Difference between revisions of "Part:BBa K3672005"
 
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A collagen binding domain (CBD) WREPSFCALS, which was identified from von Willebrand Factor (vWF), exhibits low binding affinity (Kd: 100 μM) for collagen type I  
 
A collagen binding domain (CBD) WREPSFCALS, which was identified from von Willebrand Factor (vWF), exhibits low binding affinity (Kd: 100 μM) for collagen type I  
  
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===Usage and Biology===
 
  
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===Sequence and Features===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3672005 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3672005 SequenceAndFeatures</partinfo>
  
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===References===
 
===References===
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<li>Wahyudi H, Reynolds AA, Li Y, Owen SC, Yu SM. Targeting collagen for diagnostic imaging and therapeutic delivery. J Control Release. 2016;240:323-331. doi:10.1016/j.jconrel.2016.01.007
 
<li>Wahyudi H, Reynolds AA, Li Y, Owen SC, Yu SM. Targeting collagen for diagnostic imaging and therapeutic delivery. J Control Release. 2016;240:323-331. doi:10.1016/j.jconrel.2016.01.007
 
<li> Takagi J, Asai H, Saito Y. A collagen/gelatin-binding decapeptide derived from bovine propolypeptide of von Willebrand factor. Biochemistry. 1992;31:8530–8534.  
 
<li> Takagi J, Asai H, Saito Y. A collagen/gelatin-binding decapeptide derived from bovine propolypeptide of von Willebrand factor. Biochemistry. 1992;31:8530–8534.  
 
<li> Zhao W, Chen B, Li X, Lin H, Sun W, Zhao Y, Han Q, Dai J. Vascularization and cellularization of collagen scaffolds incorporated with two different collagen-targeting human basic fibroblast growth factors. J Biomed Mater Res. 2007;82A:630–636.
 
<li> Zhao W, Chen B, Li X, Lin H, Sun W, Zhao Y, Han Q, Dai J. Vascularization and cellularization of collagen scaffolds incorporated with two different collagen-targeting human basic fibroblast growth factors. J Biomed Mater Res. 2007;82A:630–636.
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<h2>1 This could be new information learned from literature</h3>
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            <br>
 +
          <h3>1. <strong>"Analysis of the role of von Willebrand factor, platelet glycoprotein VI-,
 +
              and α2β1-mediated collagen binding in thrombus formation" (2014)</strong></h2>
 +
            <br>
 +
            <h4>Research Background and Purpose</h4>
 +
              <p>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.</p>
 +
                <br>
 +
            <h4>Experimental Methods</h4>
 +
              <p>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.</p>
 +
                  <br>
 +
            <h4>Key Findings</h4>
 +
              <p>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.</p>
 +
              <p>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.</p>
 +
              <p>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.</p>
 +
                  <br>
 +
            <h4>Conclusions</h4>
 +
              <p>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.</p>
 +
                <br>
 +
            <h4>Significance</h4>
 +
              <p>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<a href="#ref1">[1]</a></p>
 +
                <br>
 +
 +
            <h3>2. <strong>"Novel Likely Pathogenic Variant in the A3 Domain of von Willebrand
 +
              Factor Leading to a Collagen-Binding Defect" (2021)</strong></h3>
 +
              <br>
 +
              <h4>Research Background</h4>
 +
                <p>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.</p>
 +
                  <br>
 +
              <h4>Research Methods</h4>
 +
                <p>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.</p>
 +
                  <br>
 +
              <h4>Key Findings</h4>
 +
                <p>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.</p>
 +
                    <br>
 +
              <h4>Conclusions</h4>
 +
                <p>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.</p>
 +
                  <br>
 +
              <h4>Significance</h4>
 +
                <p>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.<a href="#ref2">[2]</a></p>
 +
                    <br>
 +
 +
            <h3>3. <strong>"Coarse-Grain Modeling of Shear-Induced Binding between von Willebrand
 +
              Factor and Collagen" (2018)</strong></h3>
 +
              <br>
 +
              <h4>Research Background and Purpose</h4>
 +
                <p>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.</p>
 +
                  <br>
 +
              <h4>Model and Methods</h4>
 +
                <p>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.</p>
 +
                    <br>
 +
              <h4>Key Findings</h4>
 +
                <p>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.</p>
 +
                      <br>
 +
              <h4>Biological Significance</h4>
 +
                <p>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.</p>
 +
                    <br>
 +
              <h4>Conclusions</h4>
 +
                <p>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.<a href="#ref3">[3]</a></p>
 +
                    <br>
 +
             
