Periodic Reporting for period 4 - MMA (Molecular Mechanical Adhesives)
Periodo di rendicontazione: 2021-10-01 al 2023-03-31
Bleeding is a major cause of death in cases of traumatic injury. Due to the local depletion of clotting factors, fibrin-based blood clots can be formed with reduced structural integrity in the event of severe trauma. Many different approaches for hemostasis in the clinic have been developed, but a key feature is the ability to restore the mechanical strength of blood clots formed under coagulopathic conditions. To address this challenge, the goal of this ERC project was to develop engineered proteins that alter the properties of blood clots such as stiffness, degradation rate in the body, cell adhesion/infiltration, and self-assembly rate.
This topic is important for society because the currently used protein-based blood products being used in the clinic, including fibrinogen and infused clotting factors, suffer from many limitations. For example, existing blood products purified from human donors are very costly, difficult to transport and store, and unsatisfactory in terms of clinical potency and efficacy in some cases. The ultimate envisioned applications of these materials will be in the clinic or emergency medicine scenarios where coagulopathy is problematic and enhancement of blood clotting is desirable. There must be a balance that is struck between hemorrhage and thrombus formation. Our research is poised to enhance our understanding of artificial coagulation cascades.
The conclusions of the action are as follows: new protein-based materials have been developed for binding to blood clots, stabilizing clots and changing the structure and morphology. These protein-based materials consist of genetically engineered proteins that are produced using genetic engineering with E. coli. The proteins developed consist of disordered or weakly ordered protein sequences, referred to as protein polymers. They differ from conventional therapeutic proteins such as monoclonal antibodies because they do not have well-defined 3D structure. The clot-binding function of these proteins is imparted by particular amino acid sequences that are encoded at the design stage. This work will continue along the line towards human therapeutic use by validating and testing the clot-modulating properties of this class of molecules using in vivo animal experiments.
This topic is important for society because the currently used protein-based blood products being used in the clinic, including fibrinogen and infused clotting factors, suffer from many limitations. For example, existing blood products purified from human donors are very costly, difficult to transport and store, and unsatisfactory in terms of clinical potency and efficacy in some cases. The ultimate envisioned applications of these materials will be in the clinic or emergency medicine scenarios where coagulopathy is problematic and enhancement of blood clotting is desirable. There must be a balance that is struck between hemorrhage and thrombus formation. Our research is poised to enhance our understanding of artificial coagulation cascades.
The conclusions of the action are as follows: new protein-based materials have been developed for binding to blood clots, stabilizing clots and changing the structure and morphology. These protein-based materials consist of genetically engineered proteins that are produced using genetic engineering with E. coli. The proteins developed consist of disordered or weakly ordered protein sequences, referred to as protein polymers. They differ from conventional therapeutic proteins such as monoclonal antibodies because they do not have well-defined 3D structure. The clot-binding function of these proteins is imparted by particular amino acid sequences that are encoded at the design stage. This work will continue along the line towards human therapeutic use by validating and testing the clot-modulating properties of this class of molecules using in vivo animal experiments.
The work performed under this action included design and production of recombinant proteins with engineered sequences that specifically interact with and bind to blood clots. These proteins were produced using genetic engineering in bacteria, and further purified and tested using in vitro clotting assays, along with physical and chemical characterization of the proteins and the clots formed in the presence of these proteins. Additionally, the toxicity and influence of these materials on cell growth was tested. The main results achieved include identification and production of novel protein sequences that bind blood clots and alter the physical and morphological properties of blood clots. Future work will focus on developing these proteins towards human therapeutic use.
The progress beyond the state of the art has involved taking a different approach to targeting blood clots with proteins. Instead of relying on non-covalent protein interactions as is typically the case for antibody-based drug delivery, in this project we are leveraging peptide ligation chemistry that is specifically active through the natural clotting pathway. This allows us to isolate binding proteins that are not susceptible to spontaneous unbinding or off-target binding to fibrinogen in the blood. Other novel aspects include the use of intrinsically-disordered human protein motifs in the development of coagulation therapy.