KI) have been developed to test human CLEC2-targeted therapies

• Different antibody clones recognize distinct epitopes on CLEC2, affecting their functional properties:

  • Some antibodies (like AYP1) recognize conformational epitopes

  • Others can detect both phosphorylated and non-phosphorylated forms of CLEC2

Understanding these distinctions is essential for selecting the appropriate antibody and interpreting experimental results correctly.

How can flow cytometry be optimized for CLEC2 antibody detection in platelet research?

Optimizing flow cytometry for CLEC2 antibody detection requires specific methodological considerations:

• Sample preparation protocol:

  • Use fresh blood samples collected in acid-citrate-dextrose (ACD) or sodium citrate anticoagulant

  • For platelets, prepare platelet-rich plasma (PRP) by centrifugation at 200g for 20 minutes

  • Fix samples with 1% paraformaldehyde if immediate analysis is not possible

• Antibody selection and controls:

  • Use fluorophore-conjugated antibodies (e.g., APC-conjugated anti-CLEC2) for direct detection

  • Include appropriate isotype controls (e.g., Mouse IgG2A Allophycocyanin)

  • Co-stain with platelet markers (e.g., anti-CD41/Integrin alpha 2b) to confirm platelet population

• Gating strategy:

  • First gate on platelet population based on forward/side scatter characteristics

  • Use CD41 positivity to confirm platelet identity

  • Analyze CLEC2 expression on CD41+ events

• Special considerations:

  • Unlike other platelet receptors, CLEC2 expression remains stable even after activation

  • Monitor both receptor density and percentage of positive cells

  • For microparticle analysis, use specialized protocols to distinguish CLEC2+ microparticles from platelets

This methodology has successfully demonstrated that CLEC2 expression is restricted to platelets in human blood, with no detectable expression on monocytes, neutrophils, T cells, B cells, or dendritic cells .

What are the established protocols for using CLEC2 antibodies in immunoprecipitation experiments?

CLEC2 immunoprecipitation experiments require specific protocols to effectively isolate and study this receptor:

• Cell lysis procedure:

  • Wash platelets (3×10⁸) in modified Tyrode's buffer

  • Lyse in 1% NP-40 lysis buffer containing:

    • 50 mM HEPES (pH 7.4)

    • 150 mM NaCl

    • 1% NP-40

    • Protease inhibitor cocktail

    • Phosphatase inhibitors (2 mM sodium orthovanadate, 5 mM sodium fluoride)

• Immunoprecipitation steps:

  • Pre-clear lysates with protein G-Sepharose for 1 hour at 4°C

  • Incubate with anti-CLEC2 antibody (e.g., AYP1) at 2-5 μg per sample overnight at 4°C

  • Add protein G-Sepharose and incubate for 2 hours at 4°C

  • Wash beads 3× with lysis buffer

  • Elute bound proteins by boiling in SDS sample buffer

• Detection methods:

  • Western blot with specific antibodies to detect CLEC2

  • Probe for tyrosine phosphorylation (with anti-phosphotyrosine antibodies) to assess activation status

  • For conformational epitope-specific antibodies (e.g., AYP1), use non-denaturing conditions for detection

• Critical considerations:

  • Some CLEC2 antibodies can immunoprecipitate both phosphorylated and non-phosphorylated forms

  • AYP1 can immunoprecipitate CLEC2 but does not detect it by western blot as it recognizes a conformational epitope

  • Include appropriate controls (isotype-matched antibodies) for validation

This approach has been successfully used to investigate CLEC2 signaling pathways and interactions with other proteins in platelet activation cascades.

How can CLEC2 antibodies be effectively used to study receptor internalization and trafficking?

Studying CLEC2 internalization and trafficking using antibodies requires specialized approaches:

• Time-course receptor tracking protocol:

  • Label surface CLEC2 with fluorescently tagged antibodies (e.g., AYP1-FITC)

  • Induce activation with CLEC2 agonists (e.g., rhodocytin)

  • Monitor receptor localization at fixed timepoints (0, 15, 30, 60, 120 minutes)

  • Quantify surface expression changes by flow cytometry

• Distinguishing mechanisms of receptor regulation:

  • Use broad-spectrum metalloproteinase inhibitors (e.g., GM6001) to block potential shedding

  • Apply membrane-permeable calpain inhibitors (e.g., E64d) to prevent intracellular proteolysis

  • Employ endocytosis inhibitors to block internalization

  • Measure surface versus intracellular receptor pools

• Comparative analysis with other platelet receptors:

