1 Add New Documentation to an Existing Part on that Part's Registry Page

Part:BBa_K3672005

1.1 This could be New Information Learned from Literature

1.1.1 "Analysis of the role of von Willebrand factor, platelet glycoprotein VI-, and α2β1-mediated collagen binding in thrombus formation" (2014)

Research Background and Purpose

This study investigates the role of collagen-binding mutations in the A3 domain of von Willebrand Factor (VWF) in thrombus formation. VWF is a key protein in the coagulation process, binding to exposed subendothelial collagen to initiate platelet adhesion and thrombus formation. The main goal is to analyze these collagen-binding mutants' effects on VWF function through mouse models and in vitro experiments.

Experimental Methods

Researchers created five loss-of-function mutants and one gain-of-function mutant in the VWF cDNA, including p.S1731T, p.W1745C, p.S1783A, p.H1786D, p.L1757A, and one A3 domain deletion mutant. These mutants were analyzed through collagen binding assays, platelet adhesion assays, and flow chamber assays under high shear conditions.

Key Findings

Loss-of-function mutants showed significantly reduced collagen binding capacity: ELISA experiments revealed that most loss-of-function mutants had markedly reduced binding to type I and III collagen, especially p.W1745C and p.S1783A, with collagen-binding activities only 10%-30% of the wild type.

The gain-of-function mutant p.L1757A exhibited significantly enhanced collagen binding capacity: This mutant showed increased collagen binding in vitro, which was confirmed under high shear conditions, suggesting a stronger prothrombotic effect in thrombus formation.

The p.H1786D mutant demonstrated significantly weakened thrombus formation ability in vivo: In a ferric chloride-induced mouse model, the p.H1786D mutant showed delayed platelet adhesion and reduced thrombus formation, while the p.L1757A gain-of-function mutant accelerated thrombus formation.

Conclusions

The study indicates that VWF's binding to collagen plays a crucial role in thrombus formation. Different VWF mutants display varying degrees of functional deficiency or enhancement, significantly affecting the speed and extent of thrombus formation. The study also suggests that GPVI and α2β1 receptors are important in the direct binding of platelets to collagen, although other pathways can partially compensate for platelet adhesion and thrombus formation in the absence of VWF.

Significance

This research provides new insights into the specific mechanisms of VWF in thrombus formation, particularly how collagen-binding mutations affect platelet adhesion and thrombus formation. These findings may have important implications for diagnosing and treating von Willebrand disease and could provide a basis for developing new antithrombotic therapeutic strategies[1]

1.1.2 "Novel Likely Pathogenic Variant in the A3 Domain of von Willebrand Factor Leading to a Collagen-Binding Defect" (2021)

Research Background

This study explores a newly discovered likely pathogenic variant in the A3 domain of von Willebrand Factor (VWF) that causes a collagen-binding defect. Von Willebrand disease (VWD) is a common congenital bleeding disorder primarily caused by mutations in the VWF gene, leading to quantitative or qualitative abnormalities in VWF. The 2M subtype of VWD is particularly associated with mutations in the A1 or A3 domains, with A3 domain mutations typically resulting in decreased collagen-binding activity to type I and III collagen.

Research Methods

The researchers analyzed the cases of two siblings with a bleeding tendency. These siblings had significantly reduced VWF collagen-binding activity, while other VWF parameters and multimer analysis results were normal. Next-generation sequencing (NGS) identified a heterozygous nonsynonymous single-nucleotide variant (nsSNV) in exon 30 of the VWF gene, leading to the substitution of serine with leucine at position 1731 (p.Ser1731Leu). This serine residue was previously shown to be critical for VWF collagen binding.

Key Findings

Case Analysis: The patients' VWF values were extremely low, at 0.09 and 0.11 U/mL, compared to the normal range of 0.6–1.5 U/mL. Genetic Mutation: The researchers discovered the same p.Ser1731Leu mutation in both patients, located in the A3 domain, a major collagen-binding site. In vitro functional tests indicated that this mutation caused a significant defect in VWF collagen binding. Genetic Analysis: In vitro pathogenicity predictions using multiple bioinformatics tools (e.g., SIFT, MutationTaster, and PolyPhen2) consistently suggested that this mutation might be pathogenic.

