ITGA2B Monoclonal Antibody

What is ITGA2B and why is it significant in research applications?

ITGA2B (Integrin Subunit Alpha 2b) is a membrane protein of approximately 113.4 kDa with 1039 amino acid residues that forms the αIIb subunit of the platelet integrin αIIbβ3 complex. This protein is predominantly expressed in bone marrow and placenta, with particular significance in the megakaryocyte/platelet lineage . ITGA2B is involved in critical biological processes including angiogenesis and cell adhesion, making it an important target for platelet research .

Detecting ITGA2B expression is particularly valuable for researchers studying megakaryocyte development, platelet activation, and thrombotic disorders. The protein serves as a definitive marker for identifying megakaryocytes (MK) and lung megakaryocytes . Recent research has also revealed its role in regulating platelet death during sepsis through the PTPN6 pathway, which inhibits the pro-apoptotic caspase-8 and Ripk3/Mlkl pathways, highlighting its importance in maintaining vascular integrity during inflammatory responses .

What experimental applications are most suitable for ITGA2B monoclonal antibodies?

ITGA2B monoclonal antibodies are versatile research tools applicable across multiple experimental platforms, with flow cytometry being the most prevalent application. When designing flow cytometry experiments, researchers should consider that ITGA2B is primarily a cell surface marker, requiring minimal permeabilization for optimal detection .

The applications of ITGA2B antibodies include:

• Flow Cytometry (FCM): Most commonly used for identifying and sorting megakaryocyte lineage cells and activated platelets

• Western Blotting (WB): For detecting ITGA2B protein expression levels in cell or tissue lysates

• Immunohistochemistry (IHC): For visualizing ITGA2B expression in tissue sections, particularly in bone marrow and vascular tissues

• Immunofluorescence (IF): For subcellular localization studies

• Immunoprecipitation (IP): For studying protein interactions with ITGA2B

• ELISA: For quantitative measurement of ITGA2B in various samples

For each application, optimization of antibody concentration, incubation conditions, and detection systems is essential for obtaining reliable results.

How should researchers select between different ITGA2B antibody clones?

Selection of an appropriate ITGA2B antibody clone should be based on the specific research application, species reactivity requirements, and experimental validation:

• For flow cytometry applications studying human samples, clones with demonstrated reactivity to human ITGA2B and validation in flow cytometry, such as clone KN22-20, are recommended .

• For mouse studies, particularly those examining platelet function, the MWReg30 clone has been extensively validated with numerous citations supporting its specificity and utility .

• When studying both human and mouse samples, consider antibodies with cross-reactivity to both species to maintain experimental consistency .

• For mechanistic studies of ITGA2B signaling, rabbit monoclonal antibodies like D8V7H have demonstrated utility in applications such as western blotting and immunoprecipitation .

When selecting antibodies for specific applications, researchers should prioritize clones with published validation for their particular experimental system and application.

What controls should be included when using ITGA2B antibodies in research?

Proper experimental controls are essential for validating results obtained with ITGA2B antibodies:

• Positive controls: Include samples known to express ITGA2B, such as platelets or megakaryocyte cell lines. In flow cytometry, CD42b (GPIbα) can serve as a parallel platelet marker .

• Negative controls: Include cell types that do not express ITGA2B, such as lymphocytes. For tissue staining, select tissues where ITGA2B expression is absent or minimal .

• Isotype controls: Use an appropriate isotype-matched control antibody to determine background staining levels, particularly important for flow cytometry and immunohistochemistry applications .

• Blocking controls: For specificity validation, pre-incubation with recombinant ITGA2B protein should abolish specific staining .

• Genetic knockdown/knockout controls: When available, samples from ITGA2B knockout models (such as the Q887X knockin mouse) provide definitive validation of antibody specificity .

How can researchers optimize flow cytometry protocols for ITGA2B detection?

Flow cytometry represents the most common application for ITGA2B antibodies, requiring specific optimization strategies:

• Sample preparation: For platelets, use anticoagulants that minimally affect integrin conformation (such as sodium citrate). Process samples rapidly to prevent ex vivo activation that can alter ITGA2B detection .

• Antibody titration: Determine the optimal antibody concentration by testing a range of dilutions against a fixed cell number. Plot the signal-to-noise ratio to identify the concentration yielding maximum separation between positive and negative populations .

• Panel design considerations:

  • For megakaryocyte maturation studies, combine ITGA2B with CD42b and CD61 antibodies

  • For platelet activation studies, include activation markers such as P-selectin (CD62P)

  • Consider using antibodies against the activated conformation of αIIbβ3 (PAC-1) alongside total ITGA2B antibodies to distinguish between resting and activated platelets

• Fluorophore selection: For rare cell populations like megakaryocytes, select bright fluorophores (PE, APC) for ITGA2B antibodies. Consider spectral overlap when designing multicolor panels .