  • Unlike GPVI and FcγRIIa, CLEC2 is not regulated by proteolysis upon activation

  • Rhodocytin binding to CLEC2 remains stable for up to 120 minutes

  • Cross-linked AYP1 Fab fragments remain stably bound to CLEC2 over extended incubation periods

• Methodological considerations:

  • Combine flow cytometry with confocal microscopy for spatial resolution

  • Use fluorescence quenching techniques to distinguish surface from internalized receptors

  • Apply pH-sensitive fluorophores to track movement through acidic endosomal compartments

Research has demonstrated that unlike other platelet receptors, CLEC2 is neither shed nor internalized from the platelet surface upon autoactivation or in response to activation of GPVI and FcγRIIa , making it unique among platelet receptors.

How can CLEC2 antibodies be utilized for receptor depletion studies in vivo?

Antibody-mediated depletion of CLEC2 represents a powerful approach for investigating receptor function in vivo:

• Established depletion protocols:

  • For humanized CLEC2 mouse models (hCLEC-2 KI), intraperitoneal injection of anti-human CLEC2 antibodies:

    • AYP1: Depletes CLEC2 for at least 11 days with recovery by 18 days

    • HEL1: Depletes CLEC2 for at least 11 days with recovery by 24 days

  • Dose-dependent effects: 1-2 mg/kg typically achieves significant depletion

  • Monitoring: Flow cytometric assessment of platelet CLEC2 surface expression at regular intervals

• Mechanism of antibody-mediated CLEC2 depletion:

  • Unlike receptor shedding seen with other platelet receptors

  • Potentially involves Fc-receptor-dependent clearance mechanisms

  • Different from the transient thrombocytopenia observed with anti-mouse CLEC2 antibody (INU1)

• Experimental applications:

  • Investigation of CLEC2's role in thrombosis models

  • Assessment of hemostatic function in the absence of CLEC2

  • Evaluation of potential antithrombotic strategies targeting CLEC2

  • Study of tumor metastasis and developmental processes

• Advantages over genetic models:

  • Temporary, reversible depletion allows temporal control

  • Avoids developmental defects associated with constitutive CLEC2 deficiency

  • Permits adult-specific investigation of CLEC2 function

  • Enables studies in humanized mouse models for translational research

These approaches have demonstrated that immunodepletion of human CLEC2 in humanized mouse models can be achieved, providing proof of principle for testing anti-human CLEC2 agents in vivo .

What methodological approaches exist for studying CLEC2-mediated signaling using antibodies?

Investigating CLEC2-mediated signaling requires sophisticated methodological approaches:

• Antibody-based stimulation protocols:

  • Direct activation: Cross-linked whole antibodies (e.g., AYP1 IgG) can trigger CLEC2 signaling

  • Selective blockade: Fab fragments (e.g., AYP1 Fab) can block activation without inducing signaling

  • Controls: Both AYP1 and HEL1 antibodies stimulate platelet aggregation, but their Fab fragments do not, confirming that CLEC2 dimerization at either site is sufficient to trigger activation

• Signaling pathway analysis methods:

  • Phosphorylation assays: Anti-phosphotyrosine western blotting to detect hemITAM phosphorylation

  • Kinase inhibition studies: Selective inhibition of Src (PP2) and Syk (PRT-060318) kinases to dissect pathway dependencies

  • Calcium flux measurements: Real-time monitoring of intracellular calcium release

  • Integrin activation assays: Flow cytometric measurement of αIIbβ3 activation

• Cross-talk analysis with other platelet receptors:

  • CLEC2 activation induces proteolytic cleavage of GPVI (inhibited by GM6001) and FcγRIIa (inhibited by E64d)

  • This cross-regulation is inhibited by:

    • Metalloproteinase inhibitor (GM6001) for GPVI shedding

    • Calpain inhibitor (E64d) for FcγRIIa proteolysis

    • Src (PP2) and Syk (PRT-060318) kinase inhibitors for both processes

• Advanced techniques for detailed signaling analysis:

  • Proximity ligation assays to detect protein-protein interactions

  • Phosphoproteomic analysis for comprehensive pathway mapping

  • CRISPR-edited cell lines to confirm signaling dependencies

  • Live-cell imaging with fluorescent biosensors for real-time signaling visualization

These methodological approaches have revealed that CLEC2 signaling involves a unique Src- and Syk-dependent cascade that shares components with but is distinct from ITAM receptor pathways .