Conclusions

This study is the first to describe the p.Ser1731Leu mutation in the A3 domain of VWF, which causes a collagen-binding defect, suggesting it is a new likely pathogenic variant that may lead to the development of the 2M subtype of VWD. This discovery provides new perspectives on the molecular mechanisms of VWF dysfunction and may have important implications for the diagnosis and classification of VWD.

Significance

The study highlights the importance of molecular diagnostics in identifying VWD subtypes and developing personalized treatment plans. Future research may further reveal clinical differences among patients with these mutations, helping to optimize diagnostic and therapeutic strategies.[2]

1.1.3 "Coarse-Grain Modeling of Shear-Induced Binding between von Willebrand Factor and Collagen" (2018)

Research Background and Purpose

This study aims to simulate the shear-induced binding mechanism between von Willebrand Factor (VWF) and collagen using a coarse-grained molecular model. VWF is a multimeric protein that plays a critical role in blood coagulation. When blood vessels are damaged, increased blood flow causes VWF to extend, exposing binding sites for platelets and collagen. The study uses Brownian dynamics simulations to explore VWF's binding behavior to exposed collagen on injured arterial surfaces.

Model and Methods

The study utilized Brownian dynamics simulations and a coarse-grained molecular model, focusing on VWF's behavior on static surfaces and under shear flow conditions. VWF molecules bind to collagen via reversible ligand-receptor-type bonds, following Bell model kinetics. The model was modified by introducing an additional binding criterion, ensuring binding inhibition at low shear rates and increased binding at higher shear rates.

Key Findings

Binding Depends on Shear Rate: Simulations showed that VWF binding to collagen was less likely at lower shear rates but significantly increased at higher shear rates, consistent with experimental observations of VWF unfolding and enhanced binding under high shear conditions. Effectiveness of Model Improvement: To better match experimental results, the study introduced a new binding criterion: requiring sufficient stretching of the A2 domain adjacent to the binding site before binding occurs. This modification allowed the model to exhibit shear-induced binding behavior within reasonable parameter ranges, especially under high binding energy conditions.

Biological Significance

The simulation results support the idea that VWF regulates its binding to collagen through structural changes (such as A2 domain stretching) under shear forces. This finding is significant for understanding VWF's role in thrombus formation and may provide new insights into treating related diseases.

Conclusions

The study demonstrates that introducing an A2 domain stretching criterion into the VWF molecular model can effectively simulate VWF binding behavior under high shear conditions. This model improvement provides a deeper understanding of how VWF regulates its binding to collagen in blood flow environments, explaining the observed biological activity of VWF under high shear forces.[3]

1.1.4 "The Role of the von Willebrand Factor Collagen-Binding Assay (VWF) in the Diagnosis and Treatment of von Willebrand Disease (VWD) and Way Beyond: A Comprehensive 36-Year History" (2023)

Research Background and Purpose

This article provides a detailed overview of the von Willebrand Factor collagen-binding assay (VWF) in the diagnosis of von Willebrand disease (VWD) and its development over the past 36 years. VWD is the most common inherited bleeding disorder, and the VWF assay is an important tool for assessing VWF's collagen-binding capacity. The article also discusses the assay's applications in diagnosing other diseases, such as acquired von Willebrand syndrome (AVWS).

Research Methods

The article reviews the history of the VWF assay, analyzing its development and application since its first report in 1986. The study summarizes different laboratory methods and evaluates their effectiveness in diagnosing VWD and other thrombotic diseases.

Key Findings

The Role of VWF in VWD Diagnosis: Since its first application in 1986, VWF has become one of the important tools in diagnosing VWD. The study shows that optimized VWF assays can effectively help identify different subtypes of VWD, particularly types 2A and 2B.

Diversity in Testing Methods: Due to different laboratories using collagen from different sources and methods, the VWF assay results vary. The article points out that optimized VWF methods are crucial for obtaining accurate results.

Broad Application of VWF:CB: In addition to diagnosing VWD, VWF is also used to assess ADAMTS13 activity, AVWS caused by mechanical circulatory support devices, and certain thrombotic diseases (such as thrombotic thrombocytopenic purpura caused by COVID-19). Challenges and Limitations of VWF: While VWF is a powerful diagnostic tool, its use in the U.S. is limited by FDA regulations, restricting its application in certain regions.

Conclusions

VWF plays a crucial role in diagnosing and treating VWD, and its application range is expanding as testing technology improves. The article emphasizes the historical significance of VWF and its potential in diagnosing various thrombotic diseases.

