Coagulations 'Drake Passage': The Bumpy Transition Between Fibrinogen and Fibrin

Project Summary/Abstract The long-term objective of the proposed research is to identify molecular signatures that differentiate fibrin and fibrinogen, thereby enabling clinical diagnoses and therapeutic targeting of fibrin-related pathologies. In so doing, we plan to train the next generation of scientists with interdisciplinary skills in molecular biology, biophysics, and biochemistry to tackle medical problems related to fibrin/fibrinogen. Fibrin networks act as scaffolds for blood clots, but fibrin depositions are also linked to pathological conditions such as cardiovascular disease. Fibrin only differs from its soluble precursor, fibrinogen, by a few amino acids. Thus, it is crucial to differentiate between physiological and pathological fibrin depositions, as well as between fibrin and fibrinogen, for accurate clinical diagnoses and effective therapeutic delivery. Despite the need to target fibrin, there is a lack of rationally designed fibrin-specific treatments due to a gap in understanding the chemical and physical differences between these proteins and how these differences affect their physiological functions. Our central hypothesis is that fibrinogen and fibrin exhibit distinct, regulated protein dynamics in solution, which temporally and differentially expose specific binding sites for their interacting partners and have hitherto remained unidentified due to the lack of a molecular information on soluble fibrin and fibrinogen. In this proposal, we will study the molecular details of the conversion of fibrinogen to fibrin, aiming to discover fibrin-specific epitopes that can be leveraged as therapeutic targets. Students will test our hypothesis with two specific aims: 1) Determine unique structural and dynamical fingerprints for soluble and monomeric fibrin, and 2) Elucidate the global conformer states of the central scaffold of apo and ligand-bound forms of fibrinogen. To accomplish the first aim, we will generate monomeric fibrin (mon-fibrin) by: i) using knob-mimicking GPRP peptides to prevent knob-hole interactions in native fibrinogen, and ii) expressing recombinant fibrinogen with a combination of knob and hole specific mutations. Using temperature-dependent hydrogen deuterium exchange mass spectrometry and negative stain EM, we will identify and map structural and dynamical signatures of soluble, mon-fibrin and compare them to the ligand-bound form of fibrinogen. For the second aim, we will determine and compare molecular structures of the central scaffold of apo and ligand-bound forms of fibrinogen. This information will provide detailed insights about how knob-hole interactions induce long-range changes in fibrinogen and their relationship to the initial seed-assembly steps for the formation of protofibrils. The approach is innovative because it will utilize an integrated combination of a variety biophysical methods. The proposed research is significant because it is expected to: i) develop a novel monomeric form of fibrin for molecular and biochemical studies, ii) produce the first high-resolution views of fibrinogen topology, thereby distinguishing structural fingerprints (or epitopes) as fibrinogen transitions to fibrin.