The proposed study is directed toward the development of novel technology that would enable health care professionals to more readily apply fibrin tissue sealants in clinical applications. Fibrin is the body's natural means for ensuring proper homeostasis (the prevention of blood loss) following injury. For nearly a century, physicians have been using and manipulating this natural hemostatic system as an applicable polymer for controlling bleeding and sealing tissues. The system used clinically consists of the two natural primary protein components, fibrinogen and thrombin, that have been isolated and purified from human donors or produced through recombinant technology. Currently, fibrin is the most used commercially-available tissue sealant but, despite its wide-spread use significant limitations continue to plague the utility of the system. Specifically, the rapidity with which fibrin polymer forms following the activation of the monomer results in i) polymerization prior to proper application, ii) poor reproducibility in application, and iii) insufficient working times required in many applications. In this study we propose researching a novel technology that blocks fibrin polymerization in the presence of the activator (thrombin) until a particular temperature; body temperature for instance, is reached. In order to achieve our overarching goals we have proposed two specific aims. In the first aim, we will create a recombinant protein expression system that allows us to produce proteins that are capable of displaying the fibrin blocking elements.
This aim requires the genetic mutation of current protein expression systems. Following mutation we will then clone a model protein into the expression system to determine whether we can produce the protein with the critical fibrin blocking elements. We will then test the capacity of this model protein to bind the inactive fibrin monomer, called fibrinogen, and its capacity to block fibrin polymerization in the presence of thrombin. In the second specific aim we will generate thermo- responsive proteins, based on the natural protein elastin, containing our fibrin blocking elements. These proteins, called elastin-like peptide repeats or ELPs, are soluble at lower temperatures and insoluble at higher temperatures. We can define which temperature these proteins transition from soluble to insoluble based on the sequence of the peptide repeat and the number of repeats we generate in the protein. In this aim we will generate several ELPs and test their capacity to both bind fibrinogen and block fibrin polymerization and will test how their transition from soluble to insoluble at defined temperatures regulates their binding and blocking activities. The design concept is that when the ELPs become insoluble they will no longer block fibrin polymerization, so at specific temperatures activated fibrin monomer will then form a polymer.
The proposed study will allow the development of a naturally-occurring protein-based polymer system that stops bleeding and binds tissues and, uniquely, polymerizes in response to contact with the body. This novel technology will not only allow health care professionals better control this system but, it would also allow the development of natural liquid band-aid products that are easy to use with wide-ranging application for the general public. ? ? ?