Significance

This article provides a comprehensive historical review of VWF for professionals engaged in hematology research and clinical diagnosis, highlighting its key role in diagnosing VWD and other related diseases. This summary helps researchers and clinicians better understand and apply this assay method.[4]

1.1.5 "Increased Binding of von Willebrand Factor to Sub-Endothelial Collagen May Facilitate Thrombotic Events Complicating Bothrops lanceolatus Envenomation in Humans" (2023)

Research Background

Envenomation by the Bothrops lanceolatus snake can cause severe thrombotic events in humans. Von Willebrand Factor (VWF) plays a key role in thrombus formation, particularly by mediating platelet adhesion through its binding to collagen when blood vessels are damaged. This study investigates how B. lanceolatus venom affects VWF's binding to collagen and its potential role in promoting thrombus formation.

Research Methods

The research team conducted in vitro experiments to assess the effects of B. lanceolatus venom on VWF's binding activity to different collagen types (types I, III, and VI). Additionally, the study examined the ability of Bothrofav anti-venom serum to reverse these effects.

Key Findings

CInhibition of Collagen-Binding Activity: High concentrations of B. lanceolatus venom completely inhibited VWF's binding activity to type I and III collagen. This suggests that components in the venom may block VWF's A3 domain, preventing its binding to collagen. Enhanced Binding Activity to Type VI Collagen: Conversely, low concentrations of B. lanceolatus venom significantly enhanced VWF's binding activity to type VI collagen, which may be related to the venom's potential enhancement of VWF A1 domain binding to collagen. Effect of Anti-Venom Serum: Bothrofav anti-venom serum was able to completely reverse the inhibitory effect of B. lanceolatus venom on VWF binding to type I and III collagen, but its protective effect was reduced at higher venom concentrations. Changes in VWF Antigen Levels: At low concentrations, B. lanceolatus venom increased VWF antigen levels, likely due to proteolysis induced by the venom, while at high concentrations, it reduced VWF antigen levels.

Conclusions

This study indicates that B. lanceolatus venom may promote thrombotic events by inhibiting VWF's binding to type I and III collagen while enhancing its binding to type VI collagen. This finding underscores the importance of the rapid use of anti-venom serum following envenomation to prevent severe thrombotic complications.

Significance

The study reveals the complex regulatory effects of snake venom on VWF function and suggests that specific interventions targeting different collagen types should be considered in clinical treatment to reduce the risk of thrombus formation following envenomation. [5]

1.2 This could be New Data Collected from Laboratory Experiments

We constructed a fusion protein of the vWF-A collagen-binding region WREPSFCALS (Type4 CBD probe for collagen I) with EGFP to determine the effect of this collagen-binding domain on subsequent connector protein activity (Figure 1.1). It was observed that the fluorescence intensity of EGFP was not significantly affected by the fusion with the WREPSFCALS sequence, indicating its potential use as a tag for subsequent cytokine and collagen-binding studies.

Figure 1.1 Effects of different collagen-binding domains on EGFP activity (fluorescence intensity)

Subsequently, we conducted binding experiments between VWF-A Domain-EGFP and type I collagen to determine their binding capacity. We used microthermophoresis to obtain experimental results (Figure 1.2). The dissociation constant (Kd) for VWF-A Domain-EGFP binding to collagen was found to be 8×10^-5 M, similar to the 100 μM recorded by the iGEM20_SCU-WestChina team, consistent with their literature survey results.

Figure 1.2 Binding capacity of different collagen-binding domains to type I collagen (dissociation constant) curve, with the horizontal axis representing the concentration of type I collagen in M.

1.3 Summary

We have enriched the content of Part BBa_K3672005. Through literature review and laboratory experiments , we obtained new data and insights into the von Willebrand Factor (vWF) A3 domain, which we have added to the existing part registration page.

First, we conducted a literature analysis on the role of the vWF domain in collagen binding, finding that multiple studies have demonstrated the crucial role of the vWF domain in thrombus formation and von Willebrand disease (VWD). These studies indicate that specific mutations in the vWF domain can significantly affect its binding ability to collagen, thereby influencing platelet adhesion and thrombus formation. This literature provides important information for understanding the vWF-A3's role in different pathological conditions and offers theoretical support for designing and optimizing related experiments.

