Recombinant Human Thrombomodulin

Recombinant Human Thrombomodulin (rhTM) is used in research applications for its multifunctional roles in anticoagulation, anti-inflammation, endothelial protection, and immunomodulation, making it a valuable tool for studying vascular biology, coagulation disorders, and inflammatory diseases.

Key scientific reasons to use rhTM in research include:

• Anticoagulant Activity: rhTM acts as a cofactor for thrombin, facilitating the activation of protein C, which in turn degrades coagulation factors Va and VIIIa, thereby inhibiting excessive thrombin generation and clot formation. This property is essential for studying coagulation pathways and disorders such as disseminated intravascular coagulation (DIC).

• Endothelial Protection: rhTM protects vascular endothelial cells by preserving the endothelial glycocalyx, a critical barrier that is often damaged during inflammatory or septic conditions. This protection reduces vascular permeability and tissue injury, which is particularly relevant in models of acute respiratory distress syndrome (ARDS) and sepsis.

• Anti-inflammatory and Immunomodulatory Effects: rhTM neutralizes damage-associated molecular patterns (DAMPs) such as extracellular histones and high mobility group box 1 (HMGB1), which are implicated in inflammation and tissue injury. It also suppresses excessive complement activation and modulates both innate and adaptive immune responses, making it useful for research into inflammatory and autoimmune diseases.

• Inhibition of Platelet Aggregation: rhTM can inhibit histone-induced platelet aggregation, which is relevant for studies on thrombosis and platelet function.

• Disease Modeling: rhTM has been shown to ameliorate disease severity in animal models of autoimmune diseases, sepsis, and ischemia-reperfusion injury, supporting its use in preclinical studies of these conditions.

• Bioassay Applications: rhTM is used in various bioassays to study its effects on whole cells, serum, plasma, and complex biological samples, enabling detailed mechanistic studies of coagulation, inflammation, and endothelial function.

Summary of research applications:

• Investigating mechanisms of coagulation and anticoagulation.
• Modeling and studying endothelial injury and repair.
• Exploring anti-inflammatory pathways and immune modulation.
• Testing therapeutic strategies for sepsis, ARDS, and autoimmune diseases.

Limitations: While rhTM shows promise in preclinical and some clinical studies, its efficacy in certain clinical settings (e.g., sepsis-associated DIC) remains debated, with some trials showing limited benefit. Therefore, its use in research is particularly valuable for mechanistic and translational studies rather than as a universally effective therapeutic agent.

In summary, rhTM is a versatile reagent for dissecting the interplay between coagulation, inflammation, and endothelial biology in both basic and translational research.

Yes, recombinant human Thrombomodulin can be used as a standard for quantification or calibration in ELISA assays, provided it is validated for this purpose in your specific assay system. Many commercial ELISA kits for human Thrombomodulin use recombinant protein as the standard, and these kits are designed to ensure parallelism between the recombinant standard and the natural protein in biological samples.

Key considerations and supporting details:

• Recombinant protein as standard: Multiple ELISA kits for human Thrombomodulin explicitly use recombinant protein as the calibration standard, demonstrating that this is a common and accepted practice.
• Parallelism and assay validation: For accurate quantification, the recombinant standard must show parallelism with the endogenous (natural) Thrombomodulin in your sample matrix. This means the standard curve generated with the recombinant protein should yield comparable results when measuring natural Thrombomodulin in biological samples.
• Assay specificity: Most validated ELISA kits are designed to detect both recombinant and natural forms of Thrombomodulin, ensuring that the antibodies used in the assay recognize epitopes present on both forms.
• Expression system and post-translational modifications: Be aware that recombinant proteins produced in different expression systems (e.g., E. coli, mammalian cells) may have different post-translational modifications compared to the native protein, which can sometimes affect antibody recognition or assay performance. It is important to confirm that your recombinant standard is compatible with your assay antibodies and sample type.
• Calibration and traceability: Some kits calibrate their recombinant standards against international reference materials (e.g., NIBSC/WHO standards) when available, which improves the reliability and comparability of results.

Best practices:

• Validate the use of your recombinant Thrombomodulin standard in your specific ELISA setup by performing spike-and-recovery and dilution linearity experiments to confirm parallelism and accuracy.
• Use the same buffer and matrix for standard preparation as for your samples to minimize matrix effects.
• If developing your own assay, ensure that the antibodies used recognize both recombinant and natural forms equivalently.

In summary, recombinant human Thrombomodulin is widely used as a standard in ELISA assays, but proper validation in your assay context is essential for accurate quantification.

