SERPINA10 Antibody

What is SERPINA10 and what cellular functions does it regulate?

SERPINA10 (Protein Z-dependent Protease Inhibitor) belongs to the serpin superfamily of protease inhibitors. It is predominantly expressed in the liver and secreted into plasma where it plays a critical role in regulating coagulation. SERPINA10 inhibits the activity of coagulation factors Xa and XIa in the presence of protein Z, calcium, and phospholipids . This inhibitory activity helps regulate intravenous blood clotting, and defects in SERPINA10 may increase susceptibility to venous thrombosis .

The protein functions within the serpin family, which are unusual in that they act as one-time use, non-recyclable proteins. Their native state is thermodynamically unstable, and once cleaved, they form a covalent bond with the target enzyme, rendering it inactive . This mechanism makes them particularly effective regulators of proteolytic cascades.

What is the molecular structure and weight of SERPINA10?

SERPINA10 is a 444 amino acid secreted glycoprotein. Although its calculated molecular weight is approximately 51 kDa, Western blot analysis typically shows bands at 70-75 kDa . This discrepancy is attributed to post-translational modifications, primarily glycosylation. The protein contains five potential N-linked glycosylation sites . When analyzed by Western blot:

• Human samples show bands at approximately 70-75 kDa in plasma and liver tissue

• Mouse liver tissue shows similar bands at 72-68 kDa

• Multiple bands may be observed (72 kDa, 68 kDa, 50-55 kDa) depending on glycosylation status

The protein contains a critical tyrosine at position 387, which when disrupted, renders SERPINA10 inactive .

Which species reactivity is available for SERPINA10 antibodies?

Commercial SERPINA10 antibodies show reactivity with multiple species:

ELISA kits are available with different detection ranges: human (1.25-80 ng/mL), mouse (0.94-60 ng/mL), and guinea pig (2.5-50 ng/mL) . When selecting antibodies for cross-species studies, validation in each target species is recommended to ensure specificity .

What are the optimal conditions for SERPINA10 detection by Western blot?

For optimal Western blot detection of SERPINA10:

• Sample preparation: Prepare liver tissue lysates, hepatocyte cell lysates (HepG2, L02), or plasma samples

• Protein loading: 30 μg of protein per lane under reducing conditions

• Gel conditions: 5-20% SDS-PAGE gel at 70V (stacking gel)/90V (resolving gel) for 2-3 hours

• Transfer: Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

• Blocking: 5% non-fat milk in TBS for 1.5 hours at room temperature

• Primary antibody: SERPINA10 antibody at 0.5-1 μg/mL (or 1:500-1:3000 dilution) overnight at 4°C

• Washing: TBS-0.1% Tween, 3 times, 5 minutes each

• Secondary antibody: Anti-rabbit/mouse IgG-HRP at 1:5000 for 1.5 hours at room temperature

• Detection: Enhanced chemiluminescence (ECL) detection system

Expected bands will appear at approximately 70-75 kDa, with potential additional bands at 68 kDa and 50-55 kDa depending on sample type and glycosylation status .

How should immunohistochemistry protocols be optimized for SERPINA10 detection?

For immunohistochemical detection of SERPINA10:

• Tissue fixation: Formalin-fixed, paraffin-embedded sections

• Antigen retrieval: TE buffer pH 9.0 is recommended; alternatively, citrate buffer pH 6.0 can be used

• Blocking: 10% normal goat serum to reduce background

• Antibody dilution: 1:20-1:200 dilution of primary antibody

• Incubation time: Overnight at 4°C for primary antibody

• Detection system: Appropriate HRP-conjugated secondary antibody and DAB visualization

SERPINA10 has been successfully detected in liver tissues from both human and mouse samples . Positive staining is expected primarily in hepatocytes, consistent with the liver being the main site of SERPINA10 expression .

What factors affect the reliability of SERPINA10 quantification by ELISA?

Several factors can impact the reliability of SERPINA10 quantification by ELISA:

• ELISA format: Sandwich ELISA is preferred for human and mouse samples, while competitive ELISA may be used for guinea pig samples

• Detection range: Human (1.25-80 ng/mL), mouse (0.94-60 ng/mL), and guinea pig (2.5-50 ng/mL)

• Sample types: Validated for plasma, serum, cell culture supernatant, cell lysate, and tissue homogenate, depending on species

• Cross-reactivity: Ensure antibodies don't cross-react with other serpin family members

• Acute phase response: SERPINA10 levels can significantly change during inflammation, with maximal levels occurring around day 2 post-inflammatory stimulus in mouse models

• Protein Z interaction: SERPINA10 can form complexes with Protein Z in circulation, which may affect epitope accessibility in some assay formats

For optimal results, use appropriate dilution series, include standards in each assay, and validate results across multiple methodologies when possible.