 +
 +
           
 +
           
 +
            <h3>4. <strong>"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)</strong></h3>
 +
              <br>
 +
              <h4>Research Background and Purpose</h4>
 +
                <p>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).</p>
 +
                  <br>
 +
              <h4>Research Methods</h4>
 +
                <p>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.
 +
                  </p>
 +
                  <br>
 +
              <h4>Key Findings</h4>
 +
                <p>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.
 +
                  </p>
 +
                  <br>
 +
                <p>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.
 +
                  </p>
 +
                  <br>
 +
                <p>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.
 +
                  </p>
 +
                  <br>
 +
              <h4>Conclusions</h4>
 +
                <p>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.</p>
 +
                  <br>
 +
              <h4>Significance</h4>
 +
                <p>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.<a href="#ref4">[4]</a></p>
 +
                  <br>
 +
            <h3>5. <strong>"Increased Binding of von Willebrand Factor to Sub-Endothelial Collagen May Facilitate Thrombotic
 +
              Events Complicating Bothrops lanceolatus Envenomation in Humans" (2023)</strong></h3>
 +
              <br>
 +
              <h4>Research Background</h4>
 +
                <p>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.</p>
 +
                  <br>
 +
              <h4>Research Methods</h4>
 +
                <p>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.</p>
 +
                  <br>
 +
              <h4>Key Findings</h4>
 +
                <p>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.</p>
 +
                    <br>
 +
              <h4>Conclusions</h4>
 +
                <p>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.</p>
 +
                  <br>
 +
              <h4>Significance</h4>
 +
                <p>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.
 +
                  <a href="#ref5">[5]</a></p>
 +
                  <br>
 +
              <h2>2 This could be new data collected from laboratory experiments</h2>
 +
              <br>
 +
                  <p>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.</p>
 +
                  <img src="https://static.igem.wiki/teams/5374/contribution/fig-1-1.svg" style="width: 300px">
 +
                  <p class="img-description">Figure 1.1 Effects of different collagen-binding domains on
 +
                    EGFP activity (fluorescence intensity)</p>
 +
                    <br>
 +
                  <p>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<sup>-5</sup> M, similar to the 100 μM recorded by the iGEM20_SCU-WestChina team, consistent with
 +
                    their literature survey results.</p>
 +
                    <img src="https://static.igem.wiki/teams/5374/contribution/fig-1-2.svg" style="width: 500px">
 +
                    <p class="img-description">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.</p>
 +
                      <br>
 +
              <h2>3 Summary</h2>
 +
              <br>
 +
                  <p>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.</p>
 +
                    <br>
 +
                  <p>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.</p>
 +
                      <br>
 +
                  <p>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.</p>
 +
                      <br>
 +
                  <p>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<sup>-5</sup> M. This result is consistent with the
 +
                    literature survey results of other teams, confirming the feasibility of this fusion protein in
 +
                    collagen-binding experiments.</p>
 +
                    <br>
 +
                  <p>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.</p>
 +
                    <br>
 +
              <h2>4 References</h2>
 +
              <table class="ref-table">
 +
                <tr>
 +
                    <td>
 +
                        <div class="reference" id="ref1">[1]</div>
 +
                    </td>
 +
                    <td>SHIDA Y, N RYDZ, D STEGNER, et al., <i>Analysis of the role of von Willebrand factor,
 +
                      platelet glycoprotein VI-, and α2β1-mediated collagen binding in thrombus formation.</i>
 +
                      [J]. Blood, 2014, 124(11): 1799-807.
 +
                    </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>
 +
                        <div class="reference" id="ref2">[2]</div>
 +
                    </td>
 +
                    <td>FELS S, D BOECKELMANN, H GLONNEGGER, et al., <i>Novel Likely Pathogenic Variant in the
 +
                      A3 Domain of von Willebrand Factor Leading to a Collagen-Binding Defect. </i>
 +
                      [J]. Hamostaseologie, 2023, 43(2): 122-5.
 +
                    </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>
 +
                        <div class="reference" id="ref3">[3]</div>
 +
                    </td>
 +
                    <td> WEI W, C DONG, M MORABITO, et al., <i>Coarse-Grain Modeling of Shear-Induced Binding between von
 +
                      Willebrand Factor and Collagen.</i> [J]. Biophysical Journal, 2018, 114(8): 1816-29.
 +
                    </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>
 +
                        <div class="reference" id="ref4">[4]</div>
 +
                    </td>
 +
                    <td>FAVALORO E J. <i>The Role of the von Willebrand Factor Collagen-Binding Assay (VWF:CB) in the
 +
                      Diagnosis and Treatment of von Willebrand Disease (VWD) and Way Beyond: A Comprehensive 36-Year
 +
                      History. </i> [J]. Semin Thromb Hemost, 2023, 50(01): 043-80.
 +
                    </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>
 +
                        <div class="reference" id="ref5">[5]</div>
 +
                    </td>
 +
                    <td> PIERRE-LOUIS O, D RESIERE, C ALPHONSINE, et al. <i>Increased Binding of von Willebrand Factor
 +
                      to Sub-Endothelial Collagen May Facilitate Thrombotic Events Complicating Bothrops lanceolatus
 +
                      Envenomation in Humans</i> [J]. 2023, 15(7): 441.
 +
                    </td>
 +
                </tr>
 +
              </table>
 +
</html>