Recombinant Human Thrombomodulin (rhTM) has been validated in published research for several key applications, primarily in the context of coagulation, inflammation, and endothelial function, both in clinical and experimental settings.

Key validated applications include:

• Treatment of Disseminated Intravascular Coagulation (DIC):

  • rhTM has been extensively studied and validated as a therapeutic agent for DIC, especially in sepsis-induced DIC and obstetric DIC. Clinical studies have demonstrated improvements in laboratory markers of coagulation (platelet counts, D-dimer, fibrinogen, PT-INR) and clinical outcomes in these patient populations.

• Sepsis and Sepsis-Associated Coagulopathy:

  • rhTM has been investigated as an adjunct therapy in sepsis, with studies evaluating its efficacy in reducing morbidity and mortality associated with sepsis-induced coagulopathy. While some trials have shown benefit, others have not demonstrated a significant effect on overall sepsis outcomes.

• Experimental Models of Thromboembolism and Inflammation:

  • In animal models, rhTM has been validated for its protective effects against histone-induced lethal thromboembolism, inhibition of platelet aggregation, and reduction of inflammatory responses. These studies highlight its role in modulating both coagulation and inflammation.

• Bioassays and Mechanistic Studies:

  • rhTM is used in bioassays to study its function as a cofactor for thrombin-mediated activation of protein C, its interaction with angiopoietins, and its role in endothelial cell biology. These applications include:
    • Activation of protein C in vitro.
    • Investigation of thrombomodulin-mediated anticoagulation and its inhibition by angiopoietin-2.
    • Studies on the effects of SARS-CoV-2 spike proteins on endothelial thrombogenicity.

• Liver Inflammation and Injury:

  • Experimental research has validated rhTM for reducing liver inflammation and improving survival in models of acute liver injury.

Sample types and experimental systems validated:

• Human clinical samples (serum, plasma, whole blood).
• Animal models (primarily murine).
• In vitro cell-based assays using human endothelial cells and platelets.

Summary Table: Validated Applications of Recombinant Human Thrombomodulin

In summary, recombinant human thrombomodulin has been validated for use in clinical, preclinical, and mechanistic studies focused on coagulation, inflammation, and endothelial biology, with the strongest evidence in DIC and sepsis-related applications.

To reconstitute and prepare Recombinant Human Thrombomodulin for cell culture experiments, dissolve the lyophilized protein in sterile, endotoxin-free water or buffer, then dilute to the desired working concentration using cell culture-compatible buffer such as PBS or cell culture medium.

Essential steps and best practices:

• Reconstitution:

  • Allow the lyophilized protein vial to equilibrate to room temperature before opening to minimize condensation.
  • Add sterile, endotoxin-free water or buffer (e.g., PBS, Tris-buffered saline) to the vial. The recommended volume is typically indicated on the product datasheet to achieve a specific concentration (e.g., 100 μg/mL).
  • Gently mix by swirling or inverting; avoid vigorous shaking to prevent foaming and protein denaturation.
  • Let the solution sit at room temperature for 15–30 minutes to ensure complete dissolution.

• Aliquoting and Storage:

  • Once fully dissolved, aliquot the solution into small volumes to avoid repeated freeze-thaw cycles, which can degrade protein activity.
  • Store aliquots at −20°C or −80°C for long-term stability. Avoid storing after use if the protein is not stabilized with carrier proteins or preservatives.

• Preparation for Cell Culture:

  • Dilute the reconstituted stock solution to the desired working concentration using sterile cell culture medium or buffer compatible with your experimental system (e.g., PBS, serum-free medium).
  • If required, add carrier proteins such as 0.1% BSA to stabilize the protein, especially at low concentrations.
  • Filter-sterilize the final working solution using a 0.22 μm filter if sterility is critical for cell culture applications.

• Application Notes:

  • Typical working concentrations for cell culture experiments range from 1–10 μg/mL, but optimal dosing should be determined empirically based on your assay and cell type.
  • Thrombomodulin is sensitive to proteolytic degradation; use protease inhibitors if necessary, especially in serum-free conditions.

Additional considerations:

• Always consult the specific product datasheet for recommended reconstitution buffer, concentration, and storage conditions, as these may vary depending on the recombinant protein’s formulation and intended use.
• Avoid repeated freeze-thaw cycles and prolonged exposure to room temperature to maintain protein integrity.

This protocol ensures that recombinant human thrombomodulin is prepared in a manner suitable for cell culture experiments, preserving its biological activity and minimizing contamination risks.