In our laboratory experiments, we constructed a fusion protein of the vWF collagen-binding domain (WREPSFCALS) with enhanced green fluorescent protein (EGFP) to evaluate its effect on EGFP fluorescence activity. The experimental results showed no significant change in fluorescence intensity compared to unfused EGFP, indicating that this domain does not affect EGFP's fluorescence activity. This suggests that the vWF-A3 domain can be used as a collagen-binding tag without affecting protein function in subsequent experiments and applications.

Further experiments using microthermophoresis (MST) measured the binding capacity of VWF-A Domain-EGFP to type I collagen, with a dissociation constant of 8×10^-5 M. This result is consistent with the literature survey results of other teams, confirming the feasibility of this fusion protein in collagen-binding experiments.

In conclusion, our research provides new experimental data for the application of the vWF-A3 domain and strengthens the understanding of its biological function through literature review. These findings offer valuable references for future teams in designing fusion proteins and have added detailed documentation to the part registration page to support future research and development.

1.4 References

2 Document Troubleshooting that would be Helpful to Future Teams

2.1 Problem Background

In protein research and engineering applications, the design of fusion proteins is a critical step. To enhance the functionality or specificity of a particular protein, it is often fused with other functional domains. However, choosing the right fusion tag is crucial, as some tags may negatively affect the structure and function of the fusion protein, leading to biased or failed experimental results. In this experiment, we studied the fusion of various collagen-binding domains (CBDs) with enhanced green fluorescent protein (EGFP) to evaluate the impact of these CBDs on EGFP fluorescence intensity. Changes in fluorescence intensity can indicate potential effects of these CBDs on the target protein, helping us identify tags that are unsuitable for further research. This analysis is crucial for optimizing protein fusion strategies and ensuring the success of experiments.

2.2 Experimental Methods

To evaluate the effects of different collagen-binding domains (CBDs) on the fluorescence intensity of enhanced green fluorescent protein (EGFP), we fused eight CBDs with EGFP and measured the relative fluorescence intensity of the fusion proteins under standardized conditions. First, we constructed fusion expression plasmids using genetic engineering techniques and transfected them into E. coli BL21 cells for expression. The expressed cells were preliminarily evaluated using a fluorescence microscope, and the supernatant of cell lysates with the same OD was quantitatively analyzed using a microplate reader. We used non-fused EGFP as the control group, and all experiments were repeated three times to ensure accuracy and reproducibility.

2.3 Findings and Results

The experimental results showed that certain CBDs significantly reduced EGFP fluorescence intensity, such as the DDR-EGFP and AGR-EGFP groups, where fluorescence values were significantly lower than the control group. This indicates that these tags may negatively affect the structure or function of EGFP. In contrast, other CBDs, such as DB-EGFP and FTD-EGFP, had fluorescence intensity similar to the control group, indicating that these tags have minimal impact on the fusion protein. These findings help us identify tags that may affect protein function in future experimental designs, as well as CBDs that have minimal impact on fusion protein activity.

2.4 Significance of Troubleshooting

Through this experiment, we successfully identified CBDs that may negatively impact fusion protein function. This result provides important references for future experimental designs, avoiding the use of these unsuitable tags in subsequent experiments, thereby reducing the risk of potential experimental failure and resource waste. The significance of this troubleshooting lies in its role in laying a solid foundation for our protein engineering experiments and providing clear guidance for future teams, helping them optimize fusion protein design strategies and reduce unnecessary experiments, ensuring experimental success.

2.5 Recommendations

Based on the experimental results, we recommend that future teams avoid using CBDs such as DDR and VWF-A Domain, which significantly affect fluorescence intensity, when designing fusion protein experiments. Priority should be given to tags with minimal impact on EGFP fluorescence, such as DB, MMPS, and FTD, to reduce potential interference with the structure and function of the target protein. Additionally, we recommend conducting similar pre-experiments to evaluate the impact of other possible tags on protein activity before designing new fusion proteins, ensuring the rationality and scientific nature of the experimental design.

3 Creating a 3D Printed Hardware and Documenting the Manufacturing Process

We have developed an innovative material based on a dual cytokine release system, combining a high-affinity collagen-binding domain with a low-affinity collagen-binding domain, fused with bone morphogenetic protein 4 (BMP-4) and vascular endothelial growth factor (VEGF), respectively. This design allows for stepwise release, first releasing VEGF to promote angiogenesis, followed by BMP-4 to promote bone formation. To support the development of this material, we designed a custom 3D-printed shell to house our bone repair hydrogel.

