HABP2 Antibody

What is HABP2 and what functions does it perform in physiological systems?

HABP2 (Hyaluronan Binding Protein 2), also known as Factor VII-activating protease (FSAP) or plasma hyaluronan-binding protein (PHBP), is a 63 kDa extracellular serine protease that plays multiple roles in vascular and cellular processes. HABP2 integrates into both fibrinolytic and coagulation pathways . Functionally, HABP2:

• Cleaves the alpha-chain at multiple sites and the beta-chain of fibrinogen between 'Lys-53' and 'Lys-54', but does not cleave the gamma-chain

• Converts inactive single-chain urinary plasminogen activator (pro-urokinase) to the active two-chain form

• Activates coagulation factor VII

• Functions as a negative regulator of vascular integrity through protease-activated receptor (PAR) signaling

• May function as a tumor suppressor by negatively regulating cell proliferation and migration

The protein is synthesized as a single inactive chain that undergoes autoproteolytic processing to form a functional heterodimer, with further autoproteolysis leading to smaller inactive peptides .

What are the structural characteristics of HABP2 important for antibody development?

HABP2 exhibits a complex structure with multiple domains that are critical to consider when developing or selecting antibodies:

When developing antibodies against HABP2, researchers should consider whether they need antibodies that detect specific forms (active vs. inactive) or specific domains of the protein. The full 560 amino acid sequence, as detailed in product documentation, provides multiple potential epitopes for antibody generation .

What applications are HABP2 antibodies typically validated for?

HABP2 antibodies have been validated for multiple applications, although validation varies by product:

When selecting an antibody, researchers should verify that the specific application they require has been validated for their species of interest, as reactivity often varies between human, mouse, rat, and other species .

How can researchers use HABP2 antibodies to study vascular permeability mechanisms?

HABP2 has been identified as a critical regulator of vascular integrity, particularly in acute lung injury (ALI) models. Researchers investigating vascular permeability should consider the following methodological approaches:

• Immunohistochemical analysis of HABP2 expression in vascular endothelium using validated antibodies can reveal upregulation during inflammatory conditions

• Combining siRNA knockdown of HABP2 with endothelial cell barrier function assays (e.g., transendothelial electrical resistance) can elucidate its role in barrier regulation

• Co-immunoprecipitation using HABP2 antibodies can identify interactions with PAR receptors (particularly PAR-1 and PAR-3) that mediate downstream RhoA/ROCK signaling

Research has demonstrated that HABP2 negatively regulates vascular integrity through PAR/RhoA/Rho kinase signaling pathways. This presents a potential therapeutic target for conditions characterized by increased vascular permeability .

For experimental design, researchers should note that:

• LPS induces HABP2 expression in murine lung endothelium in vivo and in human pulmonary microvascular endothelial cells in vitro

• High-molecular-weight hyaluronan (HMW-HA) decreases HABP2 expression and activity

• Low-molecular-weight hyaluronan (LMW-HA) increases HABP2 expression and activity

These findings suggest that modulation of HABP2 expression or activity could be a strategy for treating vascular leak syndromes .

What are the considerations for detecting different forms of HABP2 in experimental systems?

HABP2 exists in multiple forms due to its proteolytic processing, presenting challenges for detection and interpretation:

• Detecting inactive versus active forms:

  • The inactive single-chain precursor appears at approximately 70 kDa

  • The active form consists of a 50 kDa heavy chain and a 27 kDa light chain

  • Further processing yields two 26 kDa fragments (heavy chain) and 17 kDa and 8 kDa fragments (light chain)

• Antibody selection strategies:

  • For total HABP2 detection: Select antibodies raised against full-length protein (AA 1-560)

  • For specific domain detection: Choose antibodies targeting specific domains (e.g., serine protease domain)

  • For activation state specificity: Consider antibodies that preferentially recognize the active two-chain form

• Sample preparation considerations:

  • Protease inhibitors should be included in lysate preparation to prevent artifactual processing

  • Non-reducing versus reducing conditions in SDS-PAGE can affect epitope accessibility

  • Antigen retrieval methods may be critical for IHC detection (TE buffer pH 9.0 or citrate buffer pH 6.0)

Research demonstrates that HABP2 expression patterns differ between normal and pathological tissues. For example, immunohistochemical analysis has shown increased HABP2 protein expression in papillary thyroid cancers and follicular adenoma tumors from affected family members with the G534E variant, but no staining in normal thyroid tissue from the same individuals .

