Recombinant Viridovipera stejnegeri Coagulation factor IX/factor X-binding protein subunit B2

What is Recombinant Viridovipera stejnegeri Coagulation factor IX/factor X-binding protein subunit B2?

Recombinant Viridovipera stejnegeri Coagulation factor IX/factor X-binding protein subunit B2 is an anticoagulant protein derived from the venom of Viridovipera stejnegeri (Chinese green tree viper or bamboo viper). This protein functions as a subunit of a larger protein complex that interacts with blood coagulation factors IX and X, crucial components of the blood coagulation cascade. The protein exhibits significant anticoagulant activity by interfering with the normal blood clotting process through specific binding mechanisms.

The protein binds to the gamma-carboxyglutamic acid (Gla) domains of coagulation factors IX (F9) and X (F10) in a calcium-dependent manner, with a 1:1 stoichiometry, inhibiting their participation in the coagulation cascade. Its recombinant form is produced through genetic engineering techniques to enable detailed study and potential therapeutic applications.

What molecular characterization has been performed on this protein?

The native form of this protein, Coagulation factor IX-binding protein (TSV-FIX-BP), has been isolated from Trimeresurus stejnegeri venom and characterized through various biochemical techniques. On SDS-polyacrylamide gel electrophoresis, TSV-FIX-BP displays a single band with an apparent molecular weight of 23,000 Da under non-reducing conditions, and two distinct bands with apparent molecular weights of 14,800 and 14,000 Da under reducing conditions. This suggests a dimeric structure with disulfide bridges connecting the subunits.

Amino acid sequence analysis places this protein in the family of snake venom proteins that target coagulation factors. The protein contains multiple cysteine residues that form intramolecular disulfide bonds critical for maintaining its three-dimensional structure and biological activity.

How should researchers store and handle this recombinant protein?

For optimal stability and activity preservation, Recombinant Viridovipera stejnegeri Coagulation factor IX/factor X-binding protein subunit B2 should be stored at -20°C to -80°C immediately upon receipt. To prevent repeated freeze-thaw cycles that can compromise protein integrity, it is recommended to prepare small aliquots. When working with the protein, researchers should maintain sterile conditions and avoid prolonged exposure to room temperature.

For experiments requiring calcium-dependent binding activity, ensure appropriate buffer conditions containing calcium ions (typically 1 mM Ca²⁺) as this concentration has been shown to support maximal binding to coagulation factors, based on studies with similar proteins like IX/X-bp from Trimeresurus flavoviridis .

What are the binding kinetics of this protein with coagulation factors?

The binding properties of factor IX/factor X-binding proteins from snake venoms have been extensively studied. While specific data for the Viridovipera stejnegeri protein varies somewhat from similar proteins like those from Trimeresurus flavoviridis, the general binding characteristics provide valuable insights. Studies with IX/X-bp from Trimeresurus flavoviridis demonstrate that half-maximal binding and maximal binding to both factors IX and X occur at calcium ion concentrations of 0.4 mM and 1 mM, respectively .

The concentration of IX/X-bp at half-maximal binding to solid-phase bovine factor IX and solid-phase bovine factor X were measured at 0.4 ± 0.1 nM and 1.1 ± 0.4 nM, respectively, in the presence of 1 mM Ca²⁺ ions . The binding kinetics to bovine factors IXa and Xa and human factors IX and X typically follow similar patterns to those observed with bovine factors IX and X.

For experimental design, researchers should consider the following parameters when studying binding kinetics:

How does this protein compare with similar anticoagulant proteins from other snake species?

Anticoagulant proteins targeting factors IX and X have been identified in several snake species, with varying structural and functional characteristics. The Recombinant Viridovipera stejnegeri Coagulation factor IX/factor X-binding protein demonstrates specificity for the Gla-domain regions of factors IX and X in a calcium-dependent manner.

When comparing with similar proteins:

• Trimeresurus flavoviridis (habu snake) IX/X-bp shows high specificity toward factors IX/IXa and X/Xa, with no binding to other coagulation factors such as prothrombin, factor VII, protein C, and protein Z under experimental conditions .

• Proteins from Bothrops species often display thrombin-like enzyme activity rather than direct factor IX/X binding. For example, TLBro from Bothrops roedingeri selectively cleaves the Aα and Bβ chains of bovine fibrinogen .