How does SERPINA10 interact with Protein Z to inhibit Factor Xa?

SERPINA10 and Protein Z form a complex that dramatically enhances inhibition of coagulation Factor Xa:

• Protein Z (PZ), a vitamin K-dependent plasma protein, acts as a cofactor that dramatically enhances SERPINA10's inhibitory activity against Factor Xa

• The inhibition requires calcium and phospholipids in addition to Protein Z

• The interaction between SERPINA10 and Factor Xa involves SERPINA10 functioning as a pseudo-substrate, where once cleaved, it forms a covalent bond with Factor Xa, rendering it inactive

• SERPINA10 can also directly inhibit Factor XIa without requiring Protein Z as a cofactor

• In circulation, a significant portion of SERPINA10 forms a complex with Protein Z, which affects its half-life and functional properties

This complex formation provides a fine-tuning mechanism for regulation of coagulation, with knockout studies in mice showing enhanced responses in models of induced thrombosis, supporting a physiologically relevant role for the PZ/SERPINA10 system in coagulation regulation .

How do inflammatory conditions affect SERPINA10 expression and function?

Inflammation significantly impacts SERPINA10 expression and circulating levels:

• In mouse models of acute inflammation (turpentine-induced aseptic abscess), plasma SERPINA10 levels significantly increase, with maximal levels occurring around day 2 post-stimulus

• This increase in SERPINA10 occurs alongside classical acute phase proteins like serum amyloid A and fibrinogen

• Interestingly, Protein Z levels also increase during inflammation, but with a different time course (maximal levels around day 4)

• The increase in Protein Z levels following inflammation is dependent on SERPINA10, suggesting a regulatory relationship between these two proteins

• This differential regulation may represent a fine-tuning mechanism to prevent excessive coagulation during inflammatory responses

Researchers studying SERPINA10 in inflammatory contexts should consider these temporal dynamics and the relationship with Protein Z when designing experiments .

What are the implications of SERPINA10 mutations for thrombotic disorders?

Mutations in SERPINA10 have been associated with venous thrombosis and altered coagulation dynamics:

• Defects in the gene encoding SERPINA10 may increase susceptibility to venous thrombosis

• This association is consistent with SERPINA10's role in inhibiting coagulation factors Xa and XIa

• Knockout mice for either Protein Z or SERPINA10 show enhanced responses in models of induced thrombosis, supporting the physiological relevance of this inhibitory system

• Tyrosine at position 387 is critical for SERPINA10 activity, and mutations affecting this residue render the protein inactive

• The anticoagulant activity of SERPINA10 depends on its ability to form complexes with Protein Z, and mutations affecting this interaction may also impact thrombosis risk

Research approaches examining SERPINA10 in thrombotic contexts should include both functional assays and genetic screening to fully characterize the impact of specific mutations .

How should researchers interpret different molecular weight bands in Western blot for SERPINA10?

When analyzing Western blot results for SERPINA10, researchers may observe bands at different molecular weights:

• The calculated molecular weight of SERPINA10 is approximately 51 kDa, but observed bands typically appear at higher molecular weights

• Major bands are commonly observed at 70-75 kDa in human samples

• Additional bands may be observed at 68 kDa and 50-55 kDa

• These differences are attributed to:

  • Post-translational modifications, primarily glycosylation (SERPINA10 contains five potential N-linked glycosylation sites)

  • Species differences in glycosylation patterns

  • Potential proteolytic processing in different sample types

  • Formation of complexes with other proteins, particularly Protein Z

To confirm band specificity, researchers should:

• Include positive controls (liver tissue or hepatocyte cell lines)

• Consider deglycosylation experiments to confirm glycosylation as the source of size differences

• Compare results across multiple antibodies targeting different epitopes

• Include appropriate negative controls (non-expressing tissues or knockout samples when available)

What controls should be included when studying SERPINA10 in coagulation research?

Proper controls are essential for reliable SERPINA10 research in coagulation studies:

Positive controls:

• Human, mouse, or rat liver tissue lysates (primary site of SERPINA10 expression)

• Hepatocyte cell lines: HepG2, L02, or RAW264.7 cells

• Recombinant SERPINA10 protein (for antibody validation and assay standardization)

• Normal plasma samples (for clinical studies)

Negative controls:

• Non-expressing tissues or cell lines

• SERPINA10 knockout mice samples (when available)

• Antibody pre-absorption with immunizing peptide

• Isotype control antibodies for flow cytometry and immunostaining applications

Functional controls:

• Protein Z inclusion/exclusion in Factor Xa inhibition assays

• Calcium and phospholipid dependency tests

• Comparison of wild-type vs. mutated SERPINA10 (especially mutations affecting Tyr387)

For studies examining SERPINA10 in inflammatory contexts, appropriate controls should include time-course measurements and comparison with established acute phase proteins like serum amyloid A and fibrinogen .