What are the experimental considerations for using CLEC2 antibodies in humanized mouse models?

Working with CLEC2 antibodies in humanized mouse models requires specific experimental considerations:

• Model system characteristics:

  • Humanized CLEC-2 (hCLEC-2 KI) mice replace mouse CLEC2 with the human variant

  • Surface expression of hCLEC-2 is approximately double that on human platelets

  • Other platelet glycoprotein receptors remain comparable to wild-type mice

  • Platelet activation and aggregation responses to G protein-coupled receptors and GPVI agonists are normal

• Antibody selection criteria:

  • Human CLEC2-specific antibodies (e.g., AYP1, HEL1) are required

  • AYP1 and HEL1 act at different sites on CLEC2, providing complementary approaches

  • Both whole antibodies and Fab fragments should be prepared for different applications

  • Species-matched secondary antibodies are essential for detection protocols

• Functional assay adaptations:

  • Thrombosis models: FeCl₃-induced carotid artery thrombosis with intravital microscopy

  • Hemostasis assessment: Tail bleeding time measurement

  • Platelet function: Aggregometry with CLEC2 agonists (rhodocytin) and other platelet activators

  • Flow chamber assays: Thrombus formation on collagen under defined shear conditions

• Critical controls and validation steps:

  • Confirm human CLEC2 expression by flow cytometry

  • Verify antibody binding specificity in humanized versus wild-type mice

  • Include appropriate isotype controls for in vivo studies

  • Monitor potential immune responses against humanized proteins or administered antibodies

This model system has demonstrated that human CLEC2 can functionally replace mouse CLEC2 during development and in hemostasis, suggesting conserved interactions between CLEC2 and podoplanin in humanized mice .

How can researchers address contradictory findings regarding CLEC2 expression on immune cells?

Resolving contradictory findings about CLEC2 expression on immune cells requires methodological precision:

• Reconciling contradictory expression data:

  • Human studies show CLEC2 expression restricted to platelets

  • Murine studies demonstrate CLEC2 expression on peripheral blood neutrophils but only weakly on bone-marrow or elicited inflammatory neutrophils

  • This apparent contradiction can be explained by:

    • Species differences in CLEC2 expression patterns

    • Distinct antibody specificities and detection sensitivities

    • Sample preparation variables affecting epitope accessibility

• Standardized detection protocol:

  Cell Type	Sample Preparation	Antibody Selection	Flow Cytometry Settings	Positive Control
  Platelets	Minimal processing, gentle fixation	Direct fluorophore conjugates	High sensitivity for small cells	CD41+ gating
  Neutrophils	Gradient isolation, avoid activation	Multiple epitope antibodies	Adjusted for larger cells	Ly6G+ (mouse) or CD66b+ (human)
  Other Immune Cells	Minimal RBC lysis, maintain viability	Multiple clones and conjugates	Exclude platelet contamination	Lineage-specific markers

• Critical validation approaches:

  • Multiple antibody clones recognizing different epitopes

  • Correlation with mRNA expression data (RNA-seq or qPCR)

  • Functional assays (e.g., phagocytosis assays for neutrophil CLEC2)

  • Genetic models with conditional CLEC2 deletion in specific cell lineages

• Experimental factors affecting detection:

  • TLR activation can modulate CLEC2 expression on neutrophils

  • Cell isolation procedures may activate platelets, leading to microparticle contamination

  • Antibody cross-reactivity with other C-type lectin family members

  • Heterogeneity of neutrophil populations in different compartments

These approaches have helped clarify that murine CLEC2 has broader expression than human CLEC2, explaining some conflicting reports in the literature .

What are the technical challenges in detecting CLEC2 modifications and how can they be overcome?

Detecting CLEC2 post-translational modifications presents several technical challenges that require specific approaches:

• Phosphorylation detection challenges:

  • The single YXXL motif (hemITAM) of CLEC2 has lower stoichiometry of phosphorylation than dual ITAM receptors

  • Solution approach: Enhance sensitivity using:

    • Phosphatase inhibitor cocktails during cell lysis

    • Anti-phosphotyrosine antibodies for immunoprecipitation followed by CLEC2 detection

    • Phos-tag™ SDS-PAGE for mobility shift detection of phosphorylated species

    • Mass spectrometry for precise phosphosite mapping

• Glycosylation analysis difficulties:

  • CLEC2 contains multiple potential N-glycosylation sites

  • Solution approach: Systematic glycoproteomic analysis:

    Method	Application	Advantages	Limitations
    PNGase F treatment	Removes N-linked glycans	Simple, effective	Loses glycan information
    Lectin binding assays	Identifies glycan types	Non-destructive	Limited specificity
    Mass spectrometry	Detailed glycan structure	Comprehensive	Complex analysis
    Site-directed mutagenesis	Functional significance	Direct causality	Potential structural effects

• Conformational epitope preservation:

  • Some antibodies (e.g., AYP1) recognize conformational epitopes lost during denaturation

  • Solution approach: Modified immunodetection methods:

    • Native gel electrophoresis

    • Dot blot analysis

    • Flow cytometry of intact cells

    • Sandwich ELISA using capture antibodies against linear epitopes

• Dimerization and complex formation detection:

  • CLEC2 dimerization is critical for signaling but challenging to detect

  • Solution approach: Advanced biophysical techniques:

    • Chemical crosslinking prior to SDS-PAGE

    • Blue native PAGE for complex detection

    • Förster resonance energy transfer (FRET) for live-cell analysis

    • Single-molecule tracking in membrane microdomains

These technical approaches have revealed that CLEC2 undergoes significant post-translational regulation, including dimerization-dependent signaling that is distinct from proteolytic regulation seen with other platelet receptors .

How should researchers interpret conflicting results between different CLEC2 antibody clones?

Interpreting conflicting results between different CLEC2 antibody clones requires systematic analysis:

• Epitope-dependent functional differences:

  • AYP1 and HEL1 antibodies act at different sites on CLEC2

  • HEL1 Fab fragments neither block rhodocytin-induced platelet aggregation nor block AYP1 IgG-induced aggregation

  • This indicates distinct binding sites with different functional consequences

  • Resolution approach: Map epitopes using:

    • Competition binding assays

    • Deletion/mutation constructs

    • Hydrogen-deuterium exchange mass spectrometry

    • X-ray crystallography of antibody-antigen complexes

• Systematic antibody validation framework:

  Validation Parameter	Method	Expected Outcome	Troubleshooting
  Specificity	Western blot with KO controls	Single band at expected MW or no signal with conformational epitopes	Test multiple antibody concentrations
  Sensitivity	Titration on recombinant protein	Consistent detection limit	Optimize signal amplification
  Functional effect	Platelet aggregation assays	Clone-specific activation or inhibition	Test both whole IgG and Fab fragments
  Cross-reactivity	Testing on related proteins	No binding to other family members	Confirm with genetic knockouts

• Application-specific selection criteria:

  • Flow cytometry: Prefer antibodies recognizing extracellular epitopes (e.g., AYP1)

  • Western blotting: Select antibodies against linear epitopes

  • Immunoprecipitation: Consider ability to recognize native protein complexes

  • Functional studies: Evaluate activating versus blocking properties

• Data integration approaches:

  • Triangulate results using multiple antibody clones

  • Correlate antibody-based findings with genetic approaches

  • Consider species differences in epitope conservation

  • Evaluate potential steric hindrance between different antibodies

This analytical framework has revealed that differences between antibody clones can provide valuable insights into receptor function, as demonstrated by the distinct effects of AYP1 and HEL1 on CLEC2 signaling .

How are CLEC2 antibodies being developed as potential therapeutic agents?

The development of CLEC2 antibodies as therapeutic agents represents an emerging research direction:

• Therapeutic rationale:

  • CLEC2 deficiency reduces vessel occlusion in thrombosis models with minimal effects on hemostasis

  • Occlusion is unaltered in CLEC2 Y7A signaling-null mice, suggesting receptor presence rather than signaling affects thrombus stability

  • Immunodepletion of CLEC2 using antibodies has similar effects on thrombus formation

  • These findings position CLEC2 as a potential antithrombotic target with a favorable risk-benefit profile

• Current development approaches:

  • Humanized mouse models (hCLEC-2 KI) to evaluate human CLEC2-targeted antibodies in vivo

  • Testing of different antibody formats:

    • Whole IgG for immunodepletion strategies

    • Fab fragments for functional blocking without depletion

    • F(ab')2 fragments for receptor dimerization without Fc effects

  • Evaluation of administration routes and pharmacokinetic profiles

• Efficacy and safety assessment strategy:

  Parameter	Methodology	Key Findings	Implications
  Antithrombotic efficacy	FeCl₃-induced carotid artery thrombosis	Reduced vessel occlusion	Potential therapeutic benefit
  Bleeding risk	Tail bleeding time	Minimal prolongation	Favorable safety profile
  Immunogenicity	Anti-drug antibody detection	Model-dependent	Requires humanization strategies
  Duration of effect	Flow cytometric monitoring	11-24 days for depletion	Dosing interval guidance