How do hyaluronan (HA) forms differentially regulate HABP2 activity and what antibody-based approaches can detect these changes?

Hyaluronan (HA) regulates HABP2 in a molecular weight-dependent manner, with significant implications for experimental design:

To investigate these regulatory mechanisms:

• Antibody-based quantification of HABP2 expression:

  • Use validated HABP2 antibodies for Western blot to quantify protein levels after treatment with different HA forms

  • Include appropriate loading controls and perform densitometry

• Activity-based assays:

  • Enzymatic assays using purified HABP2 with synthetic substrates can measure activity changes

  • Combine with immunoprecipitation using HABP2 antibodies to isolate the protein from complex samples

• Domain-interaction studies:

  • Use the polyanion-binding domain (PABD) peptide as a competitive inhibitor to confirm LMW-HA interaction mechanism

  • Perform site-directed mutagenesis of the PABD followed by activity assays to identify critical residues

Research has demonstrated that the effects of LMW-HA, but not HMW-HA, on HABP2 activity can be inhibited with a peptide of the polyanion-binding domain of HABP2, indicating different mechanisms of action between these HA forms .

What controls and validation steps are necessary when working with HABP2 antibodies?

Proper validation of HABP2 antibodies is crucial for ensuring experimental reliability:

• Positive controls:

  • Human liver tissue (expresses high levels of HABP2)

  • HepG2 cell line (human hepatocellular carcinoma cells)

  • Recombinant HABP2 protein for Western blot ladder verification

• Negative controls:

  • HABP2 knockout or knockdown samples (siRNA-treated cells)

  • Tissues known to lack HABP2 expression

  • Secondary antibody-only controls to assess non-specific binding

• Specificity validation:

  • Peptide competition assays using the immunogen peptide

  • Cross-validation with multiple antibodies targeting different epitopes

  • Correlation between protein (antibody-based) and mRNA expression data

• Antibody characteristics verification:

  • Confirm expected molecular weight bands in Western blot

    • Full-length: 63-75 kDa

    • Heavy chain: 50 kDa or two 26 kDa fragments

    • Light chain: 27 kDa or 17 kDa and 8 kDa fragments

  • Verify expected cellular localization pattern

  • Check cross-reactivity with related serine proteases

When publishing research using HABP2 antibodies, include comprehensive methodology sections detailing antibody validation steps, catalog numbers, dilutions used, and incubation conditions to ensure reproducibility.

What are the optimal sample preparation methods for HABP2 detection in different applications?

Different applications require specific sample preparation approaches for optimal HABP2 detection:

• Western Blotting:

  • Sample buffer: Include protease inhibitors to prevent degradation

  • Protein extraction: RIPA buffer with protease inhibitor cocktail

  • Reducing conditions: DTT or β-mercaptoethanol required to break disulfide bonds

  • Loading amount: 20-30 μg of total protein per lane

  • Expected bands: Primary band at 63-75 kDa; processing fragments at 50, 27, 26, 17, and 8 kDa

• Immunohistochemistry:

  • Fixation: 10% neutral buffered formalin

  • Antigen retrieval: TE buffer pH 9.0 or citrate buffer pH 6.0

  • Blocking: 5% normal serum from the species of secondary antibody

  • Antibody dilution: Typically 1:50-1:500 (antibody-dependent)

  • Detection system: HRP or fluorescent secondary antibodies

• Flow Cytometry:

  • Fixation: 4% paraformaldehyde

  • Permeabilization: 90% methanol (for intracellular staining)

  • Antibody concentration: ~10 μg/mL (antibody-dependent)

  • Controls: Isotype control and unstained cells

• ELISA:

  • For serum/plasma: Dilution typically 1:2 to 1:10 in assay buffer

  • Standard curve: Recombinant HABP2 protein (0.156-10 ng/mL range)

  • Sample volume: Usually 100 μL per well

  • Detection: Biotin-conjugated antibody followed by Avidin-HRP conjugate

Experimental data shows that HABP2 expression can be dramatically altered in pathological conditions, such as increased expression in murine lung endothelium following LPS challenge. Therefore, careful consideration of sample timing and preparation is essential for accurate results .

