Applications of the Phase Separation of Intrinsically Disordered Proteins in the Design of Intravenous Hemostatic Agents

Liquid-liquid phase separation of biological macromolecules such as proteins, RNA, and DNA, is now understood to be a critical mechanism for the organization of cellular processes. The ubiquity of this phenomenon points to phase separating proteins as both potential tools and targets for the treatment of various disorders. Here, we describe the design and implementation of a particular type of phase separating, intrinsically disordered protein – an elastin-like polypeptide – as an intravenous hemostatic agent for the treatment of uncontrolled bleeding. A plurality of trauma deaths are caused by hemorrhagic shock, and there are currently limited interventions available for patients with non-compressible injuries. The current standard of care involves the transfusion of blood products, which is hampered by drawbacks such as short shelf-life, high cost, and the potential for viral transmission. Furthermore, the development of artificial intravenous hemostats is hampered by the need to achieve high specificity for blood clots at wound sites, in order to avoid the risk of off-target thrombosis. The hemostatic elastin-like polypeptides (hELPs) described in this work overcome these issues, to yield a hemostat that is specific, stable, and that can functionally improve the hemostatic properties of blood clots. The following chapters elucidate the rationale behind the development of hELPs, describe their design and effect on the biophysical properties of clots in vitro, and highlight their validation in a rat model of acute bleeding. Chapter I gives a general introduction on the concepts of biological phase separation, intrinsically disordered proteins, and elastin-like polypeptides, as well as on hemostasis and currently available hemostatic interventions. Chapter II takes a deeper look at the importance of the structure and mechanical properties of the fibrin networks that form the scaffold of blood clots, and how those characteristics affect hemostatic efficacy. Chapter III describes the design of hELPs and their effect on fibrin clots in vitro, highlighting in particular how the phase separation of hELPs is critical to their being able to improve the functionality of blood clots. Chapter IV explores alternative hELP designs, and describes the validation of hELPs in a rat model of acute bleeding. Altogether, the results outlined herein, in addition to indicating that hELPs are a hemostatic technology that could potentially be applied in the clinic, also provide a proof-of-concept for the use of phase separation of intrinsically disordered proteins as an intervention in disorders which lead to substandard mechanical properties in other tissues and extra-cellular matrix components (e.g. cartilage, bone, etc.).