Identifying the function of the Fibrin(ogen) alpha-C connector region

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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 αC connector region. In this research project, we will test this hypothesis and will also determine whether the αC 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 αC region on the mechanical and structural properties of fibrin fibers and fibrin clots. Using protein engineering, we will generate fibrin molecules with truncated αC 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 Fӧrster Resonance Energy Transfer) embedded in the αC 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.