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 Bß432A) 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.