• Translational research directions:

  • Optimizing humanized antibodies to minimize immunogenicity

  • Exploring bispecific antibodies targeting both CLEC2 and complementary pathways

  • Developing site-specific antibody-drug conjugates

  • Investigating small-molecule CLEC2 inhibitors as alternatives to antibody therapies

The humanized CLEC2 mouse model has provided proof of principle that anti-human CLEC2 agents can be tested in vivo, paving the way for preclinical evaluation of potential therapeutics .

What are the methodological considerations for using CLEC2 antibodies in microparticle and extracellular vesicle research?

CLEC2 antibody applications in microparticle and extracellular vesicle research require specialized methodological considerations:

• Isolation protocols for CLEC2-bearing microparticles:

  • Differential centrifugation: 1,500g (15 min) → 13,000g (2 min) → 20,000g (20 min) for microparticle enrichment

  • Size-exclusion chromatography for separation based on particle size

  • Immunoaffinity capture using anti-CLEC2 antibodies (e.g., AYP1)

  • Flow cytometry-based sorting of CLEC2+ microparticles

• Detection and characterization strategies:

  • High-sensitivity flow cytometry using:

    • Calibrated size beads for size determination

    • Fluoresceinated anti-CLEC2 antibodies (e.g., AYP1)

    • Co-staining for platelet markers (CD41) to confirm origin

    • Annexin V binding to detect phosphatidylserine exposure

  • Nanoparticle tracking analysis with fluorescent antibody labeling

  • Electron microscopy with immunogold labeling using anti-CLEC2 antibodies

• Functional assessment methods:

  • Binding assays to podoplanin-expressing cells

  • Signaling capacity in recipient cells

  • Procoagulant activity measurements

  • Immunomodulatory effects on target cells

• Critical experimental considerations:

  • CLEC2 is expressed on microparticles derived from activated platelets, while GPVI is not

  • Pre-analytical variables (collection, processing, storage) significantly affect results

  • Standardization using reference materials improves inter-study comparability

  • Combination of multiple techniques provides more comprehensive characterization

This research area has revealed that CLEC2 and GPVI have distinct patterns of distribution on microparticles, with CLEC2 but not GPVI being expressed on microparticles derived from activated platelets , suggesting different functional roles in intercellular communication.

What novel approaches are being developed for studying CLEC2-podoplanin interactions using antibodies?

Innovative approaches for investigating CLEC2-podoplanin interactions using antibodies are advancing our understanding of this important receptor-ligand pair:

• Advanced imaging techniques:

  • Super-resolution microscopy (STORM/PALM) with dual-labeled antibodies

  • Intravital microscopy with fluorescently tagged antibodies for in vivo visualization

  • Correlative light and electron microscopy (CLEM) for ultrastructural context

  • Live-cell single-molecule tracking of receptor-ligand interactions

• Biosensor development for real-time interaction studies:

  • FRET-based biosensors using antibody-conjugated fluorophore pairs

  • Bioluminescence resonance energy transfer (BRET) systems

  • Surface plasmon resonance with immobilized antibodies or recombinant proteins

  • Quartz crystal microbalance with dissipation monitoring (QCM-D) for binding kinetics

• Engineered antibody formats for specific applications:

  Antibody Format	Application	Advantages	Limitations
  Bispecific (CLEC2/podoplanin)	Co-localization studies	Simultaneous targeting	Complex production
  Nanobodies	In vivo imaging, therapeutics	Small size, tissue penetration	Potentially immunogenic
  scFv fragments	Biosensors, targeting	Monovalent binding	Reduced stability
  Photoswitchable antibodies	Controlled activation	Spatiotemporal control	Requires specialized equipment

• Experimental models for interaction studies:

  • 3D co-culture systems with CLEC2+ and podoplanin+ cells

  • Microfluidic platforms with controlled shear stress for dynamic interaction studies

  • Organ-on-chip models incorporating lymphatic and blood vessels

  • Humanized mouse models (hCLEC-2 KI) for in vivo investigation

These approaches have demonstrated that human CLEC2 can compensate for mouse CLEC2 during development and in hemostasis, suggesting a conserved interaction between CLEC2 and podoplanin in humanized mice and providing new opportunities for studying this critical receptor-ligand pair in health and disease.