Although we know a great deal about the structure and many aspects of the functions of fibrin(ogen), we still know very little about the microscopic and molecular structural origins of the fibrin clot's mechanical properties. Since blood clotting in vivo is essentially a mechanical task, it is important to determine how clots and thrombi respond to mechanical stresses imposed by highly dynamic conditions, such as blood flow, stretching a vessel wall and wounds, etc. In the research proposed in this application, the structural basis of the elastic and viscous properties of fibrin biopolymers is going to be examined using an integrated approach, which includes different levels of analysis, the molecular level, individual fibers, fiber network, and the whole clot, and the determination of relationships between these different levels of structure.
Specific Aim 1 : At the nano scale, the micromechanics of fibrin(ogen) will be examined by forced unfolding of its molecular domains during pulling on engineered oligomeric constructs by single-molecule atomic force microscopy, and observing the structural transitions by wide angle X-ray diffraction or Fourier Transform infrared spectroscopy while stretching of fibrin clots.
Specific Aim 2 : At the microscopic scale, the mechanical properties of fibers will be studied by bending and stretching of individual fibers in different clots by atomic force microscopy or optical tweezers, and investigating potential elongation of molecules and molecular packing by means of the small angle X-ray diffraction pattern during stretching of magnetically oriented clots. Structural changes in fiber network rearrangement, such as alignment and bundling of fibers, with clot deformation will be examined by scanning and transmission electron microscopy.
Specific Aim 3 : At the macro level, the viscoelastic properties of a variety of whole clots and thrombi extracted from patients'coronary arteries will be measured using rotational and extensional rheometry and correlated with parameters quantifying clot and thrombi structure. To build a general theory of the structural origin of clot mechanics, we will develop constitutive models that take advantage of the quantitative information derived from experiments at all the structural levels. In biological terms, fibrin(ogen) may represent one of the first clear examples of the physiological function of forced protein unfolding. On the clinical side, understanding mechanisms of fibrin deformation would explain and predict clot behavior in different physiological or pathophysiological conditions related to hemostasis, thrombosis, and wound healing and may lead to new methods of prophylaxis, diagnosis, or treatment. The proposal represents a new and promising field of biomedical research, namely biomechanics of hemostasis 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 with bleeding, while very stiff clots have been associated with thrombosis and thromboembolism, which cause heart attacks and strokes. In biological terms, fibrin(ogen) may represent one of the first clear examples of the physiological function of protein unfolding. 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
Research Project (R01)
Project #
5R01HL090774-04
Application #
8267014
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Link, Rebecca P
Project Start
2009-07-20
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2014-05-31
Support Year
4
Fiscal Year
2012
Total Cost
$388,847
Indirect Cost
$141,347
Name
University of Pennsylvania
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Cines, Douglas B; Lebedeva, Tatiana; Nagaswami, Chandrasekaran et al. (2014) Clot contraction: compression of erythrocytes into tightly packed polyhedra and redistribution of platelets and fibrin. Blood 123:1596-603
Hategan, Alina; Gersh, Kathryn C; Safer, Daniel et al. (2013) Visualization of the dynamics of fibrin clot growth 1 molecule at a time by total internal reflection fluorescence microscopy. Blood 121:1455-8
Litvinov, R I; Weisel, J W (2013) Shear strengthens fibrin: the knob-hole interactions display 'catch-slip' kinetics. J Thromb Haemost 11:1933-5
Weisel, John W; Litvinov, Rustem I (2013) Mechanisms of fibrin polymerization and clinical implications. Blood 121:1712-9
Kononova, Olga; Litvinov, Rustem I; Zhmurov, Artem et al. (2013) Molecular mechanisms, thermodynamics, and dissociation kinetics of knob-hole interactions in fibrin. J Biol Chem 288:22681-92
Sun, Jessie E P; Vranic, Justin; Composto, Russell J et al. (2012) Bimolecular integrin-ligand interactions quantified using peptide-functionalized dextran-coated microparticles. Integr Biol (Camb) 4:84-92
Tsurupa, Galina; Pechik, Igor; Litvinov, Rustem I et al. (2012) On the mechanism of ?C polymer formation in fibrin. Biochemistry 51:2526-38
Tsurupa, Galina; Mahid, Ariza; Veklich, Yuri et al. (2011) Structure, stability, and interaction of fibrin ?C-domain polymers. Biochemistry 50:8028-37
Purohit, Prashant K; Litvinov, Rustem I; Brown, Andre E X et al. (2011) Protein unfolding accounts for the unusual mechanical behavior of fibrin networks. Acta Biomater 7:2374-83

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