A new field of biomedical research, biomechanics of hemostasis and thrombosis, has been quickly developing over the past few years. The mechanical properties of fibrin are naturally variable and largely determine whether clots stanch bleeding, or lead to thrombosis or hemorrhage, and this makes them a desirable therapeutic target. In this application, fibrin mechanics will be studied with respect to structural changes during physiologically relevant fibrin deformations at increasing levels of complexity, including individual molecules, fibrin oligomers, and whole fibrin clots as well as ex vivo thrombi. The structural basis of the viscoelastic properties of fibrin is going to be examined using a uniquely broad, integrated approach based on state-of-the- art biophysical techniques, such as single-molecule optical trap-based force spectroscopy, wide angle X-ray scattering, Fourier Transform infrared spectroscopy, high-resolution rheometry, atomic force microscopy, confocal and electron microscopy, combined with computational molecular dynamics simulations and multiscale modeling.
In Specific Aim 1, the structural transitions in fibrin at the molecular level induced by mechanical force will be studied. Understanding of the unfolding of the coiled-coils, ?C regions, and ?-nodules will define the molecular changes that occur in vivo as a result of blood flow, clot retraction, and wound stretching. The ?-helix to ?-sheet transition in the coiled-coils is an important mechanism of fibrin mechanics and potentially tunable for clinical purposes. Straightening of the ?C polymers and unfolding of the ?-nodules also play major roles in fibrin mechanical properties.
In Specific Aim 2, nanomechanics of the A:a knob-hole bonds that hold fibrin together will be studied at the single-molecule level. Preliminary data show that at the A:a bonds exhibit counterintuitive """"""""catch"""""""" bond behavior, meaning that the strength of the bond increases with increasing force. This novel finding is a basis for further in depth studies because of its general importance for the field of biomolecular interactions and potential physiological significance. Using a new approach, Binding- Unbinding Correlation Spectroscopy, that we developed we will extensively characterize the two-dimensional kinetics and thermodynamics of formation and dissociation of single A:a bonds.
In Specific Aim 3, mechanical properties of clinically significant clots and thrombi will be studied, with a logical progression from the molecular and microscopic levels to increasingly complex macroscopic structures formed in vivo. Screening of chemicals and structural modifications that potentially stabilize or destabilize fibrin molecular domains will be performed to reveal potential modulators of fibrin mechanical properties for therapeutic purposes. These studies would advance the field of hemostasis and thrombosis by leading to new structure- and mechanics-based approaches to prevent and treat bleeding and thrombosis.

Public Health Relevance

The focus of the research proposed in this grant application will be on the characteristics of fibrin (ogen) molecules, fibers, and networks that give rise to blood clot mechanical properties and the determination of relationships between these different levels of structure, using a variety of biophysical techniques. The results of these studies have clinical significance since clots with low elasticity and high plasticity tend to be associated wit bleeding, while very stiff clots have been associated with thrombosis and thromboembolism, which cause heart attacks and strokes. More generally, this research involves the determination of relationships between molecular structure and the mechanical properties of a remarkable biological material, the blood clot.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
High Priority, Short Term Project Award (R56)
Project #
2R56HL090774-05A1
Application #
8903542
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Link, Rebecca P
Project Start
2007-12-01
Project End
2015-08-31
Budget Start
2014-09-01
Budget End
2015-08-31
Support Year
5
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Bannish, Brittany E; Chernysh, Irina N; Keener, James P et al. (2017) Molecular and Physical Mechanisms of Fibrinolysis and Thrombolysis from Mathematical Modeling and Experiments. Sci Rep 7:6914
Litvinov, Rustem I; Weisel, John W (2017) Role of red blood cells in haemostasis and thrombosis. ISBT Sci Ser 12:176-183
Kim, O V; Litvinov, R I; Chen, J et al. (2017) Compression-induced structural and mechanical changes of fibrin-collagen composites. Matrix Biol 60-61:141-156
Kononova, Olga; Litvinov, Rustem I; Blokhin, Dmitry S et al. (2017) Mechanistic Basis for the Binding of RGD- and AGDV-Peptides to the Platelet Integrin ?IIb?3. Biochemistry 56:1932-1942
Tutwiler, Valerie; Peshkova, Alina D; Andrianova, Izabella A et al. (2017) Contraction of Blood Clots Is Impaired in Acute Ischemic Stroke. Arterioscler Thromb Vasc Biol 37:271-279
Ponomareva, A A; Nevzorova, T A; Mordakhanova, E R et al. (2017) Intracellular origin and ultrastructure of platelet-derived microparticles. J Thromb Haemost 15:1655-1667
Nevzorova, Tatiana A; Zhao, Qingze; Lomakin, Yakov A et al. (2017) Single-Molecule Interactions of a Monoclonal Anti-DNA Antibody with DNA. Bionanoscience 7:132-147
Kim, Oleg V; Litvinov, Rustem I; Alber, Mark S et al. (2017) Quantitative structural mechanobiology of platelet-driven blood clot contraction. Nat Commun 8:1274
Tutwiler, Valerie; Wang, Hailong; Litvinov, Rustem I et al. (2017) Interplay of Platelet Contractility and Elasticity of Fibrin/Erythrocytes in Blood Clot Retraction. Biophys J 112:714-723

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