How can researchers distinguish between HABP2 and other related serine proteases in experimental systems?

Distinguishing HABP2 from related serine proteases requires strategic approaches:

• Antibody selection strategies:

  • Choose antibodies targeting unique regions of HABP2 not conserved in related proteases

  • Validate antibody specificity against recombinant related proteases (e.g., hepatocyte growth factor activator, factor XII)

  • Consider using multiple antibodies targeting different epitopes

• Activity-based discrimination:

  • HABP2 has specific substrate preferences:

    • Cleaves α and β chains of fibrinogen (but not γ chain)

    • Activates factor VII

    • Converts pro-urokinase to active form

    • Does not cleave prothrombin or plasminogen

  • Using selective substrates can help distinguish HABP2 activity

• Expression pattern analysis:

  • HABP2 shows specific tissue expression patterns

  • Normal expression detected in only 9 of 82 normal tissue types

  • Comparative expression analysis with related proteases can aid identification

• Genetic approaches:

  • siRNA knockdown of HABP2 followed by antibody detection confirms specificity

  • Overexpression systems with tagged HABP2 provide positive controls

Research has shown that HABP2 has high structural similarity to other serine proteases, particularly in the conserved serine protease trypsin domain. The G534E variant occurs in a highly conserved site not only in HABP2 but also in other serine protease domain-containing proteins such as hepatocyte growth factor activator and factor XII . This structural similarity highlights the importance of careful antibody selection and validation.

How can HABP2 antibodies be used to investigate vascular pathologies in acute lung injury models?

HABP2 plays a crucial role in vascular integrity, making it an important target for investigating acute lung injury (ALI):

• Experimental approaches:

  • Immunohistochemical staining using HABP2 antibodies can detect increased expression in murine pulmonary vasculature following LPS challenge

  • Western blotting of lung tissue lysates can quantify HABP2 upregulation during ALI

  • Plasma HABP2 levels (ELISA) can serve as a potential biomarker for vascular leak syndromes

• Mechanistic investigation strategies:

  • Combine HABP2 antibody detection with PAR-1 and PAR-3 antibodies to examine receptor activation

  • Analyze RhoA/ROCK pathway activation in conjunction with HABP2 expression

  • Use siRNA knockdown of HABP2 in animal models to assess functional impact

Research findings demonstrate that:

• LPS induces HABP2 expression in murine lung endothelium in vivo and in human pulmonary microvascular endothelial cells in vitro

• Silencing (siRNA) HABP2 expression augments HMW-HA-induced endothelial cell barrier enhancement

• HABP2 knockdown inhibits LPS and LMW-HA-mediated endothelial cell barrier disruption

• Vascular silencing of HABP2 significantly reduces LPS- and ventilator-induced pulmonary vascular hyperpermeability in murine models

These findings suggest HABP2 is a potentially useful therapeutic target for syndromes of increased vascular permeability.

What is the significance of the HABP2 G534E variant in cancer research and how can it be detected?

The HABP2 G534E variant has emerged as a potential genetic risk factor for familial nonmedullary thyroid cancer:

• Detection methodologies:

  • PCR-based genotyping (the variant results in a G→A substitution at base 1601)

  • Sanger sequencing of HABP2 exon 13

  • Mutation-specific antibodies (though not yet widely available)

  • Immunohistochemistry shows differential expression patterns in tumor tissues from variant carriers

• Research findings on the G534E variant:

  • Present in 7/7 affected members of a kindred with familial nonmedullary thyroid cancer

  • Found in 4.7% of 423 patients with thyroid cancer

  • Associated with increased HABP2 protein expression in tumor samples compared to normal adjacent thyroid tissue

