Project Summary/Abstract
The broad, long-term objective of the proposed research is to determine the mechanisms regulating
fibrin and fibrinogen function, with the goal of improving the diagnosis and treatment of cardiovascular
disease. In so doing, we plan to train the next generation of scientists with an interdisciplinary skill set
to tackle problems in medicine, physics, and biochemistry.
In this specific proposal, we will study the polymerization, mechanical properties, and enzymatic digestion of
fibrin fibers. Fibrin fibers form the structural scaffold for blood clots and are remarkably elastic, often being
compared to rubber bands, before being digested by plasmin after wound healing terminates. Understanding
how fibrin can act like a rubber is potentially important for both clinical diagnoses and the development of
treatment approaches for many cardiovascular diseases, because altered fibrin elasticity is often associated with
strokes, heart attacks, and other pathologies. However, we currently do not have a complete understanding of
which structural properties of fibrin enable its elasticity. Based on previous studies and indirect evidence, we
hypothesize that these elastic properties originate from a specific region in fibrin, the aC connector region. In
this research project, we will test this hypothesis and will also determine whether the aC connector region is
involved in fibrin polymerization, fibrin structure, and the digestion of fibrin by the enzyme plasmin. This
interdisciplinary work will rely heavily on student researchers, providing training in molecular biology,
biochemistry, biophysics, and blood coagulation.
Specific Aim 1: Determine the importance of the aC region on the mechanical and structural properties
of fibrin fibers and fibrin clots. Using protein engineering, we will generate fibrin molecules with truncated aC
connector regions. We will test the polymerization and structure of fibrin fibers composed of these molecules
using fibrinopeptide release assays, turbidity and turbidimetry, scanning electron microscopy, and permeability
assays and compare it to fibers made of native, human fibrin. We will measure the mechanical properties of
these fibers using atomic force microscopy. Additionally, we will engineer fibrin molecules with molecular tension
sensors (based on F¿rster Resonance Energy Transfer) embedded in the aC connector region. Taken together,
these experiments will reveal the extent to which this region regulates fibrin polymerization, mechanical
properties, and fiber structure.
Specific Aim 2: Determine the mechanical and structural regulators of fibrin fiber fibrinolytic rates. Little
is known about how the mechanical and structural properties of individual fibers influence their susceptibility to
plasmin lysis, even though the lysis of a blood clot occurs through the digestion of fibers. We will determine how
internal fiber structure, fiber tension, and the spacing between fibers impacts plasmin digestion using native fibrin
and the engineered fibrin molecules described in Aim 1.