3.1 Material Selection

When designing our bone repair hydrogel shell, we chose PEEK (Polyether Ether Ketone) plastic, which offers unique advantages, making it particularly suitable for applications in bone repair.

Excellent Biocompatibility

PEEK plastic demonstrates excellent biocompatibility, meaning it does not trigger immune responses or adverse reactions with body tissues. This makes PEEK an ideal material for safe implantation in the body, with good integration with surrounding tissues.

High Mechanical Strength and Wear Resistance

PEEK has extremely high mechanical strength, capable of withstanding the loads applied during bone repair. This ensures that the PEEK shell provides stable support during the patient's daily activities, maintaining the effectiveness of the bone repair material. Additionally, PEEK's high wear resistance ensures long-term structural integrity, preventing damage due to friction or stress.

Excellent Chemical Corrosion Resistance

PEEK is highly resistant to chemicals, enabling it to maintain long-term stability in the body's environment without being corroded by bodily fluids or medications. This durability helps ensure the long-term use of the implant material, reducing the risk of secondary surgeries.

X-ray Transparency

PEEK material is transparent under X-rays, meaning that during subsequent medical examinations, doctors can easily observe bone healing without interference from the implant material. This feature is crucial in clinical applications, allowing doctors to evaluate and adjust treatment effectively.

By using PEEK material, our 3D-printed shell ensures functionality and durability while significantly improving the safety and effectiveness of clinical applications. This elevates our bone repair system to a new level in terms of treatment efficacy and patient experience.

3.2 Key Points of Shell Design

Anatomical Structure of the Target Application Site

• Matching the Size and Shape of the Bone Defect: The shell's dimensions should be designed according to the specific anatomical features of the target bone defect site. Different bone sites (such as the spine, long bones, or hip joints) have different anatomical shapes and sizes. Therefore, the design of the shell needs to match the anatomical structure of the bone defect site, ensuring that the repair material fully covers and fills the defect area.
• Thickness and Curvature: The shell's thickness should be thin enough to avoid unnecessary pressure on surrounding tissues but strong enough to withstand physical loads. Additionally, the shell's curvature should match the target bone's surface shape to ensure a good fit.

Capacity for Collagen Sponge and Hydrogel

• Internal Volume: The internal volume of the shell should be designed to accommodate enough collagen sponge and hydrogel to provide continuous cytokine release and tissue support. The internal space should be slightly larger than the collagen sponge volume to prevent excessive compression after filling while allowing the free flow of cytokines and nutrients.
• Porous Structure: The design of the pores should not only prevent the collagen sponge from falling out but also ensure that the pore size is appropriate for cells and nutrients to freely pass through during the repair process. These pores should also account for the physiological load of the repair site, ensuring that the material does not collapse during application.

Biomechanical Requirements

• Structural Strength and Stability: The shell needs sufficient mechanical strength to withstand the stress loads caused by the patient's daily activities. This is especially important for load-bearing bones or spinal areas, where parameters such as strength and fill density are critical. The structural design should also avoid sharp edges to minimize irritation to surrounding tissues.
• Material Elasticity: The elastic modulus of PEEK is similar to human bone, which can help prevent stress shielding. Therefore, the shell design should consider matching the elasticity of the target bone, ensuring good stress transmission and distribution during bone repair.

Ease of Surgical Operation

• Surgical Compatibility: The shell's size should meet the needs of minimally invasive or conventional surgery, ensuring that surgeons can easily implant the shell. The design should consider how to make the shell easy to fix while avoiding damage to the shell or its internal materials during surgery.
• Positioning and Fixation: The shell's design should include structures that facilitate positioning and fixation, possibly requiring additional fixation holes or slots to securely fix the shell to the defect site during surgery.

Customization and Adjustment

• Customizability: Since each patient's bone defect situation may vary, the design should consider the shell's customizability, allowing for size adjustments based on specific clinical needs. This can be achieved through 3D printing technology, using the patient's imaging data (such as CT or MRI) to create a customized shell.

3.3 Design and Manufacturing Process

We used ZW3D software for shell design, exporting the design as an .stl file, and used Bambu X1C for 3D printing.

Figure 3.1 Shell Design

Figure 3.2 Printing Process

Figure 3.3 Printed product