• Erythrofibrase from Trimeresurus erythrurus venom demonstrates both fibrinogenolytic and fibrinolytic activities, resulting in consumptive coagulopathy and hypofibrinogenemia .

The major structural difference between these snake venom proteins and thrombin is the number of cysteine residues forming disulfide bonds. While thrombin contains 7 cysteine residues, many snake venom serine proteases possess 12 cysteine residues, which are highly conserved .

What methodological approaches are most effective for studying the mechanism of anticoagulant activity?

Studying the anticoagulant mechanism of Recombinant Viridovipera stejnegeri Coagulation factor IX/factor X-binding protein subunit B2 requires a multi-faceted approach combining biochemical, biophysical, and functional assays:

• Binding Assays: Enzyme-linked immunosorbent assays (ELISA) can determine binding affinities and calcium dependency. Solid-phase binding assays with immobilized factors IX and X can quantify the interaction kinetics and identify specific binding domains .

• Functional Coagulation Assays: Activated partial thromboplastin time (aPTT) and prothrombin time (PT) assays can measure the protein's effect on coagulation pathways. Thromboelastography provides insights into the viscoelastic properties of clot formation and dissolution.

• Inhibition Studies: Factor Xa and IXa chromogenic substrate assays can determine whether the protein inhibits the enzymatic activity of these factors or merely prevents their activation by binding to the zymogen forms.

• Structural Studies: X-ray crystallography or cryo-electron microscopy of the protein-factor complexes can reveal binding interfaces and conformational changes upon interaction.

• Domain Mapping: Testing binding to various fragments derived from factors IX and X can localize the binding sites. For example, IX/X-bp binding to factor IX can be inhibited by peptides containing the Gla domain, suggesting this is the primary interaction site .

How can researchers optimize expression and purification of this recombinant protein?

Optimizing the expression and purification of Recombinant Viridovipera stejnegeri Coagulation factor IX/factor X-binding protein subunit B2 requires careful consideration of several factors:

• Expression System Selection: E. coli systems may struggle with proper disulfide bond formation, essential for this protein's activity. Consider eukaryotic expression systems such as Pichia pastoris or mammalian cell lines (CHO or HEK293) that better support post-translational modifications.

• Purification Strategy: A multi-step chromatographic approach is recommended, similar to methods used for other snake venom proteins:

  • Initial capture using ion exchange chromatography

  • Intermediate purification using hydrophobic interaction chromatography

  • Polishing step with size exclusion chromatography

For example, a two-step chromatographic method was successfully used to purify erythrofibrase from Trimeresurus erythrurus venom to homogeneity . Similarly, TLBro from Bothrops roedingeri was effectively purified using reverse-phase HPLC on a Discovery ® BIO Wide Pore C5 analytical column .

• Activity Assessment: Throughout purification, monitor both protein concentration (Bradford or BCA assay) and anticoagulant activity (clotting assays) to ensure the purified protein remains functionally active.

• Quality Control: The final purified protein should be assessed for purity using SDS-PAGE under both reducing and non-reducing conditions, and its identity confirmed by mass spectrometry or N-terminal sequencing.

What experimental approaches should be used to evaluate potential therapeutic applications?

Evaluating the therapeutic potential of Recombinant Viridovipera stejnegeri Coagulation factor IX/factor X-binding protein subunit B2 requires a systematic research pipeline:

• In Vitro Coagulation Studies:

  • Dose-response curves in standard coagulation assays (aPTT, PT, TT)

  • Thrombin generation assays with platelet-poor and platelet-rich plasma

  • Fibrin clot formation and lysis studies using turbidimetric methods

• Ex Vivo Studies:

  • Whole blood coagulation assays using rotational thromboelastometry

  • Flow chamber studies to assess effects on thrombus formation under shear stress

• Pharmacokinetic/Pharmacodynamic Studies:

  • Half-life determination in animal models

  • Distribution studies using fluorescently labeled protein

  • Metabolism and excretion pathways

• Efficacy Models:

  • Venous thrombosis models (e.g., IVC ligation in rodents)

  • Arterial thrombosis models (e.g., FeCl₃-induced carotid artery thrombosis)

  • Pulmonary embolism models

• Safety Assessment:

  • Bleeding time assays

  • Immunogenicity studies

  • Off-target effects screening

This approach resembles the development pathway that has been suggested for de novo designed proteins targeting snake venom toxins, where computational design enables the creation of binding proteins with high affinity and specificity .