  • Functional studies show the variant results in loss of tumor-suppressive function

• Experimental considerations:

  • Verify germline versus somatic mutation status

  • Correlate genotype with protein expression using antibodies

  • Assess functional consequences through cell proliferation and migration assays

Immunohistochemical analysis showed increased HABP2 protein expression in papillary thyroid cancers and follicular adenoma tumors from G534E variant carriers, but no staining in normal thyroid tissue from the same individuals. In contrast, only 3 of 12 sporadic papillary thyroid cancers had faint HABP2 protein staining . This differential expression pattern suggests potential use of HABP2 antibodies as diagnostic markers for variant-associated tumors.

What role might HABP2 play in reproductive biology and how can antibodies help investigate this?

Research has begun exploring HABP2's potential role in reproductive biology, particularly in recurrent miscarriage:

While the exact mechanisms remain unclear, HABP2's known functions in vascular integrity and coagulation suggest potential relevance to placental development and function. Further research using HABP2 antibodies could help elucidate its role in normal reproductive processes and pathological conditions like recurrent miscarriage .

How can researchers leverage HABP2 antibodies to investigate novel signaling pathways?

HABP2's interactions with multiple signaling pathways open avenues for exploring novel regulatory mechanisms:

• PAR receptor signaling investigation:

  • Co-immunoprecipitation with HABP2 antibodies can identify interactions with PAR-1, PAR-3, and PAR-4

  • Activation of PAR receptors (N-terminal cleavage) can be detected following HABP2 addition

  • Functional significance can be assessed by silencing PAR expression in human endothelial cells

• RhoA/ROCK pathway analysis:

  • HABP2 activates RhoA/ROCK signaling downstream of PAR receptor activation

  • Phosphorylation status of ROCK targets can be assessed in conjunction with HABP2 expression

  • Inhibitor studies (ROCK inhibitors) can help establish causality in HABP2-mediated effects

• Hyaluronan-HABP2 signaling axis:

  • Differential effects of HMW-HA versus LMW-HA on HABP2 function

  • Potential receptor-independent effects of HABP2 on matrix components

  • Investigation of CD44 and RHAMM involvement in HABP2 regulation

Research has demonstrated that HABP2 activates PAR-1, PAR-3, and PAR-4, with functional significance for PAR-1 and PAR-3 in endothelial barrier disruption. Silencing PAR-1, PAR-3, and/or PAR-4 attenuates thrombin-induced endothelial cell barrier disruption, while silencing PAR-1 and/or PAR-3 also inhibits HABP2-mediated barrier disruption . These findings highlight the potential for HABP2 antibodies to help decipher complex signaling networks.

What are the challenges and solutions for detecting low abundance HABP2 in primary tissues?

Detecting HABP2 in primary tissues can be challenging due to potentially low expression levels:

• Common detection challenges:

  • Low basal expression in many normal tissues

  • Complex processing leading to multiple forms

  • Cross-reactivity with related serine proteases

  • Sensitivity limitations of standard detection methods

• Enhanced detection strategies:

  • Signal amplification techniques:

    • Tyramide signal amplification for IHC

    • Highly sensitive chemiluminescent substrates for Western blot

    • Digital droplet PCR for transcript quantification

  • Sample enrichment approaches:

    • Immunoprecipitation to concentrate HABP2 before analysis

    • Fractionation to separate cellular compartments

    • Enrichment of secreted proteins from conditioned media

• Optimized antibody protocols:

  • Extended primary antibody incubation (overnight at 4°C)

  • Higher primary antibody concentrations (antibody-dependent)

  • Optimized antigen retrieval methods (TE buffer pH 9.0)

  • Alternative fixation protocols for preserved tissues

Research has shown that HABP2 protein expression has been reported in only 9 of 82 normal tissue types . Additionally, detection patterns differ dramatically between normal and pathological states, with HABP2 overexpression observed in tumors from G534E variant carriers compared to adjacent normal thyroid tissue . These expression patterns highlight the importance of sensitive detection methods and appropriate positive controls.