Identifying structural differences between fibrinogen and fibrin to provide novel therapeutic targets

Project Summary/Abstract The broad, long-term objective of the proposed research is to identify molecular signatures differentiating 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 an interdisciplinary skill set to tackle problems in medicine, biophysics, and biochemistry. Fibrin networks act as scaffolds for blood clots, but fibrin depositions are also linked to pathological conditions such as cardiovascular disease. Fibrin only differs by a few N-terminal amino acids from its soluble precursor fibrinogen, which constantly circulates in the vasculature. Therefore, differentiating between physiological and pathological fibrin depositions and differentiating between fibrin and fibrinogen are crucially important for correct clinical diagnoses and accurate therapeutic delivery. Despite the need to target fibrin, rationally designed fibrin- specific therapies are lacking due to a gap in knowledge regarding structural differences between fibrin and fibrinogen. Our central hypothesis is that fibrinogen and fibrin exhibit distinct, allosterically-regulated structural conformations, which have hitherto remained unidentified due to the lack of a molecular fibrin structure; these conformations are anticipated to be further regulated by alterations of glycosylations at specific sites. In this proposal, we will study structural differences between fibrinogen and fibrin (and their various glycosylated forms), with the intention of discovering fibrin-specific epitopes that can be leveraged as therapeutic targets. Students will test our hypothesis with two specific aims: 1) Determine unique structural fingerprints for monomeric fibrinogen and fibrin, and 2) Elucidate the regulation of glycosylation variations upon fibrin and fibrinogen structures and fiber properties. To accomplish the first aim, we will generate monomeric fibrin (mon-fibrin) by expressing recombinant fibrinogen (mon-fgn) with specific mutations (?A364H and B?432A) that have previously been individually shown to inhibit fibrin polymerization but have not been combined to make monomeric fibrin. Polymerization capabilities of mon-fibrin will be assessed using fibrinopeptide release assays and turbidity. Using circular dichroism (CD), hydrogen deuterium exchange mass spectrometry (HDX-MS), and biological small angle x-ray scattering (BioSAXS), we will then resolve and map unique structural signatures of monomeric fibrinogen and fibrin. For aim 2, we will generate recombinant fibrinogen molecules with site-specific mutations that abolish residue-specific glycosylation sites. The structures and fibrin polymerization will be assessed by HDX-MS, BioSAXS, and turbidity measurement to determine how specific glycan sites regulate fibrin polymerization, molecular structures, and mechanical properties. The approach is innovative because it will utilize an integrated combination of methods, including multi-temperature HDX-MS. The proposed research is significant because it is expected to develop a novel monomeric form of fibrin for structural studies and will also illuminate the structural fingerprints fibrinogen and fibrin and their glycosylated variants.