• Live/dead discrimination: Include a viability dye to exclude dead cells that may cause false-positive staining .

What are the key considerations for using ITGA2B antibodies in sepsis and inflammation research?

Based on recent discoveries of ITGA2B's role in sepsis, researchers studying inflammation should consider:

• Expression dynamics: ITGA2B mRNA is actively converted into new proteins in platelets during sepsis, accompanied by increased activation of integrin αIIbβ3. Monitor both total and activated ITGA2B levels during experimental sepsis .

• Regulatory pathways: ITGA2B upregulates PTPN6 in megakaryocytes via the transcription factors Nfkb1 and Rel. This pathway inhibits platelet apoptosis and necroptosis by targeting the Ripk1/Ripk3/Mlkl and caspase-8 pathways. Consider examining these downstream molecules alongside ITGA2B .

• Functional assessments: In sepsis models, monitor:

  • Platelet viability using assays like Cell Titer Glo that measure ATP abundance

  • Markers of apoptosis and necroptosis in platelets

  • Inflammatory cytokine levels (IL-1β, TNF-α, IL-6, HMGB-1)

  • Vascular permeability using methods such as Evans Blue extravasation

• Genetic models: The ITGA2B (Q887X) knockin mouse represents a valuable model for studying decreased ITGA2B expression. These mice show exacerbated inflammatory responses during sepsis with increased cytokine production and vascular leakage .

• Platelet clearance: In sepsis models with altered ITGA2B function, assess platelet clearance, particularly in the liver, which serves as the primary site for platelet clearance in Itga2b-deficient models during sepsis .

How should researchers approach transcriptomic analysis of ITGA2B-related pathways?

When conducting transcriptomic studies related to ITGA2B function:

• Sample preparation for platelet RNA-seq:

  • Ensure high platelet purity with minimal leukocyte contamination

  • Use specialized platelet RNA isolation protocols to capture the low RNA content

  • Consider both polyA-selected and total RNA approaches to capture various RNA species

• Analysis strategies:

  • Principal component analysis can effectively separate samples based on ITGA2B expression levels

  • Gene Ontology and pathway enrichment analyses can identify biological processes regulated by ITGA2B, such as inflammation pathways

  • Generate custom gene sets focused on specific pathways (e.g., inflammation, cell death) for more targeted analyses

• Validation approaches:

  • Confirm key gene expression changes by qRT-PCR

  • Assess protein-level changes for key pathway components

  • Use gene set enrichment analysis (GSEA) to identify coherent changes in functionally related gene sets

• Integration with functional data:

  • Correlate transcriptomic changes with functional readouts such as platelet apoptosis, necroptosis, or inflammatory responses

  • Consider the temporal dynamics of gene expression changes relative to functional outcomes

What are common technical issues with ITGA2B antibody applications and how can they be resolved?

Researchers frequently encounter these technical challenges when working with ITGA2B antibodies:

• Loss of surface epitopes during sample processing:

  • Issue: Proteolytic degradation of ITGA2B during platelet isolation

  • Solution: Add protease inhibitors to buffers and process samples rapidly at 4°C

• Background staining in flow cytometry:

  • Issue: Non-specific binding, particularly with fixed/permeabilized samples

  • Solution: Optimize blocking conditions using 2-5% serum matching the species of the secondary antibody; include an FcR blocking step when working with samples containing Fc receptors

• Weak western blot signal:

  • Issue: Inefficient extraction of membrane-bound ITGA2B

  • Solution: Use specialized membrane protein extraction buffers containing appropriate detergents (e.g., NP-40, Triton X-100); avoid excessive heating which can cause ITGA2B aggregation

• Inconsistent immunohistochemistry results:

  • Issue: Epitope masking during fixation

  • Solution: Test multiple antigen retrieval methods; for formalin-fixed tissues, citrate or EDTA-based heat-induced epitope retrieval is often effective

• Ex vivo platelet activation affecting ITGA2B detection:

  • Issue: Artificial activation during sample handling changing ITGA2B conformation

  • Solution: Include prostaglandin E1 in isolation buffers to minimize activation; monitor activation markers (CD62P) as a quality control

How can researchers address data interpretation challenges in ITGA2B studies?

