Towards Establishing Methods of Synthesis, Purification, and Structural Determination for Human Fibrinogen

Abstract

Fibrinogen is a prevalent blood-based glycoprotein. In its polymerizable form, fibrin, it serves as the structural component of blood clots and helps to incorporate red blood cells, macrophages, and fibroblasts to the wound-site. Fibrinogen plays important roles in inflammation and tissue regeneration, regulating growth factor receptor pathway activation, scar formation, and immune response, yet many questions remain about fibrinogen's structure-function link in these various pathophysiological states. Studies focused on improving expression systems for recombinant fibrinogen, and developing more rapid and robust techniques for purifying fibrinogen from complex media would provide a means by which to progress mutational studies of fibrinogen. Similarly, developing methods for high-resolution imaging of fibrinogen in its native, aqueous-based environment is necessary to establish proper structure-function relationships encompassing this complex 340 kDa glycoprotein. The work described in this thesis produced advancements in each of these crucial areas of fibrinogen research. Herein, chapter 2 reports, to our knowledge, the first methodology of transient recombinant human fibrinogen expression using suspension HEK Expi293TM cells. A highly selective and robust affinity-based approach was also developed for the rapid isolation of fibrinogen from a variety of complex media. Chapter 3 describes cryogenic electron microscopy and our process of obtaining a high-resolution, in-solution structure of human fibrinogen's D region. This structure is currently the highest resolution structure of any fibrinogen domain obtained in the native, solution state and further supports evidence for fibrinogen's intrinsic flexibility, expanding the textbook representations of fibrinogen as a rigid, rod-like structure. Lastly, future directions outlining fibrinogen's role during pregnancy and unique fibrinogen-specific protofibril-like structures are discussed. In an appendix, I have included figures from an illustrated review journal article, which I participated in as a co-first author.