What are the critical considerations for designing site-directed mutagenesis studies?

Site-directed mutagenesis studies of Recombinant Viridovipera stejnegeri Coagulation factor IX/factor X-binding protein subunit B2 should focus on several key aspects:

• Identification of Target Residues:

  • Conserved residues across similar proteins from different snake species

  • Residues in predicted binding interfaces with factors IX and X

  • Cysteine residues involved in disulfide bridge formation

  • Residues likely involved in calcium coordination

• Mutation Strategy:

  • Conservative mutations (e.g., similar charge or size) to probe subtle structural roles

  • Non-conservative mutations to dramatically alter specific properties

  • Alanine scanning of putative binding interfaces

  • Cysteine pair mutations to test disulfide bond importance

• Functional Assessment:

  • Binding affinity measurements using surface plasmon resonance or ELISA

  • Calcium dependency testing with varying Ca²⁺ concentrations

  • Anticoagulant activity evaluation in clotting assays

  • Thermal stability analysis using differential scanning fluorimetry

• Structural Validation:

  • Circular dichroism to assess secondary structure changes

  • Limited proteolysis to probe conformational alterations

  • X-ray crystallography of key mutants to confirm structural hypotheses

When analyzing the results, researchers should consider that snake venom proteins often have active site residues forming a catalytic triad (His57, Asp102, and Ser195 in the chymotrypsin numbering system), which are conserved across different species . Additionally, the 12 cysteine residues typically present in snake venom serine proteases are crucial for maintaining the protein's tertiary structure .

How should researchers interpret contradictory binding data?

When faced with contradictory binding data for Recombinant Viridovipera stejnegeri Coagulation factor IX/factor X-binding protein subunit B2, researchers should implement a systematic troubleshooting and analytical approach:

• Methodological Variables Assessment:

  • Compare experimental conditions between contradictory results (buffer composition, pH, temperature, calcium concentration)

  • Evaluate protein quality in different experiments (purity, storage conditions, freeze-thaw cycles)

  • Assess binding partner quality (native vs. recombinant factors, species differences)

• Technical Analysis:

  • Re-examine raw data and curve fitting parameters

  • Consider experimental limitations (detection limits, non-specific binding)

  • Evaluate potential systematic errors in experimental setup

• Biological Interpretation:

  • Consider allosteric effects that might cause varying binding behaviors

  • Evaluate potential for multiple binding sites with different affinities

  • Assess the impact of binding partner conformational states

The binding of IX/X-bp to factors IX and X is known to be highly dependent on calcium concentration, with half-maximal binding occurring at 0.4 mM Ca²⁺ and maximal binding at 1 mM Ca²⁺ . Discrepancies in binding data might result from varying calcium concentrations or different binding methods.

Additionally, data from comparable proteins shows that binding to the Gla domain is inhibited by specific peptides derived from factor IXa β , suggesting that contradictory results might arise from differences in the conformational state of the coagulation factors or the presence of competing molecules.

What statistical approaches are appropriate for analyzing dose-response data?

Analyzing dose-response data for Recombinant Viridovipera stejnegeri Coagulation factor IX/factor X-binding protein subunit B2 requires careful selection of statistical methods:

• Non-linear Regression Analysis:

  • Four-parameter logistic (4PL) model is typically most appropriate for sigmoidal dose-response curves

  • Calculate EC50/IC50 values with 95% confidence intervals

  • Assess Hill slopes to determine cooperativity in binding

• Comparison Between Conditions:

  • ANOVA followed by post-hoc tests (e.g., Dunnett's test when comparing multiple groups to a control)

  • Consider p < 0.05 as statistically significant, indicating that observed differences are not likely due to chance

  • For multiple comparisons, apply appropriate correction methods (Bonferroni, Tukey, or false discovery rate)

• Validation Approaches:

  • Residual analysis to ensure model fit

  • Replication across independent experiments

  • Bootstrap analysis for robust parameter estimation

For example, in the characterization of TLBro from Bothrops roedingeri venom, statistical analysis involved presenting data as average values with standard error of the mean, followed by ANOVA and post-hoc Dunnett's test to determine significant differences between experimental groups and the control group .