Interpreting data from ITGA2B studies presents several challenges:

• Distinguishing between total and activated ITGA2B:

  • Challenge: Standard ITGA2B antibodies may not discriminate between resting and activated conformations

  • Solution: Use conformation-specific antibodies (like PAC-1) alongside total ITGA2B antibodies to assess activation state

• Analyzing ITGA2B in mixed cell populations:

  • Challenge: Heterogeneous expression across different cell types

  • Solution: Use multiparameter flow cytometry with lineage markers, or single-cell approaches to resolve cell-specific expression patterns

• Differentiating between membrane and internalized ITGA2B:

  • Challenge: Activated platelets may internalize ITGA2B

  • Solution: Use surface-selective labeling methods versus permeabilization protocols to distinguish localization

• Resolving contradictory survival data in sepsis models:

  • Challenge: Balancing ITGA2B's roles in hemostasis versus inflammation

  • Solution: Design time-course experiments to capture the dynamic nature of ITGA2B functions; separately assess hemostatic and inflammatory endpoints

• Accounting for compensatory mechanisms in genetic models:

  • Challenge: Other integrins may compensate for reduced ITGA2B function

  • Solution: Perform comprehensive integrin expression profiling alongside functional assays

How is ITGA2B being studied in the context of vascular integrity and inflammation?

Recent research has uncovered novel roles for ITGA2B in maintaining vascular integrity during inflammatory conditions:

• Mechanisms of vascular protection:

  • ITGA2B appears to regulate vascular wall permeability during systemic inflammatory responses

  • Studies using Evans Blue extravasation in ITGA2B-deficient mice demonstrate significantly increased vascular permeability in multiple organs following sepsis induction

  • This suggests therapeutic potential in targeting ITGA2B pathways to prevent vascular leakage in inflammatory conditions

• Platelet-dependent organ protection:

  • ITGA2B deficiency leads to increased organ damage during sepsis, particularly affecting the liver

  • Liver tissues from ITGA2B-deficient mice show ballooning degeneration, a form of apoptosis, following sepsis induction

  • The protective effect of ITGA2B appears to involve prevention of platelet death, which maintains adequate platelet function for vascular integrity

• Modulation of inflammatory signaling:

  • Transcriptomic analysis reveals that ITGA2B downregulation significantly increases inflammatory gene expression

  • Gene Ontology analysis of upregulated genes in ITGA2B-deficient platelets shows enrichment of pathways related to inflammation

  • This suggests ITGA2B may serve as a negative regulator of inflammatory processes in platelets

What new methodologies are being developed for studying ITGA2B in cellular contexts?

Emerging methodologies for ITGA2B research include:

• Genetic models with conditional ITGA2B modification:

  • The ITGA2B (Q887X) knockin mouse represents a valuable model with significantly reduced ITGA2B expression

  • This model exhibits platelet dysfunction without spontaneous bleeding phenotypes, allowing study of ITGA2B's non-hemostatic functions

• Platelet transfusion studies:

  • Transfusion of wild-type platelets into ITGA2B-deficient mice can partially rescue the heightened mortality in sepsis models

  • This approach allows for isolation of platelet-specific ITGA2B effects from other cell types

• Molecular pathway analysis:

  • Studies now link ITGA2B to regulation of PTPN6, which inhibits platelet apoptosis and necroptosis

  • This pathway represents a novel mechanism by which ITGA2B protects against excessive platelet clearance during inflammatory conditions

• Combined transcriptomic and functional analyses:

  • Integration of RNA-seq data with functional assays provides comprehensive understanding of ITGA2B biology

  • Principal component analysis of transcriptomes can effectively separate samples based on ITGA2B expression levels, accounting for significant transcriptional variation

How does ITGA2B research intersect with clinical applications in inflammatory diseases?

The translation of ITGA2B research to clinical applications shows promise in several areas:

• Biomarker potential:

  • ITGA2B upregulation in circulating platelets correlates with higher mortality rates in septic patients and mice

  • This suggests potential diagnostic or prognostic applications in monitoring sepsis progression

• Therapeutic targeting:

  • Understanding the ITGA2B-PTPN6 axis may provide new therapeutic targets for modulating platelet survival during sepsis

  • Interventions maintaining platelet ITGA2B function could potentially reduce vascular leakage and organ damage

• ITGA2B in platelet transfusion efficacy:

  • Research shows that transfused platelets with normal ITGA2B levels can improve survival in sepsis models

  • This suggests consideration of ITGA2B status in platelet products for transfusion in inflammatory conditions

• Monitoring platelet death mechanisms:

  • ITGA2B status affects both apoptotic and necroptotic pathways in platelets

  • Assessing platelet viability through ATP abundance measurement may provide insights into platelet quality in inflammatory conditions