How can researchers validate SERPINA10 antibody specificity across different applications?

Validating SERPINA10 antibody specificity across different applications requires a multi-faceted approach:

Western blot validation:

• Confirm band size (primary bands at 70-75 kDa, with potential additional bands at 68 kDa and 50-55 kDa)

• Test multiple tissue/cell types with known expression (liver tissue, hepatocytes)

• Include knockout or knockdown samples when possible

• Peptide competition assays to confirm specificity

Immunohistochemistry/immunofluorescence validation:

• Compare staining patterns with known expression profiles (primarily liver)

• Include positive controls (human/mouse liver tissue)

• Confirm subcellular localization (secretory pathway for this secreted protein)

• Compare results across multiple antibodies targeting different epitopes

Flow cytometry validation:

• Compare with isotype control antibodies

• Include unlabelled samples as blank controls

• Validate with cells known to express SERPINA10 (e.g., HEL cells, RT4 cells)

• Confirm specificity with blocking peptides

ELISA validation:

• Perform spike and recovery experiments

• Test linearity of dilution

• Compare results across multiple ELISA formats (sandwich vs. competitive)

• Validate against other quantification methods (e.g., Western blot, mass spectrometry)

Cross-application validation is also important - results from one technique should be consistent with those from others when studying the same samples .

What is the potential of SERPINA10 as a biomarker in coagulation and inflammatory disorders?

SERPINA10 shows promise as a biomarker in several contexts:

• Thrombotic disorders: Given its role in inhibiting coagulation factors Xa and XIa, altered SERPINA10 levels may indicate thrombotic risk

• Inflammatory conditions: SERPINA10 functions as an acute phase protein, with levels significantly increasing during inflammation

• Liver dysfunction: As primarily expressed in the liver, SERPINA10 levels may reflect hepatic function

• Diagnostic potential: Changes in SERPINA10/Protein Z ratio might provide more informative diagnostic value than either protein alone

For biomarker development, researchers should consider:

• Establishing reference ranges in different populations

• Determining the diagnostic sensitivity and specificity

• Comparing SERPINA10 with established coagulation markers

• Developing standardized ELISA protocols optimized for clinical samples

• Investigating post-translational modifications as potential markers of specific pathologies

The unique temporal dynamics of SERPINA10 elevation during inflammation (peaking around day 2) compared to Protein Z (peaking around day 4) may offer insights into the stage of inflammatory responses .

How can SERPINA10 research contribute to therapeutic development for coagulation disorders?

Understanding SERPINA10 biology offers several avenues for therapeutic development:

• Recombinant SERPINA10 therapy: May potentially benefit patients with thrombotic disorders, particularly those with SERPINA10 deficiency or dysfunction

• Small molecule modulators: Compounds that enhance SERPINA10's inhibitory activity against factors Xa and XIa could serve as novel anticoagulants

• Targeting the SERPINA10-Protein Z interaction: Modulation of this interaction could provide fine-tuned control over coagulation

• Personalized medicine approaches: Genetic screening for SERPINA10 mutations could identify patients who might benefit from specialized anticoagulant therapies

• Anti-inflammatory applications: Given SERPINA10's role in the acute phase response, targeting its pathway may offer new approaches to managing inflammatory conditions

Research methodologies supporting these directions should include:

• High-throughput screening for SERPINA10 modulators

• Structure-function studies to identify critical domains for therapeutic targeting

• Animal models (including existing Protein Z and SERPINA10 knockout mice) for preclinical testing

• Development of sensitive assays to monitor SERPINA10 function in clinical samples

What are the challenges in studying SERPINA10 across different model systems?

Researchers face several challenges when studying SERPINA10 across different experimental systems:

• Species differences: While SERPINA10 is conserved across mammals, there may be species-specific differences in regulation, post-translational modifications, and interaction with Protein Z

• Expression systems: Recombinant SERPINA10 production requires mammalian expression systems to ensure proper glycosylation and folding

• Half-life variations: The half-life of SERPINA10 varies depending on whether it's complexed with Protein Z (~8 hours when free, ~60 hours when complexed), complicating pharmacokinetic studies

• Post-translational modifications: The extensive glycosylation of SERPINA10 creates challenges for structural studies and recombinant expression

• Antibody cross-reactivity: Ensuring antibody specificity across species requires careful validation

• Functional assays: Assays measuring inhibitory activity against factors Xa and XIa require carefully controlled conditions including calcium, phospholipids, and Protein Z

To address these challenges, researchers should:

• Validate reagents and methods in each model system

• Consider both glycosylated and non-glycosylated forms in structural studies

• Account for the interaction with Protein Z when designing experiments

• Utilize knockout models to establish specificity of observations

• Develop standardized protocols that can be applied across different model systems