How can researchers effectively compare this protein with novel engineered anticoagulants?

Comparing Recombinant Viridovipera stejnegeri Coagulation factor IX/factor X-binding protein subunit B2 with novel engineered anticoagulants requires comprehensive assessment across multiple parameters:

• Mechanistic Comparison:

  • Binding specificity and affinity for target factors

  • Effect on different pathways of coagulation (intrinsic, extrinsic, common)

  • Dependency on cofactors (calcium, phospholipids)

  • Reversibility of anticoagulant effect

• Pharmacological Properties:

  • Potency (IC50/EC50 values)

  • Onset and duration of action

  • Species specificity

  • Half-life and clearance mechanisms

• Safety Profile:

  • Bleeding risk assessment

  • Immunogenicity potential

  • Off-target interactions

  • Antidote availability or reversal strategies

• Manufacturing Considerations:

  • Expression efficiency

  • Stability characteristics

  • Formulation requirements

  • Scalability of production

Novel approaches like deep learning-based design methods (e.g., RFdiffusion) offer advantages for developing antivenoms with enhanced tissue penetration compared to large antibodies . When comparing with such engineered proteins, researchers should consider that de novo designed proteins can offer high thermal stability and be produced using low-cost microbial fermentation strategies , potentially addressing limitations in traditional anticoagulant development.

What are the emerging technologies that could advance studies of this protein?

Several cutting-edge technologies show promise for advancing research on Recombinant Viridovipera stejnegeri Coagulation factor IX/factor X-binding protein subunit B2:

• Structural Biology Advances:

  • Cryo-electron microscopy for high-resolution structure determination without crystallization

  • Hydrogen-deuterium exchange mass spectrometry to map protein-protein interaction surfaces

  • Single-molecule FRET to study conformational dynamics during factor binding

• Computational Approaches:

  • Deep learning methods like RFdiffusion for designing proteins that can neutralize snake venom toxins

  • Molecular dynamics simulations to predict binding mechanisms and conformational changes

  • In silico mutagenesis to predict the effects of amino acid substitutions on function

• High-throughput Screening:

  • Microfluidic platforms for rapid assessment of anticoagulant activity

  • Droplet-based assays for single-molecule enzyme kinetics

  • Automated protein engineering workflows for directed evolution

• In Vivo Imaging:

  • Intravital microscopy to visualize anticoagulant effects in real-time

  • PET/SPECT imaging with radiolabeled protein to track biodistribution

  • Optogenetic approaches to control protein activity with light

Researchers exploring de novo protein design approaches have demonstrated advantages over traditional methods of antivenom development, creating proteins with high affinity and specificity without requiring extensive experimental screening programs . Similar approaches could be applied to engineer variants of the Viridovipera stejnegeri protein with enhanced therapeutic properties.

How might this protein contribute to understanding evolutionary adaptations in snake venoms?

Studying Recombinant Viridovipera stejnegeri Coagulation factor IX/factor X-binding protein subunit B2 provides valuable insights into evolutionary adaptations in snake venoms:

• Molecular Evolution Analysis:

  • Comparative genomics across snake species to trace gene duplication and diversification

  • Positive selection analysis to identify regions under evolutionary pressure

  • Ancestral sequence reconstruction to understand evolutionary trajectories

• Structure-Function Relationships:

  • Comparison of binding mechanisms across different snake families

  • Correlation between venom composition and prey specialization

  • Identification of conserved functional motifs versus variable regions

• Ecological Context:

  • Association between anticoagulant potency and feeding ecology

  • Geographic variation in protein structure related to prey availability

  • Coevolutionary dynamics between venomous snakes and prey resistance

• Convergent Evolution:

  • Comparison with factor IX/X binding proteins from distantly related organisms

  • Analysis of similar functional domains that evolved independently

  • Identification of molecular constraints that shape protein evolution

The presence of similar anticoagulant proteins across different snake species (Viridovipera stejnegeri, Trimeresurus flavoviridis, Trimeresurus erythrurus) with varying molecular structures suggests convergent evolution toward effective blood coagulation disruption strategies . By studying these variations, researchers can gain insights into the selective pressures driving venom evolution and the molecular mechanisms underlying prey immobilization and digestion.