Latest revision as of 02:59, 16 September 2024


Type4,CBD probe for collagen I

A collagen binding domain (CBD) WREPSFCALS, which was identified from von Willebrand Factor (vWF), exhibits low binding affinity (Kd: 100 μM) for collagen type I


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References

  1. Wahyudi H, Reynolds AA, Li Y, Owen SC, Yu SM. Targeting collagen for diagnostic imaging and therapeutic delivery. J Control Release. 2016;240:323-331. doi:10.1016/j.jconrel.2016.01.007
  2. Takagi J, Asai H, Saito Y. A collagen/gelatin-binding decapeptide derived from bovine propolypeptide of von Willebrand factor. Biochemistry. 1992;31:8530–8534.
  3. Zhao W, Chen B, Li X, Lin H, Sun W, Zhao Y, Han Q, Dai J. Vascularization and cellularization of collagen scaffolds incorporated with two different collagen-targeting human basic fibroblast growth factors. J Biomed Mater Res. 2007;82A:630–636.

The TestDaily-I-Beijing2024 team has improved the following content based on this part

1 This could be new information learned from literature


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]


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]


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]


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]


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]


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.


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.


4 References

[1]
 SHIDA Y, N RYDZ, D STEGNER, et al., Analysis of the role of von Willebrand factor, platelet glycoprotein VI-, and α2β1-mediated collagen binding in thrombus formation. [J]. Blood, 2014, 124(11): 1799-807.
[2]
 FELS S, D BOECKELMANN, H GLONNEGGER, et al., Novel Likely Pathogenic Variant in the A3 Domain of von Willebrand Factor Leading to a Collagen-Binding Defect. [J]. Hamostaseologie, 2023, 43(2): 122-5.
[3]
 WEI W, C DONG, M MORABITO, et al., Coarse-Grain Modeling of Shear-Induced Binding between von Willebrand Factor and Collagen. [J]. Biophysical Journal, 2018, 114(8): 1816-29.
[4]
 FAVALORO E J. The Role of the von Willebrand Factor Collagen-Binding Assay (VWF:CB) in the Diagnosis and Treatment of von Willebrand Disease (VWD) and Way Beyond: A Comprehensive 36-Year History. [J]. Semin Thromb Hemost, 2023, 50(01): 043-80.
[5]
 PIERRE-LOUIS O, D RESIERE, C ALPHONSINE, et al. Increased Binding of von Willebrand Factor to Sub-Endothelial Collagen May Facilitate Thrombotic Events Complicating Bothrops lanceolatus Envenomation in Humans [J]. 2023, 15(7): 441.